Eurotox 2005 Abstracts

Eurotox 2005 Abstracts

Toxicology Letters 158S (2005) S1–S258 Abstracts EUROTOX 2005 Abstracts IN0VITED LECTURES Sunday, September 11, 2005 Keynote Lecture Monday, Septemb...

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Toxicology Letters 158S (2005) S1–S258


EUROTOX 2005 Abstracts IN0VITED LECTURES Sunday, September 11, 2005 Keynote Lecture Monday, September 12, 2005 PS1 Plenary Session S1 Dendritic Cell in Immunotoxicology S2 Cardiovascular Tioxicity—Clinical and Molecular Aspects W1 Nuclear Receptor Responses in Food Safety S3 Low Doses Extrapolation in Carcinogenesis W2 New Approaches to Toxicity Screening in Drug Development W3 Molecular Mechanisms in Neurotoxicology Round Table Discussion Tuesday, September 13, 2005 PS2 Bo Holmsted Memorial Lecture S4 Genetics and Individual Susceptibility W4 Integratyed Exposure and Risk Assessment of PAH W5 Quantitative Risk Assessment for Contact Sensitizers—An Attainable Goal in Public Health Protection W6 Mechanisms of Hepatogenesis (Molecular Aspects) S5 Nutritional and Safety Assessment of Foods and Feeds Nutritionally Improved Through Biotechnology S6 Framework for Integrating Human Data in Risk Assessment Wednesday, September 14, 2005 PS3 Plenary Session S7 Does Modern Toxicology Allow the Safe Use of Pesticides W7 Chemical Pollution and Development of Asthma W8 Children and Health POSTER SESSION Monday, September 12, 2005 P1 Toxicity Screening P2 Toxicity Testing and Mechanisms

0378-4274/$ – see front matter © 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.toxlet.2005.05.014

1 2 3–7 8–12 13–17 18–22 23–26 27–30 31 32–35 36–38 39–41 42–45 46–50 51–54 55 56–60 61–63 64–68

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Abstracts / Toxicology Letters 158S (2005) S1–S258

P3 P4 P5 P6 P7 P8 P9 P10 P11

Mechanisms of Carcinogenesis Quantitative Risk Assessment Allergy and Asthma Persistent Organic Pollutants—Integrated Exposure and Risk Assessment Immunotoxicology Genetic Toxicology Food Safety Development Toxicology Pesticide Toxicology

Tuesday, September 13, 2005 P12 Environment and Health P13 Metals Toxicology P14 Oxidative Stress P15 Exposure Monitoring P16 Toxicokinetics P17 Alternative Methods P18 Apopthosis and Cell Death P19 Clinical Toxicology P20 Molecular Epidemiology P21 Systemic Toxicology P22 Ecotoxicology P23 Other

INVITED LECTURES Sunday, September 11, 2005 1 CLINICS AND EPIDEMIOLOGY OF HEREDITARY BREAST CANCER J. Lubinski1 , B. G´orski1 , C. Cybulski1 , T. Huzarski1 , T. Byrski1 , J. Gronwald1 , A. Jakubowska1 , M. Stawicka2 , S. Gozdecka-Grodecka3 , M. Szwiec4 , K. Urba´nski5 , J. Mitu´s5 , E. Marczyk5 , J. Dziuba1 , P. Wandzel6 , D. Surdyka7 , O. Haus8 , H. Janiszewska8 , T. D˛ebniak1 , A. Tołoczko-Grabarek1 , K. M˛edrek1 , B. Masoj´c1 , M. Mierzejewski1 , E. Kowalska1 , H. Zientek9 , J. Pamuła9 , K. Metcalfe10 , N. Tung11 , W.D. Foulkes12 , K. Offit13 , R. Gershoni14 , M. Daly15 , Ch. Kim-Sing16 , H. Olsson17 , P. Ainsworth18 , A. Eisen19 , H. Saal20 , E. Friedman21 , O. Olopade22 , M. Osborne23 , J. Weitzel24 , H. Lynch25 , P. Ghadirian26 , P. Sun10 , S.A. Narod10 , Hereditary Breast Cancer Clinical Study Group 1 Department

of Genetics and Pathology, International Hereditary Cancer Center, Pomeranian Medical University, Szczecin, Poland; 2 Prophylactic and

P3-01–P3-17 P4-01–P4-19 P5-01–P5-05 P6-01–P6-09 P7-01–P7-25 P8-01–P8-29 P9-01–P9-27 P10-01–P10-26 P11-01–P11-34 P12-01–P12-15 P13-01–P13-45 P14-01–P14-46 P15-01–P15-31 P16-01–P16-18 P17-01–P17-26 P18-01–P18-09 P19-01–P19-40 P20-01–P20-02 P21-01–P21-04 P22-01–P22-17 P23-01–P23-04

Epidemiology Center, Poznan, Poland; 3 Poznan Medical University, Poland; 4 Regional Oncology Hospital, Opole, Poland; 5 Regional Oncology Center, Krak´ow, Poland; 6 Regional Oncology Hospital, Bielsko-Biała, Poland; 7 Regional Oncology Hospital, Lublin, Poland; 8 Department of Clinical Genetics, Bydgoszcz Medical University, Poland; 9 Oncology Center, Gliwice, Poland; 10 Centre for Research in Women’s Health, University of Toronto, Canada; 11 Beth Israel Deaconess Hospital, Boston, USA; 12 Program in Cancer Genetics, Department of Oncology and Human Genetics, McGill University, Montreal, Canada; 13 Department of Human Genetics and Medicine, Memorial Sloan-Kettering Cancer Center, New York, USA; 14 Institute of Genetics, Rambam Medical Center, Haifa, Israel; 15 Division of Population Science, Fox Chase Cancer Center, Philadelphia, USA; 16 British Columbia Cancer Agency, Vancouver, British Columbia, Canada; 17 The Jubileum Institute, Department of Oncology, Lund University Hospital, Lund, Sweden; 18 London Regional Cancer Center, London, Ontario, Canada; 19 Toronto Sunnybrook Regional Cancer Centre, Toronto, Canada;

Abstracts / Toxicology Letters 158S (2005) S1–S258 20 Hereditary

Cancer Program, Division of Human Genetics, Children’s Hospital Medical Center, Cincinnati, USA; 21 Oncogenetics Unit, Chaim Sheba Medical Center, Tel-Hashomer, Israel; 22 Center for Clinical Cancer Genetics, University of Chicago, Chicago, USA; 23 Strang Cancer Prevention Center, New York, USA; 24 City of Hope Hospital, Duarte, CA, USA; 25 Department of Preventive Medicine and Public Health, Creighton University School of Medicine, Omaha, USA; 26 Epidemiology Research Unit, Centre hospitalier de l’niversit´e de Montr´eal (CHUM), HˆotelDieu, University of Montreal, Quebec, Canada Part I. Tamoxifen And Contralateral Breast Cancer In BRCA1 And BRCA2 Carriers: An Update Women with a mutation in BRCA1 or BRCA2 face a lifetime risk of breast cancer of approximately 80% and following the first diagnosis, the 10-year risk of contralateral breast cancer is approximately 30%. It has been shown that both tamoxifen and oophorectomy prevent contralateral breast cancer, but it is not clear if there is a benefit to giving tamoxifen to women who have previously undergone an oophorectomy. Furthermore, the relative degree of protection in BRCA1 and BRCA2 carriers has not been well evaluated. We studied 285 women with bilateral breast cancer and a BRCA1 or BRCA2 mutation, and 751 women with unilateral breast cancer and a BRCA1 or BRCA2 mutation in a matched case-control study. Control women were of similar age and had a similar age of diagnosis of breast cancer and had been followed for as long as the case for a second primary breast cancer. The history of tamoxifen use for the first breast cancer was compared between bilateral and unilateral cases. The multivariate odds ratio for contralateral breast cancer associated with tamoxifen use was 0.48 for carriers of BRCA1 mutations (95% CI: 0.29–0.79) and was 0.39 for carriers of BRCA2 mutations (95% CI: 0.16–0.94). The protective effect of tamoxifen was not seen among women who had undergone an oophorectomy (OR = 0.95; 9 5% CI: 0.31–2.92) but this subgroup was small. In contrast, a protective effect of tamoxifen was apparent among women who had undergone natural menopause (OR = 0.31; 9 5% CI: 0.10–0.97) (Int. J. Cancer 2005, submitted for publication). Part II. Direct-to-Patient BRCA1 Testing: The Twoj Styl Experience


Ideally, a genetic screening program for cancer should offer testing to all women who qualify, and who wish to participate, and who might benefit from the test. As the number of preventive options for women at high risk for hereditary breast cancer expands, the demand for testing increases. However, many women do not have ready access to testing because of cost, and many others have not been recognized by their physicians to be candidates for testing. It is possible to increase women’s awareness about hereditary cancer through the popular press. Genetic testing was offered to 5000 Polish women through an announcement in a popular women’s magazine (Twoj Styl) in October 2001. 5024 women who qualified received a free genetic test for three mutations in BRCA1 which are common in Poland. One hundred ninety eight (3.9%) were found to carry a BRCA1 mutation. The overall cost per mutation detected was 630 US dollars—approximately 50–100 times less than the equivalent cost in North America. Genetic counselling was offered to women with a positive test or with a significant family history of breast or ovarian cancer. The great majority of women who took part in the program expressed a high degree of satisfaction and after one year approximately two-thirds of identified mutation carriers had complied with our recommendations for breast cancer screening (Lancet 2005, submitted for publication). Part III. Breast Cancer Predisposing Alleles in Poland Mutant alleles of several genes in the DNA repair pathway have been found to predispose women to breast cancer. From a public health perspective, the importance of a given allele in a population is determined by the frequency of the allele and by the relative risk of breast cancer that it confers. In Poland founder alleles of the BRCA1, CHEK2 and NBS1 genes have been associated with an increased risk of breast cancer, but the relative contribution of each of these alleles to the overall breast cancer burden has not yet been determined. We screened 2012 unselected cases of breast cancer and 4000 population controls for seven different mutations in these genes. Overall, a mutation was found in 12% of the cases and in 6% of the controls. Mutations in BRCA1 and CHEK2 contributed in approximately equal measure to the burden of breast cancer in Poland. A BRCA1 mutation was present in 3% of the cases. The missense BRCA1 mutation C61G was associated with a higher odds ratio for breast


Abstracts / Toxicology Letters 158S (2005) S1–S258

cancer (OR = 15) than were either of the truncating BRCA1 mutations 4153delA (OR = 2.0) and 5382insC (OR = 6.2). In contrast, a higher odds ratio was seen for truncating CHEK2 mutations (OR = 2.1) than for the missense mutation I157T (OR = 1.4). This study suggests that cancer risks may be specific for particular alleles of a susceptibility gene and that these different risks should be taken into account by genetic counsellors (Breast Cancer Res. Treat. 2005, 00: 1–6). Part IV. BRCA1-positive Breast Cancers in Young Women in Poland We identified 4778 unselected incident cases of earlyonset (<51 years at diagnosis) breast cancer in 18 Polish hospitals between 1996 and 2003. We were able to obtain a blood sample for DNA analysis from 3615 of these (76%). The proportion of cases with a BRCA1 mutation was 5.5%. The presence of a mutation was significantly associated with very early age at diagnosis (less then age 40) and with medullary histology. Forty percent of the 199 hereditary cases were either medullary or atypical medullary. Hereditary cases were slightly larger, on average, than non-hereditary cases (2.4 cm versus 2.0 cm) but were less likely to be nodepositive at diagnosis (36% versus 49%; p = 0.04). 57% of the hereditary cases had a family history of breast or ovarian cancer. A significant proportion of breast cancer cases in women diagnosed in Poland under the age of 50 years are hereditary and these cases could not be reliably identified by their family histories. The association between medullary breast cancer and the presence of a BRCA1 mutation may be greater than previously thought (Br. J. Cancer 2005, submitted for publication). Monday, September 12, 2005 PS1 Plenary Session 2 BIOMARKERS IN ENVIRONMENTAL CARCINOGENESIS RESEARCH: STRIVING FOR A NEW MOMENTUM S.A. Kyrtopoulos Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece

The term “molecular epidemiology” was proposed more than 20 years ago to describe the study of the etiology of human cancer based on the application of biomarkers in population studies. Coupled with the availability of a battery of biomarkers reflecting exposure over different time windows, early biological effects and genetic susceptibility, this approach generated high hopes of rapid progress towards the elucidation of the environmental causes of cancer. During the intervening years exquisite methods have been developed for the measurement of different biomarkers, ranging from chemical-specific assays of macromolecular adducts, through the analysis of mutation spectra to the detection of early alterations in protein structure and function. Yet, despite these advances and their application in a large number of population studies, there are only few “good news” stories to tell and the promise of substantial progress in the fight against environmental cancer remains largely unfulfilled. Currently the “-omics” revolution is further enriching the arsenal of biomarkers, moving biomarker science to a qualitatively new level. In addition to developing new tools and conceptual approaches for handling the unprecedented flood of information which is being generated thanks to current technological advances, it would seem appropriate, at this threshold, to look back at the lessons that can be drawn from more than two decades’ experience with “classical” biomarkers. S1 Dendritic Cell in Immunotoxicology 3 DENDRITIC CELL BIOLOGY: AN OVERVIEW M. Kapsenberg Academic Medical Centre of the University of Amsterdam, Department of Histology and Cell Biology, The Netherlands Dendritic cells (DC) are key to the control of the immune response by initiating protective immunity to potentially dangerous pathogens, and tolerance to innocuous enteties, such as autoantigens and allergens. DC promote the selective development of protective immunity upon their activation by recognition of pathogen-associated molecules (“pathogenassociated molecular patterns” or PAMPs) via conserved innate receptors (“pattern recognition recep-

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tors” or PRRs). For example, RNA and DNA from intracellular pathogens trigger particular sets of intracellular PRRs (e.g. Toll-like receptor 3, 7, 8 and 9) that condition DC for the production of factors (e.g. IL-12 family members, interferons) supporting the development of protective type 1 T helper (Th1) cells. In contrast, helminths express certain glycoproteins that trigger a set of poorly defined cell-bound PRR that prime DC for the production of factors (e.g. OX40L) promoting the development of protective Th2 cells. In steady state conditions DC mediate tolerance via preferential induction of regulatory T cells which contribute to tolerance against self and harmless environmental antigens by suppressing effector Th1/Th2 cells directly or via infectious tolerance. Many pathogens down-regulate immunity by inducing pathogen-specific regulatory T cells by expressing compounds that bind to negatively signalling PRR (e.g. C-type lectins) expressed by DC. Paradoxically, these regulatory T cells are also required for the host to limit damage by effector T cells, whereas the slower pathogen clearance also secures the development of strong memory. In summary, DC possess a large set of innate receptors which allow them to orchestrate an optimal balance between immunity and tolerance in various conditions. 4 INTERACTIONS BETWEEN DENDRITIC CELLS (DC) AND EPITHELIAL CELLS (EC) IN ALLERGIC DISEASE E.L. Roggen1 , F. Rousset2 , M. Lindstedt3 , C.A.K. Borrebaeck3 , G.R. Verheyen4 1 Novozymes,

Sweden; 2 L’Or´eal, Sweden; 3 University of Lund, Sweden; 4 VITO

DC plays a crucial role in the sensitisation process. Upon encounter with an allergen, DC requires interactions with other cells and factors for triggering a primary or secondary immune response. EC express features of accessory cells, such as expression of HLA-DR, costimulatory molecules, functional Fc␥R, molecules of the antigen-processing machinery, and display an ability to internalise antigen. These features may authorize them to function as immunomodulators (e.g. Amplification of memory Tcells during secondary immune responses). EC may


increase chemokine (e.g. CCL20) secretion thereby attracting DC. Epithelial human TSLP activates DC, which allow them to prime naive T-cells for the production of pro-inflammatory cytokines, while downregulating IFN-␥ and IL-10. EC may also influence the local polarization of type l and type 2 antigenpresenting cells via PGE2 by impairing the ability of maturing DC to produce bioactive IL-12 p70. PGE2 is synergistic with IL-1␤ and TNF-␣ in the induction of functional and phenotypic maturation of DC and induce IL12 p40 production. Sensitisation via the respiratory route may be Th2 skewed, possibly because the antigen recognition by DC occurs in an environment rich of airway EC-product such as PGE2 . Koyama et al. (1999), Constant et al. (2000), Kalinski et al. (2001), Krakauer (2001), Soumelis et al. (2002), Lambrecht et al. (2003), Oei et al. (2004), Pichavant et al. (2005). 5 ROLE OF DENDRIC CELLS IN THE ACQUISITION OF ALLERGIC SENSITISATION A. Cavani Laboratory of Immunology and Allergology, IDIIRCCS, Rome, Italy Skin-derived dendritic cells (DCs), such as Langerhans cells (LCs) and dermal DCs, play pivotal roles in immune surveillance because of their efficient antigen uptake and the unique capacity to prime na¨ıve T cells. Following the encounter with potential sensitizers, DCs undergo complex modifications affecting their functional properties, and migrate towards regional lymph nodes where hapten determinants recovered from the skin can be presented to T cells. DC mobilization is mainly regulated by cytokines, IL-1␤, TNF and IL-18 in particular, locally released by DCs and other skin resident cells upon hapten exposure. DC mobilization is allowed by the rearrangement of adhesion molecules and chemokines receptors. E-cadherins, which mediate homotypic adhesion of LCs to keratinocytes are rapidly downregulated. The upregulation of CCR7 is involved in LCs homing to secondary lymphoid organs, where ELC/MIP-3␤, the CCR7 ligands, are abundantly expressed. DCs migration is paralleled by their functional maturation, as indicated by the increased expression of MHC class I/class II and cos-


Abstracts / Toxicology Letters 158S (2005) S1–S258

timulatory molecules, and by the downregulation of the antigen uptake capacity. Critical consequence of hapten interaction with skin DCs is the generation of hapten epitopes for CD4 and CD8 T cells. Haptens are recognized by the immune system as trimolecular complexes formed by hapten-peptides mounted in the groove of MHC molecules. Such complexes may be generated in the skin by direct interaction of the hapten with peptideMHC complexes exposed on LCs. Alternatively, free haptens or haptens coupled to extracellular proteins can be internalized in MHC compartments, processed, mounted on MHC molecules and finally exposed on LC membrane. Once primed by skin-derived DCs, effector CD8 and CD4 T cells, bearing proper skin homing receptors, can be rapidly recruited at the site of hapten challenge, and, in sensitized individuals, mediate the tissue damage by inducing keratinocyte apoptosis and through the release of pro-inflammatory cytokines. Although critically involved in the sensitization phase, DCs are not required for the expression of skin allergies to chemicals, being professional antigen presenting cells not needed for effector T cell activation. 6 USE OF DENDRITIC CELLS IN THE DEVELOPMENT OF ALTERNATIVE APPROACHES TO ALLERGEN HAZARD IDENTIFICATION E. Corsini Laboratory of Toxicology, Department of Pharmacological Sciences, Faculty of Pharmacy, University of Milan, Via Balzaretti 9, 20133 Milan, Italy Allergy is the adverse health effect, which results from the stimulation of specific immune responses. Hypersensitivity reactions are the result of normally beneficial immune responses acting inappropriately, causing inflammatory reactions and tissue damage. Chemicals, as well as proteins, are able to induce allergic reactions following the exposure of predisposed individuals to small molecular weight materials (aptens). The two most frequent manifestation of chemical-induced allergy are contact hypersensitivity and respiratory sensitization, both of which can have serious impact on quality of life and represent a common occupational health problem.

In the screening of cosmetics, topic drugs and other chemicals for human use, it is very important, both from scientific and economic point of view, to have biological markers to characterize safety and efficacy before their use in humans. Animal studies are not practical for large-scale screening purposes and their costs are high. As a consequence, there is a growing need for in vitro assay systems to assess chemicals for skin toxicity and, if possible, for allergic potential. With the increasing understanding of the complex cellular and molecular events that induce skin sensitization and elicit allergic contact dermatitis, it is becoming possible to consider alternative approaches for hazard identification and characterization. Due to their anatomical location and critical role in skin inflammatory and immunological reactions, the use of the keratinocytes and dendritic cells as a simplified in vitro model to evaluate the potential toxicity and efficacy of chemicals destined for cutaneous application is amply justified. Although there are yet no well-characterized in vitro methods and further studies are necessary for predicting in vitro the potential of chemicals to cause contact sensitization, epidermal cells might offer the possibility to discriminate between irritant and sensitizing chemicals. In particular, this presentation will focus on the use of dendritic cells as an alternative approach to allergen hazard identification. 7 DENDRITIC CELLS AS IMMUNOSUPRESSION



N.I. Kerkvliet Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, USA Dendritic cells play a vital role in the initiation of adaptive immune responses. Through processing and presentation of antigenic epitopes, DCs induce the activation of antigen-specific T cells. In turn, activated T cells promote DC maturation and survival. Prolonged contact with DCs is necessary for optimal differentiation and survival of effector T cells. Several immunosuppressive drugs have been shown to impair the function of DCs, by inhibition of antigen uptake or suppression of cytokine production. In addition, some chemicals have been shown to reduce DC survival by inducing apoptosis thereby leading to premature ter-

Abstracts / Toxicology Letters 158S (2005) S1–S258

mination of the T cell response. Apart from direct suppression of DC function or survival, an emerging topic of interest is the ability of chemicals to specifically induce tolerogenic DCs. Inhibition of DC maturation and/or induction of IL-10 results in DCs with tolerogenic properties, and, in some cases, is associated with increased frequency of CD4+ CD25+ T regulatory cells. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent immunosuppressive chemical and AhR ligand, has been shown to produce divergent effects on DCs, namely an inappropriate activation in the absence of antigen, and decreased survival via induction of apoptosis. These effects appear to be associated with inhibition of NFkB activation. Recently, TCDD has also been shown to increase the frequency of CD4+ CD25+ T regulatory cells. Potential links between these outcomes will be discussed. Supported by NIH grants ES00040 and ES00210. S2 Cardiovascular Toxicity—Clinical and Molecular Aspects 8 IN VITRO MODELLING OF THE STRUCTUREACTIVITY DETERMINANTS OF ANTHRACYCLINE CARDIOTOXICITY G. Minotti G. d’Annunzio University School of Medicine and Center of Excellence on Aging, Via dei Vestini, 66013 Chieti, Italy Anthracyclines rank among the most effective anticancer drugs ever developed but their clinical use is limited by cardiomyopathy upon chronic administration. According to the prevailing hypotheses anthracyclines would induce cardiotoxicity by forming secondary alcohol metabolites and/or reactive oxygen species (ROS). Secondary alcohol metabolites are formed through a two-electron reduction of the carbonyl group in the side chain, and ROS are formed through a one-electron redox-cycling of the quinone moiety in the planar ring system. In many cases the clinical pattern of cardiotoxicity induced by an anthracycline does not correlate with its levels of formation of ROS or secondary alcohol metabolite in laboratory animals. Much of this inconsistency is caused by interand intra-species heterogeneity in drug metabolism.


Research on anthracycline cardiotoxicity has therefore suffered from the lack of translational models. To obviate this problem we developed an in vitro model that exploits human myocardial samples and best describes the correlates between anthracycline metabolism and clinically documented cardiotoxicity. This model has been of value in screening chemical substitutions that influence alcohol metabolite formation and its modulation by chemotherapeutics often administered in combination with anthracyclines. It also helps to predict whether an anthracycline will form ROS at pertinent membrane sites or accumulate in redox-inactive cell compartments. Results obtained with this model have offered guidelines in developing less cardiotoxic anthracyclines or anthracycline-taxane regimens. 9 PRECLINICAL ASSESSMENT OF ANTHRACYCLINE CARDIOTOXICITY IN LABORATORY ANIMALS: PREDICTIVENESS AND PITFALLS J. Robert Institut Bergoni´e, 229 cours de l’Argonne, 33076 Bordeaux-Cedex, France Doxorubicin has been for 40 years one of the most prescribed anticancer drugs, due to its important activity in haematological malignancies as in solid tumours, including sarcomas and adenocarcinomas. However, its important cardiac toxicity still limits its long-term use and prevents from reaching the optimal benefits of this drug in patients. Numerous solutions have been proposed to improve anthracycline chemotherapy and avoid the limitations encountered, such as the use of protracted infusions, the development of less cardiotoxic analogues or of special formulations and the identification of cardioprotectors. One of the problems in this respect is the availability of preclinical models able to screen rapidly these different approaches of cardiotoxicity circumvention and to provide rational bases for clinical trials. The first model which was implemented to study anthracycline cardiac toxicity is the long-term rabbit model. Weanling rabbits given weekly injections of doxorubicin or daunorubicin for 4 months developed a cardiomyopathy which was obvious from a clinical (cardiac failure) as well as from a pathological point


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of view (cytoplasmic vacuolisation, myofibrillar loss). This model has been widely used afterwards for the discovery of cardioprotective molecules. Models in other animals such as rat, mouse, pig or dog were similarly implemented, also with long-term exposures to the drug, resulting in cardiac failure and severe pathological alterations which could be graded for comparison of various anthracyclines or cardioprotectors. In the 1990s, we were seeking a model that would be able to predict the cardiotoxicity of new anthracyclines or new formulations, which would be or more rapid and easier use than the classical chronic cardiotoxicity models. We became aware by analysing the literature (1) that the damage caused by anthracyclines on cardiomyocytes was immediate after each injection, even if a physiological compensation prevented for a long time from the occurrence of heart failure; (2) that the functional efficiency of the myocardium should be affected by the anthracyclines long before the morphological alterations become detectable and quantifiable. In these conditions, we analysed the cardiac performances of isolated perfused hearts of rats that had been treated within 12 days by repetitive administrations of the molecule(s) to be tested. This model revealed to be simple, easy to implement, able to give rapid results and, simultaneously, to provide the informations which were expected from the clinical experience: (1) epirubicin appeared less cardiotoxic than doxorubicin; (2) liposomal formulations appeared less cardiotoxic than free drug formulations; (3) dexrazoxane appeared as a good cardioprotector against doxorubicin cardiotoxicity. Starting from these observations, we were then able to bring original results concerning the fact that paclitaxel potentiate doxorubicvin cardiotoxicity, but that docetaxel did not so; or that daunorubicinol was not more cardiotoxic than daunorubicin, in contrast to the fact that doxorubicinol is more cardiotoxic than doxorubicin. More recently, we also showed that a high dose of dexrazoxane (20× the anthracycline dose) brought significantly higher protection than a conventional dose (10× the anthracycline dose). Based upon these various contributions, we can encourage the use of the short-term model of isolated perfused rat heart to screen the preclinical cardiotoxicity of anthracycline molecules, formulations and combinations.

10 IN VITRO/IN VIVO CORRELATIONS: PRECLINICAL VERSUS CLINICAL ISSUES IN THE EVALUATION OF CARDIOVASCULAR LIABILITIES L. Hanson Pfizer, Kalamazoo MI, USA Noncardiac drugs that affect the cardiovascular system pose potential safety risks to patients. Many of these adverse effects can be evaluated preclinically with several in vitro and in vivo models with varying usefulness to predict clinical effects. While many of these effects are monitorable in the clinical setting, small or rare effects are difficult to detect. One recent issue to receive attention is torsades de pointes [TdP] caused by drugs that prolong the QT interval of the electrocardiogram, a measure of ventricular repolarization time. QT prolongation is a poor surrogate for TdP. Nevertheless, QT prolongation is considered a risk factor for TdP. The predictiveness of assays was not well understood due in part to variability in methods, species, and consistency in the assays reported in the literature. The Health and Environmental Sciences Institute of the International Life Sciences Institute (ILSI/HESI) outlined a set of studies to determine how well selected commonly used non-clinical assays predicted whether compounds could prolong QT. Compounds known to prolong ventricular repolarization and compounds considered safe by many years of clinical use were tested in three assays: HERG, Purkinje repolarization, and in vivo QT studies in telemeterized dogs. The data suggest that compounds that may pose a proarrhythmia risk for patients can be distinguished from safe compounds. Taken collectively, the in-vitro and in-vivo preclinical results can be integrated to develop an accurate risk assessment to support clinical safety. 11 ASSESSMENT OF QT LIABILITY IN DRUG DEVELOPMENT. REGULATORY STATUS AND CURRENT PRACTICES C. Arrigoni Nerviano Medical Sciences, Milan, Italy Since the publication, in 1997, of the CPMP Points to Consider document on “The assessment of potential

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for QT prolongation by non-cardiovascular medicinal products”, both Regulatory bodies and the Pharmaceutical industry have paid more and more attention to the conduct of careful pre-clinical studies on the subject, both in vivo and in vitro. Regulatory attention has focused on the drafting of Safety Pharmacology guidelines through the ICH process, which resulted in the approval by ICH, and acceptance by the three main regions (USA, Europe and Japan), of the ICH S7A guideline. This guideline, however, does not deal only with cardiovascular studies and does not provide guidance on QT investigations. This part has been deferred to a second guideline (ICH S7B), which is still under discussion. Nevertheless, pharmaceutical Companies have implemented screening strategies aimed at selecting compounds that do not present QT liabilities. These strategies can differ according to the pharmaceutical class, while experimental model differ according to the stage of development of the compound. Several in vitro models are employed in Discovery (radioligand binding, high throughput patch clamp, efflux and fluorescence assays). Other in vitro models applied later in the R&D process are the APD in purkinje fibers or papillary muscle and the isolated heart. The most robust and accepted in vivo test is represented by the telemetry study in conscious non-rodents. 12 PROTECTION AGAINST DOXORUBICIN—INDUCED CARDIOTOXICITY BY FLAVONOIDS A. Bast Dept. of Pharmacology and Toxicology, Maastricht University, Faculty of Medicine, Maastricht, The Netherlands The mechanisms thought to be responsible for cardiotoxicity of the antitumour agent doxorubin are related to the formation of free radicals. Iron ions play a catalytic role in this radical formation. The iron chelator ICRF-187 has been studied as a protective agent against doxorubicin-induced cardiotoxicity. However, it has the disadvantage of causing bone marrow suppressioin. We therefore searched for other effective cardioprotectors. We looked for compounds with both iron chelating and antioxidant activity. Several (naturally


occurring) flavonoids possessed these characteristics. Optimalisation led to a selection of inhibitors of membrane lipid peroxidation. Subsequently, the selected compounds were tested in preventing the doxorubicininduced negative inotropic action in the electrically paced mouse left atrium. It appeared that the semisynthetic 7-monohydroxyethyl-rutoside (monoHER) almost completely prevented doxorubicin-induced cardiotoxicity. This compound was investigated further in an in vivo model for cardiotoxicity in the mouse. With telemetry the lengthening of the ST-interval of the electrocardiogram was determined as a measure for cardiotoxicity. Pretreatment with monoHER (500 mg/kg, i.p.) was able to prevent doxorubicin-induced heart damage. The antitumour activity (both in vitro and in vivo) of doxorubicin was not affected by monoHER. Pharmacokinetic studies with monoHER in the mouse revealed the effective plasma level. Subsequently we aimed for the similar plasma levels in human volunteers after i.v. administration. This phase I study has now been finished and a phase II study, i.e. in patients is now in progress. W1 Nuclear Receptor Responses in Food Safet Assessment 13 THE ERE-Luc MOUSE: A PARADIGM OF REPORTER MOUSE FOR PHARMACOLOGICAL AND TOXICOLOGICAL STUDIES A. Maggi, P. Ciana Center of Excellence on Neurodegenerative Diseases and Dept. of Pharmacological Sciences, University of Milan, I-20129 Milan, Italy During the past decade the remarkable progress made by molecular genetics, and the possibility to manipulate cells for the expression of genes reporter of drug activity determined majors changes in the strategies for drug development. We have recently generated transgenic mouse reporting on estrogen receptor activity. The mouse was engineered with a construct carrying the luciferase reporter gene under the control of an estrogen responsive element. In addition Matrix Attachment Regions were used to flank the transgene construct in order to ensure the generalized expres-


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sion of the reporter. This model system was extensively validated to demonstrate that luciferase expression is strictly associated to the state of estrogen receptor transcriptionally activation. We show that by biochemical, immuno-histochemical, and pharmacological criteria, luciferase content reflects ER transcriptional activity and thus represents a novel system for the study of ER dynamics during physiological fluctuations of estrogen and for the identification of SERMs or endocrine disruptors. The description of such a reporter mouse allowing in vivo analysis of hormone receptor activity opens new orisons for drug discovery and toxicological studies: these novel animal models in association with in vivo imaging technologies allow for a global view of the tissues target of the drug action after acute and repeated treatment thus enabling to predict potential side effects at a very early phase of the preclinical studies; in toxicological studies these animals allow for monitoring the state of estrogen receptor activation in vivo during prolonged exposure of environmental pollutants or toxic agents. It is anticipated that further improvements of transgene architecture will lead to models which will allow for a global view of endocrine disrupter activity. Our work is supported by UE (EDERA, QLRT 200102221, EMIL and CASCADE Projects), MIUR 200258785, AIRC and Telethon References: Ciana, P., Di Luccio, G., Belcredito, S., Pollio, G., Vegeto, E., Tatangelo, L., Tiveron, C., Maggi, A., 2001. Engineering of a mouse for the in vivo profiling of estrogen receptor activity. Mol. Endocrinol. 15 (7), 1104–1113 Ciana, P., Raviscioni, M., Vegeto, E., Mussi, P., Que, I., Parker, M.G., Lowik, C., Maggi, A., 2003. In vivo imaging of transcriptionally active oestrogen receptor. Nat. Med. 9, 82–86 DiLorenzo, D., Villa, R., Biasiotto, G., Belloli, S., Ruggeri, G., Alberini, A., Apostoli, P., Raviscion, M., Ciana, P., Maggi, A., 2002. Isomer-specific activity of DDT with estrogen receptor in adult and suckling mice. Endocrinology 143, 4544–4551. Maggi, A., Ciana, P., 2005. Tailoring animals to image drugs in action. Nat. Rev. Drug Discov. 4, 249–255. PATENT REQUEST: A transgenic mouse for the screening, pharmakokinetics and pharmacodynamic profiling of ligands active on intracellular receptors and estrogen receptors, methods for its preparation. Patent request n. MI2000A 001503 04/07/2000, European and USA extension PCT/EP 01/07622

14 RISK CHARACTERIZATION OF DIOXINS L.S. Birnbaum Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or “Dioxin”) is the most toxic member of a family of structurally related compounds which are ubiquitous environmental pollutants. The most potent of these classes, the polyhalogenated dibenzo-p-dioxins and furans, were never produced intentionally, but are unwanted contaminants of certain industrial and combustion processes. The polyhalogenated biphenyls, napthalenes, and azo- and azoxybenzenes were made commercially, and specific isomers are dioxin-like in their properties. The major sources of PCDDs and PCDFs in the United States today are uncontrolled incineration and other combustion processes. Other sources include metal refining and reservoir sources. Deposition worldwide is associated with atmospheric transport of new emissions as well as environmental recycling of contaminated soils and sediments. Their resistance to physical, chemical, and biological degradation has resulted in extreme persistence and bioaccumulation up the food chain. Dioxins induce a broad spectrum of biological effects in multiple species. Adverse effects in reproduction of fish, birds, and mammals in the wild have been linked to exposure to this class of chemicals. Poisoning incidents have led to loss of domestic animals including chickens, cattle, and horses. A plethora of effects have been observed in laboratory animals, ranging from lethality at high doses to subtle biochemical responses. Many of the effects are widely distributed, while others are more species, sex, organ, tissue, or age specific. Dioxin has been shown to immunotoxic, neurotoxic, developmentally toxic, hepatotoxic, dermally toxic, a reproductive toxicant, and carcinogenic. (This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.)

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15 TRANSGENIC XENOPUS AS A SMALL MODEL ORGANISM FOR DETECTING NUCLEAR RECEPTOR DISRUPTION N. Turque, K. Palmier, S. Le M´evel, C. Alliot, B.A. Demeneix UMR CNRS 5166, Evolution des R´egulations Endocriniennes, Dept. Regulations, Development and Molecular Diversity, Museum National d’Histoire Naturelle, 7 Rue Cuvier, 75231 Paris Cedex 05, France Increasing numbers of substances present in the environment are postulated to have endocrine disrupting effects on vertebrate populations. The data on disruption of thyroid signalling is fragmentary, particularly at the molecular level. Thyroid hormone (tri-iodo thyronine, T3 ) acts principally by modulating transcription from target genes, thus thyroid signalling is particularly amenable to analysis with a transcriptional assay. Also, T3 orchestrates amphibian metamorphosis, thus providing an exceptional model for identifiying thyroid disrupting chemicals. We therefore combined these two advantages to develop a method for following and quantifiying the transcriptional action of T3 in Xenopus leavis tadpoles. This technology provides a means of assessing thyroid activity at the molecular level in a physiologically relevant situation. Moreover, translucent tadpoles are amenable to “on-line” imaging with fluorescent reporter constructs that facilitate in vivo measurement of transcriptional activity. We adapted transgenesis with thyroid hormone responsive elements coupled to either luciferase or GFP to follow T3 -dependent transcription in vivo. To reduce time of exposure and to synchronise responses, we optimized a physiological pretreatment protocol that allowed us to assess T3 effects and T3 disruption within 48 h. This pretreatment protocol was based on a short (24 h), weak (10−12 M) pulse of T3 that induced thyroid hormone receptors, facilitating and synchronizing the transcriptional responses. This protocol was successfully applied to somatic and germinal transgenesis with both reporter systems. Finally, we show that the transcriptional assay allows detection of the thyroid disrupting activity of low concentrations (10−10 M) of acetochlor, a persistent herbicide. Furthermore, this approach can


be adapted to follow other endocrine signals, such as estrogen, in an in vivo context. This work is supported by CNRS, MNHN, ANVAR and EU contract no. 506319 (CASCADE). 16 RECOMBINANT CELLS AS REPORTER MICE TO ASSESS SIGNALING THROUGH THE ANDROGEN RECEPTOR O.S. Janne University of Helsinki, Helsinki, Finland Assays in cultured cells and in live animals that will permit monitoring of signaling through androgen receptor are valuable tools to assess potential androgenic and/or antiandrogenic activities of chemical compounds, such as those with putative endocrine disrupting properties. We have developed a recombinant cell assay to measure androgen bioactivity directly from human serum. This assay takes advantage of the ligand-dependent interaction of the amino-terminal region and the ligandbinding domain of the androgen receptor that were expressed in the assay as separate polypeptides together with a receptor coactivator, ARIP3, in recombinant cells. By the use of this bioassay, we have found that there is suppressed androgen bioactivity in serum of infant boys with cryptorchidism and of men with prostate cancer and that circulating androgen bioactivity mediates the tempo of pubertal maturation. In addition, androgenic and/or antiandrogenic activities of a number of potential endocrine disrupting compounds were evaluated. For in vivo studies, we have generated a transgenic mouse line that has an androgen reporter construct inserted into its germline. The reporter gene (luciferase) is driven by minimal thymidylate kinase promoter and duplicate androgen response elements of the mouse slp gene. Androgen-dependent activation of the reporter gene is observed in most of the tissues known to represent androgen targets. Collectively, these and similar other recombinant cell and reporter mouse approaches have turned out to be very useful means to examine properties of compounds that potentially interfere with of modulate sex steroid hormone action.


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17 INTERACTIONS OF SELECTIVE RETIOID RECEPTOR LIGANDS TO INDUCE SYNERGISTIC TERATOGENIC EFFECTS Heinz Nau Department of Food Toxicology, University of Veterinary Medicine Hannover, D-30173 Hannover, Germany Vitamin A and retinoids are potent teratogens in laboratory animals and the human. Most developmental effects of retinoids are mediated by the nuclear retinoic acid receptors (RAR␣, RAR␤ and RAR␥, as well as the retinoid X receptors RXR␣, RXR␤, and RXR␥). It has been shown by a number of previous studies that RAR-RXR-heterodimers may be decisive for mediating the retinoid-induced temporal- and special-specific effects in the embryo. We tested this hypothesis in vivo by administering a dose of a specific RAR-ligand which by itself induces a weak or no teratogenic response, simultaneously with a RXRligand which by itself does not elicit any teratogenic response. The compounds were administered on day 8 or 11 of gestation in the mouse. Both ligands administered together were shown to induce potent teratogenic effects such as the induction of cleft palates, limb defects and neural tube defects including a high percentage of Spina bifida aperta. Also the natural RXR-ligand phytanic acid which is an endogenous fatty acid derived predominately from milk and meat, and is present in ␮M concentrations in our blood, can synergize with a specific RAR␣ ligand (Elmazar and Nau, Arch. Tox. 78: 660, 2004). These studies indicate that teratological risk assessment of retinoids must take into account possible interactions at the molecular level, and natural RXR-ligands such as the nutritional compound phytanic acid may induce synergistic teratogenic effects if combined with an RAR ligand. Supported by the EU-programme BoneTox (QLK4CT-202-02528) and the RTN “Nutriceptors” (HPRNCT-2002-00268).

S3 Low Dose Extrapolation in Carcinogenesis 18 HYPOTHESIS TESTING AND CHOICE OF A DOSE–RESPONSE MODEL D. Oughton Department of Plant and Environmental Sciences, The ˚ Norway Norwegian University of Life Sciences, As, See Book of Late Abstracts. 19 DOSE–RESPONSE OF CHEMICALLY-INDUCED CANCER AND REGULATORY IMPACT H.M. Bolt Institut f¨ur Arbeitsphysiologie an der Universit¨at Dortmund, Leibniz Research Centre for Working Environment and Human Factors, Ardeystr. 67, D-44139 Dortmund, Germany There is agreement that it should be distinguished between genotoxic and non-genotoxic chemicals. The risk assessment approach used for non-genotoxic chemicals implies insertion of an uncertainty (safety) factor permits the derivation of permissible exposure levels at which no relevant human cancer risks are anticipated. For genotoxic carcinogens, there is an array of possibilities. Positive data of chromosomal effects only, in the absence of mutagenicity, may support the characterisation of a compound that produces carcinogenic effects only at high, toxic doses. Non-DNA-reactive genotoxins, such as topoisomerase inhibitors or inhibitors of the spindle apparatus are considered in this respect. In such cases, arguments are in favour of the existence of “practical” thresholds. It has been proposed to basically distinguish between “perfect” and “practical” thresholds. There is consensus that for non-DNA reactive genotoxins such as aneugens (aneuploidy, chromosome loss, non-disjunction) thresholds should be defined. It is discussed as to whether the identification of possible threshold effects should also include other mechanisms of genotoxicity, in addition to aneugenic effects. Oxidative stress as an important mechanism is triggered by exposure to exogenous factors such as UV and ionising radiation, anoxia and hyperoxia, and by chemicals producing reactive oxygen species. The idea is receiving increased

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support that ROS-mediated processes of carcinogenesis have practical thresholds. The existence of “endogenous” DNA adducts has been accepted, and possible regulatory implications of the presence of endogenous carcinogens have been discussed. It is becoming evident that a diversity of methods of carcinogenic risk extrapolation to low doses must be considered, dependent on modes of action. 20 CANCER RISK IN THE POPULATION OF XRAY WORKERS IN POLAND D. Kluszczy´nski, J. Jankowski ´ Nofer Institute of Occupational Medicine, ul. Sw. Teresy 8, 90-950 Ł´od´z, Poland The term “cancer” means the sentence of death for many individuals each year and worldwide. In Poland this sentence hear about 87 thousand people a year. Cancer is the second cause of the death in Poland (24% of all deaths), when the first place take circulation diseases (47%). The risk of the mortal cancer (expressed as ASR) increases in the period 1959–2002 in Poland two times in the men population and 1.3 times in the women population. It must be notice that almost all inductions of the cancers have the genesis of the environmental agents and the human habitat. International Agency for Research on Cancer (IARC) counts 95 known human carcinogenic agents and 307 probably or possibly human carcinogenic agents. Among them, ionising radiation is one of the most known agents. The observed increase of the cancer number at all and the very basic knowledge of the correlation between ionising radiation and the cancer lead more and more people to the opinion that the any work in the ionising radiation conditions equals the cancer in their older age. Nofer Institute of Occupational Medicine manages the largest Polish data base of occupationally exposed X-ray workers. The data collected in this data base extend from the year 1966 till nowadays. Each year, the annual doses of about 30 thousand workers are calculated. The paper will present the analysis of the long term doses of the occupationally exposed Polish workers with the focus on the high-exposed subgroup selected from the data base. The carried-out calculations allow quantifying the real risk of the death due to the occupational cancer. The risk calculations are based


on BEIR V and NCI-CDC reports. The conclusion is that the lifetime risk of occupationally inducted mortal cancer in the population of Polish X-ray workers is very low, even in the high-exposed subgroup. 21 WHAT CONTRIBUTES TO THE SHAPE OF A DOSE-CANCER INCIDENCE CURVE? W.K. Lutz1 , R.W. Lutz2 , M.E. Andersen3 1 Department

of Toxicology, University of W¨urzburg, 97078 W¨urzburg, Germany; 2 Seminar for Statistics, Swiss Federal Institute of Technology, 8092 Z¨urich, Switzerland; 3 CIIT Centers for Health Research, Research Triangle Park, NC 27709, USA Cancer risks at low dose must be estimated on the basis of extrapolations of dose-response curves. Mechanistic considerations are usually put forward to postulate a linear relationship for DNA-reactive carcinogens, while other modes of action may allow a discussion of nonlinearity. This focus overlooks the fundamental difference between (i) dose–response relationships for continuous measures of early biochemical endpoints, such as levels of DNA adducts or rates of DNA replication, and (ii) dose–incidence relationships for mutation or cell division. The latter are quantal (binary) effect measures that answer the question “yes or no”? Each data point of a dose-incidence curve divides the group into two subgroups, those who show the effect (e.g., individuals who manifest a tumor within a given period of observation; proportion of cells triggered to divide) and those who do not; it separates the more susceptible individuals from the more resistent. A dose–incidence curve can be obtained by superimposing the relationship between dose and early response with the tolerance distribution for the binary effect. This procedure is illustrated for the induction of cell division, using a variety of functions and parameters, and including background processes. We show that the susceptibility distribution dominates the shape of the dose–incidence relationship. Mechanistic considerations appear to be less important. On the other hand, modes of action are crucial in the search for factors that may render individuals particularly susceptible. For cancer prevention, elimination of critical susceptibility factors and exposure reduction in susceptible subgroups are options that have so far not been the given appropriate attention.


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22 LOW DOSE NON-LINEARITIES IN EXPERIMENTAL CHEMICAL HEPATOCARCINOGENESIS G.M. Williams, M.J. Iatropoulos, A.M. Jeffrey, C.E. Perrone Director Environmental Pathology and Toxicology, New York Medical College, Valhalla, NY 10595, USA Our laboratory has been investigating the possibility that rat liver carcinogenicity induced by DNAreactive carcinogens exhibits nonlinearities and practical thresholds in a low subtoxic dose range. In this research, we have conducted a series of exposureresponse studies using two well-studied hepatocarcinogens, 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN). In these studies, exact intragastric doses of carcinogen were administered during an initiation segment (IS) of 10–16 weeks followed in some experiments by phenobarbital (PB) as a liver tumor promoter for 24 weeks to elicit manifestation of initiation. The cumulative doses (CDs) at the end of ISs ranged for AAF from 0.094 to 282.2 mg/kg and for DEN from 25.5 to 306.6 mg/kg. The findings for AAF in the IS can be summarized as follows: (1) the earliest parameter to be affected with administration of low doses was the appearance of DNA adducts (around 4 weeks), followed at higher dose by cell proliferation; (2) formation of DNA adducts was nonlinear, with a noobserved effect level (NOEL) at a CD of 0.094 mg/kg and a plateau at higher doses (94.1 mg/kg); (3) cytotoxicity (necrosis) showed a NOEL at a CD of 28.2 mg/kg; (4) compensatory hepatocellular proliferation showed a NOEL at a CD of 28.2 mg/kg and was supralinear at a high CD (282.2 mg/kg); (5) formation of preneoplastic hepatocellular altered foci (HAF) showed a NOEL at a CD of 28.2 mg/kg, and was supralinear at a high CD (282.2 mg/kg); (6) a NOEL (CD 28.2 mg/kg) was found for tumor development and the exposure-response was supralinear. Similar findings were made for DEN. We interpret these findings to reflect practical thresholds for hepatocellular initiating effects of these carcinogens and exaggerated responses at high cytotoxic doses. Thus, mechanisms of carcinogenesis differed between high and low doses and extrapolation from high dose effects to poten-

tial carcinogenicity at low doses, at which no measurable effects on any parameter of liver function or structure occur, is not warranted unless similar mechanisms are demonstrated to be operative in both dose ranges. W2 New Approaches to Toxicity Screening in Drug Development 23 VALIDATION OF IN VITRO SYSTEMS T. Hartung, ECVAM Team EU Joint Research Centre, European Centre for the Validation of Alternative Methods, Ispra, Italy Starting with the animal welfare Directive from 1986 and continuing until most recent chemicals and cosmetics legislation, Europe has laid the ground for the implementation of alternative methods. In order to meet these political expectations, a couple of technical and strategic developments became necessary: • An analysis of current in vivo test performance to set benchmarks for alternatives. • An analysis of the frequency (prevalence) of toxic health effects in different areas of test application. • An inventory and database of the alternative methods available. • A coached development of lacking tests also making use of novel technologies. • An acceleration and international harmonisation of the validation process and regulatory implementation. • A development of quality assurance systems for in vitro methods such as Good Laboratory Practice and Good Cell Culture Practice. • A transition from single tests as stand-alone replacements to the composition of test strategies and their validation. The European Centre for the Validation of Alternative Methods (ECVAM) has played a proactive role in all these processes coordinating many stakeholder activities. A review of the state of these developments shall be given, in order to show how a new type of evidence-based toxicology is emerging which is based on validated and quality controlled test strategies.

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24 HEPATIC CYTOCHROME P450 REDUCTASE NULL MOUSE AS A TOOL TO IDENTIFY A SUCCESSFUL CANDIDATE ENTITY C.J. Henderson, G.J. Pass, R. Finn, C.R. Wolf Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, University of Dundee, Ninewells Hospital & Medical School, Dundee, DD1 9SY, UK Cytochrome P450s play a pivotal role in the metabolism of drugs and xenobiotics, and have been intensively studied over many years. Much of the work carried out on the role of hepatic cytochrome P450s in drug metabolism and disposition has been done in vitro, and has yielded vital information on P450 regulation and function. However, additional factors such as route of administration, absorption, drug transporters, renal clearance and extra-hepatic P450s, make it difficult to extrapolate from in vitro data to in vivo pharmacokinetics. A number of CYP knockout mice have been generated, although many have been of limited usefulness due either to embryonic/perinatal lethality, or to the functional redundancy inevitably found in a large family of isoenzymes. We have developed a mouse line (HRN) in which cytochrome P450 oxidoreductase (POR), the unique electron donor to cytochrome P450s, is deleted specifically in the liver, resulting in the loss of essentially all hepatic P450 function. The HRN mouse, although having disturbances in lipid and bile acid homeostasis, develops and breeds normally. We have used the HRN mouse as a model to establish the role of hepatic versus extra-hepatic metabolism in drug metabolism and disposition, and also to investigate the relationship between drug toxicokinetics and therapeutic effect, initially with the chemotherapeutic prodrug cyclophosphamide. 25 SHORT-TERM IN VITRO ASSAYS FOR EMBRYOTOXICITY M. Longo, S. Zanoncelli, D. Manera, M. Brughera, P. Colombo Department of Preclinical Development, Nerviano Medical Sciences S.r.l., Viale Pasteur 10, 20014 Nerviano, Milan, Italy


A panel of in vitro models has been implemented in our laboratories to provide an early insight into the teratogenic potential of new drugs. This panel was created by using complementary models that allow important stages and events of embryonic development to be mimicked. It includes the whole embryo culture model, the micromass teratogen assay, the frog embryo and zebrafish embryo teratogenesis assays. The use of complementary tests provides a broad picture of developmental hazard of a drug or it allows a case-by-case selection of the optimal screening model for different classes of compounds. The panel was utilized to evaluate a wide range of compounds from different therapeutic classes. It proved capable of identifying many substances likely to exert effects on embryonic development. It helps in designing more reliable treatment schedules for in vivo embryofetal developmental toxicity studies; it helps in ranking compounds within a homologous series and provides valuable information to investigate mechanisms of pathogenesis and to interpret data coming from in vivo animal studies. Although in vitro models do not represent replacements for current in vivo animal tests as a whole because they are not accepted by Regulatory Agencies as a basis for risk assessment, data obtained can serve to reduce the attrition rate in drug discovery and can help in explaining in vivo data and in extrapolating them to humans. 26 IN VITRO APPROACHES FOR IMMUNOTOXICITY SCREENING J. Dastych International Institute of Molecular and Cell Biology in Warsaw & Center of Medical Biology and Microbiology Polish Academy of Sciences, Poland The predictive immunotoxicity testing is a complicated process that requires the battery of tests. Most of the existing tests for immunotoxicity employ experimental animals. There is however a growing number of new in vitro tests that could be implemented in the process of safety evaluation of chemicals and drugs. Most of these assays determine changes in the phenotype of cultured immune cells upon in vitro exposure to tested chemicals. The endpoints employed include cell surface antigens expression, proliferation rate,


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and cytokine expression. Tests that releay on cytokine expression include whole blood assay, allergenicity assays in established keratinocyte cell lines, and fluorescent cell chip assay. The fluorescent cell chip assay has been developed specifically for fast screening of large number of compounds. This test is based on a panel of geneticaly modified reporter cell lines that regulate the expression of fluorescent protein in the same way as they regulate expression of cytokines. Thus changes in fluorescence intensity represent changes in cytokine expression with one cell line per cytokine. Consequently this technique conform to efficiency expected from high throughput screening assay. In a prevalidation effort we analyzed more than 60 compounds in the standardized experimental protocols. Tested compounds generated reproducible patterns of changes in fluorescence that allows for the hierarchical clustering of their expected activities based on bioinformatics approach. Resultant classification revealed good correlation with available immunotoxicity data from PubMed and the National Toxicology Program USA databases. In conclusion, there are new promising approaches for in vitro screening of chemicals for their immunotoxicity that if properly validated could be employed in chemical safety evaluation. W3 3 Molecular Mechanisms in Neurotoxicology 27 INSIGHTS INTO NEUROTOXICITY FROM GENETIC MANIPULATION OF EXPERIMENTAL MODELS P. Glynn MRC Toxicology Unit, University of Leicester, UK Neuropathy target esterase (NTE) is the putative site of action of those organophosphorus compounds which induce a degeneration of long nerves in the peripheral and central nervous systems. More than 35 years have elapsed since the discovery of NTE, but only recently has light been shed on the biological functions of this protein. Cloning NTE revealed the presence of homologues in species from bacteria to man. By studying yeast mutants, we have shown that NTE degrades phosphatidylcholine (the major mem-

brane lipid of eukaryotic cells) to soluble products. In Drosophila mutants, lack of NTE activity causes accumulation of phosphatidylcholine and progressive neurodegeneration. Similar neuropathological changes are evident in mice with brain-specific deletion of the NTE gene. Our current studies are seeking evidence that disruption of phospholipid homeostasis underlies organophosphorus-induced neuropathy. 28 ANALYZING MECHANISM OF NEUROTOXICITY AND NEUROPROTECTION USING GENE EXPRESSION AND PROTEIN PROFILING M.B.H. Youdim Eve Topf and NPF Centers of Excellence for Neurodegenerative Diseases Research Technion, Rappaport Family Faculty of Medicine, Haifa, Israel The antiParkinson’s disease (PD) drug, rasagiline and antioxidants possess concentration dependent neuroprotective and neurorescue activities In Vitro and In Vivo employing neuronal culture preparations and In Vivo models Studies have indicated that their actions is concentration dependent with involving Bcl-2 family proteins at mitochondrial permeability transition pore, PKC-dependent MAPkinase pathway and other anti and proapoptotic genes. To reveal these mechanisms two methods were adopted: genomic and proteomic profiling systems, in the neurotoxicity of MPTP mouse model of PD. RNA, and protein fraction were produced from midbrains of mice, cDNA was synthesized and was hybridized to microarray gene chip (Clontech, USA), containing ∼2000 genes, while proteins were detected and analyzed in Powerblot Proteomic array system (BD, USA) containing over 1000 mAbs. Gene analysis demonstrated that MPTP neurotoxicity involves pro-apoptotic mechanism and proteomics indicated that the JNK pathway is recruited by MPTP, in addition to Fas ligand, nNOS, glutamate receptor, and phosphorylated form of GSK-3␤. Proteomic analysis confirmed the gene expression profile related to MPTP neurotoxicity Rasagiline reversed the pro apoptotic action of MPTP, enhanced anti-apoptotic Bcl-2, AKT, PKC, growth factor and in particular ␤-NGF, its downstream transcription factors: EGR1 and EGR3

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gene expressions. Rasagiline’s ability to up regulate pro-survival proteins and signaling of Ras, PI3K, AKT and PKC indicates that its neuroprotective activity is partly mediated via neurotrophic factors.



M. Dyzma

A. Moretto

At the dawn of a new Millennium “stress” is a word on everyone’s lips. Despite 60 years of intense research effort, we are only just beginning to unravel the complex interactions between systems in the body which underlie this vital and yet tantalizingly obscure phenomenon. Selye’s description of the General Adaptation Syndrome was the driving force in stress research that led to recognition of the adrenal’s role in the stress response. Briefly—Selye’s theory based on the thesis, that stress response is a unified form of coping with adverse situations/stimuli. Stressors could be anything, actual or perceived, that threaten to disrupt the homeostasis. The stress response on the other hand is a cascade of hormonal and biochemical events that has evolved to restore homeostasis and promote survival. Successful control of the hormonal response to stress is essential for normal functioning. The hormones and other physiological agents that mediate the effects of stress on the body have protective and adaptive effects in the short run and yet can accelerate pathophysiology when they are over-produced or mismanaged. Research over the last few years has shown that glucocorticoids can damage the nervous system severely. For example, they are capable of killing neurons in the hippocampus, or lead to the apical dendrites atrophy in CA1/CA3 region. Considering recent data obtained in our laboratory, we have demonstrated that even a single experience with major stressors in the form of social defeat or immobilization may have long-term consequences ranging from hours to days and weeks. Moreover preexposure to non-chemical stressors may alter a subject’s vulnerability to the same stimulus as well as to other stimuli, unrelated to the initiating stimulus, i.e. neurotoxic agents. These findings have disturbing implications. Procedures run in laboratories concentrated on neurotoxicological studies often may include highly (or extremely) stressful conditions, i.e. immo-

Universit`a degli Studi di Padova, Dipartimento di Medicina Ambientale e Sanit`a Pubblica, Padova, Italy Alterations in the nervous system may be identified and characterised based on an integrated assessment of functional (neurological, behavioural, and physiological) and neuropathological changes using appropriate biomarkers. A number of methods can evaluate changes in sensory, motor, autonomic and cognitive functions. However, only when neurobehavioral effects are correlated with neurochemical/neuropathological effects some insight on the mechanistic events can be gained. Acute neurobehavioral effects related to acetylcholinesterase inhibition by organophosphates or carbamates are the typical example. For other (groups of) compounds such a correlation has yet to be found or demonstrated, and a mechanism of action identified. The use of neurotoxicant with known mechanism/mode of action should allow the proper interpretation of neurobehavioral tests and put them in a biologically plausible perspective. In fact, in the absence of such information the interpretation of the results of neurobehavioral tests is difficult. This is particularly true when we are confronted with statistical significant changes of isolated parameters, or with systemic toxicity which may complicate, although not always preclude, the interpretation of those changes as evidence of neurotoxicity. A large number of neurobehavioral endpoints are available in experimental animals and many compounds have been tested. However, the biological significance of subtle changes of certain end-points has not been established in the absence of proper animal models which elucidate the relationships between molecular/cellular events and clinical/neurobehavioral manifestations.

Nofer Occupational Medicine Institute, Department of Toxicology and Carcinogenesis, Sw. Teresy 8; 9 0-950 Lodz, Poland


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bilization, which has never been taken under consideration in evaluating final toxicity. Likely social interactions between animals in breeding colony as well as conditions of breeding may have considerable contribution to final result of conducted studies. Thus, understanding the principles of molecular logic that underlie signal transduction and information processing in biological systems occurring under stressful conditions may be crucial in neurotoxicity assessment. Tuesday, September 13, 2005 PS2 Bo Holmstedt Memorial Lecture

Individuals with at least three variant alleles had a statistically significant odds ratio for being in the highest tertile of adducts compared with those with undetectable adducts (three alleles, adjusted OR = 5.07, 95% CI = 1.29–19.9; four alleles, adjusted OR = 5.03, 95% CI = 1.18–21.45; five alleles, adjusted OR = 7.65, 95% CI = 0.94–62.20). The combined effect of multiple variant alleles may be more important than the investigation of single SNP for example in modulating DNA repair capacity. Hence, the importance of focussing on gene pathways in the study of gene-environment interactions.



See Book of Late Abstracts.

I. Cascorbi

S4 Genetics and Individual Susceptibility

Institute of Pharmacology, University Hospital Schleswig-Holstein, Kiel, Germany E-mail address: [email protected]

32 SINGLE AND MULTIPLE GENES AS DETERMINANTS OF INDIVIDUAL SUSCEPTIBILITY P. Vineis Imperial College London and University of Torino, Italy Most genes act in a sequence or in cascades. This is typical, for example, of DNA repair genes, but also of metabolic genes (Phase I enzymes activate and Phase II enzymes deactivate carcinogens). Genotyping according to pathways is likely to be more rewarding then genotyping for single SNP, for both biological plausibility and for statistical power. For example, Matullo et al. (2003) have investigated the role of SNPs in three DNA repair genes (XRCC1-Arg399Gln, exon 10; XRCC3-Thr241Met, exon 7 and XPD-Lys751Gln, exon 23), and their combination, in modulating the levels of “bulky” DNA adducts in a population sample of healthy individuals. DNA-adduct levels were measured as Relative Adduct Level (RAL) per 109 nucleotides by 32 P-post DNA labeling assay in white blood cells from peripheral blood. Such DNA adducts express a particular kind of damage to DNA that is associated with exposure to aromatic chemicals. They found a dose-response relationship between the number of “atrisk” alleles and the levels of adducts (p = 0.0046).

The inter-individual drug effects are subject to substantial variability, e.g. one out of 300 subjects are at high risk to develop bone marow suppression after treatment with azathioprine or 6-mercaptopurine. More than 3% of psychiatric patients suffer from clozapine-induced leukopenia or even agranulocytosis. There are multiple reasons based on pathophysiological factors and environmental interactions, but also genetic characteristics. Already in the early fifties it had been observed that patients, who received the tuberculostatic drug isoniazid, excreted the acetylated product in the urine with different rates. It was shown that among slow acetylators alternative pathways of detoxification of arylamines such as N-hydroxylation via cytochrome P4501A2 may increase the risk of peripheral neuropathies or liver injury. Since then, groundbreaking successes have been achieved in the field of pharmacogenomics and toxicogenomics. In particular, the identification of hereditary polymorphisms in genes of the cytochrome P450 system and phase II-enzymes such as TMPT contributed considerably to the explanation of the individually varying pharmacokinetics of a number of drugs. Furthermore, hereditary variations in genes of membrane drug transporters were recently discovered. Along with these factors which can influence pharmacokinetics, strong efforts have been undertaken

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to clarify the role of genetic polymorphisms in receptors or signal transduction proteins modulating drug efficacy. However, pharmacogenomics is trying to elucidate also the complex interaction of many polymorphic genes in order to explain and to develop improved therapies particularly for common diseases such as cardiovascular disorders and cancer. Particularly for malignant diseases such as bladder or lung cancer, polymorphic foreign compound metabolizing enzymes have been identified as susceptibility factors, modulating an individual’s cancer risk dependent on the extent of environmental exposure. In face of millions of single nucleotide polymorphism currently discovered, it represents a technical as well as a logistic challenge to identify suitable markers of clinical outcome or even individual risk factors. It can be expected that the early adaptation of a therapy regimen to genetic traits could help to avoid side effects and improve the clinical outcome of pharmacotherapy. 34 ETHICAL IMPLICATIONS OF GENETIC PROFILING FOR SUSCEPTIBILITY K. V¨ah¨akangas Department of Pharmacology and Toxicology University of Kuopio POB 1627, FIN-70211 Kuopio, Finland The knowledge on links between genetic defects and diseases accumulates with growing speed, increasing possibilities to detect susceptibilities by genetic profiling. This opens up new ways to improve health. If the disease is preventable, prevention measures can be implemented and if there is cure, early diagnosis probably improves the prognosis. It has also been argued that people can plan better for their life decisions, if they have a better idea of their putative future health. However, people may also get depressed and hopeless of the prospect of a serious illness. Family members free of the trait may feel guilty of their better luck. In addition to these emotional drawbacks, there are also other ethical implications that should be seriously considered before large-scale profiling studies or clinical programs. It has to be decided at which validation stage methods for profiling should before information can be given to individuals. For an adult, an informed consent both in research and in clinical care is normally sought out. It is being discussed, how the informed consent


should be handled in the case of children. Genetic profiling reveals information that sticks to the person for life. In the case of small and unborn children, it will be the decision of the parents. When the child is at the age he/she can decide, there is no longer a possibility to retract from the knowledge. The seriousness of the disease and strength of association of the genetic profile with the disease affect the potential social consequences. 35 HOW WILL GENETICS AFFECT THE USE OF DRUGS IN INDIVIDUAL PATIENTS? A. Hirvonen Biomonitoring Laboratory, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FI-00250 Helsinki, Finland Commonly, drugs are designed for the extensive metabolizing (category) of individuals that show the expected response to a standard dose of drug. However, as a result of polymorphisms in genes encoding drug receptors, drug transporters and drug metabolizing enzymes, the same drug may be metabolized at different rates between individuals. Poor metabolizers (PMs) lack active enzyme alleles and may not respond due to lower than expected concentration of active metabolite, or suffer from adverse drug reactions (ADR) due to an increased concentration of active drug. Ultrarapid metabolizers (UMs), on the other hand, may not reach therapeutic levels of active drug, or experience an ADR due to higher than expected concentrations of active metabolite. The term ‘pharmacogenetic test’ refers to an assay to study the inter-individual variations in conjunction with drug therapy. To date rather simple new techniques, such as polymerase chain reaction (PCR)based assays, enable precise and fast estimation of an individual’s metabolic capacity associated with a specific enzyme from a tiny amount of DNA. These tests could therefore easily be used to improve the safety of patients during clinical trials, together with establishing efficacy of the drug under development. The tests could also aid decision making concerning further testing of specific drug compounds in a defined patient subgroup, and speed up drug development by allowing progress of multiple compounds by rapid clarification of underlying characteristics.


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W4 Intagrated Exposure and Risk Assessment of PAHs 36 MECHANISM-BASED APPROACHES TO IMPROVE CANCER RISK ASSESSMENT OF AMBIENT AIR PAHs J.H.M. van Delft1 , S.A. Kyrtopoulos2 , the AMBIPAH project team 1 Maastricht

University, POBox 616, 9200MD Maastricht, The Netherlands; 2 National Hellenic Research Foundation, 48 Vassileos Constaninou Avenue, Athens 11635, Greece Polycyclic aromatic hydrocarbons (PAHs) are a large family of chemicals, which includes a number of powerful carcinogens. Because of the widespread human exposure to PAHs, the potential risks of adverse health effects, especially cancer, are a major public health concern. The setting of exposure limits for ambient air PAHs is complicated by the fact that they are formed and emitted as complex mixtures consisting of many hundreds of related compounds with varying structure and biological activity. The objective of the AMBIPAH project is to help improve the accuracy of the risk assessment of PAHs. Therefore, the effects of selected PAHs, individually or as mixtures, on different end-points and cellular pathways relevant to tumour-initiation and tumour-promotion are examined in vitro and in vivo. In cell lines and precisioncut slices and microsomes from rat lung and liver effects on CYP1A1 expression and binding to the Ah receptor (AhR), global gene expression profiles, PAH metabolism and DNA damage, and mutagenicity are examined. In short-term animal studies metabolism and kinetics, binding to DNA, gene expression profiling and mutagenesis in liver and lung, and binding to serum albumin is investigated. An overview will be presented of results achieved so far. EU Project no. QLRT-2001-02402.

37 THE GENOTOXIC EFFECT OF PAH MIXTURES ON HUMAN CELLS IN VITRO AND IN VIVO—EVALUATION OF BIOMARKER DATA AND SUSCEPTIBILITY FACTORS FOR RISK ASSESSMENT R.J. Sram, B. Binkova, J. Topinka, P. Rossner, O. Beskid, I. Chvatalova, O. Sevastyanova Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Prague, Czech Republic Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population as well as affect pregnancy outcomes. Organic compounds adsorbed to air particles (PM10) induced DNA adducts and embryotoxicity in vitro and in vivo studies. The carcinogenic polycyclic aromatic hydrocarbons (carc-PAHs) were mostly responsible for the genotoxic activity, contributing to 45%–50% of all DNA adducts induced by these complex mixtures. The effect of carc-PAHs in polluted air in human volunteers was studied using biomarkers: DNA adducts by 32 P-postlabeling assay, chromosomal aberrations by conventional cytogenetic analysis and fluorescent in situ hybridization (FISH), polymorphisms of metabolic genotypes (GSTM1, GSTP1, GSTT1, EPHX, CYP1A1-MspI, MS, MTHFR) and DNA repair genotypes (XRCC1 and XPD). Studies on groups environmentally exposed to carc-PAHs implied the relationship between air pollution and DNA-PAH adducts as well as chromosomal aberrations by FISH in exposed subjects, which may be further modified by metabolic and DNA repair genes polymorphisms. All results indicate that carc-PAHs represent a very significant group of air pollutants, which should be used for the risk assessment of humans. Supported by the Czech Ministry of Environment VaV/740/5/03 and by the EC IC QLRT-2000-00091. 38 PAH-INDUCED APOPTOSIS J.A. Holme, A. Solhaug Norwegian Institute of Public Health, Oslo, Norway

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In comparison to their genotoxic properties, the mechanisms involved in PAH-induced apoptosis are less characterized and their health implications are not fully clarified. PAH have been shown to induce apoptosis in immature lymphocytes, which could give an explanation to their immunosuppressive effects. Apoptosis induced by Ahr-driven Bax transcription have been suggested to be a cell-death signalling pathway responsible for PAH-induced ovarian failure. PAH-induced apoptosis may also have a role cancer initiation and promotion. Apoptosis of cells exposed to PAH is considered to function anti-carcinogenic since cells with extensive DNA-damage will be removed. On the other hand it is possible that removal of these cells may give survival and proliferating signals to surrounding cells with less DNA-damage, increasing their probability of having mutations and give promotion signals. Regarding promotion, also inhibition of the apoptotic process is considered to be an important part of chemicalinduced carcinogenesis, since the total number of cells may be increased by an inhibition of apoptosis in early cancer cells as well as by an increased proliferation. Here we characterize benzo[a]pyrene (B[a]P)induced apoptosis in Hepa1c1c7 cells. Interestingly, we find that the DNA-damage caused by reactive B[a]P-metabolite(s) results in apoptotic as well as anti-apoptotic signals, whereas the parent compound seemed to give anti-apoptotic signals. An implication of this could be that cells having B[a]P-induced DNA damage that normally would result in apoptosis nevertheless may survive, but with an increased risk of having mutations. W5 Quantitative Risk Assessment for Contact Sensitizers—An Attainable Goal in Public Health Protection 39 EPIDEMIOLOGICAL ASPECTS OF SKIN SENSITISATION A. Schnuch1 , W. Uter2 1 Information Network of Departments of Dermatology

(IVDK), G¨ottingen, Germany; 2 Dept. of Med. Informatics, Biometry & Epidemiology, Erlangen, Germany Epidemiological aspects of skin sensitization are best addressed by methods of clinical epidemiology (CE).


CE exploits the full scope of standardized routine data generated during diagnosis of allergic contact dermatitis, as collected in multicentre projects like the IVDK ( Several research issues regarding (A) the public health impact of sensitization (PHI) and prevention (Prev), (B) quantitative aspects of elicitation and (C) risk assessment, can be treated by using the large database of the IVDK (>130.000 patients). (A) The PHI of individual allergens is quantified by the frequencies of sensitization in the population of eczema patients (nickel, fragrances, and preservatives), or in selected subgroups thereof with a defined occupational exposures (e.g. dyes as ptoluylendiamine in hairdressers). The analysis of time trends indicates, when increasing, the necessity of prevention (e.g. dose reduction), and when decreasing, confirms the success of prevention (e.g. regulation of nickel or methyl(chloro)isothiazolinone). (B) Repeated Open Application testing (ROAT) together with allergy (patch) tests in different concentrations may find both, the concentration range necessary for elicitation and a non eliciting use concentration. This data helps to optimize individual diagnosis and supplies scientific evidence for regulation. (C) CE data can be used to validate preclinical risk assessment. If animal data is missing, CE may be the only available evidence. In case valid data on human exposure to a substance is available, “real-life” risk assessement is possible by relating sensitization frequency to exposure intensity and frequency. 40 CONTACT SENSITIZATION AND ALLERGIC CONTACT DERMATITIS: IMMUNOBIOLOGICAL MECHANISMS P.S. Friedmann University of Southampton, Southampton, UK The dose–response relationships of the human immune system can be defined using the induction and elicitation of lymphocyte mediated allergic reactions to experimental contact sensitisers such as dinitrochlorobenzene (DNCB). Five groups of healthy volunteers received a sensitising dose of DNCB applied to a 3 cm diameter circle on the volar forearm. Doses applied were 62.5, 125, 250, 500 and 1000 mg. Four weeks later a concentration series of 3.125, 6.25, 12.5


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and 25 mg was applied to the upper inner arm on 1 cm paper discs which were removed after 6 h. Fortyeight hour later the responses were scored clinically and thickness measured with callipers. The proportion of people reacting to the challenge doses showed a sigmoid log-dose response curve, 100% reacting to 500 mg. The responses to challenge also showed a log dose–reponse. As sensitising dose increased so more people were sensitised to a proportionately greater degree. These dose–response relationships reflect the effects of increasing the concentration of sensitiser on a fixed area. The effect was examined of keeping the concentration per sq cm constant but of varying the total area. When 35.4 m/cm2 , which sensitised 80% of people when applied to a circle 3 cm diameter (area 7.1 cm2 ), was applied on a 1.5 cm diameter circle or 4.5 cm diameter, there were no differences in the proportions sensitised or their degree of reactivity. This was clearly on the plateau for the sensitising effect. However, when the same concentration/cm2 was applied on a 3 mm diameter area much weaker sensitisation was obtained. This shows the concentration of sensitiser per unit area is the critical determinant of whether sensitisation occurs, whereas the total dose may be varied over a wide range, but if the concentration per unit area is constant there is no effect on sensitising potency. In other words few Langerhans cells presenting many antigen molecules per cell is a much more potent sensitising stimulus than the same number of molecules presented by many Langerhans cells, each presenting few molecules. These observations clearly have important implications across the whole field of risk assessment for induction of contact sensitivity. 41 CHEMISTRY OF SKIN SENSITISERS AND OPORTUNITIES FOR DEFINITION OF STRUCTURE ACTIVITY RELATIONSHIPS J.P. Lepoittevin Universit´e Louis Pasteur, Laboratoire de Dermatochimie, Strasbourg, France Chemical reactions and properties of molecules are pivotal in the biological process which results in a patient developing delayed contact sensitivity.

Four directions, in which chemistry plays a major role, are currently explored and developed. 1. 2. 3. 4.

Identification of sensitizers. How molecules are interacting with proteins? How molecules are metabolized by the skin? How modified proteins are recognized by T-cell receptors?

One of the major objectives in the next few years is the development of alternative tests for the evaluation of sensitizing potential of a molecule and many research programs have been initiated to develop in vitro techniques. In parallel with these biological studies, another approach is becoming important, namely the study of quantitative structure–activity relationships (QSARs). In order to develop accurate models, a right understanding of how molecules can interact with proteins is pivotal. This is indeed the first step in the risk-assessment process, identify a hazard, which is intrinsic to the structure of the molecule, . . . and then if possible quantify this hazard X-ray Mechanistic investigations performed using carbon 13 labeled molecules have shown, for several sensitizers ranging from moderate to strong, a reactivity pattern toward amino acids that can be in some cases combined with a mechanistic specificity. Moreover, this selectivity for some amino acids seems to be more important that the overall level of modification of a protein. These different factors will be discussed and illustrated in the perspective of QSARs developments. W6 Mechanism of Hepatocarcinogenesis (Molecular Aspects) 42 ROLE OF OVAL CELLS IN HEPATOCARCINOGENESIS S.S. Thorgeirsson National Cancer Institute, Bethesda, USA Much interest has recently been focused on the hypothesis that tissue stem cells are a major target for the origin of cancer and that “cancer stem cells” sustain neoplastic growth. We have demonstrated that primitive epithelial cells isolated from normal rat liver (often referred to as RLE cells) can be spontaneously transformed in vitro, as well as with transformed dominant oncogenes and chemical carcinogens. These cells, when transplanted,

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gave rise to essentially all tumor types known to exist in the liver. These results suggest that, at least in principle, stem/progenitor cells could constitute a target compartment for neoplastic development in the liver. We also have identified a new population of epithelial cells in non-neoplastic liver tissues from hepatitis B virus-positive human hepatocellular carcinoma with distinct structure, as well as cytokeratin and alphafetoprotein expression. These cells were characterized by oval nuclei, scant, pale cytoplasm, small cell size, and cross-reaction with a monoclonal antibody against rat oval cells. These putative human oval cells were strongly positive for cytokeratin 19 and displayed considerable heterogeneity in alpha-fetoprotein and albumin expression. The oval cells were most prominent in actively regenerating nodules and in liver tissue surrounding the cancer. Oval cells and transitional types of cells appear to be the principal producers of alphafetoprotein in the regenerating liver. Cancer cells positive for cytokeratins 8, 18 and 19 were observed in half of the hepatocellular carcinomas studied. The data suggest that a new cell population structurally similar to oval cells seen in early stages of chemical hepatocarcinogenesis in rats is consistently present in regenerating liver lesions associated with human hepatocellular carcinoma. Furthermore, it is possible that the proliferation of these oval-type cells may partly account for the elevation of serum alpha-fetoprotein frequently seen in precancerous stages of hepatitis B virus-associated human hepatocellular carcinoma. These, as well as other related issues, will be discussed in my presentation. 43 MOLECULAR MECHANISMS OF TUMOUR PROMOTION IN MOUSE LIVER M. Schwarz Institute of Pharmacology and Toxicology, University of Tuebingen, Germany Tumor promoters are non-mutagenic chemicals which increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. Model tumour promoters include phenobarbital (PB), an antiepileptic drug which promotes hepatocarcinogenesis in rodents when administered subsequent to an initiating carcinogen like diethylnitrosamine


(DEN). Based on the hypothesis that tumor promoters may act by stimulating the outgrowth of target cells of a certain, promoter-specific genotype/phenotype, we have investigated the prevalence and patterns of mutations in two genes, Ha-ras and Catnb which codes for ␤-catenin), both known mutational targets in mouse hepatocarcinogenesis. Our results showed that tumors from mice treated with DEN alone were mostly mutated in Ha-ras while no Catnb mutations were detectable in tumors of this treatment group. By contrast, Ha-ras mutations were undetectable in tumors from mice treated with DEN/PB, while ∼80% of tumors from this group showed Catnb mutations. These results demonstrate that PB strongly affects the prevalence of mutations in the two cancer-related genes, presumably by positive and negative selection for cells harboring the respective mutation. We subsequently investigated by microarray analysis the gene expression profiles in tumors of the two genotypes. Several functional clusters were identified which involve changes in amino acid utilization and ammonia disposition in Catnb-mutated tumors as opposed to alterations in lipid and cholesterol metabolism in Ha-rasmutated tumors. Moreover, several genes coding for inhibitory molecules within the Wnt-signaling pathway were upregulated in Catnb mutated tumors, suggesting induction of a negative feedback loop, whereas Ha-ras mutated tumors showed alterations in the expression of several genes functional in monomeric G-protein signaling. We conclude that mouse hepatoma cells adopt different evolutionary strategies which allow for their selective outgrowth under variable environmental conditions. Finally, we observed that many of the genes altered in expression in Catnb mutated tumors demonstrate respective changes in normal hepatocytes from the pericentral domain of the liver lobule, while gene expression patterns in Ha-ras mutated tumors resemble those in hepatocytes from the periportal domain. This indicates that hepatoma cells, depending on the type of their mutation, retrieve differentiation programs that are characteristic for pericentral and periportal subpopulations of normal hepatocytes. Acknowlegment: This project was supported by grants from the Deutsche Forschungsgemeinschaft (SCHW 329/3) and the Deutsche Krebshilfe (grant 106356).


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44 ATTENUATED p53 RESPONSE IN CARCINOGEN-INDUCED PRENEOPLASTIC LESIONS IN THE RAT U. Stenius Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Cancer risk assessment is often based on rodent bioassays and the liver is a major target organ site for tumorigenesis. The relevance of these tumors for humans is often debated and their development needs to be characterised. Preneoplastic lesions (EAF) in rodents often do not exhibit a p53 response to DNA damage. We have shown that this lack of p53 response in EAF is related to stress caused by DNA-damaging xenobiotics, and the attenuated p53 response may confer growth advantages to affected lesions. Time is an important determinant for the development of p53-negative EAF at low doses of DNA-damaging xenobiotics. Studies with p53+/− mice show that the presence of only one functional p53 allele counteracts the growth advantage. An attenuated p53 response in EAF correlates with high levels of the p53 antagonist Mdm2. We have found that EAF often arise in midzonal areas where hepatocytes constitutively express high levels of cytoplasmic Mdm2. These data indicate a role of Mdm2 in early EAF development. Many signalling pathways and stress responses converge on Mdm2 and modulate its regulation of p53 responses to DNA damage. Mdm2 might e.g. be activated by the survival factor Akt kinase in many cell types, but an analysis of Mdm2 and Akt kinase reveals a perhaps liver specific regulation. Many EAF exhibit low levels of activated Akt and abrogation of Akt activation was associated with Mdm2 activation. A liver specific regulation of Mdm2 and Akt might be crucial for the development of preneoplastic lesions, and will be further discussed. 45 DAMAGE OF ZINC FINGERS IN DNA REPAIR PROTEINS, A NOVEL MOLECULAR MECHANISM IN CARCINOGENESIS W. Bal Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland

Zinc finger motifs participate in protein–nucleic acid and protein–protein interactions in many groups of proteins, including those involved in DNA repair. The Zn(II) ion, bonded tetrahedrally to cysteine thiolates and/or histidine imidazole groups, maintains the threedimensional structure, crucial for the specificity of the domain. Its function can be thus compromised by a substitution of Zn(II) with another metal ion or by a release of Zn(II), due to the oxidation of thiolate donors. The latter may be result from an action of redox-active metals or other oxidative agents. Studies in cell cultures and ex vivo demonstrated that soluble compounds of definite carcinogenic metals and metalloids, such as arsenic, cadmium and nickel, and putative carcinogens, including cobalt and lead, inhibit zinc finger containing DNA repair proteins (Hartwig, 2001). Further experiments demonstrated that these metals, as well as endogeneous oxidative substances, including H2 O2 , nitrosoglutathione, and selenium compounds damage or distort zinc finger domains (Bal et al., 2003; Kopera et al., 2004; Blessing et al., 2004). This reactivity can therefore be regarded as a novel molecular mechanism in carcinogenesis. References: Hartwig, A., 2001. Antiox. Redox Signal. 3, 625–634. Bal, W., Schwerdtle, T., Hartwig, A., 2003. Chem. Res. Toxicol. 16, 242–248. Kopera, E., Schwerdtle, T., Hartwig, A., Bal, W., 2004. Chem. Res. Toxicol. 17, 1452–1458. Blessing, H., Krauss, S., Heindl, P., Bal, W., Hartwig, A., 2004. Eur. J. Biochem. 271, 3190–3199.

S5 Nutritional and Safety Assessments of Foods and Feeds nutritionally Improved through Biotechnology 46 ILSI TASK FORCE ON NUTRITIONAL AND SAFETY ASSESSMENTS OF FOODS AND FEEDS NUTRIONALLY IMPROVED THROUGH BIOTECHNOLOGY K.C. Glenn Monsanto Co., 800 N. Lindbergh Blvd., St. Louis, MO 63167, USA In 2001, the ILSI International Food Biotechnology Committee convened a task force and an expert

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working group to develop a framework and provide scientific underpinnings for the safety and nutritional assessment of products nutritionally enhanced through biotechnology. The expert working group of international academic scientists worked in consultation with the task force to develop a document that discusses the scientific approaches and methods needed for such evaluations. This document was written to provide guidance to product developers and serve as a key reference for scientific and regulatory considerations on general and technical issues. After extensive international peer review including an international workshop with members of the OECD Task Force for the Safety of Novel Foods and Feeds in Paris, December 2003, the Executive Summary of this document was published in the March 2004 issue of the Journal of Food Science, and the full document was published in April 2004 in the on-line journal, Comprehensive Reviews in Food Science and Food Safety. The document is available free to the public at: as a PDF downloadable file. The full publication includes terms and definitions for describing improved nutrition products, identifies the key safety and nutritional challenges, and introduces potential approaches and methods to address those challenges. The document also discusses the extensive experience available from the commercialization of GM crops to date, and focuses on the unique questions and challenges associated with nutritionally improved products. The significance and value of this ILSI document has been confirmed recently, when the European Food Standards Authority referenced it in their guidance document for the risk assessment of GM plants and derived food and feed. A second phase of this project has been initiated in which nutrition and safety assessment recommendations are developed for the “real world”, publicly available examples of products with improved nutrition properties, based on the guidelines and recommendations in the recently published ILSI document.



CropScience, Research Triangle Park, NC, USA; 2 Executive Associate Director of the Biotechnology Center, University of Illinois at Urbana-Champaign, USA Nutritionally improved GM foods have already been introduced into the marketplace after completion of pre-market safety assessments in a number of countries. The paradigm previously applied to evaluation of novel foods and feeds produced through conventional breeding is applicable to those produced through the application of biotechnology. The availability of comprehensive and robust composition and dietary intake databases greatly facilitates evaluation of nutritionally improved foods and feeds. In the case of nutritionally improved foods or ingredients introduced into the marketplace as novel products, it is assumed that regulations will require changes in nutritional content to be identified on the food product label. Nutritionally improved feeds are likely to be marketed through vertically integrated markets in order to capture the value of the improvement. Historically, nutritionally improved foods have been subjected to pre-market safety assessment but have not been required to demonstrate efficacy beyond a statement of the nature of the compositional change. No safety or health-based rationale for changing this paradigm was identified. Developers wishing to make marketing claims regarding the potential health benefits of a nutritionally improved product (food or feed) may wish to develop additional scientific support for the claim; in some jurisdictions such evidence may be required to support advertising claims and to substantiate bioavailability. Recommendations: 1. All nutritionally improved novel foods and feeds should be evaluated for the potential impact on nutrition and health regardless of the technology used to develop them. It is important to shift the primary focus from the composition of the individ-


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ual foods or ingredients to the composition of the diets of individuals. 2. Alterations in composition that change dietary intake should be evaluated regardless of the intended purpose for which the food or food ingredient was developed. 3. Pre-market assessment should be required to demonstrate that the introduction of a novel food will not significantly and adversely change nutrient intake for a large cross-section of consumers. 4. Human dietary intake data and dietary intake forecast models should be developed for all targets and at risk populations. 48 NUTRITIONAL ASSESSMENT OF FEEDS DERIVED FROM PLANTS PRODUCED BY MODERN BIOTECHNOLOGY R.H. Phipps School of Agriculture, Policy and Development, University of Reading, Reading RG6, 6AR, UK Eighty 80 million ha of GM crops (soyabean, maize, cotton and canola) modified for herbicide tolerance (Ht) and/or insect protection (Bt) are now grown annually and are all used as feed for monogastric and ruminant livestock. Chemical composition of conventional and GM feeds is the cornerstone for nutritional assessment. Once Ht and Bt crops have been established as compositionally equivalent to an appropriate counterpart, nutritional equivalence can be assumed and feeding studies have add little to the nutritional assessment procedure. Feed quality is a major constraint in livestock production and GM crops with improved nutritional characteristics are being developed and such crops will better match the nutrient requirement of livestock. For example the nutrient profile of these crops can be modified by increasing nutrient availability or specific nutrients or reducing anti-nutritional factors. With these crops, compositional analysis is still the starting point for their nutritional assessment and the need for feeding studies should be determined on a case-by-case basis. For example, crops in which nutrient availability has been increased would be assessed by in vivo studies in target species. However, the need for feeding studies designed to demonstrate the nutritional benefits that might be expected from the use

of GM crops in which either nutrient density or the concentration of a specific macronutrient is increased would be decided on a case-by case basis. 49 THE ROLE OF ANALYTICAL TECHNIQUES IN IDENTIFYING UNINTENDED EFFECTS IN CROPS A. Lommen RIKILT, P.O. Box 230, 6700 AE Wageningen, The Netherlands E-mail address: [email protected] Identifying unintended effects in crops in itself is an open scientific question. What should one measure? How do you go about this? Traditionally – on the metabolite level – a targeted approach is done in which a number of key metabolites are chosen and measured to obtain information on changes. This should then be compared with what is normally found. A more complete and cheaper way of identifying unintended metabolic effects is now coming available in the form of modern state of the art automation of comparison of raw data obtained from 1 H NMR, GC- and LC–MS. Chemical profiles from these three analytical techniques can be screened for differences using new sophisticated software packages and statistics. Real consistent differences in raw data can be pinpointed prior to identification. This shortcuts analysis time in a major way, meanwhile covering a much broader chemical composition. 50 THE FEASIBILITY OF POSTMARKET MONITORING OF FOODS DERIVED FROM BIOTECHNOLOGY TO IDENTIFY EFFECTS ON HUMAN HEALTH I. Munro Cantox Health Sciences International, 2233 Argentia Road, Suite 308, Mississauga, Ont., Canada L5N 2X7 The premarket safety assessment of foods derived from biotechnology is both rigorous and science-based, incorporating a decision strategy that varies case-bycase with the agricultural trait. The outcome of the premarket safety assessment provides sufficient information to estimate the likelihood of any adverse effects

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to consumers, including specific subsets of the population that may be at greater risk. Premarket studies are designed to identify endpoints of safety, critical to the acceptability of the food. Postmarket monitoring is not routine in supporting the safety or regulatory approval of food products, except in a few unique instances. The assessment of biotechnology-derived foods with enhanced nutritional characteristics may require additional consideration of exposure and intended positive effects on human health. However, the feasibility of identifying effects on human health must be derived from testable hypothesis and include appropriate biomarkers for measurement, along with accurate food consumption data. Current exposure assessment principles may be applied to postmarket monitoring of biotechnology-derived foods, but are dependent on the availability of adequate data to assess the fate of the product or commodity within the food supply, as well as requisite information on the consumption of relevant foods. Postmarket monitoring programs may be appropriate when specific hypotheses requiring directed investigation have been identified as a result of the premarket assessment that data generated from premarket studies are unable to address. For example, postmarket monitoring may be useful in certain instances where better estimates of dietary exposure and/or nutritional consequence of a biotechnology-derived food are required, when a potential safety issue, such as allergenicity, cannot be adequately addressed through pre-market studies, to corroborate dietary intakes of a nutritionally improved food with effects on human health, or to monitor changes in dietary exposure resulting from the introduction of a nutritionally modified food. S6 Framework for Integrating Human Data in Risk Assessment 51 ACHIEVING A CONSICTENT USE OF HUMAN DATA IN RISK ASSESSMENT AND MANAGEMENT Ch. Money Industrial Hygiene Advisor, Europe, Exxon Mobil Petroleum & Chemical, Hermeslaan 2 B-1831 Machelen, Belgium


Although it is accepted that the process for the risk assessment of chemicals will best be served by the examination of all available information, it is recognised that available human effects data are not consistently considered or interpreted. Such practice is cause in itself, but more so given society’s increasing call for risks to be properly evaluated and animal welfare concerns respected. ECETOC, together with other regulatory agencies and non-governmental bodies, has set itself the challenge of identifying mechanisms that would enable available human data to be better utilised to improve the accuracy and confidence of human risk assessments. The presentation will review some of the recent activities that aim to enable available human data to be more readily accessed and evaluated. It will describe how a recent ECETOC workshop on the topic judged the validity and value of a range of human data sources for a variety of risk assessment and management purposes. Building on these examples, it will outline a basis for a framework in which human data can be consistently and reliably used in risk assessments and which accounts for the societal expectation for a process that is straightforward, transparent and equitable. 52 ROLE OPF EPIDEMIOLOGY IN RISK ASSESSMENT R. Schnatter ExxonMobil Biomedical Sciences, Inc, East Millsone, NJ 08875-2350, USA To understand the preferred role of null data in risk assessment, one needs to: (a) define the strengths and weaknesses of this data, (b) understand the requirements and stages of the risk assessment process, and finally, (c) understand the strengths and weaknesses of complimentary data on which the risk assessment may be based. Evaluating the strengths and weaknesses of null data should not differ from the evaluation of human and/or epidemiologic data in general. The adequacy of the study design, lack of bias, control of confounders, and the adequacy of exposure and disease definitions are paramount. For null data, the precision of the risk estimate is often of heightened importance. It is argued and shown that statistical power is technically not a


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germane issue after study results are known, yet the related concept of study size and precision is. Most null epidemiologic data is better suited for use in the hazard identification, or first stage of the risk assessment process. Fewer studies directly apply to the dose/response assessment stage since exposure data is often compromised. Yet, even if exposure is not quantified within a given study, strong epidemiologic data lacking in quantitative exposure are presently underused in risk assessment validations and/or sensitivity analyses. When there is a mixture of positive and null epidemiologic data, it is essential to use the strongest data in risk assessment, regardless of whether the data is positive or null. If study outcomes are not explained by exposure levels or study strengths, it is important to identify sources of heterogeneity on the results through meta-analysis or pooled analysis techniques. Finally, when both animal and human data exist, the strengths and weaknesses of both bodies of data should be used to assess the optimal use of the epidemiologic and animal data. 53 EVALUATION OF DIFFERENT SOURCES OF HUMAN DATA A. Ferrer-Dufol Clinic Hospital, University of Zaragossa, Spain One of the main problems in Human Clinical Toxicology is to know how accurate are animal data to predict acute, subacute or chronic toxicity in humans. Many examples demonstrate that interspecies differences in susceptibility are wide enough to stress the importance of collecting as much human data as possible to approach the human risk. In the specific field of acute toxicity the examples of paraquat (much more toxic in humans) and dioxin (much more toxic in animals) are impressive enough. Sources of human data are: (1) Case-to-case clinical descriptions: unavoidable first step to know how chemicals interact with humans at high doses; retrospective collections of the cases are in the base of the description of toxic syndromes related to specific agents and permit the evaluation of the response to different treatments; an interesting example was the demonstration, in a particular patient, of metabolic production of CO in

the metabolism of methylene chloride. (2) Human toxic epidemics: produced extensively all along the second half of the XX century, its careful critical evaluation lightens the human effects of relevant families such as organochlorinated and organophosphate pesticides, methyl mercury compounds, PCBs toxic alcohols and others. The particular case of HCB epidemic in Turkey is the best example of unforeseen toxicity detected for the first time in a human population due to their consumption of seed treated for sowing. In some of these epidemics the research efforts have allowed the calculation of human toxic doses. (3) Prospective collection of cases: one of the main efforts of poison control centres since their implementation in the fifties has been the registration of human data obtained through the calls asking for toxicological information. The best database is the US toxic exposure surveillance system (TESS) developed since 1985 that has collected 35 millions of cases. Its main limitation is the source of data (85% of non-professional calls). Another important source of data are the Emergency Departments. In Spain the Health Ministry and the section of Clinical Toxicology of the Spanish Association of Toxicology have developed a collaborative program since 1999 to maintain an updated profile of the toxic incidents caused by chemical products that reach the Emergency Departments. The system has collected 3358 cases, drugs, toxic plants and animals non-included. The comparison between the same families of chemicals among the two systems show the different estimation of risk related to different sources. The integration of data obtained trough all these origins are crucial for an accurate prediction and surveillance of human acute poisoning. 54 INTERPRETATION OF HUMAN DATA FOR RISK ASSESSMENT IN THE OCCUPATIONAL SETTING IN RELATION TO ANIMAL DATA G.J. Mulder Div. of Toxicology, Leiden Amsterdam Center for Drug Research (LACDR), Leiden University, Leiden, the Netherlands, and the Dutch Expert Committee on Occupational Safety (DECOS), The Hague, The Netherlands The ideal data set for risk assessment of chemicals in the occupational environment is based on exposed

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humans. For several chemicals that have already been in use for decennia usually (some) epidemiological data are available, which may include information on potential carcinogenicity of the chemical. For chemicals more recently introduced only more or less acute toxicity data may be available in exposed workers. The most frequent problems encountered in the evaluation of such epidemiological data are: (1) uncertainties about exposure levels; (2) big differences in exposure of different groups of workers, dependent on their job; (3) frequent co-exposure to other chemicals; (4) a strong influence of life style factors, in particular smoking. In most cases animal data will have to be used because of lack of sufficient epidemiological data. Uncertainty factors compensate for extrapolation uncertainties. For medical drugs both human and animal data are available. However, even here unexpected toxicity is often discovered soon after introduction of the drug on the market. Even extensive animal data is not fool proof! The problems in the interpretation and decision making on occupational exposure of chemicals will be illustrated with some examples: nitric oxide, formaldehyde and ethanol. Wensday, September 14, 2005 55 Plenary Session 2 See Book of Late Abstracts. S7 Does Modern Toxicology Allow the Safe Use of Pesticides? 56 TOXICOLOGICAL CONCERNS OF PESTICIDES J. Kniewald Laboratory of Toxicology, Faculty of Food Technology and Biotechnology, University of Zagreb, 10000 Zagreb, Croatia Toxic residues from pesticides are found everywhere: in water systems, in animals, in the vast majority of human beings, even in that most sacred nectar—mother’s milk. The acute reactions to many pesticides are well documented, affecting the liver, kidneys, lungs, skin, eyes and brain. Long-term chronic


effects on humans include a whole series of cancers, liver and kidney disfunction, sterility, neurological disorders and birth defects. Many developmental and reproductive disorders are caused by the complex interactions of multiple pesticides with our hormone systems. Pesticides, that take a long time to biodegradate in the environment, accumulate in organisms as they move up the food chain. Humans, as top predators, can accumulate relatively high concentrations of pesticides through the food supply. New evidence indicates that proper functioning of the human body’s endocrine/hormone system can be severely altered by low-level cumulative pesticide exposure. This indicates that it can cause chronic reproductive, immunological and neurological disorders. Low-level exposure of unborn children to pesticides can affect a wide range of developmental processes, from reproductive system formation to brain function. The most common types of hormone disruption are hormone mimicking, hormone blocking or disturbing the production process of the hormones. As pesticide synergy exists, exposure to multiple pesticides may be thousands of times more potent in mimicking estrogen than it was previously thought. Does this mean that current tolerance levels (NOAEL, ADI, MRL) set for individual pesticide residues allowed in our food are, when combined, actually far above dangerous limits? What are the implications for the unborn, children and adolescents? How should the pesticides be substituted by application of modern biotechnological procedures? What are the alternatives? 57 THE ROLE OF MECHANISTICS STUDIES IN AGROCHEMICAL SAFETY EVALUATION L.L. Smith Syngenta International AG, CH-4002 Basel, Switzerland See Book of Late Abstracts. 58 LIFE STAGE CONSIDERATIONS IN TOXICITY TESTING OF AGROCHEMICALS J. Buschmann Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany


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The goal of life stage toxicity is the assessment of potential adverse effects which can occur specifically during different ontogenetic periods, such as preconception, embryo-fetal, newborn, and preweaning periods as well as in young adults and aged individuals. The process of toxicity testing of agrochemicals is ruled by a framework of guidelines (OECD, OPPTS, etc.). Amongst them, the following guidelines or draft guidelines consider life stages in the broadest sense of the term (OECD as an example): Prenatal Developmental Toxicity Study One-Generation Reproduction Toxicity Study Two-Generation Reproduction Toxicity Study Reproduction/Developmental Toxicity Screening Test Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test Carcinogenicity Studies Chronic Toxicity Studies Combined Chronic Toxicity/Carcinogenicity Studies Developmental Neurotoxicity Study Within this framework, the attempt will be made to identify existing gaps and make suggestions to close them. Examples will be provided and discussed illustrating the current situation and showing potential improvements for the future. These examples include consideration of maternal/parental toxicity, differences in the vulnerability of the juvenile organism, developmental milestones, and transplacental carcinogenicity. The discrepancy between a purely scientifically based approach and both practicality and predictibility of the available test strategies and test methods will be addressed. The advantages and shortcomings of including more tests in the most suitable study type, the two-generation study, will be discussed and compared to the approach of using (and introducing) special study types. This also includes a critical review of the triggers for different study types. 59 IMMUNOTOXIC EFFECTS THE FUNGICIDE HEXACHLOROBENZENE R. Pieters1 , J.G. Vos2 , J. Ezendam2 1 IRAS-Immunotoxicology, Utrecht University, Utrecht,

The Netherlands; 2 National Institute of Public Health and the Environment, Bilthoven, The Netherlands

Hexachlorobenzene (HCB) is an environmental pollutant that induces adverse immune effects in humans and rats. Brown Norway rats (BN) appear to be very susceptible to HCB-induced immune effects. Oral exposure causes inflammatory skin and lung lesions, enlarged spleen and lymph nodes (LN) and elevated humoral responses. This review describes recent experiments that were performed to elucidate the mechanisms underlying these adverse immune effects. The metabolites tetrachlorohydroquinone and tetrachlorobenzoquinone are capable of generating neoantigen-specific T cells in the mouse reporter antigen popliteal lymph node assay with 2,4,6-trinitrophenyl (TNP)-Ficoll. Additional experiments in BN rats have shown that T cells are involved in the development of HCB-induced skin lesions, the increase in auricular LN weight, lung eosinophilia and humoral responses. However, splenomegaly and macrophage infiltration in the spleen and lung occur independent of T cells. Remarkably, HCB does not induce T cell sensitization in BN rats, suggesting that T cells are activated non-specifically, for example by macrophages. Transcriptome profiles obtained after subchronic HCB exposure to BN rats reveal that HCB induces a systemic inflammatory response in which several innate immune cells, such as macrophages, and pro-inflammatory cytokines are involved. Additional experiments have shown that macrophages are implicated in HCB-induced skin lesions, in particular in the aggravation of skin effects. Also, HCB-induced lung eosinophilia and elevation of anti-ssDNA IgM antibodies are less pronounced after macrophage elimination. Presumably, HCB activates macrophages, thereby generating pro-inflammatory adjuvant signals that induce a systemic inflammatory response. Subsequently, this may lead to polyclonal activation of T and B cells, eosinophilia and eventually visible clinical effects. 60 A TIERED APPROACH TO ASSESSING THE TOXICOLOGY OF PESTICIDES A.R. Boobis Section of Experimental Medicine and Toxicology, Division of Medicine, Imperial College London, Ducane Road, London W12 0NN, UK

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The assessment of pesticides is based on guidelines that were developed at least 10 years ago. In the intervening period understanding of toxicological mechanisms has increased as have consumer demands for more rigorous and broader safety and health risk assessments. Hence, there is considerable international interest in improving the design of the current testing paradigms for agricultural chemicals. Recently, the ILSI Health and Environmental Sciences Institute (HESI) organised an international project involving experts from academia, regulatory agencies and industry to devise a credible testing strategy taking account of the above concerns, whilst recognising the need to better match exposure durations to the requirements of risk assessors. The aim was to improve the accuracy and efficiency of the assessment process, using fewer animals and producing fewer spurious or irrelevant results. The strategy comprises a tiered approach that incorporates existing knowledge on the chemistry, toxicology, and human exposure to the compound. Testing should be informed and interpreted by better knowledge of the metabolism and kinetics of the compound. The tiered approach is such that a relatively comprehensive base set of studies is undertaken, incorporating triggers for specific endpoints, such as immunotoxicity and neurotoxicity. These triggers serve to initiate the next tier of testing, in which the endpoint of concern is more fully characterised. The strategy integrates information on metabolism and kinetics, life stages susceptibility, and systemic toxicity much more effectively than at present. Overall, the proposed strategy would result in the more effective risk assessment of agricultural chemicals whilst using fewer animals. W7 Chemical Pollution and Development of Astma 61 CHEMICAL POLLUTION, ASTHMA AND ALLERGY: AN ECETOC PERSPECTIVE I. Kimber Syngenta Central Toxicology Laboratory, Macclesffield, UK There has in recent years been a significant increase in the prevalence of atopic disease, including allergic asthma. The bases for this increase are unclear although it is acknowledged that the changes in inci-


dence observed have been too rapid to have resulted from alterations in the gene pool. Attention has focused therefore upon the possible impact of environmental and lifestyle factors on susceptibility to allergy. Among the possible influences that have been implicated is exposure to chemical pollutants. The possible impact of chemicals on the acquisition of allergic sensitisation and asthma was the focus of a discussion earlier this year at a Workshop convened by European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) and The Long-Range Research Initiative and entitled: Chemical Pollution, Respiratory Allergy and Asthma. The results of those discussions will be summarised here. 62 CROSS-SHIFT STUDY IN MASTIC ASPHALT WORKERS: CHEMICAL-IRRITATIVE EFFECTS OF FUMES AND AEROSOLS OF BITUMEN M. Raulf-Heimsoth1 , B. Pesch1 , M. Kappler1 , K. Schott2 , B. Marczynski1 , J. Angerer3 , R. Merget1 , Th. Br¨uning1 1 Research Institute of Occupational Medicine (BGFA),

Ruhr-University Bochum, Germany; 2 TiefbauBerufsgenossenschaft, Germany; 3 Institut und Poliklinik f¨ur Arbeits-, Sozial- und Umweltmedizin, University Erlangen, Germany To assess the chemical-irritative and genotoxic effects of bitumen exposure a cross-shift study was conducted with 66 mastic asphalt workers and 49 construction workers without bitumen exposure. Based on personal exposure measurements during shift, the mastic asphalt workers were classified into a low (≤10 mg/m3 ; n = 38) and a high (>10 mg/m3 ; n = 28) exposure group. Spirometry, humoral and cellular analysis of nasal lavage fluid (NALF) and induced sputum were performed. Internal exposure was determined by urinary metabolites of polycyclic aromatic hydrocarbons (PAH). The results showed that fumes and aerosols of bitumen released under high processing temperatures can exert chemical-irritative effects on the upper and lower airways assessed with NALF and induced sputum analysis (increase of neutrophils and IL-8 during shift). The effects were supported by a moderate impairment of the lung function and a dose-dependent increase in the prevalence of respiratory symptoms.


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The airborne bitumen exposure was accompanied by a dose-dependent increase of the elimination of PAHmetabolites in the post-shift urine samples. Possible carry-over effects could be found in exposed workers indicating (sub)chronic inflammation in these groups. The results of lung function measurements, NALF and sputum biomarkers pointed to a “healthy worker effects” in the low exposure group. 63 ASTHMA DUE TO CHEMICALS EXPOSURE C. Palczynski Department of Occupational Diseases, Institute of Occupational Medicine, Lodz, Poland Occupational asthma is a disease in which exposure to chemical agents, both allergens and irritants plays an important role. It is needed to distinguish two different toxicant effects. The first one is toxicant acting as an antigen producing antibodies specific for this antigen. Second, a subsequent exposure to this antigen results in the production of the immediate and/or delayed asthmatic reaction. Chemical allergens that cause adverse health effects secondary to the stimulation of an immune response should be discriminated from the agents that may provoke similar symptoms, but via irritant, pharmacological or other non-immune mechanisms. Pathology, symptoms and diagnostics concerning occupational asthma of immunologic origin caused by chemical sensitizers with low molecular weight will be reviewed. Sensitizers of a low molecular weight are commonly encountered in the work environment as well as on the major mechanisms responsible for their effect on the immune cells of the respiratory tract. A better understanding of the pathogenesis of occupational asthma due to chemicals should facilitate the development of better diagnostics and the improvement of strategies for disease surveillance and intervention. W8 Children and Health 64 CHILDREN AS A RISK GROUP—REPORT FROM AN ECOTOC-TASKFORCE H. Autrup Institute of Public Health, University of Aarhus, Aarhus, Denmark

A wide range of environmental factors has an impact on children’s health, extending well beyond industrial chemicals. These factors include nutrition, lifestyle, socio-economic status and parent health. There is clear evidence of increasing rates of asthma in childhood, although rates in some countries have stabilised. It is important to distinguish between factors causing asthma and those exacerbating. Chemicals, which exacerbate asthma are ambient air pollutants, i.e., particulates, ETS and VOC, e.g. formaldehyde. The frequency of neurodevelopmental disorders, i.e. autism and attention deficit is commonly believed to be increasing. Exposure to lead, mercury, and PCBs is associated with neurodevelopmental disorders. The relationship of these chemicals to neurobehavioural changes at general environmental levels has not been well established except for lead. Data on reproductive effects are also limited, and large geographic heterogeneity is apparent. The risk for cryptorchidism and hypospadias appears to be related to a variety of non-environmental factors. The one clearly identified is the earlier onset of puberty in girls in the developed world, appears to be due to lifestyle changes. There is no evidence for increased frequency of childhood cancer. A gradually increasing incidence in leukaemia with a corresponding drop in the incidence of lymphoma. Alterations in brain tumour frequency may be related to the development of new diagnostic capabilities. With the increasing number of childhood cancer survivors, secondary cancers following chemotherapy appear to be increasing. The evidence of chemicals as risk factors in childhood diseases is limited, however experimental studies indicate a potential risk at non-environmentally relevant levels. 65 GENE-ENVIRIONMENT IN DEVELOPMENT OF ASTHMA AND ALLERGY M. Kabesch University of Munich, 80337 Munich, Germany Childhood asthma is thought to be caused by a genetic predisposition in combination with specific environmental exposures or the lack thereof. In asthma, genetic predisposition may alter the capability of the airways to

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protect itself against inhaled toxic substances from the environment. In turn, this may lead to airway inflammation, the damage of airway epithelium and an alteration of the airways’ barrier function. Numerous studies have reported an association between environmental tobacco smoke (ETS) exposure and respiratory diseases: Maternal smoking during pregnancy and early childhood is associated with impaired lung growth and diminished lung function, and in asthmatic children parental smoking increases symptoms and the frequency of asthma attacks. Recent studies have shown that the capability of ETS to induce asthma and asthma symptoms may be modified by genetics. Linkage studies which took smoking and passive smoking status of study subjects into account came to different results than those studies based on unstratified samples. These analyses indicated that some chromosomal regions (e.g. 5q) may harbour genes that exert their effects predominantly in combination with ETS exposure. Some of the genes modifying the effect of ETS and air pollution on the body may have been identified. When these detoxification enzymes are genetically defect or missing the capability of the lung to metabolize hazardous substances is dramatically diminished. As a consequence, pulmonary inflammation may occur and the barrier function of the lung may be compromised allowing allergens to penetrate and asthma to start. 66 JOINT EFFECTS OF LEAD (Pb) AND STRESS: CONSEQUENCES FOR BEHAVIORAL PROCESSES, NEUROCHEMICAL SYSTEMS, STRESS RESPONSIVITY AND HPA AXIS FUNCTION D.A. Cory-Slechta Environmental and Occupational Health Sciences Institute, Piscataway, NJ, USA The highest Pb burdens typically occur in disadvantaged populations, the same groups thought to sustain the highest levels of environmental stress. Socioeconomic disadvantage and stress are already associated with higher levels of disease and dysfunction, a phenomenon attributed to chronic elevation of stress hormones. Both Pb and stress (via the HPA axis) act on mesocorticolimbic and hippocampal systems of the


brain, regions critical to complex cognitive function, suggesting that combined exposures could produce greater neurotoxicity than either risk factor alone. To assess this possibility, we examined consequences in rats of: (1) maternal Pb, maternal stress or the combination on offspring, including stress challenges; and (2) chronic postweaning Pb exposures combined with intermittent stress challenges. Outcomes confirm the importance of Pb-stress interactions as manifest in neurochemical and behavioral alterations. They demonstrate permanent effects of Pb itself on HPA axis function and stress responsivity that depend upon Pb exposure parameters and the nature of the stress challenge. Finally, the profile of effects of Pb alone differs notably from that produced by Pb and stress. Collectively, these findings suggest new mechanisms for Pb-induced cognitive deficits. They raise questions about the ability of current risk assessment approaches, based on studies of chemicals in isolation, to adequately identify neurotoxic risk. Finally, they suggest a critical need for Pb screening during pregnancy to avoid maternallymediated Pb effects. 67 CHILDREN AND HEALTH. BIOMARKERS TO ASSESS EXPOSURE FOR GENOTOXICANTS: META-ANALYSIS OF EUROPEAN DATA D.F. Merlo1 , M. Neri1,2 , D. Ugolini1,3 , S. Bonassi4 , ˇ am7 , M. Ceppi4 , V. A. Fucic5 , N. Holland6 , R.J. Sr´ 1 2 Bocchini , L.E. Knudsen 1 Epidemiology

and Biostatistics, Genoa, Italy;

2 Institute of Public Health, University of Copenhagen,

Copenhagen, Denmark; 3 Molecular Epidemiology, National Cancer Research Institute, Genoa, Italy; 4 Dipartimento di Oncologia, Biologia e Genetica, University of Genoa, Genoa, Italy; 5 Institute for Medical Research and Occupational Health, Zagreb, Croatia; 6 School of Public Health, University of California, Berkeley, CA, USA; 7 Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Prague, Czech Republic A systematic review of field studies conducted in children exposed to environmental pollutants – including environmental tobacco smoke (ETS) – and incorporating biomarkers, was carried out. Chromosome aberrations (CA), micronuclei (MN), DNA-, albumin-


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and hemoglobin-adducts. were found consistently increased in exposed children. Meta-analyses performed in children exposed to ETS and in newborns exposed in utero to their mothers’ smoke showed 1.3 and 7 times higher levels of hemoglobin adducts compared to referent subjects, respectively. These increases are consistent with the epidemiological evidence of higher lung cancer risks reported in adults who had never smoked and were exposed to ETS during childhood and with 7 to 15 times higher lung cancer risks reported in smokers than in non-smokers. According to our findings (Rev. Mutat. Res., in press), future research and biomonitoring programs on children are expected to benefit from the inclusion of selected biomarkers that could provide biologically based evidence for the identification of intervention priorities in environmental health.

several studies. Although the epidemiological studies has been limited by luck of specific pesticide exposure estimation, small size of examined groups and potential for recall bias, the magnitude of the risks is often greater than among adults, indicating greater susceptibility. There is also increasing evidence on neurobehavioral effects of pesticide exposure in children. The results of studies evaluating the effects of prenatal exposure to p,p -DDE indicated delay in mental and psychomotor development at 13 months. The similar effects due to exposure to other pesticides seems likely but were not so far confirmed in epidemiological studies.



W. Hanke, J. Jurewicz

W. Roosen1 , J. Vandenberghe1 , L. Lammens1 , A. Lampo1 , W. Coussement1

Department of Environmental Epidemiology, Nofer Institute of Occupational Medicine, Lodz, Poland

1 Johnson

The amount epidemiological study on the effects of pesticide exposure is enormous. However the quality of evidence is still poor. It is mainly due to the limitations of exposure assessment, which in most studies is done retrospectively and is based only questionnaire data. On other hand in agriculture setting usually several pesticides are applied in the same time and it is difficult to separate the health effects of each of them. The epidemiological methods employed thus far have not significantly contributed to the assessment of adverse reproductive and developmental toxicity of pesticides. Although several pesticides were associated with wide range of reproductive disorders (low birthweight, small-for-gestational-age (SGA) birth, preterm delivery, stillbirth), the consistent results from more than one study were obtained only in case of phenoxy herbicides and birth defects. The association between exposure to pesticides in general and the risk of leukemia, brain tumors, neuroblstoma. Wilms’ tumor, Ewing’s Sarcoma, Non-Hodgkin’s lymphoma, soft-tissue sarcoma, Hodgkin’s disease was found in

POSTER SESSIONS Monday, September 12, 2005 P1 Toxicity Screening

& Johnson Pharmaceutical Research & Development, a division of Janssen Pharmaceutica N.V., GPCD/PCDE, Beerse, Belgium The purpose of this study was to compare three different Beagle strains (Janssen, CEDS and Marshall Beagles). Therefore, three groups of eight males and eight females (7–8.5 months old at study start) were dosed once daily by the oral route (via gavage) with demineralised water, for a period of 1 month. Following parameters were evaluated: behaviour (clinical observations), body weight, food consumption, ECG and heart rate. Additionally, ophthalmology, haematology, serum analysis, urinalysis, endocrinology, organ weights, necropsy and histopathology were performed. Compared to CEDS and Marshall Beagles, the Janssen beagle showed more inflammatory skin lesions (clinically and histologically) and warts, a higher body weight and higher food consumption and a longer serum protrombine time. CEDS beagles, when compared to the other examined strains, seem to have lower red blood cell parameters and increased number of lymphocytes at haematology, several differences in urinary parameters, inflammatory renal changes and a gener-

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ally less mature appearance. The most relevant differences between the Marshall beagle and the Janssen and CEDS beagles were lower white blood cell parameters and an increased incidence of deep S-waves at E.C.G. measurements. It was concluded that, although differences between the beagle strains were noted, their differences were of minimal relevance in view of the use in toxicology studies. P1-02 TOXICOLOGICAL ASSESSMENT OF A NOVEL NIACIN-BOUND CHROMIUM(III) (NBC) SUPPLEMENT D. Bagchi1,2 , M.A. Shara1 , R.C. Gupta3 , T. Yasmin1 , T. Canerdy3 , M. Bagchi2 1 Creighton

University Medical Center, Omaha, NE; 2 InterHealth Nutraceuticals Inc., Benicia, CA; 3 Murray State University, Hopkinsville, KY Chromium(III) is an essential trace element required for normal protein, fat and carbohydrate metabolism. Studies have shown that NBC is bioavailable and promotes lean body mass and maintains healthy lipid profile. This study assessed broad spectrum safety of NBC. Acute oral, acute dermal, primary dermal irritation and eye irritation, Ames bacterial reverse mutation assay, mouse lymphoma test and a dose-dependent 90day subchronic toxicity were conducted. Acute oral and dermal LD50 of NBC were greater than 5000 and 2000 mg/kg, respectively, in both male and female rats. No changes in body weight or adverse effects were observed following necropsy. NBC was classified as slightly irritating to the skin and practically non-irritating to the eye. NBC did not induce any mutagenic effects in Ames bacterial reverse mutation assay and mouse lymphoma test. The subchronic toxicity study demonstrated no significant changes in selected organ weights individually and as percentages of body and brain weights, hematology and clinical chemistry, histopathology, hepatic lipid peroxidation and DNA fragmentation on 30, 60 or 90 days of treatment. Furthermore, supplementation of NBC (1 mg/day) in 20 large breed dogs for 120 consecutive days did not cause


any adverse events including physical examination and serum chemistry (ALT, BUN and CK). These results suggest the broad spectrum safety of NBC. P1-03 ESTABLISHMENT OF ILLUMINA CUSTOM ARRAYS FOR HEPATOTOXICITY SCREENING N. Zidek, P.J. Kramer, P.G. Hewitt Molecular Toxicology, Institute of Toxicology, Merck KGaA, Frankfurter Str. 250, Darmstadt, Germany Drug-induced liver toxicity is a major issue for health care and drug development, and toxicogenomics is gaining importance as a tool for toxicity prediction as well as for support of classical tests for rapid and early toxicity screening. We have established a beadbased oligonucleotide microarray containing 550 genes known to be involved in liver toxicity. The major aim is to build a predictive hepatotoxicity model for rapid and early screening by using well-known hepatotoxic and non-toxic model compounds. So far, gene expression alterations in the livers of male Sprague–Dawley rats exposed to low and high doses of tetracycline (TET) or clofibrate (CF) (6 h, 24 h or 72 h (three animals per condition) were examined. Briefly, RNA was isolated, labelled and hybridized onto Illumina custom microarrays according to the manufacturer’s guidelines, and the data analyzed using various bioinformatics tools. The observed gene expression changes after treatment with either TET or CF revealed multiple genes involved in well-known cellular processes reported in the literature for these two compounds. This indicates that this custom array, with a small set of wellcharacterized genes, enables detailed analysis across a large number of samples rapidly and cheaply. The hepatotoxicant TET mainly induced genes involved in the inflammatory response and decreased xenobiotic metabolism gene expression—common for hepatotoxicants. CF showed activation of the PPAR␣ and its typical mediated responses. This supports the continuation of the construction of a predictive hepatotoxicity model based on a small subset of specific hepatotoxic genes.


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P1-04 SHORT-TERM EFFECTS OF LIPOPOLYSACCHARIDE ON GENE EXPRESSION ALTERATIONS IN VIVO TO PREDICT HEPATOTOXICITY N. Zidek, P.J. Kramer, P.G. Hewitt Molecular Toxicology, Institute of Toxicology, Merck KGaA, Frankfurter Str. 250, Darmstadt, Germany Toxicogenomics shows promise in improving the understanding of molecular and cellular (adverse) effects of substances in biological systems by the examination of gene expression alterations prior to histopathology changes. The mechanism of toxicity (hepatotoxicity) of the model hepatotoxin lipopolysaccharide (LPS), found on the outer membrane of Gramnegative bacteria, has been elucidated using highdensity oligonucleotide microarrays. To identify genes showing response to a single low (2 mg/kg) or high (8 mg/kg) dose LPS exposure (i.p.), male Sprague–Dawley rats were sacrificed after 6 h, 24 h or 72 h and livers removed. Dose and time dependent expression profiles were analyzed using the Affymetrix rat microarrays RAE230A, which includes about 15,000 well-characterized genes. Various tools of the Expressionist software (Genedata) were used for data analysis. Analysis identified multiple genes and groups of genes that were affected by LPS, supporting the practical use of high-density oligonucleotide microarrays for the elucidation of LPS hepatotoxicity. Multiple genes involved in inflammation accompanied by acute phase response were strongly up regulated, while a decrease in metabolism (xenobiotic biotransformation, lipid and energy conversion metabolism) were revealed. The strong deregulation of gene expression was mainly restricted to 6 h LPS exposure; after 24 h a clear dose-dependency was seen, with a more pronounced response after high dose treatment. Additionally, several potential safety markers for the prediction of hepatotoxicity were identified. This gene expression study facilitated the elucidation of LPS hepatotoxicity in rats in vivo, showing the validity of such toxicogenomic approaches for future testing of compounds with unknown toxicity.

P1-05 THE DOG TILT MODEL FOR EVALUATION OF ORTHOSTATIC REACTIVITY G. Froget, A.M. Betat, L. Camus, P. Lainee, R. Forster Cit, Evreux, France In the present study we have studied the effects of prazosin (PRAZ), an alpha-1-adrenergic antagonist, on orthostatic reactivity in the dog tilt model. The study demonstrates the utility of the tilt model in evaluating the autonomic involvement in cardiovascular changes. Four female beagle dogs implanted with telemetry transmitters (DSI) were submitted to a postural change (90 degrees tilt in standing position for 60 s). Arterial pressure and heart rate were monitored continuously during the tilt procedure (at least 30 s before and 30 s after). This procedure was repeated before and 30, 60, 90 and 120 min after oral administration of either the vehicle (VEH) or PRAZ at 0.03 or 0.1 mg/kg. A dose-related reduction of the orthostatic hypertensive response (OHR) was observed beginning 60 min after PRAZ administration (VEH: +36 ± 15 mmHg, PRAZ 0.03: +23 ± 17 mmHg, PRAZ 0.1: +4 ± 15 mmHg). At this time point, there were no major effects on systolic arterial pressure (SAP). At 0.1 mg/kg, PRAZ caused a progressive decrease in OHR over time, finally resulting in inversion to hypotension (of SAP) at 120 min (VEH: +31 ± 11 mmHg, PRAZ: −2 ± 13 mmHg, p < 0.05). Nevertheless, no syncope was observed during the study. Tachycardia, suggesting a hypotensive action of PRAZ was evident from 30 min after dosing, but this was insufficient to maintain SAP at the high doselevel. These investigations could complement standard telemetry studies and enhance understanding of the clinical relevance of findings. P1-06 IMPROVEMENTS TO THE DEREK FOR WINDOWS PREDICTION OF CHROMOSOME DAMAGE K. Langton, C.A. Marchant Lhasa Limited, 22–23 Blenheim Terrace, Woodhouse Lane, Leeds LS2 9HD, UK DEREK for Windows (DfW) is a knowledge-based expert system designed to predict the toxicity of a

Abstracts / Toxicology Letters 158S (2005) S1–S258


chemical from its structure. The knowledge base is composed of alerts, example compounds and rules which each contribute to the predictions made by the system. The alerts define chemical environments that are associated with toxicity. The DfW knowledge base currently contains 86 alerts for genotoxicity. The majority of these alerts relate to Ames test mutagenicity. Recent work has therefore focussed on improving coverage for chromosome damage (the term used in DfW to describe structural and/or numerical chromosomal aberrations). Two approaches are being taken. In the first, the possibility of directly extending existing DfW mutagenicity alerts to the chromosome damage endpoint has been investigated on the basis that many Ames test mutagens also cause this effect. Using databases and published collections of in vitro chromosome aberration test data, the number of active and inactive compounds matching each mutagenicity alert was established. This was then used in conjunction with mechanistic considerations to determine whether there was sufficient evidence to extend the alert. A summary of this analysis will be presented alongside examples of resulting improvements in the DfW knowledge base. A second approach will investigate the identification of new alerts for chromosome damage unrelated to mutagenic mechanisms.

The aim of this study is to examine the role of ERK 1/2 pathways in alcohol and 17-␤-estradiol treated rats by Western blot analysis. Female Wistar rats were ovariectomized (OVX) and then treated with 17-␤-estradiol (1 mg/kg, s.c.) and 30% aqueous solution of ethanol (10 ml/kg, by gavage) for 4 weeks. The results demonstrated that estrogen treatment alone activates ERK 1/2. The alcohol treated rats did not show the significant changes in the activation of ERK 1/2. When ethanol was combined with 17-␤estradiol, the results showed lower activity of ERK 1/2, compared to group treated with 17-␤-estradiol. The results suggest that estrogen effects MAPK kinases in pituitary. Moreover, alcohol treatment inhibits estrogen-activated extracellular-regulated kinases. Taken together our results showed the difference in effects of alcohol and estrogen on MAPK kinases-regulated function of pituitary.


Molecular Toxicology, Institute of Toxicology, Merck KGaA, 64271 Darmstadt, Germany

T. Shumkova1 , Boyadjieva1


Nikolova2 ,


Mitev2 ,


1 Department

of Pharmacology and Toxicology, Medical University, Sofia, Bulgaria; 2 Department of Chemistry and Biochemistry, Medical University, Sofia, Bulgaria Estrogens stimulate cell proliferation in typical estrogen-responsive tissue including the anterior pituitary gland and modulate several signaling cascades, including MAPK kinases and particularly extracellular-regulated kinases (ERK 1/2), related to cell proliferation and differentiation. On the other side, alcohol is known to cause many endocrine disfunctions including hyperprolactinemia in both humans and experimental animals.


Primary rat hepatocytes have been used as a model system for in vitro assessment of hepatotoxicity. Cells lose many of their specific functions if cultured in monolayer, but most are retained when cultivated as a sandwich, where they keep their polygonal morphology and form bile canalicular structures. Using different cell culture systems (monolayer and sandwich culture) and media formulations (with and without FCS), primary rat hepatocytes (male Wistar rats) were cultivated and changes in morphology and gene expression over time analyzed. The main aim was to elucidate the system which better maintains a differentiated state and therefore allows for prolonged hepatotoxicity in vitro studies. For the analysis of gene expression, Sentrix BeadChip Custom Arrays from Illumina were used. We examined the expression of 550 toxicologically relevant, hepatic specific genes at eight different time


Abstracts / Toxicology Letters 158S (2005) S1–S258

points for up to nine days of cell culture to assess changes in expression levels. Most changes (up to 136 deregulated genes) appeared between isolation and culturing of the hepatocytes. After plating, the cells recovered and gene expression stabilized. From 24 h to 9 day in culture, a maximum of 80 genes were differential regulated. An analysis of these genes showed that cytochrome P450 genes were more stable when cultured without serum. Under these culture conditions, only eight genes were constantly deregulated, whereas in all the other cultures there were many more. These specifically deregulated genes show the importance of the cell culture conditions, especially if primary hepatocytes are to be used for long-term hepatotoxicity screening tests in vitro.

quantification of all radiolabelled compounds by phosphorimaging. Placental microsomes were incubated with 50 nM 3 H-androstenedione for 30 min; lipophilic compounds were extracted, concentrated and separated on TLC plates. After drying the plates were exposed to 3 H sensitive phosphorimaging plates, and luminescence spots were quantitated after background correction. This method allows separation and quantitation of multiple products, e.g. from liver microsomes, quantification of androstenedione, estrogen and estrone as well as other products. Parallel processing of large sample numbers is possible, as well as using differently radiolabelled substrates. Stereospecific labelling is no precondition for the accuracy of the determinations. We will present placenta aromatase enzyme characteristics determined by this assay.



M. Schulze1 , C.P. Siegers1 , J. Schulze2 1 Institute of Experimental and Clinical Pharmacology

and Toxicology, Ratzeburger Allee 150, 23538 L¨ubeck, Germany; 2 Office of the Dean, Johann Wolfgang Goethe-University Frankfurt/Main, Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany Aromatase has gained scientific attention as an enzyme involved in hormonal regulation as well as breast cancer development and growth. Its activity is inhibited in breast cancer patients; additionally aromatase inhibition is tested for chemoprevention. Aromatase inhibitors have been found in a variety of plant extracts including e.g. soy, clover and cimicifuga. Aromatase activity is usually determined by 3 H release from stereospecifically labelled androstenedione. The accuracy of this assay depends on labelling stereospecificity and stoichiometric release. Also, other enzymatic activity must not result in 3 H release. This assay has a high validity in in vitro assays using expressed enzyme or placental microsomes; other systems may contain additional enzymatic activities. In order to circumvent these problems we have developed an alternative method based on separation of estrogen and estrone from androstenedione by thin layer chromatography and

M. Saunders, R. Davis, S. Fox, L.K. Earl Huntingdon Life Sciences Ltd., Woolley Road, Alconbury, Cambs, UK Rhesus monkeys are commonly used in non-clinical drug development. However, the animals used in these studies are typically young or adolescent, presenting problems in the assessment of compound effects on the sexually mature reproductive tract. Sexual maturity can be determined by a variety of factors, including hormonal status and anatomical development. This investigation was performed on 19 age-matched rhesus monkeys to determine if sexual maturity could be predicted by analysis of in-life parameters such as bodyweight, testicular volume and plasma testosterone concentration. Sexual maturity of the animals was subsequently confirmed by histopathological examination of the testes. Data from the variables was statistically analysed using principal component analysis. Analyses of the results indicated that there was a strong correlation between plasma testosterone concentration and testicular measurements with the sexual maturity of the animals, as confirmed by histopathology. The results also indicated that the animal bodyweight was an unreliable primary indicator of sexual maturity. It can therefore be concluded that the in-life testicular

Abstracts / Toxicology Letters 158S (2005) S1–S258


measurements and plasma testosterone levels can aid in the prediction of sexual maturity and development in rhesus monkeys.

other antiestrogens with a more or less similar intrinsic estrogenic potential have not been shown to be carcinogenic in humans.



P. Hirsim¨aki1 , A. Aaltonen2 , Y. Hirsim¨aki3 1 Turku

University Central Hospital, Department of Pathology, BioCity, Tykist¨okatu 6.B.6., FIN-20520, Turku, Finland; 2 Hormos Medical Ltd, PharmaCity, It. Pitk¨akatu 4.B, FIN-20520, Turku, Finland; 3 Orion Pharma P.O. Box 425, FIN-20101, Turku, Finland The objective of the present paper is to review the safety of some selective estrogen receptor modulators (SERMs). Tamoxifen, toremifene, droloxifene and idoxifene are polyphenylethylene antiestrogens while faslodex is a derivative of estradiol with a long, hydrophobic side chain at the seven alpha position and raloxifene is a benzothiophene. Raloxifene is approved for osteoporosis and the other antiestrogens are used in the prevention and/or treatment of estrogen— responsive malignancies such as breast cancer. Tamoxifen has to been shown to be genotoxic in several studies. It induces unscheduled DNA synthesis in rat hepatocytes and micronuclei in MCL-5a -cells in vitro. Tamoxifen also induces aneuploidy in rat liver in vivo and chromosome aberrations and micronuclei in mouse bone marrow. Toremifene has shown to be genotoxic to a far lower extent. Carcinogenicity: tamoxifen has shown to be hepatocarcinogenic in the rat in at least four independent long-term studies. The initiation of tumors in the rat is the result of metabolic activation by cytochrome P-450 isoenzymes to an electrophile(s) that binds irreversibly to DNA. In a short-term DNA microarray study this difference between tamoxifen and toremifene was not possible to distinguish. The other SERMs are not carcinogenic in rodents. In several independent clinical studies the risk of endometrial cancer has increased among tamoxifen treated women. Reviewing the available data the International Agency for Research on Cancer (IARC) concluded that there was sufficient evidence to show that tamoxifen is a class I human carcinogen. However, the

C. Garbero, M. Gosmar, V. Manfredi, N. Trucco, F. Mattioli University of Genoa, Department of Internal Medicine, Clinical Pharmacology and Toxicology Unit, Viale Benedetto XV 2, I-16132 Genoa, Italy A problem to be solved in the assessment of the genotoxic/carcinogenic risk to human in the extrapolation to humans of data provided by studies in laboratory animals. To contribute in the solution of this problem seven chemicals known to induce in the rat thyroid follicular-cell adenomas and/or carcinomas were examined for their ability to induce DNA lesions in primary cultures of human thyroid cells. Each chemical was tested on cells from at least three donors. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkalilabile sites, as measured by the Comet assay, were induced by a 20 h exposure to the following concentrations of the seven test chemicals: 4,4 -thiodianiline (THA) from 0.032 to 0.18 mM, 2,4-diaminoanisole (DAA) from 0.10 to 0.56 mM, 4,4 -methylene-bis(N,Ndimethyl)benzenamine (MDB) from 0.32 to 1.80 mM, nitrobenzene (NB) and potassium bromate (PB) from 1.25 to 5.0 mM, propylthiouracil (PTU) from 1.80 to 5.60 mM, methimazole (MZ) from 2.5 to 10 mM. At the highest concentration tested the average increase over control in the frequency of DNA lesions was: 1131% for PTU, 741% for DAA, 381% for MDB, 374% for THA, 369% for PB, 115% for NB and 88% for MZ. In conclusion, these findings suggest that the seven chemicals tested, six of which are classified by the International Agency for Cancer Research as possibly carcinogenic to humans, may cause tumors of the thyroid not only in rats but also in our species.


Abstracts / Toxicology Letters 158S (2005) S1–S258

P1-13 FORENSIC MEDICINE ASPECTS OF THE DEATHS, RELATED TO THE CONSUMPTION OF NARCOTIC SUBSTANCES IN KLAIPEDA COUNTY (LITHUANIA) G. Sniepiene Law University of Lithuania Institute of Forensic Medicine, Klaipeda Department, Lituania Drug-related mortality in Klaipeda County due to forensic medicine autopsies in 1993–2002 in average comprised 2.8 cases per 100,000 of population. The highest mortality rate was established in the city of Klaipeda during the year 2000 when there were 6.7 cases per 100,000 of population. During the investigation period, the most common death reason among the drug users was deaths due to accidental or intentional overdose—51.8%. Among the materials found in the biological samples of the deceased due to the overdose the following materials were established most often: opiates in average comprised 64.2% of all intoxication cases, benzodiazepines—50.9%, ethanol—28.3%, diphenhydramine—5.7% and psychostimulators— 5.7%. The most common narcotic and psychotropic substance combinations, which caused fatal intoxication were opiates–benzodiazepines (20.8%) and opiates–ethanol (9.4%) combinations. The average age of victims who died in Klaipeda County during the investigation period was 31.2 years. P1-14 IMMUNOLOGICAL DETECTION OF THE PEPTIDE BINDING CAPACITY OF SENSITIZING CHEMICALS P. Aeby1 , F. Python1,4 , P. Griem2 , C. Goebel3 1 Cosmital SA, Marly, Switzerland; 2 Clariant GmbH, Sulzbach am Taunus, Germany; 3 Wella AG, Darmstadt, Germany; 4 Supported by The European Cosmetic Toiletry and Perfumery Association (Colipa), Germany

In order to elicit a sensitization reaction, a chemical has to penetrate into the skin, react with proteins, be presented by antigen presenting cells and be recognized as antigenic by immune cells. Since a correlation between protein reactivity and skin sensitization

has been established, the measurement of the protein binding properties of a chemical may represent a promising endpoint for the development of an alternative approach to animal testing. We have therefore produced monoclonal antibodies with high affinity to CLIP (class II-associated invariant-chain) peptide. The synthetic CLIP peptides have been designed such that they have a target side chain to which the chemical to be tested can bind. Thus, covalent binding of the chemical or its metabolites can be detected in an enzyme-linked immunosorbent assay (ELISA) as a concentration-dependent decrease in antibody binding. The high-throughput assay under consideration consists of the CLIP peptide, which is immobilized by binding to polymer beads, and a CLIP peptidespecific monoclonal antibody conjugated with peroxidase. First results demonstrate that the assay is able to detect chemicals with protein binding properties. Further work is ongoing in order to establish a correlation between the protein binding capacity measured in our assay and the sensitization potential of a chemical. P1-15 BUILDING RELIABLE PREDICTIVE MODELS FROM HIGHLY CURATED DATABASES: NONGENOTOXIC CARCINOGEN MODELS FROM US FDA CRADA DATABASES C. Yang, G. Myatt, K. Cross, P. Blower Leadscope Inc., 1393 Dublin Road, Columbus, OH 43215, USA There are numerous challenges to the development of a comprehensive strategy for predictive toxicology. Access to high quality data, from which accurate predictive models can be generated, continues to be a major impediment. A toxicology controlled vocabulary, ToxML, based on the XML standard has been developed to expedite the integration of diverse database. High quality toxicity database was prepared using the ToxML standards. As an example, genetic toxicity and chronic databases from US FDA CFSAN and CDER via collaborative research and development agreement will be discussed. Based on the information available from these databases, models for nongenotoxic carcinogen models for drug-like compounds are developed. An approach to building and applying

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predictive model will also be described including an approach to subsetting the data into dataset from which predictive models can be built. P1-16 URINARY ENZYMES AS MARKERS OF OCCUPATIONAL NEPHROTOXICITY F.M. Metwally, K.S. Ibrahim National Research Centre, Dokki, Cairo, Department of Occupational Health and Industrial Medicine, Giza, Egypt Routine tests for measurement of renal function are insensitive for the detection of subclinical renal impairment. A marker of early renal affection is needed to be used for the screening of workers at risk. This study was designed to examine the activity of the urinary enzyme N-acetyl-B-d-glucosaminidase (NAG) as a marker of subclinical renal impairment in healthy and ill groups of workers exposed to nephrotoxic chemicals. The efficacy of NAG as a marker was also compared with that of urinary B2-microglobulin. The studied groups consisted of 132 subjects: 37 chemical laboratory workers exposed mostly to aliphatic hydrocarbon solvents, 33 car painters exposed mostly to aromatic hydrocarbon solvents, and 26 plumbers exposed mostly to lead fumes and a referent group of matched 36 clerks. Exposure indices included lifetime hydrocarbon exposure score (HES), lead in blood (ug/dL), and lifetime exposure score for lead (LES-Pb). NAG values differed significantly in all the groups compared to the control group. NAG also expressed a strong and consistent correlation with measures of cumulative exposure, unlike B2-microglobulin which failed to do so. Furthermore, an exposure-effect relationship existed between NAG and HES in solvent-exposed normotensive subjects. NAG values were higher among solvent-exposed hypertensives than among comparable normotensives. These results encourage the use of urinary NAG in the periodic screening of workers at risk, especially hypertensives and with renal disease, to avoid possible accelerated renal impairment among them.


P1-17 TOXICITY AND PHARMACOLOGICAL ACTIVITY OF TWO NEWLY SYNTHESIZED DERIVATIVES OF NICOTINIC AND ISONICOTINIC ACIDS L. Tantcheva1 , V. Petkov2 , G. Karamukova3 , Y. Chekalarova2 , S. Abarova2 , D. Tsekova4 , B. Escuder5 , J. Miravet5 , K. Lyubomirova6 1 Laboratory

of Drug Toxicology, Institute of Physiology, Bulgarian Academy of Sciences, Bulgaria; 2 Laboratory of Psychopharmacology, Institute of Physiology, Bulgarian Academy of Sciences, Bulgaria; 3 Faculty of Chemistry, Sofia University “Kliment Ohridski”, Bulgaria; 4 Faculty of Physics, Sofia University “Kliment Ohridski”, Bulgaria; 5 Dept.Organic and Inorganic Chemistry, University of Castelon, Spain; 6 Dept. Toxicology, National Center of Public Health Protection, Bulgaria Toxicity and biological activity of two newly synthesized compounds (named P-6 and M-6) were studied in ICR mice. Low oral and intraperitoneal toxicity was established (>2000 mg/kg b.w.; therapeutic index >8). Neuromuscular coordination was not influenced. Changes in the orientation, learning and memory were registered. Both compounds demonstrated good analgesic effect (>3 h). The effect of some model drugs affecting CNS also was modified. Hexobarbital sleeping time was significantly prolonged on the 1 h after P-6 administration and was available on the 24 h after. This action probably due to affinity to GABA-ergic receptors. In contrast, the compound M-6 had the opposite effect and antagonized the narcotic effect of hexobarbital. The difference in the effect of the compounds could be explained with the hiral diversity of their molecules. Pentileneterazole threshold was increased significantly 1 h after treatment with P-6 and M-6 and suggests any antiepileptic activity.The results obtained allow us making initial conclussions about their possible pharmacokinetics, mechanism of action and pharmacodynamic.


Abstracts / Toxicology Letters 158S (2005) S1–S258


Division, IRCCS Maugeri Foundation, Spain; 2 Fundaci´on Valenciana de Investigaciones Biom´edicas, Valencia, Spain; 3 University of Pavia, Pavia, Spain Cerebral cytochrome oxidase (COX) activity, nitric oxide (NO)-cyclic GMP (cGMP) pathway and cholinergic muscarinic receptors (MRs) are affected by acute carbon monoxide (CO) intoxication. The early and delayed effects of repeated and acute in vivo CO inhalation were investigated on COX activity, cGMP formation and MR binding in rat brain and lymphocytes to assess whether each endpoint was affected both centrally and peripherally. Neither CO repeated (500 ppm, 6 h/day, 5 days/week, 4 weeks) nor acute (2400 ppm, 1 h) treatment altered rat brain or lymphocyte COX activity and MR density 1 and 7 days post-exposure. In control lymphocytes cGMP levels (basal: 1.98 ± 0.99 pmol/mg protein) raised to 3.94 ± 0.55 pmol/mg protein after NO-stimulation. One day after chronic treatment cessation, the COtreated group displayed about a 50% decrease in both basal and NO-stimulated cGMP values, which persisted up to 7 days after. Acutely, CO caused a delayed enhancement (+140%) of NO-induced activation of soluble guanylate cyclase. The finding that the NOcGMP pathway is a target for the delayed effects of CO in peripheral blood cells is in accordance with our data in brain (J. Neurochem. 2004, 89, 1157–1165), and supports the use of this peripheral endpoint as a biomarker of CO central effects. (EU Grants: QLK4CT-2001-00186; FOOD-CT-2003-506543.)


Maugeri Foundation, Italy; 2 University of Pavia, Pavia, Italy

Lymphocyte cholinergic muscarinic receptors (MRs) have been proposed as surrogate markers of the same receptors in the CNS. The effects of methylmercury (MeHg) and PCB153 alone or in combination were evaluated on rat lymphocyte MR binding (using [3 H]QNB as muscarinic ligand) in vivo and in vitro. In vitro studies were also performed on human lymphocytes. In lymphocytes of adult rats orally exposed to 1 mg MeHg/kg/day for 21 days and sacrificed 14 days later, MR density was increased by 240%. PCB153, 20 mg/kg/day, fed for 7 days caused a decrease in MR density, and completely masked MeHg effect when given together with it. In in vitro studies on human and rat lymphocytes, MeHg caused 20%–40% inhibition of MR binding at 1 ␮M. Additionally, the concentration-dependent effects (0.01–100 ␮M) of PCB28, PCB77, PCB105, PCB118, PCB153, 4-OH-PCB107 were evaluated on lymphocyte MR binding in the presence or absence of 1 ␮M MeHg. In rat and human lymphocytes, no significant differences in IC50s (20–50 ␮M) were detected among the different PCBs, tested either alone or together with MeHg. At variance with in vitro data, the in vivo results provide evidence of an interaction between PCB153 and MeHg on rat lymphocyte MR binding which however does not occur directly at the receptor level. (Grants from the Italian Ministry of Health and from EU: QLK4-CT-2001-00186; FOODCT-2003-506543.)

Abstracts / Toxicology Letters 158S (2005) S1–S258


Burdan2 ,

Szumilo1 ,

Chy˙zy´nska1 ,

J. F. J. M. R. Klepacz1 , A. Korobowicz3 , L. Wronecki1 , E. Tokarska4 , E. Korobowicz1 1 Clinical Pathomorphology Department, Medical Uni-

versity of Lublin, Poland; 2 Experimental Teratology Unit of the Human Anatomy Department, Medical University of Lublin, Poland; 3 Paediatrics Pulmonology and Rheumatology Department, Medical University of Lublin, Poland; 4 Jaw Orthopaedics Department, Medical University of Lublin, Poland The potential interaction of methanol/ethanol and methanol/4-methylpyrazole within the scope of the total antioxidant status (TAS) in rat brain was investigated in the study. The experiment was carried out on rats exposed to N2 O/O2 mixture (1:1, v/v) which allow obtain similar response of rat organism on methanol poisoning to that observed in human. The rats were exposed to N2 O/O2 mixture 4 h before the administration of methanol or saline. N2 O/O2 exposure was continued throughout the experiment. Methanol was administered per os at a dose of 3 g/kg, then, after next 4 h ethanol (0.5 g/kg or 1 g/kg) or 4-methylpyrazole (15 mg/kg or 50 mg/kg) was administered by i.p. injection. The rats were decapitated 8 h after antidote treatment. TAS was determined spectrophotometrically in rat brain homogenates. The mean value concentration of TAS in rat exposed on N2 O/O2 mixture did not differ to that determined for rat control. However, decrease in TAS concentration in all groups of rats intoxicated with methanol together with ethanol or 4-methylpyrazole and methanol alone was revealed. There were no interactions between methanol and 4methylpyrazole. However, significant decrease in TAS concentration was observed in rats ingested methanol and than administered with 1 g/kg of ethanol comparing to rats receiving methanol alone (p < 0.05). It suggests an adverse interaction of methanol/ethanol within the scope of the brain antioxidant status homeostasis.


P2-04 TOLERABILITY OF SELECTIVE AND NONSELECTIVE CYCLOOXYGENASE-2 INHIBITORS IN BREAST-FEEDING RAT FEMALES F. Burdan1 , A. Chałas1,2 , J. Szumilo2 , J. Dudka3 , A. Korobowicz1,4 , B. Madej1 , L. Klepacz1 , E. Tokarska5 , R. Klepacz3 , Z. W´ojtowicz1 1 Experimental Teratology Unit of the Human Anatomy

Department, Medical University of Lublin, Poland; 2 Internal Medicine Department, Medical University of Lublin, Poland; 3 Clinical Pathomorphology Department, Medical University of Lublin, Poland; 4 Paediatrics Pulmonology and Rheumatology Department, Medical University of Lublin, Poland; 5 Jaw Orthopaedics Department, Medical University of Lublin, Poland Maternal toxicity of high doses of nonselective cyclooxygenase (COX) inhibitors was previously observed. The aim of the present study was to evaluate the late effects of nonselective (tolmetin, ibuprofen, piroxicam) and selective (DFU) COX-2 inhibitors on breast-feeding mothers. The drugs were administered orally once (DFU, piroxicam) or three times (tolmetin, ibuprofen) a day from Days 8 through 21 of gestation in three doses. The initial dose was similar to the human anti-inflammatory one and set as 8.5 (tolmetin, ibuprofen), 0.3 (piroxicam) and 0.2 mg/kg (DFU). The middle dose was increased five times and the highest one 10 times than initial dose. After spontaneous labour dams and litters were kept until Day 7 post partum without any xenobiotics. On lactation Day 7 mothers were sacrificed, blood was collected and abdominal organs were taken for pathological examination. Activity of alanine and aspartate aminotransferases and levels of total protein and urea were determined. Stomach, small and large intestines, liver and kidneys were grossly and histologically examined. Expression of COX-1 and COX-2 was evaluated immunohistochemically. Dosedependent mortality, signs of gastrointestinal toxicity, and biochemical changes were found in groups exposed to nonselective COX inhibitors. Adaptive and sporadically degenerative hepatic and renal changes were also observed. Insignificant immunoexpression changes of both COX isoenzymes were found among experimental


Abstracts / Toxicology Letters 158S (2005) S1–S258

groups. It could be stressed that tolerability of nonselective COX inhibitors, in spite of discontinuation of xenobiotics administration on the end of pregnancy, was lower compared with selective COX-2 compound. Supported by the Polish Committee of Scientific Research, Grant No. 3 P05A 048 25

P2-06 SUCCINATE DEHYDROGENASE INHIBITION BY ALKOXYACETATES J. Liesivuori1,2 , S. Pennanen1 , Paldanius2 , H. Savolainen3

J.Laitinen1 ,


1 Finnish

P2-05 DISTRIBUTION OF N-NITROSOMETHYLBENZYLAMINE-INDUCED LESIONS IN SECONDARY TARGET ORGANS IN RATS J. Szumilo1 , F. Burdan2 , J. Dudka1 , R. Klepacz1 , E. Korobowicz1 1 Clinical Pathomorphology Department, Medical Uni-

versity of Lublin, Poland; 2 Experimental Teratology Unit of the Human Anatomy Department, Medical University of Lublin, Poland N-Nitrosomethylbenzylamine (NMBA) is known to be one of the most potent carcinogens routinely used for the study of esophageal rat tumorigenesis. The secondary target organs for NMBA are: tongue, throat and forestomach. However the lesions in these locations are rarely reported. To verify organ-specificity of NMBA the mucosa of oral cavity, pharynx and forestomach of 17 male albino rats of Wistar strain was investigated. Animals were exposed on NMBA for 5 weeks (s.c.; 1 mg/kg/dose; 3 doses/week). The experiment was terminated after 22 weeks. The incidence, multiplicity, size and location of the lesions were evaluated. Histological examination was also performed. The lesions in the oral mucosa appeared in all 17 rats (100%). The average number of lesions/rat was 4.77 (range 1–8). The average size of the lesion was 1 mm (range 0.5–4 mm). All rats had lesions on the tongue (mostly on the dorsal surface of the root), four on the bucca, one on the palate, and one on the gingiva. The lesions in the throat were found in two rats (one lesion/rat). The lesions were not observed in the forestomach. Histologically, 80.72% of lesions were simple hyperplasia of squamous epithelium, 18.07%—squamous cell papilloma, and 1.2%—squamous cell carcinoma. The results indicate that NMBA-induced lesions are frequent in secondary target organs and the carcinogen can be potentially used in the study of oral rat tumorigenesis as well.

Institute of Occupational Health, Finland; of Kuopio, Kuopio, Finland; 3 Ministry for Social Affairs and Health, Tampere, Finland

2 University

Ethylene glycol ethers have been extensively used as water miscible nonodorous solvents in paints and pharmaceuticals but are being phased out because of their considerable toxic potential. It extends from neurotoxicity to haematological and reproductive effects. Some of the congeners are also suspected carcinogens. Their mechanism of toxicity is not clear while most authors agree that it is related to the biological activity of the metabolic end product, i.e. the alkoxyacetic acids. The most common acids are methoxy-, ethoxy- and butoxyacetic acid. Ethylene glycol methyl ether shows dose-dependent neurotoxicity associated with a decreased activity of succinate dehydrogenase at the complex II of mitochondria. Likewise, occupational exposure to ethyl or butyl ethers was associated with dose-dependent decrease in urinary succinate dehydrogenase activity as compared with actual alkoxy acid burden. This circumstance can be reproduced in an animal model. SDH activity of three ethylene glycol ether metabolites was determined with a microplate method. Methoxyacetic acid was the strongest SDH inhibitor, then butoxyacetic acid and ethoxyacetic acid. The inhibitory effect on the succinate dehydrogenase activity may prove very important in the light of new hypotheses on mitochondrial roles in tumorigenesis and apoptosis. Specifically, non-functional succinate dehydrogenases are directly implicated. P2-07 EFFECTS OF THE DIESTER PHTHALATE METABOLITE MEHP IN PRIMARY RAT LUNG CELLS K.E. Rakkestad, J.A. Holme, P.E. Schwarze, R. Becher Norwegian Institute of Public Health, Oslo, Norway Phthalates (dialkyl or alkyl/aryl esters of 1,2benzenedicarboxylic acid) are a class of widely used

Abstracts / Toxicology Letters 158S (2005) S1–S258

industrial compounds. They are mostly used as plasticizers, and are found in many products that we handle daily including building materials (PVC), medical products, cosmetics and children’s toys. Recent studies have suggested a link between the increased global production of these compounds and the increase in allergic symptoms in children. The most widely produced and used phthalate is DEHP (di-ethylehexyl phthalate) which is enzymatically hydrolyzed to the corresponding monoester MEHP (mono-(2-ethylhexyl) phthalate). Here we have characterized effects of MEHP on the release of cytokines and cell death in primary rat lung macrophages and type-2 epithelial cells, and examined possible roles of the MAP kinases and various PPAR isoforms. MEHP caused cell death in both cell types, with the type-2 cells as most sensitive. On the other hand, macrophages seemed more prone to die by apoptosis. Preliminary results indicate that MEHP did not affect the release of MIP-2. Interestingly, in macrophages MEHP induced formation of ROS (reactive oxygen species), and reduced phosphorylation of ERK 1/2. The role of PPAR isoforms and expression in these processes is currently under investigation. P2-08 CORRECTIVE INFLUENCE OF METABOLIC ACTION POLYVITAMIN COMPOSITION WITH EXPERIMENTAL INDUCTION OF CYTOCHROME P-450 2E1 L.G. Berezhna, A.K. Voronina, O.S. Voloshina, G.M. Shayakhmetova, V.M. Kovalenko Institute of Pharmacology and Toxicology, Academy of Medical Sciences of Ukraine, Kyiv, Ukraine Influence of metabolic action polyvitamin composition (MV) on some rat liver and serum biochemical parameters with combined introduction of cytochrome P-450 2E1 isoniazid (H) and rifampicin (R) has been investigated. Isoniazid and rifampicin were introduced to rats in doses 45 and 86 mg/kg b.w. per os 28 days (control). Against a background of preparations experimental polyvitamin composition was introduced in dose 50 mg/kg b.w. Introduction of H and R caused increasing of CYP 2E1 marker enzyme p-nitrophenolhydroxylase activ-


ity (to 2.5 times), cytochrome P-450 contents (to 1.3 times) and NADPH-dependent lipid peroxidation (to 66%) in liver microsomal fraction. Serum bilirubin level was increased 6.5 times in control group in comparison with intact rats. Combined introduction of MV with tuberculostatics produced decreasing of p-nitrophenolhydroxylase activity (to 46.5%), cytochrome P-450 contents (to 15%) and NADPHdependent lipid peroxidation (to 45%) comparison with control. Use of MV caused also serum bilirubin level normalization to the level of intact animals. Thus, experimental polyvitamin composition could inhibit cytochrome P-450 2E1 induction by decreasing speed of highly reactive intermediates formation and processes of free radical oxidation, causing hepatoprotective action in conditions of combined isoniazid and rifampicin introduction to rats. P2-09 DEGRANULATION OF CARDIAC MAST CELLS IN RATS ACUTELY POISONED BY T-2 TOXIN: INVOLVEMENT IN CARDIOTOXICITY V. Jacevic1 , L. Zolotarevski2 , K. Jelic2 , Milosavljevic2 , D. Bokonjic1 , M.P. Stojiljkovic1


1 National

Poison Control Centre, Serbia and Montenegro; 2 Insitute of Pathology and Forensic Medicine, Military Medical Academy, Belgrade, Serbia and Montenegro We have already shown that administration of T-2 toxin, a cardiotoxic mycotoxin, lead to diffuse accumulation of mast cells (MCs), preferentially with perivascular localization. The aim of this study was to investigate a total number of degranulated MCs (DMCs) in rats poisoned with single injection of 1 LD-50 of T-2 toxin (0.23 mg/kg sc) and its potential contribution of these cells to its cardiotoxicity. Twenty four hours, 3, 5 and 7 days after T-2 toxin application cardiac MCs, previously stained by Giemsa method, were counted in whole visual fields, magnification 40. In the control rats majority of cardiac MCs were tiny and hypogranular. Twenty four hours after administration of T-2 toxin more than 60% of the counted MCs showed degranulation. These large, round or ovoid cells were diffusely spreaded in epicardium, myocardium and endocardium, and secreted a large amount of different


Abstracts / Toxicology Letters 158S (2005) S1–S258

granules. After 5 days T-2 toxin lead to the strongest degranulation of MCs (82%), what was accompanied with capillary damage, haemorrhage and focal polymorphonuclear cell infiltrations. At the end of experiment T-2 toxin increased the total number of heart DMCs by 415% tissue comparing with the control. These results have shown that diffuse tissue accumulation of MCs has probably significant role in cardiotoxic effects of T-2 toxin in rats, due to degranulation and its active mediators involvement in acute inflammatory reaction caused by this mycotoxin. P2-10 CHANGES IN NEUROBEHAVIOURAL PARAMETERS AND SPONTANEOUS CORTICAL ACTIVITY IN RATS AFTER ACUTE AND SUBCHRONIC ADMINISTRATION OF MK-801 A. Luk´acs, A. Szab´o, L. Nagymajt´enyi Department of Public Health, University of Szeged, Hungary Systemic administration of the non-competitive NMDA antagonist MK-801 prevents NMDA-induced neuronal degeneration. Adult male Wistar rats were treated intraperitoneally with low (0.05 mg/kg) or high (0.1 mg/kg) dose of MK-801 acutely and subchronically. In acute treatment (10 per group) behavioural measurements (open field, acoustic startle response) were done before and 90 min after the treatment; and electrophysiological examinations were done 24 h later. In subchronic treatment (11 control and 2 × 14 treated), rats were injected every other day, altogether seven times. In the 1st, 5th and 10th weeks, open field activity was measured. Pre-pulse inhibition (PPI) of the acoustic startle response (ASR) was tested at the end of the experiments. Following the behavioural tests, spontaneous somatosensory, visual and auditory cortical activity was recorded. Horizontal and local activity of the animals increased significantly, but vertical activity decreased, after the acute treatment. In the 5th and 10th week of the subchronic experiment, exploratory activity (grooming, run length, run speed) decreased significantly. Number of noise-positive responses in the ASR after PPI was not changed significantly after acute and subchronic treatment. In the spontaneous cortical activity,

there were non-significant changes after single dose of MK-801. Subchronic administration of the drug resulted in more marked alterations. PPI and spontaneous cortical activity in the treated rats suggested the involvement of the brainstem cholinergic systems in the action of MK-801. The alterations in open field activity suggested the change of certain neurotransmitter levels (dopamine, glutamate) in the centers responsible for the behavioural functions. Supported by the Hungarian ETT grant No. T08356. P2-11 EFFECTS OF ANNULATOPHENONE (Hd15), ISOLATED FROM HYPERICUM ANNULATUM, ON ISOLATED HEPATOCYTES AND SYNAPTOSOMES FROM OLD RATS M. Kondeva1 , M. Mitcheva1 , P. Nedialkov2 , G. Kitanov2 1 Department

of Pharmacology and Toxicology, Faculty of Pharmacy, Medical University, Sofia, Bulgaria; 2 Department of Pharmacognosy and Botany, Faculty of Pharmacy, Medical University, Sofia, Bulgaria The aim of the study was to investigate the effect of 100 ␮M Annulatophenone (Hd15) on isolated rat hepatocytes and synaptosomes. Our experiments were carried out on old male Wistar rats, weighted 300–350 g. Two-stepped collagenase perfusion for isolation of hepatocytes and a Percol reagent for the synaptosomes isolation were used. In isolated hepatocytes cell viability was assessed by Trypan blue (0.05%)-test and in synaptosomes by MTT-test. The hepatocytes and synaptosomes were incubated with Hd15 and the toxic agents for 1 h. Carbon tetrachloride (CCl4 )—86 ␮M was used as a toxic model in isolated hepatocytes and itself decreased cell viability by 51%. In synaptosomes Dopamine—150 ␮M, as a toxic model it decreased viability by 84%. The effects of Hd15—100 ␮M administered in combination with CCl4 protected hepatocytes viability by 52% and in combination with Dopamine, protected synaptosomes viability by 75% statistically significant. According to our results, we could suggest protective effects of Hd15 on CCl4 -induced toxicity in isolated hepatocytes and Dopamin-induced toxicity in synaptosomes.

Abstracts / Toxicology Letters 158S (2005) S1–S258




M. Tang1 , N.A. Helsby2,3 , W.R. Wilson3 , S. Al-Ali4 , M.D. Tingle1

G. Gribilas1 , H. Karantonis2 , N. Tsantila2 , G. Kouraklis1 , C. Demopoulos2 , C. Spiliopoulou1 , S. Theocharis1

1 Department

of Clinical Pharmacology and Pharmacology, New Zealand; 2 Department of Molecular Medicine and Pathology, New Zealand; 3 Auckland Cancer Society Research Centre, New Zealand; 4 Department of Anatomy, University of Auckland, Auckland, New Zealand

CB 1954 is currently in early clinical trials in a genedirected enzyme prodrug therapy (GDEPT) context in combination with E. coli nitroreductase for cancer chemotherapy. However, a Phase I trial of the prodrug alone revealed dose-limiting gastrointestinal and liver toxicity [1]. Previously we have shown that human liver preparations can nitroreduce CB 1954 to its 4hydroxylamine and 2-amine metabolites, both of which are highly cytotoxic. Initial studies revealed that mouse liver preparations produce similar metabolites in vitro. A mouse model was used to determine the toxicological consequences of hepatic bioactivation of this prodrug. CB 1954 was administered to mice at the maximum tolerated dose (MTD) of 562 ␮mol/kg. At various time points up to 21 days post-dose, plasma and liver samples were collected for analysis of liver toxicity. Clinical chemistry evaluation demonstrated that serum levels of ALT and AST were raised up to 6 and 16 times normal levels, respectively, between 3 and 6 days post-dose, after which levels returned to normal. Histopathological examination of the livers revealed focal changes mainly in the midzonal and centrilobular areas. At 6 days post-dose, two animals had areas of severe necrosis and extensive cellular damage throughout the liver. There was a good correlation between elevated transaminase levels and histopathological changes. Combined with in vitro studies with mouse liver preparations, these results suggest that CB 1954 may cause hepatotoxicity in the mouse as a consequence of reductive bioactivation of the prodrug by endogenous enzymes.

1 Department

of Forensic Medicine and Toxicology, School of Medicine, Greece; 2 Laboratory of Biochemistry, Faculty of Chemistry, University of Athens, Greece Platelet-activating factor (PAF) is a potent lipid inflammatory mediator acting on cells through its specific receptor. Plasma PAF-acetylhydrolase (PAF-AH) is the main enzyme that inactivates PAF in blood, participating in its homeostasis. To investigate the involvement of PAF in liver fibrotic process using an experimental model. Liver fibrosis was induced on adult male Wistar rats by thioacetamide (TAA) administration in drinking water (300 mg/L) for 3 months. The animals were sacrificed at 0 (control group), 1, 2 and 3 months later on. PAF levels in liver and blood as also PAF-AH activity in plasma were determined. TAA administration resulted in progressively increased liver fibrosis, leading finally to the formation of cirrhotic nodules in liver. Throughout the experimental time PAF levels in liver tissue remained stable. “Total” (“free” plus “bound”) PAF levels in blood were decreased, reaching statistically significant differences at the 1st and 3rd month compared to control group (p <0.05). “Free” PAF levels in blood were increased at the 1st month (p < 0.05) and decreased gradually thereafter. In all treated groups “bound” PAF levels in blood were decreased and plasma PAF-AH activity was increased statistically significantly, compared to control group. Alterations of PAF levels in blood and PAFAH activity during fibrosis induction were observed, implicating the participation of PAF in liver fibrotic process.


Abstracts / Toxicology Letters 158S (2005) S1–S258



M.M. Szutowski1 , J.S. Rakoto2

N. Aubert1,2 , A. Falluel-Morel1 , D. Vaudry1 , A. Fournier 3 , C. Fisch2 , S. De Jouffrey2 , J.F. Le Bigot2 , H. Vaudry1 , B.J. Gonzalez1

1 Department of Toxicology, Medical University of War-

saw, Poland; 2 Valeant Pharmaceuticals International, Warsaw, Poland The metabolic profile of n-heptane observed in rat urine include heptanols, heptanediols, heptanones, hydroxyheptanones, ␥-valerolactone and heptanediones identified as 2,5-heptanedione and 2,6-heptanedione. Metabolites that posses gamma diketone configuration, together with capability for pyrrolic adducts formation with primary amines of neurofilamental protein, are considered to be responsible for neurotoxic effects of n-hexane and n-heptane metabolites. Experiments with rat liver microsomes and reconstituted monooxygenase system with cytochrome P450 indicated that these biotransformation models catalyze formation of other diketones: 2,4-heptanedione and 3,5-heptanedione. These metabolites are not neurotoxic. The catalytic activity of the liver reconstituted system from rats inhaled with C6 –C9 petroleum fraction differs from that of control rats, in a significant decrease in hydroxylation and oxidation to ketone at the second carbon and to some extent in a decrease at 1hydroxylation and 3-oxidation. At the fourth carbon of n-heptane molecule slight increase in hydroxylation and oxidation was observed. The increase in the formation of 2,4-heptanedione but not 3,5-heptanedione was evident however less marked than the change in the activity at the fourth carbon. In conclusion, the heptanediones formed by rat liver monooxygenase system with cytochrome P450 were not neurotoxic and induction of cytochrome P450 during inhalation with C6 –C9 petroleum fraction also did not support formation of the neurotoxic metabolites.


U413, IFRMP 23, University of Rouen, France; 2 CIT, Evreux, France; 3 INRS-Inst. A. Frappier, University of Quebec, Canada The neuropeptide pituitary adenylate cyclaseactivating polypeptide (PACAP) prevents ceramideinduced neurotoxicity and acts as a potent stimulator of the expression of both c-Fos and the anti-apoptotic factor Bcl2. As Bax and Bcl2 are known to be regulated by the transcription factor AP-1 (Fos/Jun member dimers), the aim of the present study was to determine whether ceramides and PACAP affect the formation of AP-1 complexes. Gel Shift experiments indicated that PACAP and C2-ceramide stimulated AP-1 binding in a dose- and time-dependent manner. Co-incubation of granule neurons with PACAP and C2-ceramide resulted in an additive stimulation of AP-1 DNA binding. Characterization of AP-1 complexes indicated that PACAP-induced AP-1 were enriched in c-Fos while C2-ceramide increases the proportion of c-Jun. Western blot analysis showed that PACAP increased c-Fos expression within 30 min and C2-ceramide stimulated c-Jun phosphorylation within 1 h of treatment. The effect of C2-ceramide on c-Jun phosphorylation was blocked by PACAP, the JNK inhibitor and a PP2A blocker, while the action of PACAP on c-Fos was abrogated by C2-ceramide, an ERK blocker and a PKA inhibitor. Altogether the present results demonstrate that PACAP and C2-ceramide induce the formation of different AP-1 complexes through JNK/PP2A- and ERK/PKA-dependent pathways, respectively.

Abstracts / Toxicology Letters 158S (2005) S1–S258




G. d’Annunzio University School of Medicine & Center of Excellence on Aging. Via dei Vestini, 66013 Chieti, Italy

Department of Biochemical Toxicology, Faculty of Pharmacy Collegium Medicum, Jagiellonian University, Krak´ow, Poland

Cardiotoxicity limits the clinical use of anticancer anthracyclines like doxorubicin (DOX) or epirubicin (EPI), but EPI causes cardiac events at doses higher than equimyelotoxic to DOX. This suggests that EPI might be less active than DOX at forming toxic species like a secondary alcohol metabolite or reactive oxygen species (ROS). To probe this hypothesis in preclinical settings we obtained myocardial samples from cardiac-surgery patients and exposed them to pharmacokinetically relevant concentrations of anthracyclines (1–10 ␮M). Metabolites were measured by HPLC, and ROS were measured by a dichlorofluorescein assay adapted to studies in whole tissues. DOX and EPI exhibited a similar uptake in human myocardium but EPI formed less alcohol metabolite than DOX (nmol/g/4 h: 0.03 ± 0.002 versus 0.06 ± 0.002 at 10 ␮M; n = 6, P < 0.001). This was due to increased km and reduced Vmax of EPI for the reductases that form anthracycline secondary alcohol metabolites in cytoplasm. EPI also formed much less ROS than DOX. Subcellular fractionation, confocal microscopy, and pharmacologic inhibition of vacuolar H+-ATPase showed that this was due to an entrapment of EPI in acidic vesicles and consequent reduced access of EPI to mitochondrial reductases that convert anthracyclines to ROS. These results uncover molecular determinants of the reduced cardiotoxicity of EPI and may serve guidelines to further improve its cardiac safety in patients. (Supported by AIRC, MIUR Cofin 2004, and FIRB)

Methoxy-, ethoxy-, n-propoxy- and n-butoxy acetic acids are known urinary metabolites of the corresponding alkoxyethanol solvents. It is well documented that these chemicals cause acute hemolytic anemia in laboratory animals and in humans. Current studies were designed to compare the hemolytic activity of four alkoxyacetic acids in erythrocytes (RBCs) derived from the health volunteers and male Wistar rats. The RBCs from blood samples were washed in a buffer pH 7.4, adjusted to a PCV of 45% and incubated in the presence of different concentrations of alkoxyacetic acids for 0–3 h at 37 ◦ C in a water bath. The mean cellular volume (MCV), free hemoglobin (HGBfree ), and lactate dehydrogenase (LDH) activity in the incubative medium were used as the indices of hemolytic activity of examined acids. On the basis of dose–effect relationship of these indices the EC50 values were calculated. Human RBCs were relatively more resistant to the hemolytic action of alkoxyacetic acids than rat one. EC50 values of the alkoxyacetic acids in human RBCs were 4–10 times higher than in rat erythrocytes. HGBfree concentrations correlated well with LDH activity. EC50 values of alkoxyacetic acids correlated well with the total number of ether group carbons, log P values, and pKa values. These results exemplify appropriate end-points for demonstrating species and compound differences in hemolytic activity.


Abstracts / Toxicology Letters 158S (2005) S1–S258



H. Raunio, S. Neshybova, R. Juvonen

1 Department

Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland CYP2E1 metabolises a number of toxic agents, including alcohols, low molecular weight solvents, several procarcinogens, as well as some clinically used drugs. The majority of substrates are neutral, small molecular weight compounds with relatively low log P values. The enzyme is also inhibited by numerous compounds, e.g. diethyldithiocarbamate. We developed a rapid and sensitive 96-well plate assay for measuring catalytic activity of recombinant human CYP2E1 using 7-methoxy-4trifluoromethylcoumarin as substrate. 28 structurally diverse compounds were tested with this system for their inhibitory potency against CYP2E1, and a SAR analysis was carried out. Measured as the IC50 value, aliphatic alcohols inhibited CYP2E1 in the following order: 1-pentanol (IC50 = 2 ␮M), 2-butanol, 1butanol, 2-propanol, 1-propanol, ethanol, methanol (IC50 = 1900 ␮M). The IC values of several solvents ranged from 150 ␮M (acetone) to 6000 ␮M (acetonitrile). Thus, increasing length of hydrocarbon chain of 1-alcohols increases inhibition potency. Several compounds with aromatic and lactone structures were also assayed. Inhibition potency increased in ␥-lactones with increased length of alkyl side chain in position 5. The most potent inhibitor was 2,3-dihydrobenzofuran (IC50 = 0.2 ␮M), which thus constitutes a novel potent inhibitor of CYP2E1. The data obtained in this study will be used to construct a 3D-QSAR model of the human CYP2E1 enzyme. The method used (Comparative Molecular Field Analysis, CoMFA) will take into account both steric and electrostatic interactions between the inhibitors and the CYP2E1 enzyme.

L.E. Korhonen1 , M. Rahnasto1 , C. Wittekindt2 , A. Poso2 , H. Raunio1 , R.O. Juvonen1 of Pharmacology and Toxicology, Finland; 2 Department of Pharmaceutical Chemistry, University of Kuopio, POB 1627, 70211 Kuopio, Finland

In the uninduced state CYP2B6 represents less than 0.2% of total human hepatic P450, but its amount is increased by e.g. phenobarbital type compounds. It is involved in the metabolism of 3% of clinically important drugs as well as numerous other chemicals. Currently, substrate/inhibitor structure-activity relationships of CYP2B6 are not well characterised. Thus, the aims of this study were (1) to determine the IC50 values of 45 structurally related compounds toward CYP2B6 catalyzed 7-ethoxy-4-trifluoromethyl coumarin hydroxylation and (2) to create a comprehensive 3DQSAR (CoMFA) model of these CYP2B6 inhibitors. The lowest IC50 values were obtained with benzylpyridine derivatives (<1 ␮M). High IC50 values were obtained for compounds containing a lactone structure, e.g. 28 mM for ␧-caprolactone. The created CoMFA model was of high quality with the following statistical values with two components: q2 = 0.67, SPRESS = 0.50, r2 = 0.85. The results indicate that heterocyclic nitrogen in benzylpyridine increases inhibition potency. The inhibition potency is increased by addition of bulk to carbon 4 of the benzyl ring of benzylpyridine. Broad partial negative charge near to a nitrogen atom and around benzyl tends to increase the inhibition potency. CoMFA modelling of CYP2B6 is an excellent method to understand the molecular properties of CYP2B6 inhibitors. P2-20 ALTERATIONS OF MICROTUBULE CONTENT IN CEREBRUM, SPINAL CORD AND SCIATIC NERVE OF 2,5-HEXANEDIONE INTOXICATED RATS G. Li1,2 , J. Tian2 , B. Li1 1 Marine

Drug and Food institute, Ocean University of China, Qingdao, Shandong, PR China; 2 School of Pharmacy, Yantai University, Yantai, Shangdong, PR China

Abstracts / Toxicology Letters 158S (2005) S1–S258

To investigate whether the alterations of microtubule content are responsible for the neurotoxicity of 2,5hexanedione. 2,5-hexamedione was administered by intraperitoneal injection to Wistar rats with daily dose of 200 and 400 mg/kg for continuous 8 weeks (five times every week). Cerebrum, spinal cord and sciatic nerve were excised and homogenized, then centrifuged to prepare the supernatant and pellet sample. The relative levels of a7tubulin and rtubulin were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Westen-blot. The contents of ␣tubulin and ␤-tubulin in the cerebrum were not obviously affected with the relative contents from 87% to 114% of the control. For the spinal cord, they kept unaffected in the pellet, but dropped to 59% and 47% on the supernatant of 200 mg/kg group, while they increased to 126% and156% of the control in 400 mg/kg group. In addition, 2,5-hexanedione intoxication dose-dependently reduced the tubulin content to 22%–70% of the control in the supernatant of the sciatic nerve while they kept unaffected in the pellet. 2,5-hexanedione intoxication results in alternations of microtubule content and the alternations might be related to its peripheral neurotoxicity. P2-21 EFFECT OF BUTENOLIDE ON THE RESPRATORY CHAIN OF MYOCARDIUM MITOCHONDRIA J. Tian1,2 , F. Fu1 , M. Geng2 1 Marine

Drug and Food institute, Ocean University of China, Qingdao, Shandong, PR China; 2 School of Pharmacy, Yantai University, Yantai, Shangdong, PR China To study the mechanism of the cytotoxicity induced by butenolide on myocytes. Cultures of myocytes were performed by differential plating method and myocyte viability was tested by applying the MTT-test. Mitochondria were isolated by the conventional differential centrifugation method from the hearts of Wistar rats. Malondialdehyde (MDA) was quantified by the thiobarbituric acid (TBA) method Activities of respiratory chain complexes was determined by an UV spectrophotometer. Butenolide showed toxicity in a dose-dependent manner to myocyte and the LC50 value was about 5.3 ␮g/ml. The activities of complexes I and


II decreased in treated mitochondrial suspension with butenolide when compared with controls. On the contrary, the activities of complexes III and IV increased in treated mitochondrial suspention. Primary culture myocytes are sensitive to the toxicity of butenolide. Our results indicate that heart is one of the major organs damaged by lipid peroxidation caused by butenolide. P2-22 ATROPINE PREVENTS AMPHETAMINEINDUCED HYPERTHERMIA IN MICE F. Carvalho1 , J.A. Duarte2 , C. Sousa1 , F. Remi˜ao1 , H. Carmo1 , M. de Lourdes Bastos1 1 REQUIMTE,

Department of Toxicology, Faculty of Pharmacy, University of Porto, Portugal; 2 Department of Sport Biology, FCDEF, University of Porto, Portugal Both amphetamine and atropine are reported to be used in war zones. The intake of any of these drugs may lead to variations in body temperature. However, the possible toxicological effects resulting from their concomitant use is yet to be determined. Thus, the aim of the present study was to evaluate the effect of atropine in d-amphetamine-induced hyperthermia in mice. The potential toxicity of both compounds to the liver, kidney and heart was also determined, by using the glutathione content as a biomarker of effect. Charles River CD-1 mice were used. Atropine (5 mg/kg, i.p.) was administered 10 min before damphetamine (10 mg/kg, i.p.). Temperature readings were obtained during a period of 3 h with a noninvasive technique using implanted transponders and a temperature probe reading device. d-Amphetamine induced an hyperthermic state with a maximum effect at 60 min. Atropine, by itself induced a slight hypothermic effect. Pre-treatment with atropine completely prevented the hyperthermic effect of d-amphetamine. Both drugs induced a significant depletion of hepatic glutathione when administered separately. However, glutathione depletion was prevented when atropine and d-amphetamine were administered together. In conclusion, the present study indicates that atropine prevents d-amphetamine-induced hyperthermia and liver toxicity. Authors are thankful to FCT for financial support (PRAXIS XXI/BD/20088/99).


Abstracts / Toxicology Letters 158S (2005) S1–S258



´ c2 , S. Dobri´c3 , N. Krleska Veleva4 R. Velev1 , V. Cupi´

G.M. Prodanchuk, S.G. Shandrenko, T.O. Kishko, M.P. Dmitrenko

1 Faculty

Institute of Ecohygiene and Toxicology, Ukraine

of Veterinary Medicine, Lazar Pop Traajkov 5-7, 1000 Skopje, Republic of Macedonia; 2 Faculty of Veterinary Medicine, Belgrade, Yugoslavia; 3 National Poison Control Center, Military Medical Academy, Belgrade, Yugoslavia; 4 Replek AD, Kozle 188, 1000 Skopje, Republic of Macedonia E-mail address: [email protected] The use of nonsteroidal anti-inflammatory drugs (NSAIDs) for the treatment of inflammatory conditions is limited by their significant untoward effects on the gastric mucosa. The ulcerogenic effects of NSAIDs are at least in part attributable to increase of total acid output of gastric juice and disturbances in gastric blood flow. In an attempt to develop NSAIDs which do not cause gastric injury, we compared the intensity of histopathological changes in gastric mucosa of two NSAIDs (indomethacin, nimesulide) when there are given alone or concomitant with donors of nitric oxide (glyceryl trinitrate or l-arginine). Two groups of rats were treated single with ulcerogenic doses of tested NSAIDs (25 mg/kg p.o.). Other groups were treated with GTN (6.25 mg/kg p.o.) or l-arginin (100 mg/kg i.p.) immediately after tested NSAIDs. Four hours after the treatment the animals were sacrificed and severity of gastric mucosal damage was determined by measuring the length, area and intensity score of gastric lesions, as well as by their pathomorphological analysis. The results showed that single administration of nimesulide produces significantly less pronounced gastro toxic effects than indomethacin given in the same dose. GTN significantly reduced all parameters of ulcerogenic activity of tested NSAIDs and l-arginine also produced a significant gastro protective effect that was, in the presence of l-NAME (1 mg/kg i.p.), an inhibitor of NO-sinthase, completely abolished. Our results confirm the significant role of NO in gastric mucosal defense and imply the possibility of therapeutic use of NO-donors as antiulcer drugs.

Hydroxylamine (HA) acute poisoning could result in life threatening methemoglobinemia and collaptoid state due to HA induced NO formation. That is why HA poisoning treatment programs are predominantly oriented for reduction of NO toxic influence. In vitro experiments using thymocytes with introduced NO trap – a complex of diethyldithiocarbamade Na with iron – showed that cytotoxic influence of HA sulfate (HAs) was only partly (less than 40%) connected with NO formation. With the help of electron spin resonance method it was established, that during Per Os HAs introduction to rats in dose 400 mg/kg a fast and intensive formation of methemoglobin (metHb) and hemoglobin nytrosyl complex (Hb–NO) is noticed. However, dynamic of this changes indicates that there are significant differences between mechanisms of toxic influence of HA and NO. Thus, rats that received HAs showed decreasing of metHb level three times higher than Hb–NO complex level. Whereas, after introduction classical NO donors (nytroprussidum or sodium nitrite) complete degradation of Hb–NO complex precedes a normalization of metHb level. When HAs is introduced to the rat liver tissues the intensity of electron spin resonance signal of nonhaemical iron nytrosyl complexes with proteins SH-groups, cytochrome P-450 and mitochondrial Fe–S centers, together with the level of nitrites did not change substantially. On the other hand rats that received equivalent by NO nytroprussidum doses or sodium nitrite showed manifold increasing of nitrate quantity. At the same time the intensity of electron spin resonance signal of nonhaemical iron nytrosyl complexes with proteins SH-groups increased, while cytochrome P450 and mitochondrial Fe–S centers signals decreased. Rats that received HAs with drinking water during 10 days did not show a formation of metHb, but there was present a substantial electron spin resonance signal of Hb–NO.

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It was shown that HAs forms with Hb nytrosyl complex with the same electron spin resonance signal as the complex Hb–NO has. Life time of the latter does not exceed 30 min, while HA–Hb complex is rather persistent. It saves its stability during longtime (more that 7 days) storage of erythrocytes and its lysates in a broad scale of pH (2–10) and in solutions of 9 M urea. Thus, the main target of HA toxic influence is Hb and, consequently, blood oxygen transport function. Hb serves as powerful trap for HA, and prevents significant NO formation in blood and tissues. Findings should be taking into consideration while developing preventive and treatment measures of HA poisoning, as well as definition of metHb. P2-25 ELECTROCARDIOGRAPHIC EFFECTS OF HIGH INTRAVENOUS DOSES OF ASTEMIZOLE IN DOGS G. Hanton, P. Bonnet, C. Loiret, G. Bazin Pfizer Global Research and Development, Amboise, France The aim of the current study was to determine the effects astemizole on the ECG of dogs, in particular, on cardiac repolarisation. One male and two female Beagle dogs received single intravenously injections of astemizole at increasing doses of 6, 9 and 15 mg/kg. ECG recordings (six limbs leads and four precordial leads) were performed before study start and on each day of administration, before and 15 min, 30 min, 1 h and 3 h after treatment. The beat-to-beat variability of QT was evaluated with the coefficient of variation of QT measured over 40 beats and with the formula of QT temporal dispersion provide by the literature: QTdt = log10 (CV QT/CV RR)2 . The transmural variability of cardiac repolarisation was assessed by the changes in the morphology of the T wave. At all dose levels, the treatment produced a moderate increase in QTc duration, which was most marked 1 and 3 h after treatment and was associated with an increase in the coefficient of variation of QT indicating an effect on the beat-to-beat (temporal) variability of repolarisation. Inconsistent results were found when the temporal variability of QT was assessed with the QTdt formula, which was found to reflect much more the changes in


the amplitude of sinus arrhythmia that a real affect on QT variability. The treatment also produced a notching of T wave recorded in CV5RL precordial lead. The change consisted of the appearance of a second peak on the T wave, with in some occasions, the trough between the two peaks reaching the isoelectric line. This modification of the T wave is indicative of an increased heterogeneity of repolarisation across the cardiac wall. In conclusion, astemizole increased as expected the duration of QT. In addition the treatment increased transmural and beat-to-beat variability of cardiac repolarisation. These changes are consistent with the inhibitory effects of the compound on IKr channels and are indicative of a proarrhythmic potential. We have shown that they may be assessed by examining the modification of T wave morphology and calculating the coefficient of variation of QT interval. P2-26 ELECTROCARDIOGRAPHIC EFFECTS OF BETHANECHOL AND METHACHOLINE IN DOGS G. Hanton, P. Bonnet, G. Bazin, C. Loiret Pfizer Global Research and Development, Amboise, France In this experiment, we evaluated the electrocardiographic effects of administration of bethanechol–HCl or methacholine–HCl, two agonists of the parasympathetic system, in dogs. The purpose of the study was to determine the consequences of changes in the vagal tone on QT interval, which may help the interpretation of drug effects on QT in toxicity studies. Beagle dogs, one male and two females/compound received single subcutaneous injections of bethanechol or methacholine at increasing doses, over 5 days. Doses of bethanechol were 0.1, 0.2, 0.4, 1.0 and 2.0 mg/kg; those of methacholine were 0.04, 0.08, 0.15, 0.40 and 0.80 mg/kg. ECG recordings were performed before study started and every day, before dosing and around 15 min, 30 min, 1 h and 3 h after dosing. Both compounds produced emesis, salivation and diarrhoea, which are consistent with stimulation of the parasympathetic system in the gastro-intestinal tract. Bethanechol produced a decrease in heart rate, which corresponds to the direct negative chronotropic effect of a muscarinic agonist. In contrast methacholine produced an increase in heart rate, which was assumed


Abstracts / Toxicology Letters 158S (2005) S1–S258

to be due to a reflex cardiac stimulation in response to drug-induced hypotension. Indices of sinus arrhythmia tended to inversely correlate with the changes in heart rate. The major effects of the treatment were increases in QT and QTc intervals that occurred with both compounds, but were generally more marked with bethanechol (up to 32 ms increase in QTc 1 h after administration of 2 mg/kg of bethanechol). These changes were considered to be the consequences of the stimulation of the parasympathetic system. There was no clear doserelationship, but the effects of QT or QTc intervals were overall most marked at the two highest doses that also produced emesis. In conclusion, stimulation of the parasympathetic system and associated emesis produced a prolongation of the QT or QTc interval in dogs. P2-27 INTESTINAL TOXICITY STUDIES OF SODIUM NITRITE I.P. Grudzinski Military Institute of Hygiene and Epidemiology, Warsaw, Poland Sodium nitrite, NaNO2 (CAS No. 7632-00-0) was nominated by the U.S. Food and Drug Administration (FDA) for toxicity and carcinogenesis studies based on its widespread used in foods and drinking water and the fact that many nitroso compounds are carcinogenic (Natl. Toxicol. Program Tech. Rep. Ser. 2001, 495: 7–273). In 14-weeks and 2-year drinking water studies, exposure to sodium nitrite resulted in increased incidences of epithelial hyperplasia in the forestomach of male and female F344/N rats and in the glandular stomach of male B6C3F1 mice. In the present studies, my interests have been further in training to evaluate the mechanisms of the toxic potential of sodium nitrite in the intestinal mucosa of male Wistar rats and male B6C3F1 mice. Based on the comprehensive results from subchronic drinking water experiments in vivo, the primary biochemical targets of sodium nitrite-induced toxicity were examined, including induction of lipid peroxidation in brush border membranes, direct inhibition of mitochondrial respiratory chain enzymes in enterocytes, and disturbances of proliferation processes in crypts. A novel approach to elucidate a potent mechanism of the toxic action of sodium nitrite was also provided on the basis

of intestinal immune biomarkers and histopathology studies. P2-28 THE MECHANISM BY WHICH ADENOSINE RECEPTOR MIGHT BE INVOLVED IN THE EFFECT OF FLUVOXAMINE ON ISOLATED GUINEA-PIG ATRIA A. Pousti, T. Deemyad, G. Malihi, K. Brumand Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Fluvoxamine (FLV), a selective serotonin reuptake inhibitor (SSRI) antidepressant caused a dosedependent decrease in rate and contractile force of the isolated guinea-pig atria. These effects significantly blocked by l,3-dipropargyl-8-cyclopentylxanthine (DPCPX), a specific adenosine A1 receptor antagonist. Theophylline, a non-selective adenosine A1 /A2A receptor antagonist also prevented the inotropic and chronotropic effect of FLV. The guinea-pig atrium was dissected out and suspended in modified Krebs solution under physiologic conditions. Drug was added into the solution. The changes in rate and force of contractions were measured using a physiograph. Our data indicate that DPCPX and theophylline prevent the inotropic and chronotropic effects of FLV on isolated guinea-pig atria, but these effects were not prevented by atropine and 3,7-dimthyl-l-propargylxanthine (DMPX), an adenosine A2 receptor antagonist. The effect of FLV on isolated guinea-pig atria is probably mediated through an inhibition of the uptake of adenosine or the A1 receptor mechanism. P2-29 EFFECTS OF LOVASTATIN ON TERT-BUTYL HYDROPEROXIDE INDUCED CELL TOXICITY A. Krasteva1 , M. Mitcheva1 , V. Descatoire2 1 Laboratory

of Toxicology and Drug metabolism, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Medical University-Sofia, Bulgaria; 2 Faculty of Medicine Xavier Bichat INSERM 481, Hopital Bichat, Paris, France The aim of this study was to evaluate the lovastatin effects, a HMG-CoA reductase inhibitor, on human

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hepatoma cells (Chang liver cells). tert-Butyl hydroperoxide (t-BuOOH) was used as a model for toxic cell injury. Chang liver cells were treated with t-BuOOH in concentrations 37.5 and 75 ␮M and with lovastatin 2.5, 5 and 10 ␮M in vitro. The cell viability was assessed using flow-cytometry. t-BuOOH decreased cell viability concentrationdependently—by 31% for the concentration 37.5 ␮M and by 66% for 75 ␮M, respectively. Lovastatin did not affect cell viability in the applied concentrations. Nevertheless lovastatin potentiated the toxicity of lower dose of t-BuOOH. Only the highest concentration of lovastatin (10 ␮M) statistically significantly potentiated the toxicity induced by 75 ␮M t-BuOOH. Statin-induced liver injury is not fully understood, but we suggest that lovastatin could intensify the tBuOOH—induced oxidative stress. P2-30 ESTABLISHMENT OF RISK CRITERIA AND THRESHOLD EFFECTS OF ANTI-DIABETIC DRUGS UNDER THE INHALATORY EXPOSURE M. Kudrya, I. Palagina, N. Ustenko Institute of Endocrine Pathology Problems, Kharkov, Ukraine Diabetes mellitus is one of the most frequent diseases in Ukraine (its rate exceeds 1 mln persons). Among the new anti-diabetic drugs (ADDs) good prospects belong to dicarboxilic acids derivatives. Aim of this study was to assess ADDs’ risk level under their respiratory introduction, and design safe conditions of their production. Objects of study were the derivatives of succinic (Phensuccinalum, PhS) and camphoric (Diacamph, DC) acids. Experiments were conducted over 120 Wistar rats and engaged toxicologic, physiological, biochemical methods. Inhalation of PhS and DC was accomplished one-fold in concentrations of 4.5–53.4 and 24–169 mg/m3 , respectively. We found that inhalation of PhS and DC in toxic concentrations (53.4 and 90 mg/m3 , respectively) activates lipoperoxidation (LPO) and inhibits antioxidant protection system. At threshold levels (27.9 and 24 mg/m3 , respectively) PhS and DC lead to LPO inactivation. Based on this we con-


firmed that under inhalation the limiting index of PhS and DC toxicity is defined by their LPO effects. Study concluded in establishment of risk criteria of dicarboxilic acids derivatives at toxic and threshold levels of impact, and in forecasting of their potential side effects. P2-31 INFLUENCE OF NALOXONE PRETREATMENT IN PARAQUAT NEUROTOXICITY ON NITRATE LEVELS M. Curcic, M. Djukic Institute of Toxicological Chemistry, Faculty of Pharmacy, Belgrade Nitrate levels in brain structures can be used as a marker of paraquat (PQ) neurotoxicity. Among the other mechanisms, PQ exerts its effect throughout opioide receptors, therefore in order to reveal mechanisms of PQ neurotoxicity, naloxone, an opioid receptor antagonist, was used in pretreatment of PQ poisoning. Influence of naloxone pretreatment in PQ poisoning was estimated by measuring nitrate levels in cortex homogenates of Wistar rats. PQ was intrastriatally (i.s.) applied in single dose of 2.5 g/10 L, and naloxone was administrated, in the same manner, in single dose of 5 g/10 L, immediately before PQ administration. It is known that naloxone exerts its effects via cGMP signalization, similar to NO. Moreover, i.s. PQ administration promotes NOS activity, and on the top of the results obtained in the experiment, anticipated high nitrate concentration occurred in cortex. Achieved nitrate levels (NO3 − nmol/mg of proteins) in Wistar rats’ cortex homogenates depend on treatment and time. Group pretreated with naloxone showed statistically lower nitrate concentration (4–20 times lower) in both, ipsilaterali and contralateral cortex unlike control group (18.78 ± 6.09 nmol/mg of proteins), 30 min, 24 h and 7 days after the treatment. Contrary, nitrate levels in the selected brain structures in Wistar rats poisoned with PQ were up to two times higher, 30 min and 7 days after the treatment. Extremely low nitrate levels in that group was obtain 24 h after the treatment (nine times lower). That extreme decreased value suggests that probably some other mechanisms along with opioide receptors are involved in PQ neurotoxicity.


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P2-32 USE OF EBSELEN TO MODULATE THE TOXICITY OF MECHLORETHAMINE ON HEALTHY AND TUMOR-DERIVED CELL LINES B. Billack, D. Hardej College of Pharmacy and Allied Health Professions, St. John’s University, Jamaica, NY, USA Mechlorethamine (HN2) is a nitrogen mustard compound and vesicant that affects the skin, eyes and respiratory tract. The cytotoxicity of HN2 is attributed to free radical generation, lipid peroxidation and DNA alkylation. Leucocytes are particularly sensitive to HN2 toxicity. No effective antidotes presently exist for HN2 poisoning. In vitro studies revealed that concentrations of HN2 that were lethal to human T cells (LC50 ∼4 ␮mol/24 h), showed no effect on cellular viability in cultured rat hippocampal astrocytes (RHA), human colon cancer cells and human embryonic kidney cells (LC50 >10 ␮mol/24 h). Ebselen, a selenium-containing antioxidant, was assessed for its ability to block the cytotoxic effects of HN2-induced cell death in T-cells. Our results demonstrated that this compound protected against HN2-induced cell death in a concentration- and time-dependent manner. Cell viability results were confirmed using light and scanning electron microscopy (SEM). SEM revealed significant membrane damage in HN2-treated human T-cells (4 ␮mol/24 h). RHA cells treated with sub-lethal concentrations of HN2 also exhibited membrane blebbing but to a lesser degree than the T-cells. In both cases, membrane damage was reduced by ebselen pretreatment (30 ␮mol/24 h). Our results demonstrate that ebselen can block the toxic effects of HN2 in vitro and comprise a significant firststep toward antidote development. P2-33 THERAPEUTIC RESPONSE TO SINGLE INTRAVENOUS BOLUS ADMINISTRATION OF FORMATE DEHYDROGENASE IN METHANOL-INTOXICATED RATS M. Arumughan, R. Sheeladevi Department of Physiology, Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences, University of Madras, Chennai 600113, Tamil Nadu, India

Formic acid is the toxic metabolite responsible for the metabolic acidosis and neurotoxicity observed in methanol poisoning in humans. Hemodialysis facility is not readily available in all the places, in developing countries like India. Formate dehydrogenase (EC acts directly over formate and converts formate into CO2 in the presence of NAD. Effect of single intravenous bolus infusion of formate dehydrogenase, obtained from Candida boidinii, in methanolintoxicated folate deficient rat model was evaluated. Carbicarb (Carb) (equimolar solution of Sodium carbonate and Sodium bicarbonate) was used to treat metabolic acidosis, because, Carbicarb besides increasing the pH, consumes CO2 . Tetrahydrofolate depletion was induced by methotrexate (MTX) treatment. Experimental design consists of seven groups namely saline control, methanol control, MTX control, enzyme control, MTX-methanol control, MTX-methanol-Carb, MTX-methanol-Carb-Enz group. Male Wistar rats were treated with MTX (0.3 mg/kg) (s.c.) for a week, were injected with methanol (i.p.) (4 g/kg), 12 h latter, Carbicarb solution was infused, following this enzyme was infused (i.v.) in bolus through rat tail vein. Blood samples were collected at every 15 min, for an hour, from the cannulated left jugular vein without anesthesia. Blood methanol, formate were estimated respectively with HPLC and fluorimetric assay. Blood pH, blood gases pO2 , pCO2 and bicarbonate levels were measured with blood gas analyzer. Results obtained showed that there was significant elimination of formate within 15 min, restoration of pH and insignificant changes with pCO2 . It may be concluded that single bolus intravenous infusion of formate dehydrogenase facilitates rapid elimination of formate. P2-34 RENAL TOXICITY IN RATS CAUSED BY AN OLANZAPINE INTERMEDIATE PRODUCT ´ R´onai, M. Albert, D. D´anyi E. Egis Pharmaceuticals Ltd, Department Of Toxicology, Budapest, Hungary Acute oral toxicity study with an olanzapine intermediate (4-amino-2-methyl-10H-thieno-[2,3-b][1,5] benzodiazepine HCl) (AMTB) was performed in rats, according to the OECD 423 Guideline because no LD50 value was found in the literature. The method uses pre-

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defined doses and the results allow a substance to be classified according to the Globally Harmonised System. Single 2000 mg/kg dose of AMTB caused delayed death (4–8 days of study). All of the treated animals died after decrease body weight, without clinical symptoms. Gross pathology revealed severe nephropathy and increased weight of kidneys. The rats given 300 mg/kg dose failed to produce mortality or clinical symptoms. Necropsy detected similar alteration of kidneys on the 15th day after treatment. Analysing delayed toxicity of AMTB blood was sampling from animals treated per os 300 and 2000 mg/kg doses of test item, 4 days after dosing. Marked elevation of serum creatinine, blood urea nitrogen (BUN), cholesterol, total bilirubin and glutamate-dehydrogenase (GLDH) could detected among blood parameters. Light microscopy revealed tubular dilatation and cell necrosis, crystaloid deposits with granuloma formation in the cortex and medulla as well. According to lethality of rats AMTB should be ranked for GHS category 4 (LD50 >300–2000). On the basis of our results misclassification (classifying into a less toxic category) possible in those substances causing delayed toxicity, so procedures used lethality as an endpoint for ranking should not be handle rigorously! P2-35 HORMONE INVESTIGATIONS WITH OMC S. Schulte, G. Cunha, B. van Ravenzwaay BASF-AG, Ludwigshafen, Germany Some UV absorber has been associated with weak estrogenic activity, particularly after in vitro investigations (Schlumpf et al., 2001). To investigate potential adverse effects on hormone homeostatis, the UV absorber Uvinul MC 80 N® (octyl-methoxycinnamate, OMC) was administered to groups of 13 female Wistar rats at dietary doses of 0 and 1000 mg/kg bw/d. Food consumption and body weights were determined weekly. Signs of toxicity and status of estrus cycle were determined daily. From Day 28 onwards blood was taken from 10 rats/group during proestrus for hormone determination: triiodothy-


ronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), prolactin, follicle-stimulating hormone, luteinizing hormone, estradiol and progesterone. Treatment resulted in a slight reduction of food consumption and body weight. The only statistically significant change was a slightly increased T4 concentration (+19%). The absence of any effect on TSH and T3 indicates that a potential perturbation of thyroid hormone homeostasis was minimal at the most. Many chemicals can alter thyroid hormone levels in rodents without resulting in adverse effects in long-term studies (O’Connor et al., 1999, 2002). Thus, due to the small magnitude of the effect and because a minimal increase in T4 is generally not associated with functional changes, OMC is considered not to be a relevant thyroid toxicant. Hormone investigations and estrus cycle revealed no treatment-related effects. It is therefore concluded that OMC does not affect hormone homeostasis even after 28 day administration of 1000 mg/kg b.w. P2-36 PROTECTIVE EFFECT OF METRONIDAZOLE LOW CONCENTRATIONS ON THE CELL MEMBRANES S. Rogacheva1 , E. Popyhova2 , P. Kuznetsov2 , K. Evlakov2 1 Department

of Ecology, Saratov State Technical University, Saratov, Russia; 2 Department of Biochemistry and Biophysics, Saratov State University, Saratov, Russia Metronidazole (MN) 1-(2-oxyethyl)-2-methyl-5nitroimidazole is an antimicrobial drug with effective doses 0.5–1 g/day. Besides, MN is known to protect the mammal cells. The goal of the work was to investigate the mechanism of MN protective effect on the cell membranes. Firstly using erythrocytes we have demonstrated, that the rate of their hemolysis by sodium dodecylsulfate in the presence of MN in concentration range 10−2 to 10−11 g/l substantially decreased. To clear up if the effect was specific or nonspecific the liposomes stability towards detergent action in the presence of the same MN concentrations was examined and was shown to increase. For the MN protective effect was noted at low concentrations it can not be explained by the direct substance action


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on the bilipid layer. We suggested it to be caused by the formation of slow-moving water layer near cell membranes. The fluorescent sounding of the liposome emulsions by 4-dimethylaminohalkone showed the decreasing of water diffusion mobility induced by MN input. The latter may be due to the formation of “giant fluctuations” of water density, which appear during phase conversion of the 2-d type “order-disorder”. The results of molecular dynamics computation of parameters of hydrogen bonds net and clusters of water molecules in the solutions of MN and inactive compounds proved the proposal. P2-37 HEPATOTOXIC EFFECT OF REPEATED ADMINISTRATIONS OF HIGH DOSE OF PROPYLENE GLYCOL IN RATS G.M. Shayakhmetova, I.S. Blazchuk, V.M. Kovalenko Institute of Pharmacology and Toxicology of Academy of Medical Sciences of Ukraine, Kyiv, Ukraine Propylene glycol (PG) is frequently used as a vehicle in high concentrations in the pharmaceutical and the food industry. However, acceptable doses of intake of PG has discussed to date. The effect of repeated high dose of PG on serum protein, total bilirubin, urea contents, and alkaline phosphatase and aminotranferases activities was studied. Male Wistar rats received vehicle with propane-1,2-diol 600 g/L at a dose of 13.13 ml/kg b.w. by oral administration for 90 days. Consequently total daily dose of PG was 8 g/kg b.w. The treatment produced no significant differences in serum concentration of urea and aminotranferases activity. But, it did cause an increase of serum total bilirubin level and alkaline phosphatase activity by 8.4 and 1.3 times, respectively. Total protein content in serum was decreased by 1.5 times in comparison with intact rats. We conclude that a significant increase of total bilirubin and alkaline phosphatase activity against a background of normal aminotranferases indicators is an evidence of intrahepatic cholestasis. Moreover, a decreasing of serum protein level may by denote a suppression of liver proteosynthetic function. Capacity of development hepatotoxic effects should be taken into consideration under overdosage of PG.

P2-38 RESPONSE OF PROSTACYCLIN TO LOW DOSE IRRADIATION IN THE DEVELOPMENT OF RADIATION MYELOPATHY A. Shirazi1 , S.R. Mahdavi1 , K. R Trott2 1 Department of Medical Physics, Faculty of Medicine,

Tehran University of Medical Sciences (TUMS), Iran; Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood HA6 2JR, UK

2 Gray

The priority of vascular and its secretory profile changes in pathogenesis of radiation myelopathy have recently been discussed. In this study the model of prostacyclin concentration changes after low doses of X-irradiation within a short period of time was studied. Wistar rats were irradiated with doses of 2, 4 and 6 Gy’s of X-rays. After 24 h, 2 and 13 weeks post-irradiation, samples of spinal cord were prepared for evaluation of prostacyclin and histopathologic changes. Prostacyclin content was determined by quantification of 6-keto-prostaglandin-F1␣ (prostacyclin stabilized metabolite). Irradiated segments of spinal cord were stained routinely for histological studies. Twenty four hours post-irradiation, finding shows decrease in the content of prostacyclin after doses of 0.5 and 1 Gy with 91.67 ± 1.47% 96.80 ± 2.17% of agematched control group. After 2 weeks concentration of prostacyclin shows significant decreases after 6 Gy. After 13 weeks irradiation shows marked differences even after a small dose of 2 Gy (p < 0.001) and after doses of the low dose group. In the vascular theory, circulation disturbance following vascular injury secondarily induces white matter lesions. The interpretation of this finding can be that radiation affects the synthesis of prostacyclin at both vascular and parenchymal sources responsible to produce prostacyclin in the spinal cord P2-39 SOME BEHAVIOURAL AND ELECTROPHYSIOLOGICAL ALTERATIONS IN RATS TREATED ACUTELY AND SUBCHRONICALLY BY MK801, A NON-COMPETITIVE NMDA ANTAGONIST A. Szab´o, A. Luk´acs, L. Nagymajt´enyi Department of Public Health, University of Szeged, Hungary

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N-Methyl-d-aspartate (NMDA) receptor channels play important roles in various physiological functions. Adult male Wistar rats were treated i.p. with low (0.05 mg/kg) or high (0.1 mg/kg) dose of the NMDA channel blocker, MK-801, acutely and subchronically. In acute treatment, behavioural test (acoustic startle response, ASR) was done before and 90 after the treatment, where no noteworthy change was observed. Twenty-four hours after the administration, electrophysiological recording (somatosensory, visual and auditory evoked potentials) was done, where mainly the latency of the evoked responses increased significantly. During subchronic treatment rats (11 controls 14 treated each) were injected every other day, altogether seven times. Control animals were administered saline. After the administration period, the animals were kept for 3 weeks to recover before the neurophysiological tests. From the 5th week, spatial maze learning and memory tests were done. Working memory slightly decreased in both treated groups but low-dose treated animals showed minor increase in reference memory. On the 10th week, ASR parameters were measured, but no significant changes were found. During the experiment, the rats were monitored and general toxicology parameters were measured. Body weight, e.g., dose-dependently decreased from the last day of the treatment. After the electrophysiological tests the animals were dissected and their organs were weighed. The weight of the liver decreased significantly after high dose MK-801, and weight of the kidney decreased. The relative brain weight also increased significantly after the high dose. The effects of MK-801 were reflected more in electrophysiological changes and general toxicological parameters, rather than in the behavioural tests. P2-40 INFLUENCE OF REFRACTORY CERAMIC FIBRES EXPOSURE COMBINED WITH CIGARETTE SMOKE ON SOME CYTOTOXIC PARAMETERS OF RAT LUNGS ˇ a, M. Hurb´ankov´a, Z. Kov´acˇ ikov´a, M. Beˇno, S. S. Cern´ Wimmerov´a Slovak Medical Republic





The viability of alveolar macrophages (AM), lactate dehydrogenase (LDH), acid phosphatase and cathepsin D activities in lungs after 6 month exposure to refractory ceramic fibres (RCF) and cigarette smoke (S) were studied in male Wistar rats. Four group of rats weighing at the beginning of the experiment 152.9 ± 6.6 g were treated as follows: 1) intratracheally instilled by saline solution (2 × 0.2 ml); 2) intratracheally instilled by 4 mg (2 × 2 mg) of RCF suspended in saline solution; 3) exposed only to S (85 mg of total particulate matter/m3 air = eight cigarettes) for 2 h daily; 4) exposed to RCF and S. After 6 months the animals were exsanguinated in anaesthesia and the bronchoalveolar lavage (BAL) was perfumed. Viability of AM was strongly depressed by exposure to RCF as well as to S. The combined effect was at least additive. No significant changes in LDH activity were found. The activities of lysosomal enzymes in cell-free BAL fluid increased after smoking. In the BAL cells they increased after smoking and combined exposure, cathepsin D also after CRF exposure. The combined effect was more than additive. The results indicate that the cytotoxic effects of RCF may be persistent and amplified by cigarette smoke. P2-41 SUBCHRONIC EFFECT OF REFRACTORY CERAMIC FIBRES (RCF-S) ON THE SELECTED PARAMETERS OF BRONCHOALVEOLAR LAVAGE (BAL) IN W-RATS M. Hurbankova1 , S. Cerna1 , Gergelova2 , S. Wimmerov´a1


Sulcova1 ,


1 Research Base of the Slovak Medical University, Insti-

tute of Preventive and Clinical Medicine, Bratislava, Slovakia; 2 Trnava University, Faculty of Health, Care and Social Work, Trnava, Slovakia The aim of present work was to find out the subchronic effect of RCFs (asbestos substitute) on selected parameters of BAL in rats. W-rats were intratracheally instilled with 4 mg of RCFs in 0.4 ml of saline solution/animal (exposed group) and with 0.4 ml saline solution/animal (control group). Animals were


Abstracts / Toxicology Letters 158S (2005) S1–S258

sacrificed after 6-month exposure. Bronchoalveolar lavage (BAL) was performed and selected BAL parameters (mainly inflammatory and cytotoxic) were examined: the leukocyte count, the count of alveolar macrophages (AM), the differential cell count (% of AM, % of polymorphonuclears—PMN, % of lymphocytes—LY), proportion of immature AM, the percentage of binucleate cells—BNC, the viability and the phagocytic activity of AM. Following treatment with RCFs was observed: statistically significant increase of proportion of LY and PMN as well as % of immature AM, and statistically significant decrease of viability of AM and proportion of AM in comparison with the control. The results of this study indicated that RCFs even 6 months after intratracheal instillation very significantly changed the majority of examined BAL parameters. The presence of inflammatory and cytotoxic response in lung may signalize beginning or developing disease process.

extracted, processed and hybridised to Affymetrix HG U133 Plus 2.0 arrays. Raw data was normalised using RMA and further analysed for robust changes using Genespring. To enable a direct comparison to this study, the raw data from Spira et al. (2004) were analysed in the same manner. Of 45 genes significantly (>two-fold) up-regulated in smokers compared to non-smokers, 22 were similarly regulated by TPM in H292 cells. Likewise 2/9 genes were down-regulated in H292 cells when compared to the human study. Hence, despite marked differences in the smoke exposure studies there was some similarity in the response. This suggests that the regulation of the above 24 common genes may be a direct effect of exposure to components of the particulate phase of cigarette smoke in the lung epithelium of smokers. Reference Spira, A., et al., 2004, PNAS, 101, 10143–10148.



H. Maunders1 , E.D. Massey2 , A. Richter2

R. Radykewicz1 , P. Reitmeir2 , V. Harder3 , H. Schulz3 , A. Stampfl1

1 Advanced

Technologies Cambridge, 210 Cambridge Science Park, Cambridge CB4 OWA, UK; 2 R&D Centre, British American Tobacco, Regents Park Rd., Southampton, SO15 8TL, Hants, UK Cigarette smoking is associated with development of lung cancer and chronic obstructive pulmonary disease. Published Affymetrix data detailing gene expression in the lung epithelium of non-smokers and smokers has shown that smoking-regulated genes served antioxidant, metabolising and host defence functions (Spira et al., 2004). In this study the effects of cigarette smoke total particulate matter (TPM) on the transcriptome of NCI-H292 human lung epithelial cells was investigated using Affymetrix arrays. The data were compared to the effects of smoke on the lung epithelial transcriptome to investigate whether the in vivo findings can be predicted in vitro. Confluent H292 cells were incubated in serum-free medium (SFM) for 24 h and then exposed to SFM ± TPM for 8 or 24 h. Total RNA was

1 GSF—National

Research Center for Environment and Health, Institute of Toxicology, Germany; 2 GSF—National Research Center for Environment and Health, Institute of Health Economics, Germany; 3 GSF—National Research Center for Environment and Health, Institute for Inhalation Biology, Neuherberg/Munich, Germany Epidemiological studies have shown that air ultrafine particles (UFP; diameter <100 nm) can cause diseases in the cardiovascular system. It is still unknown if these impacts have a systemic background or if they are evoked by a direct action on the heart itself. The aim of our studies was to analyze the influence of UFP on the heart without systemic interactions. Retrograde perfused hearts from guinea-pigs and Printex 90 (black carbon; diameter 14 nm) were used. The influence of the UFP at different concentrations on the heart rate was investigated during 1 h. Using

Abstracts / Toxicology Letters 158S (2005) S1–S258

8 × 108 UFP/ml the heart rate increased by 6.3%. After 1 h the amount was doubled to 1.6 × 109 UFP/ml and the increase was by 8.5%. Starting the experiments with 1.6 × 109 UFP/ml the rise was by 7.5%, after doubling to 3.2 × 109 UFP/ml by 14%. During the following washout period the beating rate decreased nearly to the initial value. Under the influence of UFP the ECG changed, i.e. a decoupling of the P–QRS complex (AV block) was observed. These results show that the UFP have a dose dependent direct influence on the heart rate. Further investigations are necessary to clarify the mechanisms and to elucidate if chemical properties of particles evoke these effects. P2-44 3D-QSAR OF CYP2A6 INHIBITORS Rahnasto1 ,

M. C. R.O. Juvonen1

Wittekindt2 ,


Poso2 ,


located near amino acid residue Leu370, which restrict binding of inhibitors/substrates. Steric favoured areas are found near amino acid residues Ile300 and Ile366. In conclusion steric factors around binding site are the key determinants of interaction between inhibitor/substrate and CYP2A6. P2-45 ALTERED GENE EXPRESSION IN THE OLFACTORY BULB FOLLOWING EXPOSURE TO THE OLFACTORY TOXICANT 2,6-DICHLOROPHENYL METHYLSULFONE U. Bergstr¨om1 , J.A. Olsson1 , T.R. Hvidsten2 , J. Komorowski2 , I. Brandt1 1 Department


Raunio1 ,

1 Department

of Pharmacology and Toxicology, Finland; 2 Department of Pharmaceutical Chemistry, University of Kuopio, POB 1627, 70211 Kuopio, Finland

The human CYP2A6 enzyme oxidizes several toxic xenobiotics e.g. tobacco-specific nitrosamines and many other toxic compounds. Nicotine, the addictive component in tobacco, is mainly eliminated also via oxidation by the CYP2A6 enzyme. The purpose of this study was to elucidate 3D-QSAR of the human CYP2A6 enzyme, with the ultimate aim of developing a potent chemical inhibitor of CYP2A6. Such an inhibitor could be used to modify nicotine kinetics in smoking cessation therapy. Our inhibitor test set contained >80 derivatives of lactones, quinoline and naphthalene. Based on a homology model of CYP2A6, these inhibitors were docked using the GOLD program. Comparative Molecular Field Analysis (CoMFA), a 3D-QSAR method taking into account steric and electrostatic interactions, was performed to obtain detailed structure-activity relationships. The analysis yielded good statistics: q2 = 0.74, SPRESS = 0.54, r2 = 0.95 with three components. This model was used to predict binding of ligands in the active site of CYP2A6. The CoMFA contour maps included negative charge favoured areas near pyrrolidine ring of nicotine. This indicates that this partial negative charge increases inhibitory activity. Sterically disfavoured areas are

of Environmental Toxicology, Sweden; Linnaeus Centre for Bioinformatics, Uppsala University, Sweden 2 The

The olfactory mucosa (OM) is a sensitive target for CYP-catalysed toxicity. Its close proximity to the brain implies that OM toxicants can indirectly affect also the ongoing regeneration/rewiring of neurons in the olfactory bulb (OB). 2,6-Dichlorophenyl methylsulphone is a model chemical to examine the complex toxic effects caused by several related OM toxicants in mice. Originating from a local metabolic activation and subsequent necrosis in the Bowman’s glands, 2,6-dichlorophenyl methylsulphone induces metaplastic remodeling of the OM in mice. In addition, a longlasting induction of the astrocyte marker glial fibrillary acidic protein (GFAP) develops in the OB, along with bone formation between the cribriform plate and the OM. To explore effects on the neuronal population in the OB, groups of mice were exposed to a single i.p. dose of 2,6-dichlorophenyl methylsulfone (32 mg/kg) or vehicle. One week later the OBs were dissected for analysis of altered gene expression using microarrays and real-time RT-PCR. Analysis of the microarray data at the LCB Data Warehouse showed a number of genes with altered gene expression. Preliminary real-time RTPCR results verify altered expression of for instance the GFAP, Tax1bp1, and Olfml3 genes. Some of the genes that were identified in the microarray analysis may provide information about mechanisms underlying the effects of 2,6-dichlorophenyl methylsulfone in the olfactory system.


Abstracts / Toxicology Letters 158S (2005) S1–S258

P2-46 UTERINE MYOMAS AND IRON STORES ´ atkowska2 , A. M. Nasiadek1 , T. Krawczyk2 , E. Swi˛ 1 Sapota 1 Medical

University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland; 2 Polish Mother’s Memorial HospitalResearch Institute, Rzgowska 281/289, Lodz, Poland In clinical studies anaemia is often observed in woman with uterine myomas. It has been suggested that it results from more intensive menstrual bleeding, impaired Fe absorption and accumulation of Fe in the myoma. The aim of this study was to investigate parameters of Fe stores both in myoma tissue and in the serum and morphological parameters as indicators of anaemia. The research was conducted on 40 women with uterine myomas, before operations and 28 women (control group). Blood samples were collected from both groups to perform the following assessments: Fe and ferritin concentrations, soluble serum transferring receptor (sTfR), morphological assays. Concentration of iron in myoma and non lesion tissues collected inoperatively from the same patients was performed by means of AAS. Morphological assays in the group of women with myomas show decreased values of haemoglobin, red blood cells and MCV in comparison with control group. In the serum of these patients no differences were detected in iron and ferritin concentrations, but increase in the sTfR concentration. In myoma tissue no changes in iron concentration were detected in comparison with non lesion tissue. The obtained data suggest that anaemia in patients with uterine myomas caused by iron deficiency does not result from accumulation of iron in growing myoma or from iron stores depletion. The increase in sTfR concentration suggests early cellular iron deficiency. P2-47 THE EFFECT OF 1,2,3,4,5-PENTACHLORONAPHTHALENE ON CYP 1A ACTIVITY IN THE LIVER OF RATS A. Kilanowicz, A. Galoch, E. Bruchajzer, A. Sapota Department of Toxicology, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland

Severe skin reactions and liver disease have been reported after occupational exposure to chlorinated naphthalenes (PCNs). The clinical and toxicological symptoms of PCNs are very similar to those caused by PCBs, PCDDs and PCDFs. The aim of this study was to assess the effect of 1,2,3,4,5-pentachloronaphthalene (PCN) on the activity of CYP 1A in the liver of rats. The experiments were performed on male Wistar rats. The investigated compound was administered intragastrically in the three different doses (1, 10, 100 mg/kg b.w.) for 7–21 days. The parameters used as indicators of hepatic microsomal cytochrome levels were: the concentrations of total cytochrome P-450 and the activity of CYP 1A. Additionally the level of MDA was determined in the liver. After PCN administration (in doses 10 and 100 mg/kg b.w.) we observed body weight loss (to 30% in comparison with control group) in all time points. We also observed the increase of relative liver mass in each dose and all time points (even to 150% after the highest dose). The exposure to PCN evoked increase in the level of total cytochrome P-450 as well as activity of CYP 1A in all time points and each dose. The maximum of CYP 1A activity (about 18-fold in comparison with control group) was observed after 21 doses. The level of MDA increased even by several times, depending on the number of doses. The study was performed under a KBN grant No 3P05D 010 25. P2-48 THE EVALUATION OF ADDICTION TO SOME OPIOID SUBSTANCES USING THE LEVEL OF NEURONAL GABA. THE ASSESSEMENT OF OXIDATIVE STRESS AFTER THEIR CRONIC ADMINISTRATION A.L. Nitulescu-Arsene1 , R. Andritoiu2 , D. Gradinaru1 , N. Mitrea1 , D. Balalau2 , D. Margina1 1 “Carol

Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Biochemistry, Bucharest, Romania; 2 “Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Toxicology, Bucharest, Romania

Abstracts / Toxicology Letters 158S (2005) S1–S258


The present study aims to evaluate the evolution of addiction within chronic administration to opioid substances in mice. For this purpose we used four groups of mice that received opioid substances, over a period of 4 weeks, as follows: first group—control; second group—morphine; third group—codeine and gluthetimide at once; fourth group—codeine and methadone at once. The evolution of addiction was evaluated weekly using a biochemical parameter involved in the neuronal metabolism: the endogenous neuronal GABA concentration. The determination of endogenous neuronal GABA concentration is based on a fluorimetric assay that depends on the formation of a fluorescent product from the reaction between GABA and ninhydrin at alkaline pH and in the presence of glutamate. Simultaneously we evaluated the oxidative stressalong the period of chronic administration of the opioid substances-using malondialdehyde (MDA) as biomarker.

uated by determining intensity of gastric lesions in treated animals. Flurbiprofen and NCX2216 were given in doses of 0.5–20 mg/kg per os 60 min before tests. The results showed that NCX2216 produced higher anti-inflammatory and analgesic efficacy than its parent compound. Anti-inflammatory and analgesic ED-50 values of NCX2216 (2.82 and 2.2 mg/kg, respectively) were about twice lower than those of flurboprofen. Moreover, NCX2216 produced negligible effects on gastric mucosa. After the dose of 20 mg/kg only in 60% animals slight changes of gastric mucosa were found (gastric damage score of 0.25 at the 8-point scale), while in 100% of animals given flurbiprofen changes of gastric mucosa were detectable already after the dose of 2.5 mg/kg; the gastric damage score after the dose of 20 mg/kg was 10 times higher than that produced by NCX2216. Our results suggest that NCX2216, besides better gastric tolerability, also produces higher antiinflammatory and analgesic activity compared with those of the parent compound flurbiprofen.




Pejci´c1 ,


Dobri´c2 ,

´ c1 , Z. Milovanovi´c2 V. Cupi´

1 Faculty

K.S. Borowiak1 , Białecka2


Machoy-Mokrzy´nska2 ,


1 Department of Forensic Medicine, Pomeranian Medi-

of Veterinary Medicine, Belgrade, Serbia & Montenegro; 2 National Poison Control Centre, Military Medical Academy, Belgrade, Serbia & Montenegro

cal University, Szczecin, Poland; 2 Department of Pharmacology, Pomeranian Medical University, Szczecin, Poland

It has been demonstrated that nitric oxide (NO) may play an important role in gastric mucosal defense and that NO-donors may give protection against experimentally induced gastric lesions including those produced by NSAIDs. The aim of this study was to compare anti-inflammatory, analgesic and ulcerogenic effects of the NSAID flurbiprofen and its NO-releasing derivative NCX2216 in rats. The experiments were performed on adult male Wistar rats. For estimating anti-inflammatory and analgesic activity the carrageenan-induced rat paw oedema test and the acetic acid-induced writhing test were used, respectively. Their ulcerogenic action was eval-

The increasing availability, acceptance and use of recreational drugs (marijuana, amphetamines) in society is escalating and is not unique to any specific country but, rather is a common problem encountered around the world. The potential influence of centrally acting drugs on the human status has been a growing concern to all specialists involved with clinical toxicology, addiction problems and traffic safety. In last five years the group of 1000 young patients (13–25 year old) were examined for tetrahydrocannabinoids (THC) in our department. In 485 cases (48.5%) the positive result for THC and amphetamines have been noted. Parallels, near 800 drivers (man 65%,


Abstracts / Toxicology Letters 158S (2005) S1–S258

woman 35%), have been examined for narcotics level (average 20% positive results). On the other hand, several studies have recently examined the ability of THC usage in therapy e.g. to protect from neuronal injury. The results have been somewhat conflicting, possibly due to the range of models and dosages used, as well as partially undetermined mechanism of neuroprotection. The number of clinical trials investigating the therapeutic potential of THC is increasing in a number of CNS disorders including emesis, pain, neurodegeneration and brain trauma. Cannabinoids may also play a role in neuroprotection in disorders such as stroke, Parkinson and Huntington’s diseases, cerebral trauma and epilepsy. There is a better side of THC usage possibilities resulting in benefits and hope for several hundred patients. P3 Mechanism of Carcinogenesis P3-01 DIFFERENT SUSCEPTIBILITY OF Min/+ MICE WITH C57BL/6J, AKR/J OR A/J BACKGROUNDS TO SPONTANEOUS OR PhIP-INDUCED INTESTINAL TUMORIGENESIS I.L. Steffensen, J. Alexander Department of Food Toxicology, Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, NO-0403 Oslo, Norway The objective of the present study was to evaluate the impact of genetic background on susceptibility to spontaneous or 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP)-induced intestinal tumorigenesis in multiple intestinal neoplasia (Min)/+ mice. Females resistant (AKR/J) and susceptible (A/J) to intestinal carcinogenesis were crossed with male C57BL/6JMin/+ mice. Three-day old Min/+ F1 offspring of C57BL/6J, AKR/J or A/J backgrounds were given a single s.c. injection of 50 mg/kg of the heterocyclic amine PhIP or left untreated as controls, and were terminated at 11 weeks. We found that both numbers of spontaneous and PhIP-induced tumors were lower in Min/+ mice with either AKR/J or A/J backgrounds compared with C57BL/6J background. However, the increase in tumor number after PhIP exposure was 3.8fold and 3.7-fold above the spontaneous levels in mice

with AKR/J and A/J backgrounds, respectively, compared with only three-fold in C57BL/6J mice. However, in the colon, PhIP increased the number of tumors slightly more in Min/+ mice of C57BL/6J background (3.3-fold) than of A/J background (three-fold). AKR/J mice had no colonic tumors. Most of the tumors were located in the distal two-thirds of the small intestine in all three strains. Our results show that genetic background influences the susceptibility towards spontaneous and PhIP-induced intestinal tumorigenesis in a murine adenomatous polyposis coli (FAP) model. P3-02 INCREASED 2-AMINO-1-METHYL-6-PHENYLIMIDAZO[4,5-B]PYRIDINE (PHIP)-DNA ADDUCTS AND INTESTINAL TUMORS IN MULTIPLE INTESTINAL NEOPLASIA (MIN) MICE CROSSED WITH HOMOZYGOUS XERODERMA PIGMENTOSUM GROUP A (XPA) DEFICIENT MICE I.L. Steffensen1 , H.A.J. Schut2 , J.M. Nesland3 , K. Tanaka4 , J. Alexander1 1 Department

of Food Toxicology, Norwegian Institute of Public Health, Oslo, Norway; 2 Department of Biochemistry and Cancer Biology, Medical College of Ohio, Toledo, USA; 3 Department of Pathology, The National Hospital—The Norwegian Radium Hospital, University of Oslo, Norway; 4 Department of Frontier Biosciences, Osaka University, Osaka, Japan C57BL/6J-Min/+ mice are heterozygous for a germline nonsense mutation in the tumor suppressor gene adenomatous polyposis coli, develop spontaneous intestinal tumors and show increased intestinal tumorigenesis after exposure to the food mutagen PhIP. Xpa−/− mice are defective in nucleotide excision repair (NER) and highly susceptible to UV or chemical carcinogenesis. The Xpa−/− Min/+ mice were more susceptible to acute toxicity of PhIP, had more PhIP-DNA adducts in middle and distal small intestine and liver, and higher numbers of small intestinal tumors at 11 weeks, compared with Min/+ mice with one or two intact Xpa alleles. The location of tumors was not affected, being highest in the middle and distal small intestine in all genotypes. The number of spontaneous intestinal tumors at 11 weeks was not significantly affected by homozygous loss of Xpa, but untreated Xpa−/− Min/+ mice had significantly shorter life-spans than their littermates. In

Abstracts / Toxicology Letters 158S (2005) S1–S258

conclusion, the Xpa gene is involved in repair of bulky PhIP-DNA adducts in Min/+ mice, and loss of both Xpa alleles is necessary for increased intestinal tumorigenesis. P3-03 BENZO(a)PYRENE INDUCED p53 PROTEIN AND CASPASE-ACTIVITY IN MCF-7 AND A549 CELLS M. Tampio1 , A. Mertanen1 , J. Loikkanen1 , M. H¨oyhty¨a2 , K.H. V¨ah¨akangas1 1 Department

of Pharmacology and Toxicology, University of Kuopio, Finland; 2 Medix Biochemica, Finland p53 protein is involved in many cellular functions: apoptosis, DNA repair and synthesis, cell cycle control and senescence. p53 protein induces apoptosis by increasing Bax expression and dimerisation. Bax on the outer mitochondrial membrane leads to cytochrome c release from mitochondria, which starts caspase chain reaction leading to apoptosis. Both MCF-7 breast adenocarcinoma and A549 lung carcinoma cell lines express wild type p53 protein. We have shown earlier that p53 protein is induced in these cells after benzo(a)pyrene exposure for 24–48 h. Aim of this study was to clarify the consequences of p53 induction in MCF-7 and A549 cells. In accordance with earlier studies, p53 immunoblotting showed a dose-dependent increase in the amount of p53 protein after exposure to BP. Caspase-3-like protease activity increased dosedependently after exposure to different concentrations (0.1, 1, 2.5, 5, 10, 20 and 40 ␮M) of benzo(a)pyrene (BP) regardless of the time point studied (3, 6, 24, 48 and 72 h). According to the specificity of the used substrate, this activity represents mainly, but not only caspase-3-activity in A549 cells. However, other caspases must be involved in MCF-7 cells which have a functional deletion in CASP-3 gene. P3-04 TUMOR MARKERS AS CRITERIA FOR EVALUATION OF CARCINOGENIC ACTION OF AGROCHEMICALS H. Avagyan, D. Dumanyan National Institute of Health, Armenia


Long-term exposures to low levels of agrochemicals can have delayed consequences, including carcinogenesis. Our previous experimental studies in laboratory animals showed that molecular mechanisms of carcinogenic, mutagenic and delayed toxic effects of organochlorine and phosphorous pesticides are conditioned by formation of intermediate oxygen and other reaction-grade radicals. A number of pesticides are not considered to be direct carcinogens for humans, but due to metabolism in microsomal fractions of liver there are formed carcinogenic compounds. Long-term pesticide application probably brings forth elevated risk of gastrointestinal and pancreatic cancer incidence. For a long time until tumor markers were developed there was no real opportunity to confirm this idea. The high effectiveness of tumor markers was shown for monitoring of disease follow-up and treatment courses. In our study tumor markers AFP, CA-125, CA19-9, CA-242, CA15-3, Calcitonin, CEA, PSA, SCC, TPA were used to perform screening aimed to early reveal the probable carcinogenic effect of pesticides. Screening investigation involved over 5000 individuals from regions of Armenia, where agrochemicals were intensely used for decades. Data obtained demonstrates that certain tumor markers can be efficiently applied as criteria for evaluation of carcinogenic action of agrochemicals. Complex investigation with the help of CEA, CA19-9 and CA242 is useful for differential diagnostics of pancreatic and gastrointestinal cancer, as early diagnostics of pancreatic cancer can be performed with high level of precision before clinical manifestation of this state. P3-05 LEUKEMIA BY BENZENE—IS THERE ROOM FOR THRESHOLD VALUES? A. Kotalczyk1 , C.P. Siegers1 , J. Schulze2 1 Institute

of Experimental and Clinical Pharmacology and Toxicology, University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany; 2 Office of the Dean, Johann Wolfgang Goethe-University Frankfurt/Main, Theodor Stern-Kai 7, 60590 Frankfurt/Main, Germany Benzene is carcinogenic and has been shown to induce leukemia in animal experiments whereas toluene and xylene, structurally related aromatic compounds, do not induce any cancer in animals. Epidemiology indi-


Abstracts / Toxicology Letters 158S (2005) S1–S258

cates a correlation between benzene exposure only and leukemia induction. Consequently benzene has been classified as a human carcinogen both by international (WHO, IARC) and national agencies; uncommon for a carcinogenic compound for human exposure a limit of 50 ppm has been set. On the other hand, benzene exposure in industrialized societies is nearly ubiquitous with gasoline as the leading exposure source. Clinically benzene exposure also has been linked to myelodysplastic syndrome and osteosclerosis indicating a toxic mechanism rather than carcinogenic; therefore it remains unclear whether leukemia is a primary response to benzene or secondary e.g. as the result of bone marrow proliferation. Sensitivity towards benzene toxicity has been linked to high activities of the isoenzyme CYP2E1 and bone marrow myeloperoxidase, and low activities of quinone oxidoreductase NQ01 and GSH-S-transferase. Benzene is metabolically activated to benzene epoxide mainly in the liver with very low concentrations of P450 2E1 in the bone marrow, therefore the reactive intermediate(s) likely have to be transported to the bone marrow for leukemia induction. This mode of action may justify a threshold; an uncommon mode of DNA damage besides direct structural changes may also be responsible, since the redox active intermediates hydrochinone and benzochinone are particularly toxic to the DNA introducing nonspecific oxidative damage. Based on likely mechanisms of leukemia induction and mechanism of benzene toxicity we will reevaluate whether a threshold for this carcinogen is appropriate. P3-06 SENSITIVITY OF HUMAN PROGENITORS CORD BLOOD CELLS TO TETRACHLOROETHYLENE: CELLULAR AND MOLECULAR END-POINTS C. Diodovich1 , D. Ferrario1 , B. Casati2 , I. Malerba1 , E. Marafante2 , L. Gribaldo1 1 ECVAM,

Institute for Health and Consumer Protection, J.R.C., Ispra, Italy; 2 PCE, Institute for Health and Consumer Protection, J.R.C., Ispra, Italy The International Agency for Research on Cancer (IARC) currently designates tetrachloroethylene (perchloroethylene, PCE) as carcinogenic in animals. PCE is considered to be possibly carcinogenic to humans

with regard to occupational exposure. Human exposure to PCE can produce oesophageal cancer, cervical cancer, non-Hodgkin’s lymphoma, urinary bladder cancer and leukaemia. The data presented showed that PCE modulates the expression of some genes implicated in cancer induction, cell differentiation, cell-cycle progression, survival and clonogenic potential of human cord blood cells. The genes modulated, after exposure to the compound, were involved in inflammatory responses like the mitogen-activated protein kinase14 (MPK 14), or in tumor and metastasis progression like the matrix metalloproteinase 17 (MMP 17), in cell proliferation like c-jun and c-fos and moreover in the apoptotic process like interferon alpha-inducible protein (IFI), BAX and BCL-2. Flow cytometry analysis of cord blood cells showed that PCE treatment induced a statistically significant increase of necrosis after 24 h, while the clonogenicity of Human Colony Forming UnitGranulocyte/Macrophage (CFU-GM) and of Burst Forming Unit-Erythrocyte (BFU-E) progenitors did not change. In conclusion, these results showed that PCE affected different pathways involved in cancer induction, but its action on cell proliferation and differentiation is not clearly understood. P3-07 THE INFLUENCE OF PLATINUM-BASED DRUGS ON THE AMOUNT OF METALLOTHIONEIN R. Prusa1 , O. Blastik2 , J. Kukacka1 , J. Zehnalek2 , V. Adam2 , J. Petrlova2 , R. Kizek2 1 Department

of Clinical Biochemistry and Pathobiochemistry, 2nd Faculty of Medicine Charles University, V Uvalu 84, 150 06 Prague 5; 2 Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zemedelska 1, 611 37 Brno, Czech Republic Neoplastic diseases represent one of the most prevalent human disorders. Many of those are generally treated by platinum-complexes. Effectiveness of such treatment depends on the therapeutic concentration of the drug, commonly influenced by binding to proteins

Abstracts / Toxicology Letters 158S (2005) S1–S258

(e.g. metallothionein). Metallothionein (MT) is a wellknown molecular protein maintaining metal ion homeostasis. The aim of this work was to study the interaction of MT with Pt compounds. The proper interaction was observed in our experiments in vitro by means of electrochemical techniques using the oxidationreduction and/or catalytic signals (metallothionein-SH groups reduction, Brdicka reaction, and H-peak). Our suggested technique was used for blood serum analysis from the patients treated with standard platinum compounds. The MT amount was increased more than 10 times in these patients’ serum in comparison with non-treated patients. The results thus show that the MT amount has a significant negative influence on the therapy of neoplastic diseases. This work was supported by grants: GACR 525/04/P132, RASO 2005.


orescence assay after incubation of the human hepatoma cell line HepG2 and primary rat hepatocytes with TCDD. Additionally, the amount of the oxidative damage marker 8-oxo-2 -deoxyguanosine (8-oxo-dG) was measured after incubation of cells with TCDD using HPLC-MS/MS. Incubation of cells with TCDD for 48 h caused increased levels of ROS in primary rat hepatocytes as well as increased levels of 8-oxo dG in DNA compared to untreated cells. In the HepG2 cell line no or much lower effects were observed for both the H2 DCFDA assay and 8-oxo-dG levels. P3-09 EFFECT OF TUMOR PROMOTERS ON DNA DEMETHYLASE ACTIVITY AND DNA HYPOMETHYLATION M.A. Pereira1,2 , P.M. Kramer,2 L. Li1,2 , and L. Tao1


Chemistry and Environmental Toxicology, University of Kaiserslautern, Kaiserslautern, Germany; 2 Department of Toxicology, University of Wuerzburg, Wuerzburg, Germany 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), was classified as a group I carcinogen by the International Agency for Research on Cancer (IARC). It acts through an aryl hydrocarbon receptor (AhR) mediated mechanism, active in humans and experimental animals. The activated AhR, a nuclear transcription factor, forms in the presence of TCDD an active heterodimer with the aromatic hydrocarbon nuclear translocator (ARNT) protein and induces (or suppresses) the transcription of numerous genes, including cytochrome P450 1A1 (CYP1A1). It has been hypothesised that TCDD may be indirectly genotoxic via generation of reactive oxygen species (ROS) by inducing CYP1A enzymes. This may lead to DNA damage via direct interaction or via generation of reactive metabolites from endogenous compounds. We determined the formation of ROS with the 2 ,7 dihydrodichlorfluorescein diacetate (H2 DCFDA) flu-

1 Ohio

State University, College of Medicine, Columbus, OH, USA; 2 Medical University of Ohio, Toledo, OH, USA DNA hypomethylation is found in most tumors including colon and liver cancer and has been proposed as a mechanism for tumor promoters. Colon (deoxycholic acid and quercetin) and liver (dichloroacetic acid and Wy-14,643) tumor promoters have demonstrated the ability to induce DNA hypomethylation. To determine whether the DNA hypomethylation resulted from increased DNA demethylase activity, male F344 rats were administered 300 mg/kg deoxycholic acid or 50 mg/kg quercetin by oral gavage for four days. Some rats also received 8.0 gm/kg methionine in their diet concurrently with the tumor promoters to determine whether it would prevent any increase in demethylase activity similar to its ability to prevent DNA hypomethylation. After two but not four days of treatment, deoxycholic acid significantly increased DNA demethylase activity in nuclear protein extracts from colonic mucosa. Methionine decreased the enhancement by deoxycholic acid of demethylase activity. In B6C3F1 mouse liver, both 500 mg/kg dichloroacetic acid and 50 mg/kg Wy-14,643 increased DNA demethylase activity concurrently with their ability to induce DNA hypomethylation. Administering methionine (450 mg/kg) 30 min after each dose of the promoters, prevented their induction of DNA


Abstracts / Toxicology Letters 158S (2005) S1–S258

hypomethylation and demethylase activity. Hence, enhancement of DNA demethylase activity could be one of the mechanisms by which colon and liver tumor promoters induce DNA hypomethylation and enhance tumor development. P3-10 ZIZYPHUS SPINA-CHRISTI EXTRACT PROTECTS AGAINST AFLATOXIN B1 -INITIATED HEPATIC CARCINOGENICITY M. Abdel-Wahhab1 , M.M. Abdel-Galil1 , A.M. Hassan2 , N.S. Hassan3 , S.A. Nada4 , A. Saeed5 1 Food

Toxicology & Contaminants, Al-Azhar University, Naser City, Cairo, Egypt; 2 Foresic Medicine & Toxicology Dept., Faculty of Midecine for Girls, Al-Azhar University, Naser City, Cairo, Egypt; 3 Pathology Department, National Research Center, Dokki, Cairo, Egypt; 4 Pharmacology Department, National Research Center, Dokki, Cairo, Egypt; 5 Pharmaceutical Science Department, National Research Center, Dokki, Cairo, Egypt Aflatoxins (AF), a group of closely related, extremely toxic mycotoxins, produced by Aspergillus flavus and A. parasiticus can occur as natural contaminants of foods and feeds. Aflatoxins have been shown to be hepatotoxic, carcinogenic, mutagenic, and teratogenic to different animal species. Zizyphus spina-christi L. extract was investigated for its antifungal and antimicrobial activities. The aim of the present work was to evaluate the antioxidant activity of the methanol extract of Zizyphus spina-christi L. leaves against aflatoxin B1 -initiated hepatic carcinogenicity in rats. Forty male Sprague–Dawley male rats were divided into four groups include the control group, the group fed aflatoxin-contaminated diet (3 mg/kg diet) and the groups treated with Zizyphus extract (5 mg/kg b.w) alone or in combination with AF for 15 days. Biochemical analysis revelled that treatment with AF resulted in a significant increase in ALT, AST, cholesterol, triglycerides, uric acid, TNF␣, LPO, NO and CEA, whereas it decrease significantly GPX and SOD. The histopatholgical examination of the liver, kidney and testis showed sever histological changes typical to those reported for aflatoxicosis. Animals treated with Zizyphus extract alone or plus AF showed a significant improvement in all biochemical parameters, tumor markers and histological picture of liver, kidney and testis. We con-

cluded that Zizyphus extract have a power protective role against aflatoxin B1 -induced hepatic carcinogenicity. P3-11 THE MODULATING EFFECTS OF THYME AND ITS MAJOR INGREDIENTS ON FOOD MUTAGENS IN HUMAN LYMPHOCYTES S. Aydın1 , A.A. Bas¸aran2 , N. Bas¸aran1 1 Department

of Toxicology, University of Hacettepe, Ankara, Turkey; 2 Department of Pharmacognosy, Faculty of Pharmacy, University of Hacettepe, Ankara, Turkey Thyme has been commonly used in foods mainly for the flavour, aroma, and preservation and also in folk medicine. Genotoxic potential of major compounds of thyme oil, i.e. thymol, carvacrol, and ␥-terpinene and the methanolic extracts of thyme were investigated in human lymphocytes by COMET assay and also the effects of these substances on the induction of DNA damage by 2-amino-3-methyl-3H-imidazo[4,5f]-quinoline (IQ) were evaluated. No increase in DNA strand breakage was observed at thymol and ␥-terpinene concentrations below 0.1 mM, but at the higher concentration of 0.2 mM significant increases in DNA damage were seen. Thymol and ␥-terpinene significantly reduced the DNA strand breakage induced by IQ at the lower concentrations studied. Carvacrol, which is an isomer of thymol, seemed to protect lymphocytes from the genotoxic effects of IQ at the nontoxic concentrations below 0.05 mM, but at the higher concentration of 0.1 mM carvacrol itself induced DNA damage. Also the constituents of the n-hexane and ethyl acetate fractions prepared from the concentrated aqueous methanolic extracts of Thymus spicata protected lymphocytes against IQ-induced DNA damage in a concentration-dependent manner. Although the role of diet in the etiology of cancer is complex, our results indicate that thyme and its compounds protect lymphocytes against the mutagenic effects of IQ in vitro and highlight the potential benefit as dietary supplements of thyme and its components. But additional animal and human studies should be performed to clarify the anti-mutagenic potential of thyme and its components. This study was supported by Hacettepe University Research Foundation (HUAF 01-02-301-003).

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P3-12 ˙ ˙ SER326CYS POLYMORPHISM IN HOGG1 GENE AND ITS SUSCEPTIBILITY TO BARRETT’S ESOPHAGUS AND ESOPHAGEAL CARCINOMA E. Kadioglu1 , A.K. Daly2 , S. Sardas1 , A.E. Karakaya1 1 Department of Toxicology, Faculty of Pharmacy, Gazi

University, Ankara, Turkey; 2 School of Clinical and Laboratory Sciences, University of Newcastle upon Tyne, Medical School, Newcastle upon Tyne, UK Ser326Cys polymorphism in the hOGG1 gene, which is involved in the excision repair of 8-hydroxyguanine in oxidatively damaged DNA, has been identified and the variant genotype appears to be related to susceptibility to certain cancers. To investigate its association with esophageal cancer and premalignant Barrett’s epithelium, we conducted a case-control study of 101 patients with Barrett’s epithelium, 101 patients with esophageal cancer and 108 healthy controls in a British population. DNA was extracted, and PCRRFLP method was used to detect genetic polymorphism. We found that the distribution of hOGG1 Ser326Cys genotype among controls (Ser/Ser, 61%; Ser/Cys, 35%; and Cys/Cys, 4%) was not significantly different from that among esophageal cancer patients (Ser/Ser, 56%; Ser/Cys, 40%; and Cys/Cys, 4%) and patients with Barrett’s epithelium (Ser/Ser, 69%; Ser/Cys, 26%; and Cys/Cys, 5%). These results suggest that the hOGG1 Ser326Cys polymorphism is probably not a major contributor to individual Barrett’s esophagus or esophageal cancer susceptibility. P3-13 p53 DEFICIENT BONE-MARROW CELLS TO ASSURE NON-THRESHOLD CHEMICAL LEUKEMOGENESIS Y. Hirabayashi1 , K. Yoshida2 , J. Kanno1 , Y. Kodama1 , I. Yoshimura3 , T. Inoue4 1 Cellular & Molecular Toxicology Division, Center for Biological Safety and Research, National Institute of Health Sciences, Tokyo, Japan; 2 Division of Radiation Biology and Oncology, National Institute of Radiological Sciences, Chiba, Japan; 3 Faculty of Engineering, Tokyo University of Science, Tokyo, Japan; 4 Center for Biological Safety and Research, National Institute of Health Sciences, Tokyo, Japan


p53 deficient C3H/He mice are highly susceptible to genotoxic carcinogens due to insufficient repair activity and faster cell-cycling, thus more sensitive to chemical tumorigenesis at the lower dose where regular wild type (WT) animals show a minimum neoplastic response. Would this exaggerated tumorigenesis in the p53 deficiency contribute to assure whether threshold or non-threshold tumorigenesis due to genotoxic carcinogen? Present study was aimed to answer this question because of current accumulated arguments based on a large number of carcinogenicity studies addressing “practical threshold” in the genotoxic carcinogens. By means of study on chemical leukemogenesis using p53deficient mice, here we show that the dose-response curve for the WT can be assumed to be a sequel due, in part, to the repair form for the curves of p53 deficiencies, and the leukemogenic response not to have seen at the low-dose level in the WT mice may not be because of statistical insufficiency, but probably due to a “practical thresholdness” derived from repair mechanism. Therefore, such a hazard-repair relationship can not be ignored even in the WT animals. P3-14 INHIBITORY EFFECTS OF FERMENTATION SUPERNATANTS OF CARBOHYDRATES ON GROWTH OF COLORECTAL CARCINOMA CELLS AND ON HISTONE DEACETYLASES DEPENDS NOT ONLY ON THEIR BUTYRATE CONTENT T. Kautenburger1 , Ch. Pohl1 , Kozianowski2 , D. Schrenk1


Theis2 ,


1 Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Germany; 2 S¨udzucker AG Mannheim/Ochsenfurt, ZAFES/BPS/AHP, Offstein, Germany

Dietary fibre sources, i.e. undigestible carbohydrates are fermented by the gut flora to yield short chain fatty acids (SCFA). Butyrate, a major SCFA, is potentially chemoprotective by suppressing the growth of tumour cells and acting as inhibitor of histone deacetylases (HDAC). Although butyrate was shown to possess some health benefits on the intestine, including its function as HDAC-inhibitor, the effect of dietary undigestible carbohydrates needs further clarification. Two dietary undigestible carbohydrates (resistant starch (RS) and fructooligosaccharides) were fer-


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mented with human faecal slurries in vitro (n = 4). Fermentation supernatants (FS) were analysed for SCFA and pH. Growth of HT29 and Caco2 cells was determined after treatment with FS. A reporter gene assay using transfected Hela cells was performed to investigate the HDAC-activity. Fermentable carbohydrates are potent sources of SCFA. The FS had only slight effects on the growth of both cell lines up to a concentration of 10%, in higher concentrations, a significant growth inhibitory effect was observed. FS of RS and fructooligosaccharides were less toxic in comparison to the controls (fermentation without carbohydrate). A correlation between growth inhibition and content of SCFA was not found. Butyrate as well as the FS inhibited HDAC significantly. The effect was more pronounced with fermented samples of the carbohydrates than in fermentation controls or with butyrate alone and was correlated to the butyrate content. However, butyrate alone could not fully explain the observed effects. Our results indicate that other HDAC inhibitors are formed and/or act synergistically together with butyrate during fermentation of carbohydrates in the human colon. P3-15 TOXICOGENOMICS AND BIOMARKER DISCOVERY FOR THE PREDICTION OF LONG TERM TOXICITY H. Gmuender, C. Pallez, A. Hohn Genedata AG, CH-4016 Basel, Switzerland Toxicogenomics is a novel approach for predicting a compound’s toxicity using gene, protein or metabolite expression information. Changes to the expression profile following exposure to a drug are interpreted using knowledge of gene function and other biological information, including conventional long term toxicology results. We present evidence that toxic exposure to known genotoxic hepatocarcinogens is manifest in the expression profile following just 10 days of chronic exposure. To gain information on the toxic mode of action (MOA) we applied promoter and pathway analysis software tools for the identification and characterization of novel gene regulation mechanisms that are relevant for the cell’s response to DNA damage. The results demonstrate that expression data from genes, proteins and metabolites can be used to build reference

libraries of known compounds against which novel compounds can be tested. Biomarker candidates can then be deduced from such libraries and further characterized to determine the causal relationship between the biomarker and toxicity in animal models and in humans. P3-16 COMPARISON OF HISTOLOGICAL AND TOXICOGENOMIC QUANTIFICATION OF GST-P IN LIVER OF NNM-TREATED RATS A. Oberemm1 , H.-B. Richter-Reichhelm1 , U. GundertRemy1 , K. Tenz2* , E. Krause2 , M. Kr¨oger3 , K. Fella3 , J. Hellmann3 , P.-J. Kramer3 , G. Scholz4** , H. EllingerZiegelbauer4 , H.-J. Ahr4 , E. Kiss5 , C. Ittrich6 , A. KoppSchneider6 1 Federal Institute for Risk Assessment, 14195 Berlin, Germany; 2 Research Institute for Molecular Pharmacology, 13125 Berlin, Germany; 3 Merck KGaA, Institute for Toxicology, 64293 Darmstadt, Germany; 4 Bayer Health Care AG, Molecular and Genetic Toxicology, 42096 Wuppertal, Germany; 5 Biostatistics; 6 Department Cellular and Molecular Pathology, DKFZ, 69009 Heidelberg, Germany

A common animal model of chemical hepatocarcinogenesis was used to demonstrate the utility of genomic and proteomic data to identify early biomarkers related to carcinogenesis. N-Nitrosomorpholine (NNM) was applied via 120 mg/L drinking water to male Wistar rats for 7 weeks followed by an exposure-free period of up to 43 weeks. A second group of untreated animals served as control. Five specimens of each treatment group were sacrificed at nine time points during and after NNM-treatment. Individual samples from the left liver lobe were prepared for histological and toxicogenomic analyses. For histological detection of foci of altered hepatocytes (FAH), tissue sections were stained using antibodies to glutathione-S-transferase, placental form (GST-P). Gene and protein expression of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat genome chips and 2-D electrophoresis (2-DE) based proteomics. Among other differentially expressed candidate genes and proteins, GST-P was identified by both approaches. In order to compare results obtained by methods of histology and toxicogenomics, GST-P-stained liver sections of all animals

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were evaluated morphometrically. A strong correlation between area fraction of FAH and both, gene and protein expression as well as between gene and protein expression could be demonstrated. On the transcriptional level, an increase of GST-P expression was detectable as early as 3 weeks after the beginning of treatment compared to 8 and 20 weeks on the morphological and translational level, respectively. These findings confirm the utility of toxicogenomic approaches to detect molecular changes of toxicological relevance. * Present

address: Federal Institute for Risk Assessment, 14195 Berlin, Germany. ** Present address: Nestl´ e Research Center, Quality and Safety Department, 1000 Lausanne 26, Switzerland. P3-17 NEW TOXIC EFFECTS OF CEREULIDE, THE EMETIC TOXIN OF BACILLUS CEREUS, RECORDED IN BOAR SPERMATOZOA AND Caco2 CELLS M. Andersson, M. Salkinoja-Salonen Department of Applied Chemistry and Microbiology, POB 56, Fi-00014, University of Helsinki, Finland Cereulide, the emetic toxin of Bacillus cereus, is a known potassium ionophore and a mitochondrial toxin. We investigated the toxic effects of nanomolar (nM) concentrations of cereulide on boar spermatozoa and Caco2 cells (human cancer cell line). The effects measured after exposure to cereulide were: inhibition of sperm motility, hyperpolarisation of the plasma membrane, depolarization of the mitochondria and induction of apoptosis measured as fragmentation of the nucleus in cells where the plasma membrane was still intact. Boar spermatozoa lost motility and their plasma membrane became depolarised within 30 s of exposure to cereulide. After 10–15 min of exposure the mitochondria became depolarized. Hyperpolarisation of the plasma membrane did not occur when the potassium ion concentration of the semen extender was raised from 2–4 to 60 mM before exposure. The results indicate that hyperpolarisation of the plasmamembrane and the inhibitions of sperm motility were caused by an efflux of potassium ions from the cytoplasm of the sperm cells. Caco2 cells responded to cereulide exposure of 3 days by depolarisation of the mitochondria and


by fragmentation of the cell nucleus, wherase no damage to the plasma membrane integrity was observed. Complete apoptotic cell death followed after 5 days of exposure. Our results demonstrate novel toxic effects of cereulide: efflux of potassium ions from the cell cytoplasmas an immediate response to exposure and induction of apoptosis after long exposure time (5 days). P4 Quantitative Risk Assessment P4-01 FREQUENCY DEPENDENCE OF THE CENTRAL AND PERIPHERAL SOMATOSENSORY EVOKED ACTIVITY IN RATS TREATED WITH XENOBIOTICS A. Papp, Z. Lengyel, Z. Fazakas, A. Szab´o Department of Public Health, University of Szeged, Hungary The complex chemical pollution of our environment results in large-scale human exposure to a number of proved or supposed neurotoxicants. The long-term, low-level exposure causes mostly no overt symptoms of nervous system damage, which indicates the need for new, more sensitive means of early detection. Sensory evoked potentials are readily recorded in experimental animals and in humans and have been shown to be sensitive of toxic effects. In the present work, male Wistar rats were exposed to various xenobiotics, insecticide agents and heavy metals, in different timing schemes. For recording, the rats were prepared in urethane anesthesia, and evoked potentials form the somatosensory cortex as well as action potentials of the tail nerve were elicited by electrical stimulation and recorded. The frequency of stimulation was varied to se if the dynamic properties of the somatosensory system, reflected in the ability to follow rapid stimulation, were impaired by the xenobiotics administered. The shape and numerical parameters of the cortical and peripheral evoked response changed with increasing stimulation frequency also in untreated control rats. Among the heavy metals tested, lead (alone and combined with e.g. mercury or cadmium) caused the most profound change in the frequency dependence. The effect of insecticides (dimethoate, propoxur, cypermethrin) was stronger on the cortical response compared to the peripheral action potential.


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Frequency dependence of the evoked activity proved to be sensitive to the influence by environmental neurotoxicants. This parameter is thus a candidate for functional effect biomarker of nervous system damage also in humans. Supported by the Hungarian OTKA grant No. 042955. P4-02 RECENT ADVANCES IN QUANTITATIVE RISK ASSESSMENT (QRA) FOR INDUCTION OF DERMAL SENSITIZATION FOR FRAGRANCE INGREDIENTS A.M. Api1 , D.A. Basketter2 , P.A. Cadby3 , M.F. Cano4 , G. Ellis5 , G.F. Gerberick6 , P. Griem7 , P.M. McNamee8 , C.A. Ryan6 , B. Safford2 1 Research

Institute Fragrance Materials, Inc., New Jersey, USA; 2 Safety and Environmental Assurance Center Unilever, Sharnbrook, UK; 3 Firmenich, Inc., Geneva, Switzerland; 4 LVMH, Orleans, France; 5 Givaudan France Fragrances SAS, Argenteuil Cedex, France; 6 The Procter & Gamble Company, OH, USA; 7 Clariant, Sulzbach, Germany; 8 The Procter & Gamble Company, Egham, UK Some of the chemicals in common use today may have the potential to cause dermal sensitization. However, the fact that a chemical is a skin sensitizer does not mean it cannot be formulated into consumer products at safe levels. This is also the case for fragrance ingredients. Based on the chemical, cellular and molecular understanding of dermal sensitization, it is possible to conduct an exposure-based quantitative risk assessment (QRA) to determine safe levels of fragrance ingredients in different consumer product types. Key steps of the quantitative risk assessment process are determination of known safe benchmarks; application of sensitization assessment factors and calculation of consumer exposure through normal product use. Using these parameters, an acceptable exposure level (AEL) can be calculated and compared with the consumer exposure level (CEL). The ratio of the AEL to CEL must be favorable to support the safe use of the skin sensitizer. This ratio must be calculated for the skin sensitizer in each product type. This presentation provides an overview of the principles of exposure-based QRA as applied to fragrance ingredients and provides

a practical example using a fragrance ingredient in different product types. P4-03 ADVANCED INTERNATIONAL TRAINING IN HEALTH RISK ASSESSMENT Ch. Rud´en, J. Zilliacus, H. H˚akansson Karolinska Institutet, Institute of Environmental Medicine, Sweden High standard education in toxicology and risk analysis is available at the Master of Science level in several European countries, but a more advanced and comprehensive, education in health risk assessment of chemicals is lacking. Within the EU CASCADE network ( the planning of such a training programme is ongoing. The course programme is intended to include about ten independent, one-week, full-time courses given over a four-year period. Some courses will be introductory, while others will be indepth courses, focused on specific topics. The course programme will be open for PhD and post-doc students, senior scientists, and other professionals. Examples of courses under planning are: Environmental Health Risk assessment I and II, including basic concepts in toxicology, epidemiology, exposure and health risk assessment. Environmental chemistry and metabolism including exposure assessment. Statistical methods and soft ware, including experiment design and quantitative risk assessment methods. Case-course, including practical risk assessment exercises. Reproductive and developmental toxicology, including endocrine disruption. Epidemiology and biostatistics. Regulatory toxicology, including presentations of European authorities and organizations. Philosophy of risk, including risk communication, theory of science, ethics, and risk-benefit analysis. Collaboration with ongoing educational activities forms an important basis of this initiative and stakeholders are hereby warmly invited to take part in the planning of the courses.

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P4-04 RISK ASSESSMENT OF PESTICIDES IN SIMPLE MIXTURES H.R. Pohl Agency for Toxic Substances and Disease Registry (ATSDR), U.S. Department of Health and Human Services, Atlanta, GA 30333, USA The agency for toxic substances and disease registry’s (atsdr) program for chemical mixtures encompasses research on chemical mixtures toxicity, health risk assessment, and development of innovative computational methods. Atsdr’s mixtures risk assessment program will be discussed using two examples. Atrazine, deethylatrazine, simazine, diazinon, and nitrate were chosen as a subject mixture because the chemicals frequently occur together in rural well water. Atrazine and simazine are triazine herbicides, deethylatrazine is a metabolite and an environmental degradation product of atrazine and other triazine herbicides, diazinon is an organophosphorus insecticide, and nitrate is a common contaminant resulting from fertilizers and human and animal waste. The weight-of evidence (WOE) analysis for interactions among these components indicates high confidence in the additivity assumption that is the basis for the hazard index. However, the chemical interactions of atrazine and simazine with nitrite can result in the formation of Nnitrosoatrazine and N-nitrososimazine, which are more genotoxic than the parent triazine compounds. Chlorpyrifos, lead, and mercury/methylmercury were chosen as a subject mixture because of concerns about neurological effects in children co-exposed to these chemicals. Chlorpyrifos is an organophosphorus insecticide widely used in the United States for agricultural and indoor and outdoor residential applications. The most important pathway of exposure to mercury is via ingestion of methylmercury in fish. Exposure to lead is mostly because of its release from mining, deteriorating lead paint, and historical use in gasoline. The WOE analysis for interactions among these components indicates that joint toxic action is primarily less than additive or additive and therefore does not increase the concern for potential health hazard above that indicated by the hazard index.



Quality of Life, P.O. Box 360, Utrechtseweg 48, Zeist, 3700 AJ, The Netherlands; 2 TNO Science and Industry, P.O. Box 155, Stieltjesweg 1, Delft, 2600 AD, The Netherlands In this study we estimated the within-animal variation in routinely studied continuous toxicological parameters from temporal fluctuations in individual nonexposed animals. Assuming that these fluctuations are non-adverse, this within-animal variation gives an indication of the minimal size of the critical effect size (CES). The CES is the breaking point between adverse and non-adverse changes in a continuous toxicological parameter, at the level of the individual organism. The total variation in the data from individual non-exposed animals was split up in variation parts due to known factors (sex, animal and day) and a residual variation, by means of analysis of variance (ANOVA). Assuming measurement errors are small, the residual variation is considered the best estimate of the within-animal variation. The data showed within-animal variations varying between 1 and 60% for different clinical chemistry and haematological parameters in 90 day rat studies. This indicates that different (minimal) CES values may have to be used for different parameters. Further studies are necessary to elucidate whether different (minimal) CES values should be derived for different sexes, species, strains, and ages. P4-06 THE DEVELOPMENT AND APPLICATION OF BIOMARKERS OF TOXICITY J.F. Ciaran, C. Faherty Biomarkers Technical Committee, ILSI Health and Environmental Sciences Institute (HESI), One Thomas Circle, NW, Ninth Floor, Washington, DC 20005, USA The development of new biomarkers has been constrained by an unclear definition of the process by


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which candidate biomarkers can be validated and accepted in a scientific and regulatory context The mission of the HESI Biomarkers Technical Committee is to advance the scientific basis for the development and application of biomarkers of target organ toxicity; to develop a systematic approach for the evaluation of biomarkers that bridge from the preclinical to clinical stages of drug development; and to provide a scientific forum for building consensus regarding how to apply biomarkers of toxicity in risk assessment. This mission has been realized through the support of multinational member companies reflecting the pharmaceutical industry, and significant input from academic and government colleagues. The committee has formed three working groups to evaluate: (1) the use of serum Troponins as a biomarker of cardiotoxicity; (2) the use of Inhibin B as a biomarker for testicular toxicity; and (3) to develop biomarkers of nephrotoxicity. An accurate process map for the validation of these biomarkers will accelerate their scientific and regulatory acceptance. Each working group proposes to draft such a process map for the development of analytical methods and evaluation and validation protocols using a subset of nominated bridging biomarkers. The results of these evaluations may be submitted to the U.S. Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) and/or the European Committee for Validation of Alternative Methods (ECVAM) for regulatory acceptance. P4-07 INITIAL DATA ON THE EFFECTS OF LONG-TERM CIGARETTE MAINSTREAM SMOKE EXPOSURE AND HIGH FAT DIET IN APOLIPOPROTEIN E-DEFICIENT MICE S. Lebrun, W. Stinn, H. Weiler, P. Kuhl, B. Friedrichs, H.-J. Urban, Klaus von Holt, T. Wallerath, R. Schleef Philip Morris Research Laboratories GmbH, Cologne, Germany To further define the mechanisms underlying the progression of atherosclerosis mediated by cardiovascular risk factors, apolipoprotein E-deficient mice were whole-body exposed for 12 months (6 h/day, 5 days/week) to diluted cigarette mainstream smoke (MS; 100 and 200 ␮g total particulate matter/l) or to filtered, fresh air (sham) in combination with three

diets differing in cholesterol/fat content: (i) chow (0.02%/4.5%), (ii) milk fat (0.17%/21%), and (iii) beef fat (0.14%/21%). Here we present data from the first 9 months. Both high-fat diets increased aortic plaque and cholesterol content. High MS effects on atherosclerosis include an elevated cholesterol content of the aortic arch over the entire time course for the high MS-milk fat group versus sham milk fat (p < 0.05, linear regression). There was also a weak trend towards increased cholesterol by MS for all diet groups (statistically significant when data for the two MS high fat diet groups were combined at 9 months). Low MS appeared to have a different affect: an increase in calcium content in the brachiocephalic artery, represented by a significant increase in calcium in the low MS chow group compared to sham chow (6 months). These data suggest that MS exposure affects the composition of plaques in a complex manner. P4-08 CONDUCTING A COMPREHENSIVE TOXICOLOGICAL EVALUATION OF NANOMATERIALS M.P. Holsapple ILSI Health and Environmental Sciences Institute, One Thomas Circle, NW, 9th Floor, Washington, DC 20005, USA The use of nanotechnology in consumer and industrial applications will likely have a profound impact on the utility of a number of products from a variety of industrial sectors. Nanomaterials exhibit unique physical/chemical properties and impart enhancements to engineered materials, including better magnetic properties, improved electrical activity, and increased optical properties. Consequently, the use of nanotechnology has the potential to facilitate substantial improvements in several critical societal functions such as energy generation and distribution, food processing, and building construction. Given the impending widespread use of nanotechnology, a systematic approach needs to be developed for evaluating the risk to human health and the environment from exposure to nanomaterials. Some of the uncertainties associated with exposure to nanomaterials include the extent to which these materials interact with cellular organelles, the biological impact of those interactions, and the

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long-term health effects from acute and chronic exposure. There is also very little known about the environmental implications of the use of nanomaterials. Accordingly, the ILSI Health and Environmental Sciences Institute (HESI) has developed a comprehensive program to improve the science associated with developing toxicological evaluations for engineered nanomaterials and to improve the understanding of the behavior of these materials in biological systems and the environment. The broad goal of the effort is to develop a systematic approach for conducting comprehensive toxicological evaluations for nanomaterials. The specific project areas that form the basis of the program activities, and that are the focus of this poster presentation, include nanomaterial characterization, toxicity, dissolution, and life-cycle analysis. P4-09 A DATABASE FOR THE ASSESSMENT OF POTENTIAL INTERACTIVE EFFECTS OF CHEMICAL MIXTURES AT WORKPLACE A.Vyskocil1 , D. Drolet2 , C Viau1 , F. Lemay2 , G. Lapointe3 , R. Tardif1 , G.Truchon2 , M. Baril2 , N. Gagnon3 , F. Gagnon1 , D.Begin1 , M. G´erin1 1 Universit´ e

de Montr´eal, Montr´eal, Canada; 2 IRSST, Montr´eal, Canada; 3 CSST, Montr´eal, Canada

This project was undertaken to develop a toxicological database allowing the identification of possible additive or other interactive effects of mixtures present in the work environment. In the first phase of the project, standard general literature references were used to compile critical data such as target organs, effects in target organs, mechanisms of action, and toxicokinetic characteristics for each of the 668 chemical substances of the Quebec Regulation. The results of the first phase allow the prediction of potential additivity among components of a mixture. In the second phase of this project, the types of interaction for mixtures most likely to be found in workplaces and for which primary literature data are available were specified. The toxicological data were evaluated only for realistic exposure concentrations up to the STEL or ceiling value or five times the 8 h TWA PEL in humans and up to 100 times the 8 h TWA PEL or ceiling value in animals. In total, 675 studies were evaluated for 209 binary mixtures of substances. For


the majority of cases where potential additivity was identified in phase 1, there is a lack of supporting toxicological data in the primary literature. The resulting database integrates the results from both phases of project. P4-10 EVALUATION THE ULCEROGENIC EFFECT OF METHANOLI EXTRACT OF ANACARDIUM OCCIDENTAL IN RAT’S STOMACH M.R. Heidari, G. Fatemi Department of Toxicology and Pharmacology, Faculty of Pharmacy, Neuroscience and Physiology Research Center, Kerman, Iran In traditional Iranian medicine, the core of the fruit of anacardium occidental (A.o) were used in treatment of pain. In this research ulcerogenic effect of percolated extract of A.o was investigated. The extract and indomethacine was administrated with dose of 200, 300, 400 and 800 mg/kg. In control group normal salin was administrated with volume of 5 ml/kg. The animals were killed 4 h. After receiving indomethecin or extract or normal salin. The stomachs were removed and 10 ml formalin 2% injected in to stomach to fix the inner layer of the gastric wall. The stomach were incised along the greater curvature and lesion in the glandular portion were evaluated, 20 min. after formalin exposure. The Ulcer index was calculated, using the J-score. The results showed that the oral dose of 200 mg/kg of the extract did not induced any ulcerogenic effect in the rat’s stomach. The dose of 300, 400 and 800 mg/kg of extract induced lower ulcerogenic effect than the same dose of indomethacine (P < 0.01). Therefore, it seems that the A.o is a suitable plant for continuing investigation for introducing an analgesic drug for clinical use. P4-11 GROUP HOUSING OF CYNOMOLGUS MONKEYS FOR REGULATORY TOXICOLOGY STUDIES P. Lainee, C. Nanuel, Ch. Thill, S. de Jouffrey, R. Forster CIT, Evreux, France


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Cynomolgus macaque monkeys (Macaca fascicularis) are highly social animals and feral populations live in stable packs of 20–30 animals. In contrast, when these monkeys are held in a laboratory environment, housing is often in individual cages. These do not provide for rich social interaction, and as a consequence of the social deprivation stereotypic behaviour patterns are commonly observed. For our work in safety evaluation studies using young adult purpose-bred cynomolgus monkeys, we have developed housing units which permit the group housing of 4–6 animals of the same sex, so that we can work with the animals while meeting their basic needs for socialisation. The units are designed to provide some environmental enrichment in terms of perches, feeding procedures and toys. They permit easy isolation of individuals when required. The units are aimed also to comply with evolving regulations regarding primate caging and physical space. The design of the units and the corresponding working procedures for animal care and handling were progressively refined through experience with prototypes over a period of 24 months. These units permit the performance of most kinds of regulatory toxicology studies and the recording of the principal routine parameters that are measured in these studies (with some brief periods of isolation). We have recently equipped a new dedicated building with these units. P4-12 E-LEARNING AND RISK COMMUNICATION. REPORT OF AN EXPERIENCE AT THE FACULTY OF PHARMACY OF THE UNIVERSITY OF PORTO F. Remi˜ao, H. Carmo, F. Carvalho, M. Lourdes Bastos Requimte, Servi¸co de Toxicologia, Faculdade de Farm´acia, Universidade do Porto, Porto, Portugal Risk communication has been one of the main concerns in toxicology and different strategies have been adopted to better elucidate the public about risk perception and risk behaviour. Pharmacists are in a privileged position as risk communicators and risk managers. These professionals have a deep knowledge in toxicological concepts, public’s trust and easy access to the population. Thus, it will be very important to teach pharmacy students how to get quick and correct data about the possible toxicological risks and how to assimilate and transmit that information to the public.

With that purpose, the toxicology department of the Faculty of Pharmacy has been associated with the elearning project of the University of Porto, through which students develop WebPages that communicate risks associated to environmental pollution, food and water contamination, chemicals or drugs. In this project the students use an e-learning platform from which the toxicology contents, discussion and chat tools, books and databases could be easily accessed. The WebPages (in Portuguese language) are linked to our Website after an adequate correction by the Department staff ( The present communication will address the results of this project concerning the quality of risk communications in the WebPages, the use of the e-learning platform, the related improvement in the scores of the toxicology exam and the results of the student’s survey. P4-13 ESTIMATION OF RADIOACTIVE DOSE FROM OYSTERS CONSUMED AT HIROSHIMA FOLLOWING A BOMB DETONATION B.N. Noller, il1 , R. Kleinschmidt2 , M.R. Moore1,2 1 EnTox, 2 QHSS,

University of Queensland, Qld, Australia; Coopers Plains, Brisbane, Qld, Australia

We have estimated of the maximum radiation dose received from consuming an oyster at Hiroshima following the A-bomb detonation in 1945. Many of the Australian troops stationed at Hiroshima were from Sydney and familiar with oyster consumption; they asked for the dose estimate to be made. This had not been previously estimated, due to a lack of recognition that oysters were a significant item of diet for troops at Hiroshima. Australian veterans of the British Commonwealth Occupational Forces (BCOF) who arrived at Hiroshima on 1 February 1946, 178 days after the atomic bomb detonation on 6 August 1945 raised the following question: did any of the occupation forces who were stationed at Hiroshima receive a significant dose of radioactivity (compared against current ICRP recommendations) from the atomic bomb detonation products by consuming oysters taken from Hiroshima Bay? The investigation considered a hypothesis that consumption of oysters which concentrated significant radioactivity following the detonation gave a significant contribution to the

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radiation dose of the BCOF. This dose was calculated by examining uptake of the main radionuclides Sr-89, Sr-90, Zr-90, I-131, Cs-137 and Ce-144, derived from a worst-case scenario that: (i) all fission products were based on the yield of weapon being 15kt; (ii) all radionuclides were assumed to be available for uptake after detonation; and (iii) all were transferred to Hiroshima Bay. A plausible estimate of dose was calculated and was found to be an insignificant addition to total when compared with current ICRP guidelines.



Low dose radiation exposures during specific gestational periods can result in permanent neuronal perturbations and eventual abnormalities in behavior and mental activity. It is hypothesized that the mechanism underlying these effects includes radiation-induced cell death among neuronal precursors in the proliferative regions of the neocortex. We used a systems biology approach to develop a new computational model based on diverse data sets to describe the extent and pattern of cell death in the neocortex and altered patterns of migration. The model predicts the subsequent effects of cell death on neuron number at the end of neurogenesis. Our results indicate induced death of a small fraction of neuronal precursors, dependent on the dose and time of exposure, leads to a significant decrease in the final neuron count and provides a means for comparing differences in absorbed dose, time of exposure, and duration of exposure. The model predicts a 1% decrease of neuronal precursor cells during the most sensitive time window of neurogenesis about a 0.5% decrease per cGy during other times of neurogenesis. This model is of utility for risk assessment as it allows for the evaluation of alterations in specific developmental dynamic processes across times and doses. (ER63674, ES09601, ES012762, ES07032.)

D.A. Basketter1 , C. Clapp1 , B. Safford1 , D. Jefferies1 , I. Kimber2 , R. Dearman2 , C.A. Ryan3 , F. Gerberick3 1 SEAC,

Unilever, Sharnbrook, UK; 2 Syngenta CTL, Macclesfield, UK; 3 Procter & Gamble, Cincinnati, OH, USA For years, methods have been available for the predictive identification of chemicals that possess the intrinsic potential to cause skin sensitization. However, many of these methods have proven less capable in terms of the determination of relative sensitizing potency. In this respect, the local lymph node assay (LLNA) has been shown to have a number of advantages. Through interpolation of LLNA dose response data, the concentration of a chemical required to produce a threshold positive response (the EC3 value) can be measured. The robustness of this measure has been thoroughly demonstrated in terms of inter and intra laboratory reproducibility. Additionally, the relationship between potency estimates from the LLNA and an appreciation of human potency has been reported. In the present work, we have sought to enhance this by undertaking a thorough and extensive analysis of existing human predictive assays. Information on the approximate threshold for the induction of skin sensitization in the Human Repeat Insult Patch Test was characterised for 24 chemicals. This was compared to LLNA EC3 values. The results from each assay (dose per unit area) showed a clear correlation, substantiating the utility of LLNA EC3 values for prediction of the relative human sensitizing potency of skin sensitizers.

W.C. Griffith, N.M. DeFrank, J.M. Gohlke, E.J. Gribble, E.M. Faustman Institute for Risk Analysis and Risk Communication, University of Washington, Seattle WA 98105, USA

P4-16 REGULATION OF COMPLEMENTARY MEDICINES IN AUSTRALIA D. Sevior, J. Ahokas Key Centre for Applied and Nutritional Toxicology, School of Medical Sciences, RMIT University, PO Box 71, Bundoora, Vic. 3088, Australia ‘Complementary Therapies’ or ‘Alternative Therapies’ are increasing in popularity. Over 52% of the Australian


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population use complementary medicines at an estimated cost of $1671 million in the year 2000. Australia is one of few individual countries to have implemented reforms in the regulation of complementary medicine. The Office of Complementary Medicines (OCM) is a body within the Therapeutic Goods Administration (TGA) that regulates complementary healthcare products. Another body, the Complementary Healthcare Consultative Forum (CHCF) is a high level forum aimed at facilitating consultation between government, the complementary health care industry and consumers. In spite of the regulatory efforts of a number of countries, the safety of the products used by consumers cannot be guaranteed. Domestically produced products may be relatively well regulated. However, internet sales and private importation of these products have changed the whole landscape. It is easy to obtain products from ill-defined sources that in many cases the efforts of local regulation are significantly compromised. In addition the international discrepancies in regulation contribute to consumer confusion. Major issues continue to be associated with the use of complementary medicines. These include public perception that natural products are inherently safe. With the increased use of a wide range of complementary medicines it is possible that previously unrecognized of delayed toxicity may emerge and it is certain that there will be increasing numbers of interactions between complementary and conventional medicines. P4-17 ENVIRONMENTAL ENRICHMENT FOR PRIMATES IN REGULATORY TOXICITY STUDIES F. Bellebeau1 , J. Silvano1 , N. Pickersgill1 , J. Descotes2


Mtimeth1 ,

1 MDS Pharma Services, 69210 St Germain s/L’Arbresle, France; 2 Lyon Poison Center, 69424 Lyon Cedex 03, France

Environmental enrichment is an important consideration when providing species-specific living conditions for primates. A study in Cynomolgus monkeys has been performed to evaluate various treats and toys. Three types of ice cubes (tap water, tap water with raisins and orange juice) were distributed on three occasions. The animals were found to prefer ice cubes with raisins

and these were then distributed over five consecutive days. Interest was obvious throughout the observation period as the animals played with, licked and ate the ice cubes for 10–15 min at a time. Six cages were equipped with removable metallic swings. Four animals were housed singly and four in pairs. Behavioural observations were performed by the same investigator four times a day over one week. Fifty percent of the observations showed animals using the swings. No differences were observed between singly housed and socialised animals. The addition of swings in the cages did not prevent the technicians from performing their routine tasks. It is concluded that ice cubes with raisins are valuable enrichment tools. It is proposed that they be used during the training of animals to specific handling conditions before study and as a reward during the treatment period. It was concluded that swings afford suitable enrichment for monkeys without impeding routine animal handling. P4-18 INTRAOCULAR PRESSURE MEASUREMENT: COMPARISON OF TWO TONOMETERS L. Bonnet1 , F. Bellebeau1 , N. Crettet1 , H. Voute1 , J. Descotes2 1 MDS

Pharma Services, 69210 St Germain s/L’Arbresle, France; 2 Lyon Poison Center, 69424 Lyon Cedex 03, France Intraocular pressure measurement can be performed in toxicity studies in several animal species using different types of tonometer. Several are commercially available and two were compared: Medtronic Tonopen® and the more recent Icare® . Repeatability, reproducibility and ease of use were evaluated. Several series of measurements were performed using both devices in different species: Beagle dogs, New Zealand White rabbits and Cynomolgus monkeys. In both conscious dogs and rabbits, intraocular pressure was measured three times a day, for three consecutive days and by two operators on the first day. In monkeys, measurements were made on only one occasion on anaesthetised animals by two operators. In all species, the Medtronic Tonopen® gave higher values than Icare® . There were neither statistically significant differences between operators nor differences in nycthemeral rhythm with either instrument. Although both the Tonopen® and Icare® were

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easy to use, the latter required no local anaesthesia of the cornea because of a limited contact of the probe with the eye. It was concluded that both tonometers were of equal value in toxicity studies, provided that the same one is used throughout the study and that the corresponding historical background data are referred to. P4-19 RISK ASSESSMENT GUIDANCE ENZYME-CONTAINING PRODUCTS


F.H. Kruszewski1 , B.A. Concoby2 , K. Sarlo3 1 The

Soap and Detergent Association, USA; 2 Genencor International, USA; 3 The Procter & Gamble Company, USA The purpose of this guidance is to describe the potential health hazards of enzymes present in consumer products and provide a framework for manufacturers of these products to conduct risk assessments to help ensure the safety of new products containing enzymes. Enzymes generally have good safety profiles. However, enzymes like many other proteins can act as allergens and induce the production of allergen-specific IgE antibody upon repeated inhalation or exposure to mucous membranes that may lead to allergy symptoms, including asthma. The primary challenge associated with enzyme use is preventing the generation of allergenspecific antibody and the development of symptoms of Type 1 hypersensitivity. This hazard is the primary focus for the risk assessment for enzymes and must be managed carefully. Another hazard that also should be addressed is primary irritation of the eye and skin. However, most uses of enzymes in consumer products do not pose a likelihood of causing irritation. If the risks posed by enzymes are not managed appropriately, the consequences may spread beyond a single product or company. This could lead to unwarranted limitations on the use of enzyme technology in other consumer applications. Therefore, it is recommended that companies using enzymes responsibly consider how they are managing enzyme safety including the conduct of appropriate risk assessments and risk management programs. The preferred approach is for product manufacturers to develop comprehensive programs to assess and manage the risks of using enzymes in consumer products. The program design should be developed on a case-bycase basis to address parameters specific to the type of product and its applications. Experience in the clean-


ing products industry demonstrates that the potential risk of adverse effects can be successfully managed by identifying the hazards, carefully assessing exposure, characterizing the risk and then applying appropriate risk management. Good understanding of these areas will lead to informed decisions about the potential risks and the development of sound approaches to manage these risks. This guidance document outlines strategies and methods that have been used successfully by the cleaning products industry. P5 Allergy and Asthma P5-01 PREVALENCE OF DRUG’S ALLERGY AMONG PATIENTS IN HOSPITALS IN AHVAZ, IRAN P. Afshari1 , S. Sadeghi2 1 Islamic

Azad University Dezful Branch, Iran; 2 Ahvaz Jondishapour Medical Sciences University, Iran Some of the allergic reactions occure after using special foods or drugs. Only 6% of drug side effects have allergic background. Clinical signs are different in children, most common drug reactions is skin rashes but it can cause other signs, and even systemic anaphylactic reactions. Methods and subjects: This is a descriptive study. We interviewed 160 patients who where admitted to our hospitals. A questionaire sheet consisted of 22 questions. Validity and reliability were checked by content validity and test retest methods respectively. The results showed 60% of patients were female and 40% male. The mean age was 38 years. Twenty six percent of samples were hospitalized due to chronic diseases. Drug allergy was seen in 10.41% that 6.25% of them had allergy to penicillin. Allergic reactions were presented in 4.17%. Twenty five percent of subjects had seasonal allergy. 12.5% of them had food allergy. Twenty eight percent of subjects had allergic history during their childhood, 52% of them in the recent years and 20% were affected recently. Young people demonstrated more allergies. The most common signs of allergic reactions were skin’s complications (itching, burning) in 13% and ophtalmic complications (tear, eye burning) in 12%. In comparison with other studies, our results showed more drug allergy (10.41%) In addition we saw allergic reactions more in the young people.


Abstracts / Toxicology Letters 158S (2005) S1–S258



R. Hagemann1 , G. Neverdal1 , A. Nilsen2 , I. Solvang2 , R. Bogen Hetland3 1 Statoil

Research Centre, Trondheim, Norway; 2 Norwegian University of Science and Technology, Trondheim, Norway; 3 Norwegian Institute of Public Health, Oslo, Norway Particulate air pollution may cause respiratory and cardiovascular diseases. The composition of the particulate mixture may vary between different locations and different seasons. Most toxicological studies are on ambient air or diesel particles. However we investigated the effects of particles derived from combustion of two different heating oils, wood pellets and standard diesel exhaust particles (SRM 1650a) in two different cell lines. The IL-8- and IL-6-inducing potential of the particles was studied in the human alveolar epithelial cell line A549. The DNA transcriptional activity (RNA) and IL-6, IL-10, IL-12 p40 and TNF␣ production was studied in the human monocyte cell line Mono-Mac-6. In the alveolar epithelial cell line, the heating oil particles were more potent on a weight basis to induce release of IL-8 and IL-6 than the diesel and wood pellets particles, all significantly different from control. In the monocyte cell line, all types of particles induced changes in DNA transcription and production of cytokines, heating oil particles being the most potent. However, it is important to emphasise that particles were compared on a mass basis and heating oil combustion emits very low particle mass pr energy unit (mg/MJ). P5-03 STIMULATION OF IL-8 IN AIRWAY EPITHELIAL CELLS BY CRUDE SALMON ORGAN EXTRACTS AND PURIFIED SALMON TRYPSIN B. Bang, L. Aasmoe, M. Larsen Department of Occupational and Environmental Medicine, University Hospital North, Norway

We have previously shown that workers in the fish processing industry have increased prevalence of airway symptoms, not linked to IgE—mediated allergy. Samples from the work environment show that the workers are exposed to endotoxins as well as proteases from the fish juice and process water. The aim of the present study was to show that fish constituents may activate inflammatory responses linked to the chemokine IL-8 in airway epithelial cells. A549 cells were stimulated by crude fish organ extracts and purified salmon trypsin for 24 h. The organs used were skin, mucus lining of the skin, gills, muscle, liver, pylorus, stomach and colon. The activity of trypsin- and chymotrypsin-like proteases was measured in the fish organ extracts. Bovine trypsin and bacterial endotoxin were used as positive controls for chemokine activation. Levels of IL-8 in cell supernatants were determined by enzyme-linked immunosorbent assay (R&D system). Preliminary results show that salmon trypsin is able to increase the level of IL-8. The maximal response, demonstrated with a trypsin activity of 0.0097 U/ml was eight times the unstimulated level. Of the fish extracts, the stomach and colon showed the greatest IL-8—stimulating activity. In conclusion, the preliminary results show that salmon organ extracts as well as purified salmon trypsin are able to stimulate IL-8 in the airway epithelial cell line A549. Further results will be presented at the meeting. P5-04 CYTOKINE RELEASE FROM ALVEOLAR MACROPHAGES EXPOSED TO AMBIENT PARTICULATE MATTER: RELATION TO SIZE, CITY, SEASON AND METAL CONTENT R.B. Hetland1 , M. Refsnes1 , F.R. Cassee2 , M. L˚ag1 , E. Dybing1 , P.E. Schwarze1 1 Norwegian

Institute of Public Health, Oslo, Norway; Institute of Public Health and the Environment, Bilthoven, The Netherlands

2 National

Studies have demonstrated associations between exposure to ambient particulate matter (PM) and respiratory and cardiovascular diseases. Inflammation seems to be important in the observed health effects. However, the predominant particle component(s) that drives the inflammation is still not fully clarified. In this study

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coarse (2.5–10 ␮m) and fine (<2.5 ␮m) PM-samples from a western, an eastern, a northern and a southern European city (Amsterdam, Lodz, Oslo and Rome) were collected during spring, summer and winter. All fractions were investigated with respect to cytokineinducing potential in primary macrophages isolated from rat lung and related to particle size and metal content. Both site-specific and seasonal variations in cytokine responses were demonstrated. The samples collected in Lodz and Rome appeared to be the most potent. Seasonal variation was most obvious with the samples from Lodz, with the highest responses induced by the spring and summer samples. Coarse fractions from all cities were more potent to induce interleukin6 and tumour necrosis factor-␣ than the corresponding fine fractions. Higher levels of specific metals such as iron and copper seemed to be prevalent in the coarse fractions. However, variations in the content of these metals did not reflect the differential cytokine release. P5-05 IS SMOKING HABIT MASKING AIRWAY MINIMAL PERSISTENT INFLAMMATION (MPI) PRESENT IN SENSITISED NONASTHMATICS? M. Miri´c, I. Maloˇca, J. Macan, D. Plavec Department for Occupational and Environmental Health Institute for Medical Research and Occupational Health, Zagreb, Croatia To examine the relationship and confounding factors of sensitization to house-dust mites with nonspecific bronchial reactivity (NBR) in nonasthmatic asymptomatic subjects. Retrospective study (2001–2003) included all subjects that underwent medical history, physical examination, spirometry, nonspecific bronchoprovocation, skin prick test to Dermatophagoides pteronyssinus (DP) and measurements of total and specific IgE. There were 234 (114 women; 146 smokers) subjects included into analysis. The mean (S.D.) age of the subjects was 29.4 (6.4) years. None of them had asthma. All subjects had lung function parameters inside normal range. Sixty-nine subjects (29.5%) had clinically relevant positive skin prick test (3 mm). The number of subjects who had specific IgE to DP >0 kU/L


was 98 (41.9%). When two subgroups (specific IgE 0 or >0 kU/L) were compared, the second one had all lung function parameters significantly lower with NBR significantly higher (P < 0.05 for all). Multiple regression analysis in the second subgroup revealed significant correlation between NBR and specific IgE level (r = 0.52, P < 0.001). Smoking habit was a significant independent predictor of NBR (OR, 0.16; 95% CI, 0.031–0.828; P = 0.0256). It seems that smoking habit can mask the existence of MPI present in sensitized individuals without clinical expression of asthma. Further studies are needed to completely elucidate the underlying pathophysiological mechanisms. P6 Persistent Organic Pollutants—Integrated Exposure and Risk Assessment P6-01 LEVELS AND TRENDS OF PERSISTENT ORGANOCHLORINE COMPOUNDS IN HUMANS IN TURKEY, 1976–2004 E. Durmaz, T. C¸alık Durmaz, ˙I. C ¸ ok Gazi University, Faculty of Pharmacy, Department of Toxicology, 06330, Hipodrom, Ankara, Turkey Persistent OCPs and PCBs are the most important groups of persistent organic pollutants (POPs). OCPs were produced in large number in the 1940–1950s and global production increased year by year. Some of the members of OCPs such as DDT have been transported throughout the global environment for many decades. PCBs were first manufactured commercially in 1929 and concern about the presence of PCBs in environment began in the 1960s. They have been used widely in heat exchangers and dielectric fluids; as stabilizers in paints, polymers, and adhesives; and as lubricants in various industrial processes. Because of the bioaccumulation and toxicity, usage of these chemicals for different purposes has been restricted or banned in most countries, since the beginning of the 1970s. In Turkey, OCPs have been started to used against to pests in 1945, large quantities of these chemicals were used during 1960s and 1970s, and since 1983 usage of these chemicals, excluding Endosulfan, have been severely restricted. In Turkey, OCP residues have been monitored in the


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Turkish population by carrying out regional surveys at given time intervals since 1976. But there are very few data of PCB contamination levels in Turkey. PCBs are restricted for use in closed systems and banned after 1996 in Turkey. The presenting study aims to determine the changes of OCP levels from past to present and current levels of PCBs in humans in Turkey. The results have been discussed in terms of regions and OCPs and PCBs in which analyses had been made. P6-02 ENVIRONMENTAL AND BIOLOGICAL MONITORING OF POLYCYCLIC AROMATIC HYDROCARBONS IN AN AIRPORT I. Iavicoli1,2 , G. Carelli1 1 Universit` a

Cattolica del Sacro Cuore, Istituto di Medicina del Lavoro, Largo Francesco Vito 1, 00168 Roma, Italy; 2 Universit`a degli Studi di Milano, Dipartimento di Medicina del Lavoro “Clinica del Lavoro L. Devoto”, Via San Barnaba 8, 20122 Milano, Italy Polycyclic aromatic hydrocarbons (PAHs) can be found as airborne substances in workplaces following combustion. PAHs include compounds that are carcinogenic and mutagenic in humans. The aim of this study was to evaluate occupational exposure to PAHs in an airport by means of environmental and biological monitoring. Twenty-three airborne PAHs were sampled using organic polymer filters and adsorbents. Analysis was carried out by gas chromatography in conjunction with mass spectrometry. In order to measure the 1-hydroxypyrene metabolite, urine samples obtained at the end of the workshift from 49 workers (13 controls and 36 exposed subjects) and were analysed by high performance liquid chromatography with fluorescence detector, after enzymatic hydrolysis. Benzo[a]pyrene levels were found to be extremely low in the airborne PAHs, especially when compared to the Technical Exposure Limit proposed by the Deutsche Forschungsgemeinschaft (0.005 mg3 /m). Moreover, urinary 1-hydroxypyrene concentrations were below the 0.76 ␮mol/mol creatinine limit, the only biological index currently established on the basis of epidemiological studies of occupational exposure to PAHs.

P6-03 RESIDUES OF ORGANOCHLORINE COMPOUNDS IN BREAST MILK FROM WOMEN OF PALIZADA, CAMPECHE, MEXICO J. Rend´on-von Osten, M. Memije Canepa Centro EPOMEX, Universidad Aut´onoma de Campeche. PO Box 520, 24030 Campeche, Campeche, Mexico During 1980s and 1990s in Campeche State was sprayed almost 10 t/year of DDT to combat malaria vectors. There are not data about the presence of DDT residues in inhabitants of Campeche exposed to this compound; therefore, the aim of this study was to determine DDTs levels in breast milk of mother residents of Palizada, Campeche. The donors were asked to express by a breast pump of breast milk to a glass bottle, and kept frozen at −20 ◦ C until analyzed. Each mother completed a questionnaire to provide personal information such as time of residence and number of children. The analysis of organochlorine compounds was carried out by GC-ECD. The preliminary results indicate that average concentration of -DDT in breast milk is 35.78 mg/L (lipid basis), and the percentage of each compound is p,p DDE = 18.8%, p,p -DDT = 18.6%, o,p -DDD = 18.8%, o,p -DDE = 11.3% and o,p -DDT = 32.5%. All the samples exceeded the acceptable daily intake (ADI) for -DDT (0.005 mg/kg/day) in cow milk, according to place of residence, the estimated daily intake (EDI) of -DDT by the infants from Palizada the EDI of children is estimated to exceed the ADI of -DDT by 2–40 times. Actually the real effects of this contaminants on infants is unknown, therefore, is necessary to identify biomarkers of exposure, susceptibility, and effects in humans to predict potential health effects associated with environmental chemicals to breast-fed. P6-04 PERSISTENT ORGANIC POLLUTANTS IN BLOOD SERUM AND SEMEN QUALITY IN FIELD WORKERS FROM BONFIL, CAMPECHE, MEXICO J. Rend´on-von Osten, M. Memije Canepa Centro EPOMEX, Universidad Aut´onoma de Campeche, Apdo, Postal 520, 24030 Campeche, Campeche, Mexico

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In Mexico a previous monitoring study of human adipose tissue of Mexican residents who lived in areas where DDT has been sprayed, revealed great contamination and exposure to their vapors during spraying and elevated DDE deposits in adipose tissue. In Bonfil town there are around 10,000 ha of field crops where many pesticides were applied. The aim of the present study was to determine the levels of persistent organic pollutants (POPs) in blood serum of field workers living in Bonfil, Campeche. Field workers were volunteers from an agricultural organization. Blood samples were collected by venipuncture and the serum was separated by centrifugation. Determinations of persistent organic pollutants were carried out by GC-ECD. All the field workers presented residues of POP’s. Average concentrations of -DDT in serum blood was of 2.07 mg/L with a range from non detected (N.D.) to 13.16 mg/L, and an average of -Aroclores (PCBs) of 0.95 mg/L (N.D. to 9.02 mg/L). The main compounds detected in serum blood were o,p -DDD and p,p -DDE and DDT. Concentrations of POPs and workers age has a good correlation with -DDT (r = 0.46) and -Aroclores (r = 0.32). Likewise, it was found a high correlation between -DDT/-PCB concentrations (r = 0.95). Spermatobioscopy results show that only 60% of field workers have a spermatic count considered above the level considered as sub-fertility level. It is necessary to carry out permanent monitoring surveys for POPs in order to prevent potential risk to human health because of accumulating properties of organochlorine pesticides in human fat tissue P6-05 RESIDUAL LEVELS OF POLYCHLORINATED BIPHENYLS IN HUMAN LIVER IN VOJVODINA (SERBIA) U. Milosevic1 , J. Adamov2 , V. Milankov1 1 University

of Novi Sad, Faculty of Medicine, HajdukVeljkova 3, 21000 Novi Sad, Serbia and Montenegro; 2 University of Novi Sad, Faculty of Sciences, Chemistry Department, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia and Montenegro In this paper the content of polychlorinated biphenyls (PCBs) was determined in the samples of human liver, collected during obductions from random donors of both gender and different ages. Residues of PCBs were


determined by GC/ECD. Concentration of total PCBs was in the range 3.6–47.8 ng/g of liver. The most abundant were congeners PCB 138, PCB 153 and PCB 180, comprising >80% of the total content of PCBs in the liver. There is no significant correlation between age and gender of the donors and the levels of PCBs in their tissue. Although PCBs have been banned in Serbia for two decades, this study shows that the exposure to these contaminants seems to continue in Serbia in spite of the fact that it was carried out on the limited number of subjects and because of the selected population results are not representative for the whole Serbian population. This research was financially supported by the Ministry of Science and Environmental Protection of the Republic of Serbia, Project No. 1622. P6-06 RESIDUAL LEVELS OF POLYCHLORINATED BIPHENYLS IN HUMAN LIVER IN VOJVODINA (SERBIA) V. Milankov1 , U. Milosevic1 , J. Adamov2 1 University

of Novi Sad, Faculty of Medicine, HajdukVeljkova 3, 21000 Novi Sad, Serbia and Montenegro; 2 University of Novi Sad, Faculty of Sciences, Chemistry Department, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia and Montenegro Although the application of most organochlorine pesticides was banned in 1970s due to their toxicity and possible adverse health effects to wildlife and humans, residues of these hazardous chemicals are still present in the environment. Man, as the top of the food chain, accumulates most of these toxicants. While there is evidence that persistent organic pollutants (POPs) concentrations in the environment have generally been declining in the last decade, data available on humans suggest that the declining was much slower to respond to the restriction in use or incident. Presumably, this is due to the high persistence of PCBs in the fatty tissues in which they are deposited. Data on human contamination with PCB and pesticides are focused on blood or milk, the only biological materials conveniently available from healthy subjects. In this paper content of POPs was determined in blood of 15 patients, obtained during vascular and abdominal surgeries at Clinic of Surgery, Novi Sad Hospital. POPs were extracted from


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blood with acetone:hexane mixture, and their content eas determined using gas chromatography (GC/ECD). P6-07 EFFECTS OF TCDD IN THE OSTEOBLASTIC CELL LINE UMR-106 C. Wejheden, S. Brunnberg, A. Hanberg, P.M. Lind Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disrupting environmental pollutant that induce skeletal abnormalities in rat pups. TCDD induce transcription of the enzymes CYP1A1 and CYP1A2, which are suggested to increase the metabolism of estrogen. Estrogen is one of the hormones involved in bone tissue regulation. Bone loss and impaired bone formation are well known effects of estrogen deficiency. Increased metabolism of estrogen and probably also other interactions between the aryl hydrocarbon receptor (AhR) and the estrogen receptors (ER) could thereby affect the bone tissue formation and regulation. The aim of this project was to characterize the osteoblastic cell line UMR-106 and examine if it can be used as an in vitro model system for studies on mechanisms behind the effects of dioxin on bone tissue. Cell viability, cell proliferation and expression of AhR, CYP1A1 and osteopontin were examined after exposure to TCDD. No effects were observed on cell viability or cell proliferation during exposure to TCDD. The results show that UMR-106 expresses the AhR and during exposure to TCDD the expression of CYP1A1 is induced. Preliminary results indicate that the expression of osteopontin is decreased after 24 h exposure to 10−11 M TCDD. However, no effect was seen in either higher or lower concentrations of TCDD or after 6 h exposure. These findings indicate that UMR-106 can be used as a model system for osteoblasts. P6-08 CYTOCHROMES P450 AS BIOMARKERS OF PAH EXPOSURE IN ANIMAL MODELS AND MAN P.H. Roos Institute of Occupational Physiology, Dortmund, Germany

PAH are ubiquitous in the environment, in the diet and in tobacco smoke and enter the body by dermal absorption, inhalation and ingestion. Ingestion of PAHcontaminated soils by mammalians induces CYP1A1. While minipigs show a large response in the duodenum, rats respond by low CYP1A1-induction in this organ but by a high increase in the liver. Inductioneffective concentrations of PAH even reach kidneys, lung and spleen. Inhalation of tobacco smoke characteristically alters the CYP profile in rat lung: CYP1A1 is induced, constitutive CYP1B1-levels are elevated, CYP2B1 is largely suppressed and CYP2E1 remains unaffected. A down-stream organ affected by tobaccosmoke is the urinary bladder. We studied CYP expression in human urothelial cells from various sources by RT-PCR. Transcripts of CYP1B1 and of arylaminemetabolising CYP4B1 are dominant in urothelial cells while CYP1A1-levels are low. In urothelial cell lines, CYP1A1 is PAH-inducible. In leukocytes of occupationally PAH exposed persons, we determined transcript levels of CYP1A1 and CYP1B1 as exposure biomarkers. As an effect of PAH exposure, constitutive CYP1B1 transcript-levels were suppressed while CYP1A1 was slightly induced. In summary, PAHs lead to specific effects in the tissues of entry which in spite of their enzyme activities cannot prevent systemic PAH distribution. Affected down-stream organs respond to PAH by modulating their specific CYP profile. Knowledge of the basal CYP profiles and their xenobioticdependent modulations in the entry and target tissues helps to understand PAH-dependent adverse effects. P6-09 IMPACT OF BROMINATED DIPHENYL ETHERS ON AH-RECEPTOR ACTIVATION IN 5L RAT HEPATOMA CELLS S. Strack, M. Wahl, H.F. Krug Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, Karlsruhe, Germany 2,2 ,4,4 -Tetrabromodiphenyl ether (BDE47), a worldwide used flame retardant in consumer goods, is found ubiquitously in environmental samples and can be traced in human blood sera. For ecological and human risk assessments, however, the toxicological data base is still insufficient. The aryl hydrocarbon receptor (AhR) has been identified to be responsi-

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ble for the induction of xenobiotic metabolism and for the expression of several regulatory mediators, including the Phase I enzyme CYP1A1 and p27Kip1 , a cyclin/cyclin dependent kinase inhibitor. In rat hepatoma cells (5Lwt and BP8AhR−/− ) cytotoxic effects (MTT-assay) of BDE47 could be observed at concentrations above 50 ␮M (25 ␮g/ml) in Dulbecco’s modified eagle medium (DMEM) with 5% fetal calve serum. Cell cycle regulation is altered in 5L wild type cells in a dose dependent manner. At concentrations higher than 50 ␮M 5L cells arrested in the G1 phase. An activation of the Ah receptor by BDE47 was indicated by an induction of a CYP1A1 expression in 5L but not in BP8 cells, an AhR deficient sub clone. Posttranscriptional gene silencing of the AhR showed a clear reduction of CYP1A1 protein levels. Our results suggest that BDE47 may act as a weak dioxin-like Ah receptor ligand, however, further studies are needed to get more insights into the involvement of the dioxin receptor in toxic responses of BDE 47 in mammalian cells. P7 Immunotoxicology P7-01 POLYMORPHISM OF IL-1a, IL-1b, AND IL1RA AND ITS EFFECT ON IMMUNOGLOBULIN TITERS IN PESTICIDE EXPOSED AND NON-EXPOSED WORKERS M. Tarkowski1 , M. Bara´nska1 , S. Birindelli2 , E. Corsini3 , C. Colosio2 , T. Vergieieva4 , J. Liesivuori5 , L. van Amelsvoort6 , H. van Loveren7 1 Nofer

Institute of Occupational Medicine, Departement of Immunotoxicology, Lodz, Poland; 2 International Centre for Pesticides and Health Risk Prevention, Busto Garolfo, Italy; 3 Universita’ degli Studi Milano, Department of Pharmacological Sciences, Milan, Italy; 4 National Center of Hygiene, Medical Ecology and Nutrition, Sofia, Bulgaria; 5 University of Kuopio, Department of Pharmacology and Toxicology, Kuopio, Finland; 6 University of Maastricht, Department of Epidemiology, Maastricht, The Netherlands; 7 National Institute of Public Health and the Environment, Laboratory for Pathology and; Immunobiology, Bilthoven, The Netherlands


The principal objective of these studies was to determine if genetic polymorphism play a role in determination of immune changes among workers exposed to pesticides. In this study we investigated whether the polymorphism of IL-1␣, IL-1␤, and IL-1RA should be taken into consideration while determining the immunoglobulin and anti-Hepatitis antibody titers in subject’s exposed and non-exposed to pesticides. Analyses were performed in 373 workers exposed to pesticides and controls from 4 different countries. We show that the frequency of the polymorphisms of selected genes are similar between exposed and controls. Statistical analyses do not show significant correlation with immunoglobulin levels and anti-Hb titers, Our analyses indicate that genetic polymorphism of IL-1 group of genes do not affect the conclusions on the effect of pesticide exposure on immunoglobulin levels. P7-02 EFFECTS OF THE FISH OIL AND ASPIRIN ON THE IMMUNE AND OXIDATIVE RESPONSE IN CHROMIUM INSTILLED RATS D. Surcel, M. Butan, R. Ghitulescu, M. Botic, S. Ramboiu Institute of Public Health, Cluj-Napoca, Romania The prolonged inhalation of hexavalent chromium (Cr) is associated with alteration of the immune and oxidative reactions. The aim of this study was to investigate the effect the associated administration of the fish oil and aspirin on the Cr induced diseases, in which immune and oxidative reactions are involved, but the exact mechanism of these effects are unknown. In vivo experiment was carried out on the Wistar rats, that were divided into seven groups as following: (1) Controlgroup; (2) Fish oil-group; (3) Aspirin-group; (4) Cr– group; (5) Cr + fish oil-group; (6) Cr + aspirin-group; (7) Cr + Fish oil + aspirin-group. The animals belonging to 4–7-groups were intratracheally instilled with Cr; the animals were given fish oil and aspirin, daily, 9 months according to outline of study. The animals were sacrificed at 9 months and bronchoalveolar lavage was carried out. The following parameters were assessed: (1) 3HTdR incorporation test; (2) IL1-assay; (3) TNFassay; (4) Chemiluminiscence assay; (5) Lipid peroxides (LP). In Cr– group, 3HTdR incorporation was decreased and values of the IL-1, TNF, ROS and LP


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were increased. A partial reversibility of these parameters was noted in the Cr + Fish oil-group. Association of the fish oil and aspirin induced the most important reversibility of the parameters. (1) Cr interferes with immune and oxidative reactions; (2) association fish oil + aspirin may have an important protective effect in Cr exposure by immunomodulatory and antioxidant effects. P7-03 EFFECTS OF ARSENIC, ENDOSULFAN AND THEIR COMBINATION ON INDUCTION OF IMMUNOTOXICITY AND APOPTOSIS IN BROILER CHICKS M. Aggarwal1 , J.K. Malik1 , G.S. Rao1 , N. Suresh Babu1 , A.K. Tiwari2 , S. Dandapat2 1 Division

of Pharmacology and Toxicology, Izatnagar 243 122, UP, India; 2 Division of Animal Biotechnology, Indian Veterinary Research Institute, Izatnagar 243 122, UP, India Arsenic has been found in higher concentrations in drinking water in various parts of the world. Endosulfan an organochlorine compound is being widely used as insecticide and acaricide. Apoptosis of the immune system has recently drawn interest since immune dysfunction can affect the survival of the host in various ways. It is evident that the living organisms are being exposed to multiple chemicals rather than the single chemical. The present study was carried out to investigate the effects of arsenic, endosulfan and their combination on induction of immunotoxicity and apoptosis in chickens. Forty day-old chicks were randomly divided into four groups each comprising of 10 chicks. Group I was kept as control whereas groups II, III and IV received arsenic, endosulfan and their combination at 3.7 ppm, 30 ppm and 3.7 + 30 ppm, respectively for 60 days. Arsenic and endosulfan were given in water and feed, respectively. Immune responses of the birds of different treatment groups were accessed by LST, DTH, ELISA and mononuclear cell function test at 10 day intervals, while apoptosis was detected in peripheral blood lymphocytes of the treated chicks as accessed by cell viability, DNA fragmentation, fluorescence microscopy, flowcytometry and scanning electron microscopy at the end of the study. Arsenic, endosulfan and their combination caused induction of higher level of apoptosis in

peripheral blood lymphocytes of chickens. The effects of co-exposure of arsenic and endosulfan on induction of apoptosis and immunotoxicological indices were relatively less as compared to the expected additive effects of individual chemicals. P7-04 ALTERED CYTOKINE PRODUCTION IN WORKERS EXPOSED TO TRICHLOROETHYLENE I. Iavicoli1,2 1 Universit` a

Cattolica del Sacro Cuore, Istituto di Medicina del Lavoro, Largo Francesco Vito 1, 00168 Roma, Italy; 2 Universit`a degli Studi di Milano, Dipartimento di Medicina del Lavoro “Clinica del Lavoro L. Devoto”, Via San Barnaba 8, 20122 Milano, Italy Trichloroethylene (TCE) is a volatile organic compound widely used as an industrial solvent. Although the principal concern with regard to TCErelated health effects is its potential carcinogenic action, TCE has also been involved in the development of autoimmune disorders and immune system dysfunction in humans. In humans, environmental exposure to TCE has been associated with systemic lupus erythematosus, systemic sclerosis, fasciitis and altered T-lymphocyte ratios. Moreover, an analysis of the occupational histories of patients has linked occupational exposure to TCE to the development of sclerodermia. Studies conducted on animals have reported immune system effects in mice exposed orally to TCE. These included inhibition of bone marrow stem cell colonization, humoral and cell-mediated immunity. Female mice appeared to be more sensitive than male mice. To investigate TCE-induced alterations of the immune system in humans, the levels of interleukin2, interleukin-4 and interferon-␥ in sera obtained from workers exposed to TCE were determined and compared with those of internal and external control subjects. In workers with a mean urinary trichloroacetic acid concentration of 13.3 ± 5.9 mg/g creatinine, exposed to a mean environmental TCE level of 35 ± 14 mg/m3 , we observed a significant increase in sera interleukin-2 and interferon-␥ levels and a reduction in interleukin-4

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concentrations compared with those of workers from the internal and external control groups. This study provides the first report on quantitative immune changes induced by occupational exposure to low levels of TCE and strongly suggests that exposure to this substance alters immunohomeostasis in humans with possible effects on health. P7-05 N-ACETYLTRANSFERASE 2 ACETYLATION GENOTYPES AND SERUM IGE LEVELS A. Khan1 , H. Dickel2 , T. Bruckner3 , H.F. Merk1 , B. Bl¨omeke4 1 Department

of Dermatology and Allergology, University Hospital of the RWTH Aachen, Pauwelsstr. 30, 52057 Aachen, Germany; 2 Department of Dermatology and Allergology, Ruhr-University Bochum, Gudrunstrasse 56, 44791 Bochum, Germany; 3 Department of Social Medicine, Occupational and Environmental Dermatology, University Hospital of Heidelberg, 69115 Heidelberg, Germany; 4 Department of Ecotoxicology and Toxicology, University of Trier, Am Wissenschaftspark 25-27, 54296 Trier, Germany IgE mediated allergic reactions play a crucial role especially in industrialized societies. A role of the N-acetyltransferase 2 (NAT2) phenotype and genotype has been investigated previously in allergic diseases such as food allergies, allergic rhinitis, and atopic asthma. We studied the NAT2 genotypes in persons with ‘high’ IgE levels. In 218 cases with high total serum IgE-levels (>200 IU) and 192 controls the genotypes coding for slow and fast acetylator NAT2 phenotypes were identified by real-time polymerase with hybridization probes. Mutations at positions 341 (C → T), 481 (C → T), 590 (G → A) and 857 (G → A) of the NAT2 gene were examined. The acetylation phenotype based on genotyping was defined as the occurence of at least one wild type allele. Allele frequencies were calculated according to the Hardy–Weinberg equilibrium. Statistical analysis showed that 39.9% of the cases and 47.4% of the controls were fast acetylators based on genotyping, while 60.1% of the cases and 52.6% of the controls were classified as slow acetylators. Five cases (2.3%) and 16 controls (8.3%) were classified as wildtypes,


whereas 59 cases (27.1%) and only 31 controls (16.1%) had a homozygous mutation at nucleotide position 481 (C → T). Allele frequencies for cases were: NAT2*4 (wild type): 17.2%, *5A/B: 48.9%, *5C: 5.7%, *6A/B: 25.9% and *7A/B: 2.3%. Allele frequencies for controls were NAT2*4: 25.6%, *5A/B: 42.4%, *5C: 4.7%, *6A/B: 24.7% and *7A/B: 2.6% compared favourly to published data. In conclusion, we found higher frequencies of phenotypically slow acetylators and genotypically homozygous mutations at position 481 (C → T) in individuals with high total serum IgE levels compared to controls. These results suggest that certain mutations in the NAT2 gene may be associated with high total IgE levels. P7-06 EFFECTS OF PERINATAL EXPOSURE TO LOW DOSES OF PCB 153 AND PCB 126 ON IMMUNE FUNCTIONS IN KIDS J.L. Lyche, H.J.S. Larsen, J. Utne Skaare, A. Tverdal, E. Dahl,1 Grethe M. Johansen, E. Ropstad National Veterinary Institute, Oslo, Norway In this study we report the effects of perinatal exposure to environmental levels of PCB 153 and PCB 126 on postnatal immune responses by assessing white blood cell counts, in vitro lymphocyte proliferations, the levels of total IgG and immunoglobulins to specific microbes, and immune responses following immunization of the kids at 2 weeks of age. PCB 153 suppressed the maternal immunity demonstrated by reduced transfer of maternal immunoglobulin G (IgG) and specific antibodies to Arcanobacterium pyogenes, Mannheimia haemolytica and Reo virus (REO-1), Mycobacterium avium ssp. paratuberculosis and Equine influenza virus (EIV-1) in the newborn kids. Furthermore, the antibody response against EIV-1 was significantly higher two weeks following immunization. The same group had significantly higher numbers of white blood cells, neutrophils, and lymphocytes at 2 week of age and significantly lower lymphocyte response to the mitogens PHA and Con A. PCB 126 exposure reduced the levels of maternal antibodies to REO-1, increased the concentrations of maternal antibodies to tetanus toxoid and reduced the postnatal levels of monocytes in plasma. The results suggest that PCB 153 and PCB 126 at environmental relevant levels modulate immunologic funtions.


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P7-07 INTERLEUKIN-15 SERUM LEVELS IN WORKERS EXPOSED TO CHEMOTHERAPEUTIC AGENTS C. Fenga, G. Spatari, C. Abbate, C. Costa, I. Polito, A. Familiari, D. German`o Section of Occupational Medicine, Department of Social and Environmental Medicine University of Messina, Italy Cytostatic anticancer drugs are known as carcinogenic, mutagenic, and teratogenic risk factors for health care workers occupationally exposed. It has been demonstrated that the administration of interleukin-15 in rat models of colon carcinoma protects against chemotherapy-induced gastrointestinal toxicities. We evaluated if occupational exposure to chemotherapeutic antiblastic agents in vivo modified circulating levels of interleukin-15 in seventeen health care workers exposed to antineoplastic drugs in relation to their jobs and in as many healthy, age and sex matched subjects. Health care workers displayed significantly higher circulating interleukin-15 levels compared to their agematched controls. If this increase represents an anticancer response remains to be established, but these findings strengthen the idea of a therapeutic use of interleukin-15 in the field of cancer. P7-08 CYTOKINE PROFILING OF CHEMICAL ALLERGENS IN MICE: MEASUREMENT OF MESSAGE VERSUS PROTEIN R.J. Dearman1 , B.F. Flanagan2 , C.J. Betts1 , H.T. Caddick1 , I. Kimber1 1 Syngenta Central Toxicology Laboratory, Macclesfield, UK; 2 University of Liverpool, Liverpool, UK

Topical exposure of BALB/c strain mice to the contact allergen 2,4-dinitrochlorobenzene (DNCB), or to the respiratory allergen trimellitic anhydride (TMA) induces selective type 1 or type 2 cytokine secretion profiles, respectively. Lymph node cells (LNC) were prepared 13 days after the initiation of topical exposure to allergen. Total

RNA was prepared from freshly isolated cells and cytokine gene expression was analyzed by ribonuclease protection assay (RPA). Cells were cultured for 24–120 h and supernatants analyzed for cytokine protein by ELISA. DNCB-activated LNC secreted high levels of the type 1 cytokine interferon (IFN)-␥, compared with TMA-stimulated LNC. The converse type 2 patterns were induced by TMA, with vigorous secretion of the type 2 cell products (interleukins [IL]-4, 5, 10 and 13) observed. LNC from TMA-treated mice displayed a similar, albeit less clearly contrasting, preferential type 2 cytokine profile for cytokine mRNA. In contrast, RNA isolated from DNCB-activated LNC displayed a mixed cytokine phenotype with relatively low levels of transcripts for both type 1 and type 2 cell products. These data indicate that IFN-␥ production by DNCB-stimulated LNC is controlled mainly at the level of secretion or translation of previously transcribed mRNA. These data suggest that cytokine profiling by RPA is not appropriate for the prospective characterization of chemical respiratory and contact allergens. P7-09 LOCAL LYMPH NODE ASSAY (LLNA) RESPONSES TO HEXYL CINNAMIC ALDEHYDE (HCA): IMPACT OF AGE I. Kimber, C.J. Betts, A. Banyard, L. Beresford, R.J. Dearman Syngenta Central Toxicology Laboratory, Macclesfield, UK The murine LLNA is an established method for the assessment of skin sensitizing activity, measured as a function of induced proliferative responses in draining lymph nodes. Skin sensitizers are defined as materials that provoke a three-fold or greater increase in proliferation compared with concurrent vehicle treated controls (stimulation index [SI] > 3). It has been reported that immune responses, including some aspects of allergen-induced lymph node activation, may be affected by age. CBACa strain mice (n = 4 per group) of average age of 8, 15 or 21 weeks were exposed to 25% HCA, a positive control for skin sensitization testing, or to vehicle alone, in two or three independent experiments. Mean SI values of 7.8, 6.7, and 6.6 were achieved for 8, 15 and 21 week old mice,

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respectively. Dose responses were performed (using 5%, 10% and 25% HCA) in order to determine mathematically using linear interpolation the estimated concentration of chemical necessary to induce the threshold SI of 3 (EC3 value). Values obtained for 8, 15 and 21 week old animals were 13.0, 10.7 and 13.6, respectively. In previous publications using animals in the age range of 8–12 weeks, EC3 values of 10.3 ± 0.6 have been reported. At least with respect to HCA, LLNA responses appear to be stable across mice with ages ranging from 8 to 21 weeks.

issues; an assessment of preclinical data needs for human immunogenicity of large molecules; an appreciation of clinical immunotoxicology—liabilities and ‘bridges’ between nonclinical and clinical approaches; and an update on the progress of creating a nonhuman primate immunotoxicology database. The success of the HESI ITC is based on an approach where international experts from academia, industry and government come together in a neutral forum and a cooperative atmosphere to engage in an open and transparent exchange of scientific ideas.



C. Faherty Immunotoxicology Technical Committee, ILSI Health and Environmental Sciences Institute (HESI), One Thomas Circle, NW, Ninth Floor, Washington, DC, USA The mission of the HESI Immunotoxicology Technical Committee (ITC) is to identify and address scientific issues related to the development and application of immunotoxicology to public health and human health risk assessment; to promote the understanding and appropriate use of immunotoxicologic data to protect human health; and to contribute substantively to the scientific decision-making processes relative to the development of guidelines and regulations for immunotoxicology testing at the local, national and international levels. This mission has been realized through the support of global member companies reflecting the agrichemical, chemical, consumer products, petrochemical and pharmaceutical industries, and significant input from academic and government colleagues. The focus of this poster will be to highlight some of the recent activities of the ITC including the following: a better understanding of how to increase research opportunities to address immune-mediated drug hypersensitivity reactions (IDHR); a proposed testing framework for developmental immunotoxicology; an identification of the priority research needs in respiratory allergy reflecting protein-specific, chemical-specific, and drug-specific

M. Takeyoshi, K. Iida, K. Shiraishi, S. Hoshuyama Hita Laboratory, Chemicals Evaluation and Research Institute, Japan The murine local lymph node assay (LLNA) is currently recognized as a stand-alone test method for determining the sensitizing potential of chemicals. It has an advantage to give quantitative endpoint that can be used for predicting the sensitization potency of chemicals. For this purpose, EC3 value has been proposed as a parameter for classifying the chemicals according to the sensitization potency. We have previously developed a non-radioisotopic endpoint for the LLNA using 5-bromo-2 -deoxyuridine (BrdU) incorporation (Non-RI LLNA). We propose here a novel strategy to predict the sensitization potency of chemicals by relative comparison with known human contact allergens. Nine chemicals categorized from class 1 to class 5 human contact allergen, diphencyclopropenone, p-phenylenediamine, glutaraldehyde, cinnamicaldehyde, citral, eugenol, isopropyl myristate, propyleneglycol, hexane, were tested by the non-RI LLNA with the reference allergens, 2,4dinitrochlorobenzene (DNCB) as the class 1 human contact allergen, isoeugenol as the class 2 human contact allergen and ␣-hexylcinnamicaldehyde (HCA) as the class 3 human contact allergen. Consequently nine


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test chemicals were assigned into nearly correct allergen classes that their own have. The results suggested that the new strategy for non-RI LLNA could provide a correct sensitization potency data. The sensitization potency data is useful to appraise the sensitization risk of new chemical products for human. Accordingly this strategy would be effective modification of LLNA with regard to the experimental design. Moreover, this strategy can be applied also in the standard LLNA using radioisotopes and the other modification of this method. P7-12 RESPONSES OF NCI-H292 LUNG EPITHELIAL CELLS TO INHALED TOXICANTS, A COMPARATIVE STUDY N. Newland, E. Massey, A. Richter British American Tobacco, R&D Centre, Regents Park Road, Southampton, UK The induction of interleukin-8 (IL-8) in lung epithelial cells in vitro is frequently used to investigate the toxicity of inhaled agents. We have previously reported that cigarette smoke total particulate matter (TPM) stimulates gene expression of matrix-metalloprotease1 (MMP-1), the gel-forming mucin MUC5AC and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in NCI-H292 cells. These targets are associated with overproduction of mucin and tissue remodelling in lung disease and are potentially useful biomarkers of response in vitro. In this study we have compared the gene expression profile of H292 cells exposed to TPM to the profile induced by other inhaled toxicants, namely lipopolysaccharide (LPS), bleomycin and vanadium sulphate (VSO4 ). Polymethyl-methacrylate (PMM) and the TPM vehicle, dimethyl-sulphoxide (DMSO) were used as negative controls. Confluent monolayers of H292 cells were exposed to serial dilutions of test agents for 24 h. The levels of expression of IL-8, MMP-1, HBEGF, MUC5AC and the cytochrome P450 s CYP1A1 and CYP1B1 were then determined by quantitativepolymerase chain reaction. TPM stimulated the expression of all of the targets genes. Bleomycin, LPS and

VSO4 upregulated IL-8, MMP-1, HB-EGF, MUC5AC, but had minimal effect on CYP1A1 and CYP1B1. The negative controls were relatively ineffective with the exception of IL-8 induction by DMSO. We conclude that the use of such a panel of in vitro biomarkers could give a more comprehensive picture of potential toxicity than the determination of IL-8 alone, particularly in the case of agents such as TPM, where the conventional vehicle is found to have some biological activity. P7-13 EVALUATION OF THE MUTZ-3 CELL LINE AS A MODEL SYSTEM FOR IN VITRO SENSITIZATION TESTING G.R. Verheyen, I. Nelissen, I. Selderslaghs, E. Schoeters, R.L. Van Den Heuvel, H. Witters, G. Schoeters Centre of Expertise in Environmental Toxicology, Flemish Institute for Technological Research, Mol, Belgium The early detection of the sensitizing potential of chemicals is of great importance to the industry. The number of animals that are currently used for these experiments has to be reduced, so in vitro alternatives are necessary. A promising alternative is an in vitro test system based on a model of human dendritic cells (DC), cultured from CD34+ progenitors. However, this DC model has several drawbacks: human cord blood has to be collected/available to obtain CD34+ stem cells, primary DC cultures are time-consuming, and experiments are hampered by the inter-individual differences that exist between donors. MUTZ-3 is a human, acute myeloid leukemia cell line with the potential to differentiate into immature DC and may therefore be used to overcome many of these difficulties. We optimized the culture conditions of the MUTZ3 cell line, allowing stable propagation of naive cells and differentiation into immature DC. The phenotypes of the naive cells and immature DC were characterized by means of the expression of relevant surface markers. To further characterize the MUTZ-3 cell line, both naive cells and MUTZ-3-derived immature DC were exposed to maturation stimuli, such as LPS, and the

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maturation response was analyzed by flow cytometry. As a preliminarily evaluation of the impact of sensitizing chemicals on maturation of MUTZ-3-derived DC, immature DC were exposed to different concentrations of nickel sulphate and DNCB. Additional experiments will be required to reveal whether the MUTZ-3 cell line is a valid model system for in vitro testing of sensitization. P7-14 MECHANISMS AND TARGET CELLS UNDERLYING LEAD-INDUCED MODULATIONS IN THE ALLOGENEIC IMMUNE RESPONSE D.G. Farrer, M.J. McCabe Jr. Department of Environmental Medicine, University of Rochester, Rochester, New York, USA Lead (Pb) is a widespread environmental contaminant and is toxic to most body systems including the immune system. In vivo, Pb is functionally immunosuppressive with a decrease in host resistance to intracellular pathogens and decreased pathogen clearance. These findings implicate the CD4+ T cell as the functional target of Pb-induced immunotoxicity. Pb markedly enhances the proliferative response of allo-reactive CD4+ T cells in mixed lymphocyte culture (MLC), a process we have termed Pb allo-enhancement. Interestingly, this enhancement in T cell proliferation is mediated through an adherent antigen presenting cell, probably a macrophage. Our studies suggest that Pb relieves a macrophage-dependent suppression of the allo-proliferative response in MLC. Since Pb alloenhancement occurred in the presence of allogeneic B cells and syngeneic macrophages but not allo-B cells alone, we hypothesized that Pb enhanced cross-priming of alloantigen through a semidirect alloantigen presentation pathway. CD4+ T cells primed against alloantigen in the presence of Pb demonstrated an enhanced secondary response to whole allo-B cells relative to control allo-primed T cells and relative to their secondary response to allo-B cell lysates and syngeneic macrophages. This suggested that T cells had originally been primed against allo-peptide:allo-MHC complexes (semidirect) rather than allo-peptide:syn-MHC com-


plexes (indirect). These findings further elucidate target cells and mechanisms involved in Pb-induced immunotoxicity and may have implications in transplantation immunology. Supported by R29-ES07365, and P30-ES01247. P7-15 SHORT-TERM TREATMENT WITH METHOTREXATE-LOADED LIPOSOMES IN A MURINE MODEL OF ARTHRITIS M. Barca1 , A.-M. Ciobanu1 , D. Balalau1 , D. Baconi1 , M. Ilie1 , M. Neagu2 , G. Manda2 1 “Carol Davila” University of Medicine and Pharmacy, Toxicology Department, Bucharest, Romania; 2 “Victor Babes” National Institute, Bucharest, Romania

Methotrexate remains the golden therapeutical tool in rheumatoid arthritis, despite its severe side-effects. It enters the cells by active or receptor-mediated transport and inhibits the inflammatory/autoimmune responses by several mechanisms. In a rat model of arthritis we studied the effects of methotrexate-injection or -loaded liposomes on several parameters characterizing the inflammatory response. Hydro-soluble and hydrophobic methotrexate were used. Three different doses from the formulations were administered weekly (for 3 weeks) and the following parameters were evaluated 24 h and 7 days after the last dose: number of peripheral leukocytes, number of splenocyte per spleen weight and the activation/proliferation capacity of splenocytes experimentally activated with Concanavalin A. Results indicate the effects of the drugs on leukocyte traffic between periphery and spleen. Elevated percentages of peripheral granulocytes were registered for all the investigated formulations depending on the administered dose. Small doses of hydrophobic methotrexate-loaded liposomes and of methotrexate injection inhibit the recruitment of leukocytes in the spleen. The treatments might inhibit leukocytes recruitment in the inflamed joint, thus limiting the inflammatory response. The functional suppression of splenocytes is registered only 7 days after the last dose


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for the animals treated with middle doses of hydrosoluble methotrexate (injection or loaded in liposomes), whilst hydrophobic methotrexate-loaded liposomes are active at the lowest dose. P7-16 METALLIC HAPTENS INDUCE DENDRITIC CELLS ACTIVATION S. Kerdine-R¨omer, D. Antonios, A. Larang´e, Ch. Espinasse, M. Pallardy INSERM UMR-S 461, Facult´e de Pharmacie Paris XI, 5 rue J.B. Cl´ement F-92296 Chˆatenay-Malabry Dendritic cells (DCs) play a major role in the regulation of immune responses to a variety of antigens (Ag) and haptens which participate to the process of dendritic cells maturation. Indeed, metallic haptens are able to induce full DCs maturation in vitro. However, the mechanism of this maturation is not well understood. We have already shown that NiSO4 activates p38 mitogen-activated protein kinases (MAPK), c-jun N-terminal kinase (JNK) and extracellular signalregulated kinase (ERK) during early events of DCs maturation. Therefore, in the present study, we analyzed the effects of MAPKs inhibitors on human DCs derived from cord blood CD34+ treated with NiSO4 . Our results show that NiSO4 induces DCs maturation with the emergence of the following phenotype CD86high , CD40high /HLA-DRhigh and CD83+ CCR7+ . The inhibition of MAPKs with pharmacological inhibitors (SB203580 for p38 MAPK, PD98059 for ERK, SP600125 for JNK) reduced significantly the upregulation of CD86, CD83 and CCR7. We also show that NiSO4 induces IL-6 and IL-12 p40 mRNAs, two cytokines playing fundamental roles in DCs functions. IL-12 p40 induction upon NiSO4 addition is inhibited with all three inhibitors whereas IL-6 mRNAs induction is only inhibited with p38 MAPK inhibitor (SB203580). These data suggest that MAPKs play a crucial role in maintaining DCs maturation allowing them to be fully activated in response to metallic haptens.

P7-17 Zn SUPPLEMENTATION EFFECTS OVER THYMUS CELLS APOPTOSIS IN MICE PERINATAL STAGES M.D. Lastra1 , A.E. Aguilar1 , N. Mungu´ıa2 , G. Fragoso3 , L. Sald´ıvar2 1 Department

of Biology, Research Laboratory in Immunology, University of Mexico, Mexico, D.F. 04510, Mexico; 2 Deparment of Analytical Chemistry, School of Chemistry, University of Mexico, Mexico, D.F. 04510, Mexico; 3 Department of Immunology, I.I.B.M., University of Mexico, Mexico, D.F. 04510, Mexico Zinc is an essential trace element for humans. Thus, lack of zinc is associated with immunodeficiency conditions that include thymic atrophy and a variety of syndromes affecting particularly T cells response. This is important in perinatal stages when zinc levels are critical in various aspects, particularly in the immune response. It is a known fact, that oral zinc supplementation increases and restores both humoral and cellular immune responses in normal individuals and those with marginal zinc deficiencies. This work deals with the effect of in vivo zinc supplementation (during perinatal stages) over thymus cells apoptosis in BALB/c mice (21, 28, 35, 42 and 49 days old). The mice received zinc acetate (500 mg/L) in drinking water administered from the day of mating throughout gestation, lactation and postweaning. Mice were divided into five groups according to age and Zn administration. Zinc intracellular concentrations were determined by atomic absorption spectrometry (AAS) and apoptosis was assessed by Fluorescense Activated Cell Sorting (FACS). Zinc intracellular concentration in the thymus cells was significantly higher in treated mice 42 and 49 days old, which coincides with cell apoptosis inhibition. There appears to be a connection between zinc cellular concentrations and cell apoptosis in the murine thymus. The zinc intracellular concentrations relates to the apoptotic cells number, in mice 42 and 49 days old. Our results suggest that in vivo zinc supplementation throughout gestation, lactancy and postweaning, delays thymic involution through apoptosis modulation. On view of these findings, we conclude that oral

Abstracts / Toxicology Letters 158S (2005) S1–S258

zinc supplements should be carefully monitored during perinatal stages. P7-18 IMMUNE SIGNALS DELIVERED IN VITRO BY THE OPIATE ANTAGONIST NALOXONE IN RHEUMATOID ARTHRITIS Manda1 ,

G. Baconi2


Neagu1 ,


Radu1 ,


Constantin1 ,


1 Victor Babes National Institute, Immunology Depart-

ment, Bucharest, Romania; 2 University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania Immune cells express opioid receptors and produce opioid peptides in inflammatory conditions, without inducing tolerance. We investigated the in vitro immunomodulatory action of the opiate receptor antagonist naloxone on peripheral lymphocytes from rheumatoid arthritis (RA) patients. Peripheral mononuclear cells from RA patients and normal subjects (N), were cultivated with naloxone and/or PHA. Cellular activation was evaluated as uridine incorporation, the number of living cells by the MTS reduction test, membrane integrity as LDH release and apoptosis by the annexin V-propidium iodide method. In the presence of naloxone, uridine incorporation is statistically diminished in resting and PHA-activated mononuclear cells from RA, whilst only PHA-treated cells from N are inhibited. Reduction of uridine incorporation is associated neither with the decrease of living cells, nor with a clear increase of LDH release. Naloxone rapidly triggers moderate apoptotic signals to normal and RA lymphocytes, confined mainly to resting cells. Apoptosis is apparently not correlated with uridine incorporation restraint, as long as naloxone raises the proportion of apoptotic cells even when uridine incorporation is intensified. We report that naloxone can deliver by itself inhibitory signals to human lymphocytes, being not only an antagonist displacing opioid drugs from cellular receptors. Therefore, targeting of opioid receptors with antagonists might be therapeutically relevant in RA.


P7-19 DISTRIBUTION OF ACUTE OPIOIDS AND OTHER PSYCHOACTIVE DRUGS POISONINGS J. Radenkova-Saeva National Center of Clinical Toxicology, Emergency Hospital “Pirogov” Sofia, Bulgaria During the past few years, drug abuse amongst the world’s youth as well as in Bulgaria has become a troubling phenomenon, engaging not only the user himself, but also his surrounding environment and society at large. This study was performed epidemiological data for drug abusers, treated in the National Center of Clinical Toxicology in Sofia between 1992 and 2003 years. The number of drug abusers, treated in the National Center progressively increased during the mentioned period. In this investigation, 3469 cases of acute poisoned patients have been studied. 1129 cases of them were opioids poisoning, and 2340 cases were poisoning by other psychoactive drugs. The most prevalence of poisoning was occurred in age group 19–25 years. 78.6% were males and 21.4% females. The following parameters were represented: social and psychological status, postintoxicational complications. P7-20 A DNA ARRAY USED FOR IN VITRO IMMUNE TOXICITY TESTING S. Szameith, H. Tuschl, Ch. N¨ohammer ARC research Seibersdorf GmbH, A2444 Seibersdorf, Austria The technology of gene expression analysis by microarrays is well suited to monitor immune function and immune pathological processes with special impact in allergy, both for the identification of sensitising agents and the development of new therapeutics. The ARC Seibersdorf research developed a DNA microarray containing oligonucleotides of 65 immune relevant genes, several housekeeping genes, as well as the Lucidea Universal Score Card System (Amersham Biosciences) for normalisation. The array was evaluated in different in vitro cell systems. Immature


Abstracts / Toxicology Letters 158S (2005) S1–S258

dendritic cells cultured from human peripheral blood monocytes were used as an in vitro test system for the detection of contact allergens. It has been proposed that contact allergen exposure can induce maturation of immature dendritic cells and changes in dendritic cell gene expression profiles. The new chip has been tested after application of NiSO4 and model allergens and an enhanced expression of certain cytokines, chemokines and chemokine receptors was established. The array was also used with the monocytic cell line THP-1 and changes in the transcriptional activity of immune genes followed after treatment of the cells with lipopolysaccharide or the immune suppressant cyclophosphamide. Further studies with the immune array will establish candidate genes for the identification of immunotoxic chemicals. Especially the application of expression profiling in monocyte-derived dendritic cells will provide an in vitro alternative to animal test methods currently available to predict the sensitising effects of chemicals. P7-21 THE PREDICTION OF THE RESPIRATORY SENSITISING POTENTIAL OF CHEMICALS IN A MODIFIED LOCAL LYMPH NODE ASSAY H. Tuschl, A. Hrdina, B. Fekete ARC research Seibersdorf GmbH, A2444 Seibersdorf, Austria The local lymph node assay is now widely used to identify the sensitizing potential of chemical contact allergens: Mice are dermally exposed to test substances and lymphocyte proliferation induced in the draining lymph node assessed by incorporation of radiolabelled thymidine. Improvements of this assay, like flow cytometric determination of B lymphocytic phenotype or T-cell activation/memory phenotype have been described. In addition, differential cytokine profiling, based on expression analyses of cytokine mRNAs or ELISAs of cytokines produced by lymph node cells ex vivo have been applied. It is now generally agreed that respiratory sensitizers can be differentiated from contact allergens by the induction of distinct cytokine profiles. In the present investigation we applied a modified local lymph node assay and measured cytokine production of ex vivo stimulated lymph node cells by a cytometric bead array and flow cytometry. The respiratory

sensitizers trimellitic anhydride, phthalic acid anhydride and toluene 2,4-diisocyanate could be very well distinguished from the contact sensitizers: oxazolon, 2,4-dinitrochlorobenzene or ␣-hexyl cinnamaldehyde and the irritant sodium dodecyl sulphate. Interleukin-4 is a differential marker well suited to define respiratory sensitization after topical application of chemicals in the modified local lymph node assay. Regarding the low numbers of animals used and the possibility to differentiate between contact and respiratory sensitizers, this test fulfils the criteria of reduction and refinement according to the principles of the 3Rs. P7-22 CHANGES OF T- AND B-CELLS POPULATION IN THE PERIPHERAL BLOOD IN CIGARETTE SMOKERS ˙ nski2 J. Rutowski1 , P. Moszczy´nski2 , Z. Zabi´ 1 E.Szczeklik’s Specjalistic Hospital in Tarn´ ow, 13 Szpi-

talna St., 33-100 Tarn´ow, Poland; 2 Regional Hospital in Brzesko, 68 Ko´sciuszki St., 32-800 Brzesko, Poland The study of cell-mediated immunity comprised 76 men, aged from 21 to 50 years (mean = 34), smoking 15–25 cigarettes daily during 2–25 years, divided into two groups: (I) 37 men aged 21–37 years (27.9), smoking cigarettes <10 years (6.9) and (II) 39 men aged 29–50 years (37.6) smoking cigarettes >10 years (19.9). The control group comprised 40 men, aged from 24 to 47 years (36.7), clinically health, who had never been smokers. The study of humoral-mediated immunity and acute phase blood proteins comprised 85 men, 18–42 years old (34.0), smoking also 15–25 cigarettes daily during 2–25 years. These smokers were divided: (1) 38 men smoking cigarettes <10 years (6.9) and (2) 47 men being smokers smoking cigarettes >10 years (18.5). The control group comprised 49 men, aged from 17 to 50 years (31.0), who had never been smokers. For the determination of T- and (CD19+)Bcells population in the peripheral blood, the Behring (Germany) monoclonal antibodies were used in indirect immunofluorescence tests: (CD3+)T-cells—BMA 030, (CD4+)T-helper—BMA 040, (CD8+)Tsuppressor—BMA 081, (CD16+)NK—BMA 070, and (CD19+)B-cells—BMA 0130. The percentage proportions of T-cells subpopulations and B-cells were calculated with a fluorescence microscope

Abstracts / Toxicology Letters 158S (2005) S1–S258

(Zeiss Jena, Germany). The serum concentration of immunoglobulins: A, D, G and M was determined by nephelometry, using Behring Nephelometer Type 100. The (CD3+)T- as well as (CD4+)T-helper percentages in subjects smoking longer—group II (>10 years) was significantly lower in comparison to the group I of smokers. In group II, percentage of (CD4+)T-helper was lower. In contrast, to the T-helper cells percentage, the percentage of (CD8+)T-suppressor cells increased in group I by 15% and in group II by 23% in comparison to the control. The absolute number (a.n.) of total lymphocytes increased only in group I, a.n. of (CD3+)T- and (CD4+)T-helper increased in group I, but decreased in group II of smokers, smoking >10 years. The a.n. of (CD8+)T-suppressor cells increased much more and was in group I higher by 64% and in group II by 24% higher than in control. Lower increase of (CD4+)T-helper cells population than (CD8+)Tsuppressor cells population was the cause of decreased value of the (CD4+)T-helper/(CD8+)T-suppressor ratio by about 13% in the group I, and by about 26% in the group II in comparison to the control. The percentage of (CD19+)B-cells was higher only in group II. IgA and IgG concentrations was lower in subjects smoking >10 years in comparison with non-smokers and with subjects smoking for <10 years but IgM was lower in smokers from group II in comparison with the control group. P7-23 THE “FLUORESCENT CELL CHIP” TECHNOLOGY AS AN EMERGING IN VITRO TOOL FOR IMMUNOTOXICANTS’ FINGERPRINT SEARCH W. Wagner1 , A. Walczak-Drzewiecka1 , A. ´Slusarczyk1 , J. Wycz´ołkowska1 , L. Rychlewski2 , P. Biecek3 , J. Dastych1 1 Centre for Medical Biology in Lodz, Polish Academy of Sciences, Poland; 2 BioInfoBank Institute in Poznan, Poland; 3 Institute of Mathematics, Wroclaw University of Technology, Poland

Fluorescent cell chip (FCC) is a new system for in vitro immunotoxicity testing, that employs changes in cytokine expression as an endpoint indicating potential for perturbation of the immune system in vivo. FCC is based on a number of genetically modified cell lines


that regulate the expression of a transgene coding for enhanced green fluorescent protein (EGFP) in a similar way as they regulate expression of IL-2, IL-4, IFN␥, IL-10, and ␤-actin. It has been previously reported that FCC could be successfully applied as a predictive screening test for immunomodulatory activities of chemicals. We combined this technology with flow cytometer equipped with multi plate loader with sampling rate 5 × 103 cells/sample/30s to conform the test to high throughput system efficiency. Complete assay for one compound to be tested included range of seven concentrations, five reporter cell lines, either activated, and three replicates. We pre-validated FCC test with over 60 reference compounds of known immunotoxicity (based on National Toxicology Program USA data base). Each of substances gave the unique pattern of changes in cytokines expression and resultant data showed good correlation with in vivo observations. We have also explored possibility for the bioinformatic approach of data handling and hierarchical clustering of substances that share activity patterns. Thus, we believe that FCC could be employed as a predictive tool for evaluation of chemical immunotoxicity. P7-24 IMMUNE ACTIVATION AND DENTAL AMALGAMS G. Guzzi1 , L. Soldini2 , Pigatto PD3 , G. Rossi4 , M. Passoni1 , G. Severi4 1 Italian

Association for Metals and Biocompatibility Research, AIRMEB, Milan, Italy; 2 Laboraf San Raffaele Hospital IRCCS, Milan, Italy; 3 Department of Dermatological Sciences, University of Milan, Maggiore Hospital IRCCS, Milan, Italy; 4 Fleming Research Laboratory, Milan, Italy; 5 Cancer Epidemiology Centre, The Cancer Council Victoria, Melbourne, Australia Activation of immune system in individuals with mercury dental amalgams is unclear. Interleukin-2 and its receptor – CD25 – are able to induce immune activation. We investigated whether serum soluble interleukin2 receptor (sIL2R) in vitro and in vivo—could be used as marker for exposure to mercury vapor and its effects. Methods: Since 1997, serum samples were collected from a group of patients (n = 24) with subjective


Abstracts / Toxicology Letters 158S (2005) S1–S258

symptoms allegedly to dental amalgam. Soluble IL2R was measured by enzyme-linked immunoassays after lymphotransformation test. Recently, we also assessed sIL2R in individuals with oral diseases associated with systemic dermatological manifestations and who displayed allergy to mercury by using patch-test (n = 8) and/or in combination with lymphocyte transformation test (n = 4). In vitro, 18 patients (75%) had mitogenic response to mercury chloride and up-regulated soluble IL2R expression, revealing mercury sensitization. Of 18, 10 (60%) had strong sensitization and 8 (40%) had mild sensitization to mercury. Six had no immunoallergic reactions to mercury (15%). Our preliminary data suggest that levels of soluble IL2R increased after exposure to mercury vapor from amalgams (e.g., amalgam removal). The significantly raised concentration of soluble IL2R in subjects with allergic sensitization to mercury and after exposure to mercury indicate that soluble IL2R may be investigated as a possible marker of response to mercury. Converging evidence from diverse methodologies demonstrate that exposure to mercury appear to be related with IL2R up-regulation. Exposure to mercury vapor from amalgam and/or its sensitization may lead to increases in serum soluble IL2R, implying an early immune activation. P7-25 INHIBITION OF HUMORAL RESPONSE TO KLH IN THE MONKEY BY CYCLOSPORINE F. Horand1 , G. Ravel1 , F. Condevaux1 , J. Descotes2 1 MDS Pharma Services, les Oncins, 69210 St Germain

sur l’Arbresle, France; 2 Lyon Poison Centre, 69424 Lyon cedex 03, France The antibody response to a T-cell dependent antigen is a widely accepted screen for detecting immunosuppressive drugs and chemicals. Although the plaque-forming cell (PFC) assay is the first-line assay, it tends to be replaced by ELISA measurement of KLH antibodies due to inter-individual variability and the lack of antigen standardization in the PFC assay. ELISA offers the advantage of measuring primary (IgM) and secondary (IgG) responses in the same animal on several occasions. Although the rat is the preferred species,

there is a need to use non-rodent species. In this study, the effect of cyclosporine on KLH antibody responses was tested in Cynomolgus monkeys. Eight monkeys received 100 mg/kg/day cyclosporine orally – a dose previously shown to prevent graft rejection – over a 28-day period. Four animals received the vehicle. KLH was injected on days 14 and 21, and blood samples collected on days 14 and 21 before the KLH administration and then on day 28. IgM levels were slightly decreased by cyclosporine (−15%), whereas IgG levels were markedly lower (−40%) in treated animals than in controls. These results suggest this assay is suitable for the detection of immunosuppression in the monkey. The relevance of the different effects on primary and secondary responses for risk assessment should be evaluated. P8 Genetic Toxicology P8-01 EFFECTS OF EXCESS DIETARY INORGANIC PHOSPHATE ON THE BRAIN OF YOUNG TRANSGENIC MICE EXPRESSING BICISTRONIC LUCR-C-MYC-IRES-LUCF REPORTER GENE H. Jin1 , S.K. Hwang1 , Y.S. Lee2 , K.H. Lee3 , A.C. Prats4 , D. Morello5 , G.R. Beck Jr.6 , M.H. Cho1 1 Laboratory

of Toxicology, College of Veterinary Medicine, and School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea; 2 Department of Food and Nutrition, College of Human Ecology, Seoul National University, Seoul 151742, South Korea; 3 Laboratory of Molecular Oncology, Korea Institute of Radiological & Medical Sciences, Seoul 139-240, South Korea; 4 INSERM U397, Endocrinologie et Communication Cellulaire, C.H.U. Rangueil, Avenue Jean Poulh`es, 31403 Toulouse Cedex 04, France; 5 Centre de Biologie du D´eveloppement, CNRS-UMR5547, IFR 109, Universit´e Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse, France; 6 Laboratory of Cancer Prevention, National Cancer Institute, Frederick, MD 21702-1201, USA Inorganic phosphate (Pi) plays a key role in diverse physiological functions. Recently, the brain-specific Na-dependent phosphate transporter (NPT) has been identified and considerable progress has been made

Abstracts / Toxicology Letters 158S (2005) S1–S258

in our understanding of the function and regulation. Potential importance of Pi as a novel signaling molecule and presence of brain-specific NPTs with poor prognosis of diverse neurodegenerative diseases have prompted us to begin to define the pathways by which Pi affects young mouse brain. High Pi increased the protein expression of NPT1 significantly in both cerebrum and cerebellum. High Pi increased Akt phosphorylation at Ser473 in cerebrum and cerebellum whereas suppression of the Akt phosphorylation at Thr308 was observed in cerebellum only. Selective suppression of eIF4E-BP1 in cerebrum was induced by high Pi. Excess Pi induced cap-dependent and capindependent protein translation in cerebrum and cerebellum, respectively. ERK2 phosphorylation compared to ERK1 phosphorylation was clearly reduced in both cerebrum and cerebellum. High Pi did not affect the activity of Mnk1. High Pi reduced the protein expressions of PCNA and cyclin D1 in cerebrum and cerebellum. In summary, we were able to demonstrate that Pi could activate the Akt-ERK pathway in young mouse brain. We believe that the Akt-ERK signaling pathway can be involved in many critical biological processes during development, and may modulate various neurodegenerative diseases of the brain. P8-02 EFFECT OF NARINGIN ON THE DNA DAMAGE INDUCED BY DAUNORUBICIN IN MOUSE HEPATOCYTES AND CARDIOCYTES ´ R. Cari˜no-Cort´es1,2 , I. Alvarez-Gonz´ alez2 , L. Martino2 2 Roaro , E. Madrigal-Bujaidar , E. Ruiz3 1 Laboratorio de Farmacolog´ıa, Instituto de Ciencias de la Salud, Universidad Aut´onoma del Estado de Hidalg, M´exico; 2 Laboratorio de Gen´etica, Escuela Nacional de Ciencias Biol´ogicas, IPN, M´exico; 3 CEACA, Universidad Aut´ onoma de Quer´etaro, M´exico

Naringin (Nar) is a flavonoid that has shown antigenotoxic effect in in vivo assays, and probably participates in the chemoprotection reported for grapefruit. The aims of this investigation were: (a) to determine the DNA breaking potential of Nar in mouse hepatocytes and cardiocytes, (b) to evaluate its capacity to inhibit the DNA damage induced by daunorubicin (Dau) in the same tissues, and (c) to determine its capacity to


trap free radicals in vitro. For the first purpose, three groups of animals received Nar by the oral route (50, 250, and 500 mg/kg), and the comet assay was performed before the chemical administration, and at 3, 12, and 21 h postadministration. As regard to the second purpose, three groups received the same doses of Nar, but 1 h later, they were i.p. injected with 1 mg/kg of Dau. Mice were also studied with the comet assay in the same schedule mentioned before. Finally, the free radical potential of Nar was determined with the DPPH method. The results showed that Nar was unable to induce DNA deterioration in both types of cells studied. However, a significant damage induced by Dau was observed in hepatocytes and cardiocytes. The coadministration of Nar with the mutagen produced an important protection of the DNA damage. A maximum reduction of 71.3% was determined at 12 h with 500 mg/kg of Nar in hepatic cells, while the lowest effect of the antimutagen corresponded to the low dose tested. In the case of cardiocytes, the administration of Nar also produced a significant inhibition of the damage induced by Dau. The highest reduction was 51.2% obtained with 250 mg/kg of Nar at 12 h of treatment. In this tissue, the lowest effect of the antimutagen corresponded again to the low dose tested. Finally, with respect to the antioxidant potential, the response obtained with Nar (logarithmic type) showed an activity of about 90%. Our study established the DNA breaking potential of Dau in mouse hepatocytes and cardiocytes, and its inhibition by the administration of Nar, probably related with its capacity to trap free radicals. P8-03 ASSESSMENT OF DNA DAMAGE IN POSTMENOPAUSAL WOMEN UNDER HORMONE REPLACEMENT THERAPY E. Ozcagli1 , S. Sardas1 , A. Biri2 1 Department

of Toxicology, Gazi University, Turkey; of Obstetric and Gynecology, Gazi University, Turkey 2 Department

To evaluate the possible DNA damage in peripheral blood leukocytes of postmenopausal women under different hormone replacement therapies (HRT), comet assay, a standard method for assessing genotoxicity has been used.


Abstracts / Toxicology Letters 158S (2005) S1–S258

Forty-six women were categorized in three groups—Group A: 15 surgical menopausal women who underwent surgery for benign conditions, receiving conjugated equine estrogen, 0.625 mg/day (CEE) for 2.3 ± 1.5 years; Group B: 16 spontaneous menopausal women receiving conjugated equine estrogen, 0.625 mg/day plus medroxyprogesteron acetate, 5 mg/day (CEE + MPA) for 2.4 ± 1.0 years and Group C: 15 spontaneous menopausal women receiving tibolone, 2.5 mg/day for 2.4 ± 1.3 years. Control group consisted of 15 spontaneous menopausal women who never had HRT. Significant differences in terms of DNA damage were observed between Groups A and B with controls as mean total comet scores 23.93 ± 5.84, 19.44 ± 6.19 and 10.07 ± 2.40, but no significance (P > 0.05) were detected between Group C and controls as mean total comet scores 12.07 ± 3.65 and 10.07 ± 2.40, respectively. Reduced DNA damage were observed with tibolone compared to CEE or CEE + MPA therapy. Studies of this approach are needed. P8-04 MICRONUCLEI FREQUENCIES IN ORAL MUCOSA CELLS FROM INDIVIDUALS USE PEROXIDE CONTAINING WHITENERS Burgaz1 ,

S. O. Mıhcıo˘glu3

Erdem2 ,


Dolar3 ,


Karakaya1 ,


1 Gazi

University, Faculty of Pharmacy, Department of Toxicology, Hipodrom, Ankara, Turkey; 2 G¨ulhane Military Medical Academy, Department of Toxicology, Etlik, Ankara, Turkey; 3 Gazi University, Faculty of Dentistry, Department of Restorative Dentistry, Emek, Ankara, Turkey

The important ingredient in tooth bleaching is hydrogen peroxide (H2 O2 ). The International Agency for Research on Cancer (IARC) concluded that there is limited evidence in experimental animals and inadequate evidence in humans for the carcinogenicity of H2 O2 and classified the chemical into Group 3 (IARC, 1999). There has still been no human study regarding to the genotoxic potential of carbamide peroxide. Micronucleus test as a genotoxic endpoint for the detection of site-specific exposure was carried out in exfoliated cells from buccal gingival samples of 19 subjects worn night guard vital bleaching containing

10% carbamide peroxide. The samples were taken at the day 0 as a control group and 7, 14, 21 and 30 days as experimental groups. The mean micronucleus frequency (‰) (±S.D.) for 0 day was 0.73 ± 0.26 and the mean micronucleus frequency (±S.D.) for seventh day was 1.10 ± 0.22, hence there was a significant statistical difference between these two groups (p < 0.001). The mean micronucleus frequency (±S.D.) for 14th day was 1.17 ± 0.22 and there was a significant statistical difference between control and 14-day groups (p < 0.001). The frequency of micronuclei (±S.D.) for 30th day (0.92 ± 0.28) were shown to have significantly decreased when compared to 14th day values (p < 0.05). Furthermore the frequency of micronuclei for 30th day was no longer significantly different from the frequency of micronuclei of the controls (p > 0.05). Our results demonstrate that cytogenetic response to the use of at home whiteners among patients is generally characterized by an increase in MN reaching a peak value and then decreasing in the following weeks. P8-05 FREQUENCY OF SISTER CHROMATID EXCHANGE IN CIGARETTE INDUSTRY WORKERS ˇ R. Rozgaj, V. Kaˇsuba, N. Kopjar, D. Zeljeˇ zi´c, M. Mili´c Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia Occupational exposure of cigarette industry workers includes exposure to numerous volatile components that can cause adverse health effects such as allergies and respiratory disorders, as well as malignant diseases. To evaluate the genotoxic effect of tobacco dust the frequency of sister chromatid exchanges (SCE) was analyzed in peripheral blood lymphocytes of cigarette industry workers. The number of high frequency cells (HFC) was also determined. The study comprised 30 tobacco industry workers of both sexes who were, in addition to tobacco dust, exposed to a source of strontium 90, used to control the density of tobacco filler in cigarettes, and 30 control subjects matched for sex, age and smoking habit. The statistical analysis was performed using the ANOVA. The results showed a significant difference in both SCE frequency and the number of HFC between the exposed and control group. Exposure duration did not influence the SCE frequency, nor

Abstracts / Toxicology Letters 158S (2005) S1–S258

did sex and age. On the other hand, smoking proved to influence SCE frequencies in both groups. Smokers from the exposed group showed a significant increase in SCE frequency in respect to smokers in the control group. P8-06 INDUCTION OF DNA FRAGMENTATION IN CALF THYMOCYTES BY SULFUR MUSTARD M. Jafari Baqiyatallah University, Faculty of Medicine, Military medicine Institute, Department of Biochemistry, Chemical Research Center, Tehran, Iran Sulfur mustard (SM) or mustard gas is a highly reactive bifunctional alkylating agent. It has been frequently used as a chemical warfare agent. SM reacts with a wide range of biological molecules. DNA is the most sensitive molecular target of SM. The mechanism to toxic action of SM is not fully understood. In this study, the effect of SM on calf thymus DNA was investigated. For this propose, DNA was incubated with various concentrations of SM (25–1000 ␮M) and the samples were subjected to UV–vis spectroscopy, gel electrophoresis and digestion with different restriction endonucleases. The results of difference spectra show that the absorbance is decreased by gradual increase in the SM concentration. Between SM values of 100 and 250 ␮M, DNA represented a smear pattern on agarose gel electrophoresis, while higher concentrations of SM (>250 ␮M) caused disappearances of DNA from the gel pattern. The results of DNA digestion with EcoRI, HindIII and BamH1 show that as the SM concentration (50 and 100 ␮M) was increased, the average size of DNA fragments apparently decreased and BamH1 digestion of DNA was greater than other restriction endonucleases. The data suggest that DNA fragmentation patterns depend upon the concentration of SM. Alkylation of DNA results in DNA strand breaks that results from direct interaction of the SM with DNA molecule.


P8-07 GENE AND PROTEIN EXPRESSIONS IN HUMAN CORD BLOOD CELLS AFTER EXPOSURE TO ACRYLONITRILE I. Malerba1 , C. Diodovich1 , D. Ferrario1 , G. Bowe1 , M.G. Bianchi2 , F. Acquati2 , R. Taramelli1 , L. Gribaldo1 1 ECVAM,

Institute for Health and Consumer Protection, J.R.C., Ispra, Italy; 2 Department of Structural and Functional Biology, University of Insubria, Varese, Italy Acrylonitrile is a very high volume industrial chemical used primarily in the manufacture of plastics and rubber, which displays a pronounced acute toxicity and may be carcinogenic. The damage to the haematopoietic function by acrylonitrile may result from interference with cytokine production and cytokine receptor binding. The results presented showed that acrylonitrile modulates the expression of some genes implicated in cell differentiation, cell-cycle progression, and clonogenic potential of human cord blood cells. A macroarray hybridisation analysis showed that expression of the CXCR4, MCP-1 and MRP8 genes was modified by acrylonitrile exposure. Moreover, the myeloid compartment seems to be the acrylonitrile cell target, as assessed by a CFU-GM assay. In particular, the down-regulation of CXCR4, MCP1 and MRP8 can be responsible for the observed reduction of cell proliferation and clonogenic capability of CFU-GM precursors. A western blot assay showed an exposuredependent induction of bax, while bcl-2 expression changes only after 48 h of chemical treatment. The different expression of these two genes, related to the apoptosis and necrosis processes, could be responsible for the increase in cell death after 24 h of treatment as seen by flow cytometry. In addiction, western blot analysis revealed that acrylonitrile treatment affects p53, p21, c-jun and c-fos expression. In conclusion, these data support strongly the toxicity of acrylonitrile, more than its potential activity to promote the tumour progression, as well as the evidence for its selective activity towards a specific bone marrow sub-population, the committed myeloid compartment.


Abstracts / Toxicology Letters 158S (2005) S1–S258



C. Lee1 , H.M. Kim1 , IL-Chan Kim2 , J.S. Lee2 , K.S. Choi1 , J.G. Na1

E. Coskun1 , D.G. Cakmak1 , F. Cetindag2 , O. Sunter2 , H. Edinsel2 , A.N. Kocabas1 , S. Burgaz1

1 Environmental

Risk Research Department, National Institute of Environmental Research (NIER), South Korea; 2 Department of Environmental Science, Hanyang University, South Korea

1 Gazi

University, Faculty of Pharmacy, Dept. of Toxicology, Hipodrom, Ankara, Turkey; 2 Abdurrahman Yurtaslan Oncology Hospital, Department of Radiation Oncology, Head and Neck Group, Ankara, Turkey

Alachlor is an acetanilide herbicide used to control many annual broad-leaved weeds and most grasses in field corn, soybeans, peanuts, cotton, milo and sunflowers. It has a relatively low acute toxicity, however, repeated exposure has been reported to cause hepatotoxicity, irreversible uveal degeneration and tumour formation in some animals. Besides alachlor is one of the herbicides reported to have endocrine disrupting effects. Alachlor is a strongly suspected endocrine disruptor in that it is listed by EPA, the Centers for Disease Control and Prevention, and the WWF as a potential endocrine disrupting chemical. 2,4-D, 2,4,5-T, amitrole and atrazine also belong to these types of herbicides. Toxicogenomics is emerging field combining genomics and bioinformatics to identify and characterize mechanisms of toxicity of drugs and environmental hazardous compounds. Studies of the metabolism of xenobiotics through the gene expression profiling using toxicogenomics such as DNA microarray techniques contribute to a scientific approach to risk assessment. Therefore, in this study we analyzed the gene expression patterns of the liver toxicity by alachlor using the cDNA microarray. These results found that a total of 15 genes were up-regulated in the alachlortreated male medaka at the concentrations of 20 and 100 ␮g/L. Of the genes that displayed significant changes in expression, several cytochrome P450 isozymes were induced by alachlor. Alachlor was shown to have not estrogenic potency, so it may disrupt endocrine system through another mechanism.

Chromosomal damage and its repair assay as the micronucleus (MN) assay has been used to evaluate the radiation sensitivity and to predict cancer risk of human subjects. In the present study, we have investigated the chromosomal radiosensitivity of head and neck cancer (HNC) patients (n = 40) and normal healthy controls (C) (n = 22) using the MN assay. For the micronucleus assay, blood samples were exposed in vitro to 2 Gy ␥ rays (60 Co) at a dose rate of 0.62 Gy/min. The mean ± S.D. frequencies (%) of radiation-induced MN were 16.31 ± 4.53 and 14.82 ± 4.67 in HNC patients and C subjects, not different significantly. Baseline MN frequencies of HNC patients (2.81 ± 1.04) were significantly higher than C subjects (0.82 ± 0.64) (p = 0.05). Using cut-off value at the 90th percentile (20.37 MN per 100 cells), the proportion of sensitive patients and healthy subjects were 17.5% and 10%, respectively. There was no correlation with age and MN frequencies, both for patients and controls. Cancer patients older than 40 years were more sensitive than normal subjects of the same range (p = 0.074), whereas there was no difference between patients and normal subjects of younger than 40 years. MN assay seems to be useful for the categorization of normal subjects and cancer patients to their radiation sensitivity. This study was supported by Gazi University Scientific Research Fund (Project No. 02/2004-17).

Abstracts / Toxicology Letters 158S (2005) S1–S258


of Pharmacy, Islamic Azad University, Tehran, Iran; 2 Molecular Research Laboratory, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 3 Department of Anatomy and Embryology, Tehran University of Medical Sciences, Tehran, Iran; 4 Department of Pathology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Mutations of p53 as an essential molecular event in the development of colorectal carcinoma can affect the expression of certain other genes. In this respect, the expression of Bcl-2, P-glycoprotein, Topo II alpha, TS and TP proteins were evaluated in relation to p53 protein expression. Out of 129 surgically resected colorectal tumors, 54 cases were randomly analyzed by IHC techniques. Among six analyzed markers, P-gp and p53 with 77% of expression were highest and the lowest one was Topo II with 35% of expression. The prevalence of lack of expression of Bcl-2, Topo II, and TS were significantly higher in p53+ tumors (p = 0.0001, 0.0001 and 0.007, respectively). Present data showed a significant correlation (p = 0.021) between p53/Bcl2 coexpression and mean age of patients (63.5 ± 10.1 years versus 52.3 ± 15.2 years). A significant correlation (p = 0.022) between p53/TP coexpression and sex (66.7% male versus 33.3% female) was also observed in present study. Overexpression of mutated p53 seen in our tumor samples may alter the expression pattern of other molecular markers, which are predictor of tumor response to chemotherapy regimens.


P8-11 STUDIES ON THE GENETIC, REPRODUCTIVE AND BIOCHEMICAL TOXICITY OF YOHIMBE IN SWISS ALBINO MICE O.A. Al-Shabanah, A.A. Al-Majed, A.A. Al-Yahya, A.M. Al-Bekairi, S. Qureshi Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia In the last decades there has been worldwide use of yohimbe, a commercial preparation, of the bark of African plants (Corynanthe yohimbe and Pausinystalia johimbe) to enhance sexual performance. The aphrodisiac activity of yohimbe is ascribed to its yohimbine content. Although, both pure compound (yohimbine) and the natural product (yohimbe) are used against sexual dysfunction, there is a paucity of literature on the toxicity of the natural product. In view of its immense use and dearth of literature, yohimbe was evaluated for its effects on spermatogenesis and reproduction in male Swiss albino mice. The mice were orally treated with different doses (188, 375 and 750 mg/kg/day) of the aqueous suspension of yohimbe for 90 days. Animals in different (control and treatment) groups were used to conduct the following parameters: (1) reproductive organ weight; (2) motility and content of sperms; (3) spermatozoa morphology; (4) cytology of the testis chromosomes; (5) histopathology of testis; (6) study on reproduction and (7) biochemical study of proteins, nucleic acids, malonaldehyde (MDA) and nonprotein sulfhydryl (NP-SH) and hormones. The treatment caused significant reduction in motility and content of spermatozoa, reproductive organ weight and some adverse effects in the histopathology of testis. Although the percent pregnant female mice was reduced in both the weeks of mating, significant pre- and post-implantation losses were observed only in mating week 2. These results are confirmed by our data on the variations in hormonal profile, spermatozoa abnormalities and chromosomal aberrations in the testis. Our study on biochemical parameters showed increase of MDA and depletion of NP-SH, proteins, RNA and DNA in the testicular cells. These data elucidated the role of free radical species in the spermatogenic and reproductive function. Our results warrant careful use of yohimbe as a remedy for impotence and/or erectile dysfunction.


Abstracts / Toxicology Letters 158S (2005) S1–S258


ent constituents might be responsible for the observed changes. In view of the observed oxidant potentials, our study warrants careful use of Ginkgo biloba.

O.A. Al-Shabanah, A.M. Al-Bekairi, A.A. Al-Majed, A.A. Al-Yahya, S. Qureshi


Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

D. Bauer1 , C. McGinnis2 , F. Staedtler1 , L. Urban2 , W. Suter1 , P. Grass1

Ginkgo biloba has immense folkloric significance in the treatment of Alzheimer’s disease and age-related dementia. The present study on the genetic and biochemical effects of Ginkgo biloba was undertaken, in view of the reports on the carcinogenic effects of the dietary supplements containing Ginkgo biloba, the cytotoxic and mutagenic potentials of its constituents and a paucity of literature on the genotoxicity. The protocol included the oral treatment of mice with different doses (100, 200 and 400 mg/kg) of Ginkgo biloba for 7 days. The following experiments were conducted: (i) cytological studies on micronucleus test, (ii) cytological analysis of spermatozoa abnormalities, (iv) quantification of proteins and nucleic acids in hepatic and testicular cells and (v) estimation of malonaldehyde (MDA) and nonprotein sulfhydryl (NP-SH) in hepatic and testicular cells. The results obtained in this study clearly demonstrate lack of any effect of Ginkgo biloba on the frequency of micronuclei, ratio of PCE/NCE, spermatozoa abnormalities and hepatic concentrations of nucleic acid, while the nucleic levels of testicular cells were significantly depleted. The results on testicular nucleic acids failed to reflect our data on spermatozoa abnormality. The discordance may due to the difference in the time of harvest of spermatozoa and the germ cells and the possible repair of the epigenetically mediated changes in the sequence of base pair in the DNA during the development of sperms. The observation on MDA and NP-SH showed Ginkgo biloba to cause genesis of lipid peroxides. This study indicates that the changes observed in the somatic and germ cells were independent of the Ginkgo biloba-induced genesis of lipid peroxides. The effect of Ginkgo biloba on cytochrome P450 and the interactions between differ-

1 Novartis

Pharma AG, 4002 Basel, Switzerland;

2 Novartis Institutes of Biomedical Research Inc., Cam-

bridge, MA 02139, USA Testing chemical compounds for genotoxicity is often performed using a few highly standardized in vitro assays. Nevertheless, relevance and predictivity regarding in vivo results is limited. Thus, an assay based on existing knowledge (e.g. well-known reference substances) and allowing optimization towards correct classification would be of clear advantage. Therefore, we performed a feasibility study in order to elucidate whether an unbiased supervised class prediction approach based on large scale gene expression profiles may lead to a stable prediction model for pre-selected classes and based on selected marker genes. Transcription profiles were obtained from TK6 human lymphoblastoid cells treated with several known genotoxic, direct-acting DNA damaging compounds, as well as several non-genotoxic compounds. Treatment was performed at nearly equicytotoxic conditions using three to six replicates. Total RNA was hybridized to genome-wide human DNA microarrays (HG–U133-2.0+, Affymetrix) and expression data was further analyzed using the software packages MAS5 (Affymetrix), Genespring-7 (Silicon-Genetics) and SIMCA-P-10 (Umetrics-AB). This extended study demonstrates the general feasibility of a genotoxicity test system based on only a few selected marker genes. In addition, it allows in depth analysis of critical parameters and limitations such as the range of cytotoxicity, usefulness of different class prediction algorithms, and essential number of replicates and adequate selection of reference compounds.

Abstracts / Toxicology Letters 158S (2005) S1–S258

P8-14 THE GENOTOXICITY STUDY OF GARLIC AND PASIPAY HERBAL DROPS BY PERIPHERAL BLOOD MICRONUCLEUS TEST H. Kalantari, A. Larki, S.M. Latifi School of Pharmacy, Jundishahpur University of Medical Sciences, Ahvaz, Iran The in vivo rodent micronucleus test is widely used as a genotoxic assay to detect the clastogenic activity of chemicals. In this study the genotoxic effects of herbal drops of garlic and pasipay were evaluated using the micronucleus test. Maximum tolerated dose (MTD) was determined by a dose–response test. For each medicine three test groups were considered with doses of MTD, \ bd MTD and \ bcMTD according to the CSGMT protocol (1995 Japan). Then the mentioned doses were administered per os (p.o.) to mice. Mitomicin C was used as a known genotoxic agent as a positive control group. The peripheral blood samples before treatment (zero time samples) were considered as negative control. The presence and appearance of a micronucleus is used as an index for genotoxic potential. The herbal drops genotoxicity in oral administration seems to be dose-dependent. As compare to the negative control group the herbal drops of Garlic and Pasipay showed significant genotoxicity (p < 0.05), but was found to be less genotoxic than that of positive control. Therefore the genotoxic effects of Garlic and Pasipay were “not significant” compared to historical negative control (p > 0.05). P8-15 IN VIVO LUNG GENOTOXICITY OF QUARTZ USING THE MICRONUCLEUS ASSAY G.D. Cakmak1 , E. Coskun1 , C. Albrecht2 , A.M. Knaapen2 , A. Becker2 , S. Burgaz1 , P.J.A. Borm2 , R.P.F. Schins2 1 Department

of Toxicology, Gazi University, Turkey; Research, Environmental Health Research Institute (IUF), Heinrich-Heine University, Germany 2 Particle

Particle surface properties are considered to play an important role in respirable silica (quartz) genotoxicity. We evaluated micronucleus (MN) formation in pulmonary macrophages and epithelial cells (ECs) from rats, at 3, 7, 21, and 90 days after a single


intratracheal-instillation of saline, quartz (DQ12) or quartz coated with either polyvinyl-pyridine-N-oxide (PVNO) or aluminium lactate (AL). MN frequency was significantly higher, at each time point, in macrophages obtained by bronchoalveolar lavage (BAL) from rats treated with DQ12, when compared to the macrophages from animals that received saline (control), or PVNOor AL-coated DQ12. MN-frequency was not significantly enhanced in the lung macrophages from rats treated with AL-coated DQ12 or PVNO-coated DQ12. In the isolated-interstitial lung ECs, the findings on MN-frequency were in line with those in the BALmacrophages. Treatment of rats with DQ12 also tended to increase MN-frequency in the isolated-interstitial macrophages, although these values did not reach statistical significance. In conclusion, quartz causes both acute and subchronic genotoxicity in target and inflammatory cells in the rat lung when measured by MN test in vivo; its surface properties appear to play a dominant role in this effect. P8-16 N7-ALKYLGUANINES IN THE URINE OF MICE EXPOSED TO STYRENE ˇ Vodiˇckov´a3 I. Linhart1 , J. Nov´ak1 , P. Vodiˇcka2 , L. 1 Institute

of Chemical Technology Prague, 166 28 Prague, Czech Republic; 2 Institute of Experimental Medicine, Academy of Sciences of Czech republic, 142 20 Prague, Czech republic; 3 National Institute of Public Health, 100 42 Prague, Czech Republic Styrene, a large scale industrial monomer, is metabolised to styrene 7,8-oxide (SO), a mutagenic metabolic intermediate, capable of binding to nucleic acids. Among DNA adducts, N7-alyklguanines, are the most abundant. They are cleaved off from the DNA molecule and, finally, excreted in urine. Therefore, they may serve as specific biomarkers of exposure to styrene as well as indicators of DNA damage. An analytical method for the determination of SO derived N7 guanine adducts, i.e., 7-(2-hydroxy1-phenylethyl)guanine (N7␣G) and 7-(2-hydroxy2-phenylethyl)guanine (N7␤G) based on a two step extraction from urine and determination by HPLC–tanem MS was developed. The detection limit was nearly 0.5 ng per injection corresponding to 5 ng/ml of the original urine sample. Authentic samples


Abstracts / Toxicology Letters 158S (2005) S1–S258

of the analytes N7␣G, N7␤G and the internal standard (isomeric guanine derivative N9␤G) were synthesised on preparative scale. Urine samples from mice exposed to styrene for 21 consecutive days were analysed. Nearly 1 × 10−5 % of absorbed dose was excreted in urine as guanine adducts N7␣G + N9␤G. Unusually high excretion of the urinary adducts was observed at the first day of exposure. The financial support by grants no. 310/03/0437 and MSM 223100001 is gratefully acknowledged. P8-17 GENETICALLY DETERMINED SUSCEPTIBILITY TO TOXICANTS: IMPLICATIONS OF ETHNICITY K. Golka1 , J.H. Shen2 , M. Angeli-Greaves1,3 , M. Aslam4 , I. Romics5 , R. Thier6 1 Institute for Occupational Physiology at the University of Dortmund, Dortmund, Germany; 2 Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China; 3 Department of Pharmacology, Universidad Central de Venezuela, Caracas, Venezuela; 4 Department of Pharmaceutics, University of Karachi, Karachi, Pakistan; 5 Department of Urology, Semmelweis University Budapest, Budapest, Hungary; 6 University of Queensland, Brisbane, Australia

Molecular epidemiological studies point to differential impacts of genetic factors in different ethnicities, including Caucasians, Africans and East Asians. In particular, genetic differences between Caucasians and Asians in xenobiotic-metabolizing enzymes are relevant for the processing of industrial and environmental toxicants. In the case of N-acetyltransferases the distribution is different between Caucasians and Chinese whereas the distribution in Central Asian populations is more similar to Caucasians. Accordingly, toxic effects are differentially expressed. The slow NAT2acetylation state is associated with a higher susceptibility to bladder cancer in Caucasians but not in Chinese. Also, the GSTP1 polymorphism seems to be associated with a higher susceptibility to bladder cancer in aromatic amine exposed Chinese but not in Caucasians. As differential susceptibilities are not restricted to phase

II enzymes, as exemplified by the different impacts of CYP2E1 on the toxicity of acrylonitrile in Caucasians and Japanese, influences of ethnicity must better be considered. Supported by Deutsche Forschungsgemeinschaft (SFB 475). P8-18 ANALYSIS OF DNA DAMAGE IN WHITE BLOOD CELLS OF WORKERS EXPOSED TO FUMES AND AEROSOLS OF BITUMEN IN A CROSS-SHIFT STUDY B. Marczynski1 , M. Raulf-Heimsoth1 , M. Kappler1 , K. Schott2 , B. Pesch1 , T. Mensing1 , H.U. K¨afferlein1 , T. Br¨uning1 1 Research

Institute of Occupational Medicine, Ruhr University Bochum, Bochum; 2 TiefbauBerufsgenossenschaft, Munich, Germany We conducted a cross-shift study with 66 bitumenexposed mastic asphalt workers and 49 construction workers without exposure to bitumen. To assess the genotoxic risk, DNA adduct levels of 8-oxo-7,8dihydro-2 -deoxyguanosine (8-oxodGuo) and the formation of DNA strand breaks and alkali-labile sites were determined in white blood cells of workers before and after shift. Significantly more 8-oxodGuo adducts were observed after the shift in both groups (P = 0.02 in bitumen-exposed workers and P = 0.001 in the reference group). Levels of 8-oxodGuo were higher before and after shift in bitumen-exposed workers compared to the reference group. However, the differences were statistically marginal significant (P = 0.01 and P = 0.11, respectively). DNA strand break frequencies measured as Olive tail moment in the Comet assay were significantly higher in workers exposed to bitumen than in the reference group (P < 0.0001 both before and after shift). Paradoxically, levels of DNA strand breaks were higher in both groups (not significant) before shift. The changes between 8-oxodGuo and DNA strand breaks during shift were not associated (rs = −0.12; P = 0.21). Our results support the hypothesis that fumes and aerosols of bitumen can cause increased DNA damage.

Abstracts / Toxicology Letters 158S (2005) S1–S258

P8-19 HOGG1 SER326CYS GENETIC POLYMORPHISM IN A TURKISH POPULATION B. Karahalil, N.A. Kocabas¸ Gazi University, Faculty of Pharmacy, Department of Toxicology, 06330 Hipodrum, Ankara, Turkey Oxidative DNA damage, caused by either endogenous or exogenous sources of reactive oxygen species (ROS), has been linked to aging, chronic degenerative diseases, inflammatory diseases and cancers. 8hydroxydeoxyguanine (8-OHdG) is a major lesion produced by ROS. 8-OHdG among various types of DNA base modifications, has been the most widely studied and considered as a key biomarker of oxidative DNA damage. Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a key component of base excision repair (BER) pathway and catalyzed the removal of 8-OHdG. Ethnic and inter-individual differences in hOGG1 activity and several kinds of polymorphisms at hOOG1 gene locus have been observed in the different populations studied so far. Since no information is available on inter-individual variability of hOGG1 genotype in Turkish population, we genotyped 206 healthy, unrelated Turkish individuals. The allelic frequencies of hOGG1 gene in the Turkish population were found 0.50, 0.41 and 0.09 (Ser/Ser, Ser/Cys and Cys/Cys, respectively). Our results are similar to Caucasian studied previously but different from Asian populations. It seems that still there is a growing need for expanding genotype studies with respect to hOGG1 gene because of its importance in various cancer types, and furthermore in smoking habits. P8-20 ATM IS INVOLVED IN THE REPAIR OF ALKYLATING DNA LESIONS M. Debiak, B. Kaina Institute of Toxicology, University of Mainz, Obere Zahlbacher St. 67, D-55131 Mainz, Germany A critical DNA lesion induced by alkylating agents is O6 -methylguanine (O6 MeG). O6 MeG incorporated in DNA is highly mutagenic and genotoxic and triggers apoptosis. Despite the potency of O6 MeG to induce cell death, the mechanism of toxicity is only poorly understood. Here we provide evidence that ATM kinase is


involved in processing of alkylating lesions, and protects against alkylating genotoxic stress. We show that mouse fibroblasts deficient for ATM are hypersensitive to DNA methylating agents such as MNNG and MMS and the anticancer drug temozolomide. Inactivation of the DNA repair enzyme MGMT by a specific inhibitor (O6 BG) increased cell death after low dose treatment with MNNG and MMS and decreased survival, whereas transfection with a MGMT expression plasmid provoked an opposite effect. From this we conclude that O6 MeG is a critical DNA lesion being responsible for death of ATM cells in case of MNNG and partially in case of MMS. Finally we show that resistance of wild-type cells is due to ATM kinase activity as treatment with caffeine (ATM inhibitor) elevated the level of apoptosis of ATM proficient but not mutated cells. Apoptosis was a late effect starting 48 h after treatment with both agents, which was shown by caspase activation, suggesting the primary alkylating lesion (O6 MeG) has to be converted during DNA replication into a critical secondary one. The protective effect of ATM was not due to cell cycle progression, since after treatment with MNNG or MMS a transient G2 block accompanied by chk1 activation was observed independent of ATM. We postulate that O6 MeG is transformed into DNA double-strand breaks (DSB) in a replication dependent manner. DSBs will be repaired in G2 phase by homologous recombination which is impaired in ATM cells. P8-21 CONCORDANCE OF THE ACETYLATION STATUS BETWEEN HUMAN NAT2 PHENOTYPES AND DEDUCED GENOTYPES OF SEVEN SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs) ANALYSED BY REAL-TIME PCR H.P. Rihs1 , A. Seidel2 , M. Scherenberg3 , T. Br¨uning1 1 BGFA,

Institut der Ruhr-Universit¨at Bochum, Germany; 2 Biochemisches Institut f¨ur Umweltcarcinogene, Grosshansdorf, Germany; 3 AMD der Bau-BG Rheinland und Westfalen, Oberhausen, Germany This study aimed to evaluate the concordance between human NAT2 phenotypes and deduced genotype results of seven human NAT2 SNPs generated by real-time PCR analysis using the LightCyclerTM system (Roche, Mannheim, Germany). After approval by the ethic


Abstracts / Toxicology Letters 158S (2005) S1–S258

commission and written consent, the NAT2 phenotypes of 40 PAH-exposed workers were determined by a defined caffeine test method. Genomic DNA was used for the determination of seven human NAT2-specific SNPs (G191A, C282T, T341C, C481T, G590A, A803G, G857A). Phenotypic results based on the molar ratio of 5-acetylamino-6-formylamino3-methyluracil (AFMU)/1-methylxanthine (1X) with 0.3 as a cut-off point between slow (<0.3) and rapid acetylators (≥0.3). Consequently, 28 samples belonged to the slow (range AFMU/1X ratio: 0.03–0.2) and 12 to the rapid (range AFMU/1X ratio: 0.3-0.5) acetylators. Analyses of the LightCyclerTM results revealed eleven different NAT2 variant combinations, whereby * 5B/* 5B (20%), * 4/* 5B or * 5A/* 11 (17.5%), * 6A/* 6A (12.5%) were the most frequent. The deduced acetylation status (28 slow/12 rapid) of the seven NAT2 SNPs matched perfectly with the 40 phenotypically determined results. Although this study showed a 100% concordance between NAT2 phenotypes and the deduced NAT2 genotypes, further comparisons with more samples including scarce variant combinations are necessary to fully elucidate the degree of concordance in the future. P8-22 COMPARATIVE STUDIES OF P-GLYCOPROTEIN AND TOPO ISOMERASE II ALPHA EXPRESSION AS MULTIDRUG RESISTANCE PROTEINS IN GI AND HEPATOCELLULAR CARCINOMA

35 gastric, 32 esophageal and 13 liver carcinoma) were randomly and retrospectively obtained from patients who underwent surgery during the years 2000–2003. Tissue expression of P-gp was 77% in colorectal and hepatocellular carcinoma, 43% in gastric and 48.5% in esophageal carcinoma. The expression patterns of Topo II were also 34.6% in colorectal, 17.2% in gastric carcinoma, 21.5% in esophageal and 46.2% in hepatocellular carcinoma. Out of four different immunophentypes, P-gp+/Topo II− in colorectal and esophageal carcinoma and P-gp−/Topo− (48.6%) in gastric carcinoma were also diagnosed significant. High expression of P-gp altered the expression of Topo II and significantly affected the clinicopathological features of our patients. These findings further emphasize on the importance of immunophenotypes as valuable prognostic or predictive markers to avoid adverse reactions of drugs and to decide about effective chemotherapeutic regimen for patients with GI and hepatocellular carcinoma. P8-23 POLYMORPHISM OF GLUTATHIONE-STRANSFERASE P1 IN BLADDER CANCER CASES WITH SUSPECTED AROMATIC AMINE RELATED OCCUPATIONAL DISEASE M. Angeli-Greaves1,2 , S. Kopps2 , H.M. Bolt2 , H.M. Prager3 , H.C. R¨omer4 , D. L¨ohlein4 , K. Golka2 1 Universidad

1 Faculty

Central de Venezuela, Caracas, Venezuela; 2 Institute for Occupational Physiology at the University of Dortmund, Germany; 3 Institute for Occupational, Social and Environmental Medicine Castrop-Rauxel, Germany; 4 Department of Surgery, Klinikum Dortmund, Germany

Comparative studies of multidrug resistance markers in cancer tissues could be helpful in reducing the target organ toxicities and selection of more effective chemotherapeutic regimen. The expression and significant immunophenotypes of P-glycoprotein and Topo II alpha were determined and compared using IHC techniques. Tissue samples of 132 patients (52 colorectal,

The genotype of glutathione-S-transferase P1 (GSTP1) influences the risk of bladder cancer among Chinese workers formerly occupationally exposed to benzidine (Ma et al., 2003. Pharmacogenetics 13, 409). Two studies of Caucasian bladder cancer cases without known occupational exposure show conflicting results. This research was conducted to define the role of the GSTP1 genotype in Caucasian bladder cancer cases supposed to be caused by aromatic amines. Methods: DNA from 143 subjects reported as affected of urothelial cancer due to occupational exposure, and 196 surgical patients without known malignancy in

S. Arbabi Bidgoli1 , E Azizi2 , F Khodayariyan1 , M. Shiri1 , B. Minaee3 of Pharmacy, Islamic Azad University, Tehran, Iran; 2 Molecular Research Laboratory, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 3 Department of Anatomy and Embryology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

Abstracts / Toxicology Letters 158S (2005) S1–S258

their medical history were genotyped by means of real time PCR (LightCycler) in relation to GSTP1 A1578G (Ile105Val) polymorphism. Results: Among the subjects with bladder cancer 46% presented the AA genotype, 39% the AG genotype, and 15% the GG genotype. In the surgical (non cancer) control group analyzed, 42% presented the AA genotype, 42% the AG genotype and 16% the GG genotype. In part of the bladder cancer group, represented by 46 painters, the genotyping showed a distribution of 41% of the AA genotype, 48% of the AG genotype and 11% of the GG genotype. Conclusions: The data point out that in Caucasians the GSTP1 A1578G polymorphism in subjects exposed to aromatic amines does not play a substantial role as a predisposing factor for bladder cancer. P8-24 VARIATION OF NAT2 IN KYRGYZ AND ROMANIANS: FROM SNP VIA HAPLOTYPE TO PHENOTYPE B. Pesch1 , S. Rabstein1 , K. Unfried2 , T. Illig3 , H.P. Rihs1 , Ch. Mambetova4 , M. Vlad5 , U. Ranft2 1 BGFA,

Institute of Ruhr University of Bochum, D44789 Bochum, Germany; 2 IUF, D-40225 D¨usseldorf, Germany; 3 GSF, D-85764 Neuherberg, Germany; 4 Kyrgyz Scientific Center of Hematology, KG-770055 Bishkek, Germany; 5 Institute of Public Health, RO400349 Cluj, Germany This study aimed to evaluate ethnic differences in NAT2 genotypes. Nine SNPs were analysed in 290 unrelated Kyrgyz and 140 unrelated Romanians by MALDI-TOF MS. 341C, 481T and 803G were less and 857A more prevalent in Kyrgyz (p < 0.0001). 857A indicates Asian descent. 282C > T and 590G > A showed no significant variation. 364G > A and 411A > T were monomorphic. Database comparisons showed that Romanians belong to Caucasians and Kyrgyz are in-between Caucasians and East-Asians. Due to a large proportion of subjects with more than one heterozygous SNP (67.6% Kyrgyz, 60.0% Romanians), haplotypes could only be predicted. The haplotype distributions differed significantly between both ethnicities. The Kyrgyz showed


a higher genetic diversity but a higher frequency of the unmutated haplotype. Accordingly, the imputed slow acetylator phenotype was less prevalent in Kyrgyz (35.2% versus 51.4% in Romanians). We found pronounced ethnic differences in NAT2 haplotypes with yet unknown impact on health risks. P8-25 GLUTATHIONE S-TRANSFERASE M1, P1, T1 AND CYTOCHROME P450 1A1 GENETIC POLYMORPHISM IN PATIENTS WITH NEOPLASMS OF THE HEAD AND NECK E. Reszka1 , P. Czekaj2 , J. Adamska2 , P. Urbaniec2 , A. Ziolkowski2 , G. Namyslowski2 , W. W˛asowicz1 1 Nofer

Poland; Poland

Institute of Occupational Medicine, Lodz, 2 Medical University of Silesia, Katowice,

Individual susceptibility to different environmental agents is expected to be associated with genetically determined differences in metabolism of xenobiotics. The investigated population was consisted of 68 diagnosed larynx cancer patients and 36 non-cancer individuals. We employed PCR technique to determinate GSTT1 polymorphisms and PCR-RFLP to distinguish GSTM1 intron 6/exon 7, GSTP1 exon 5 and CYP1A1 exon 7 genotypes. In larynx cancer patients group there were 16.2% individuals with CYP1A1 Ile/Val genotype, while in controls it was 5.6%. Defective GSTP1 Val/Val genotype was at similar frequency in cancer group (8.8%) and control group (8.3%), like in the case of GSTT1 null genotype (26.5% and 27.8%, respectively). Homozygous deletion of GSTM1 gene was present in 51.5% of patients and 44.4% of controls. The results of these studies are the part of ongoing investigations which will include much larger number of analyses. However, these preliminary results already show some pattern that when analyzed together with smoking and drinking habits, occupation may indicate important risk factors in neoplasms of the head and neck. This work was financially supported by the grant of Polish Committee for Scientific Research, Grant No. 0630/P05/2003/24.


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Molecular Biology Unit, Department of Biochemistry, University of Otago, Dunedin, New Zealand; 2 Department of Biochemistry, University of Otago, Dunedin, New Zealand Facial eczema disease is a hepato-mycotoxicosis in ruminants caused by exposure to a fungal toxin called sporidesmin. Currently the most effective prophylaxis for facial eczema is zinc sulphate pre-treatment, which forms the basis of the Time Capsule® intraruminal device. However, the mechanism by which zinc acts as a prophylactic is unknown. Elucidation of the protective role of zinc would provide insights into the mechanism of sporidesmin toxicity, and facilitate the development of alternative therapeutic approaches. Since whole animal studies are impractical, an in vitro approach using HepG2 cells was used to investigate the mechanism of zinc protection against sporidesmin toxicity. HepG2 cells are a human liver carcinoma cell line that retains many of the morphological features and gene expression characteristics of primary hepatocytes. It was demonstrated that sporidesmin is toxic to HepG2 cells in a concentration dependent manner. The cells could be significantly protected by pre-treatment with zinc sulphate; the level of protection was dependent on zinc concentration and was effected within 2 h. Zinc has many biological functions and one of the major cellular effects is to modulate gene expression. To assess the role of transcription in protection by zinc, cells were treated with the transcriptional inhibitor actinomycin D. Treatment of cells with actinomycin D did not significantly interfere with the protection by zinc, which suggests that the mechanism of protection is not mediated through de novo gene transcription.


of Pharmacology, FMRP-USP, Av. Bandeirantes, 3900, Ribeirao Preto-SP, Brazil; 2 FORPUSP, Ribeirao Preto-SP, Brazil Lead (Pb) exposure is widely recognized as a serious environmental health problem. A causal association between Pb exposure and increased cardiovascular risk has been strongly suggested. Clinical and experimental evidence is accumulating in support of an important role for lead-induced depressed nitric oxide (NO) availability. This reactive molecule is produced in endothelial cells and platelets by endothelial NO synthase (eNOS). Thus, due to the major importance of eNOS in the regulation of NO availability, many studies addressing the clinical relevance of polymorphisms in the eNOS gene have been carried out. Based on this, we investigated whether there is an association between the circulating concentrations of nitric oxide products (NOx) and the concentrations of lead in whole blood (B-Pb) and in plasma (P-Pb) from lead-exposed subjects. In addition, we also evaluated whether eNOS genotype for the T-786 C polymorphism affects NOx concentrations. As a result, we found a significant negative correlation between NOx concentrations and B-Pb in carriers of the “C” allele, and a trend for a negative correlation between NOx concentrations and P-Pb. Therefore, our data strongly suggest that Pb exposure may decrease NO availability in carriers of the “C” allele, thus suggesting a possible mechanism involved in Pb exposure-induced increase in the susceptibility to cardiovascular diseases. P8-28 MICROSATELLITE ANALYSES OF HELIANTHUS ANNUUS L. GENOME EXPOSED TO CADMIUM AND LEAD ˆ Gomes, H. Azevedo, C. Santos, T. Lopes A.

Laboratory of Biotechnology and Cytomics, University of Aveiro, Campus University de Santiago, 3810-193 Aveiro, Portugal

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Cadmium and lead are two heavy metals that are very toxic to plants. They may reach the soil by several ways including soil-applied chemicals such as fertilizers. Depending on their concentration, both cadmium and lead could be extremely cytotoxic, carcinogenic and mutagenic leading to high levels of genetic instability. However, this genetic instability does not always correspond to changes at the phenotypical level. Therefore in this study we used a PCR-based technique, microsatellite analysis, to evaluate the existence of genetic instability. Microsatellites, also known as simple sequence repeats (SSRs), are composed of tandem repeat of a sequence of two to seven nucleotides. As DNA markers, SSRs are highly polymorphic, reproducible and abundant, being extremely useful for fine-scale genetic analysis. In this work we have evaluated the mutagenic effects of cadmium and lead in the important crop species, Helianthus annuus L. Sunflower plants germinated in distilled water were exposed for 21 days to medium containing different concentrations of Cd(NO3 )2 or PbCl2 under hydroponic conditions. Genomic DNA was extracted from sunflower leaves and roots, harvested at day 21, and 10 nuclear SSR loci were tested, amplified and analysed to evaluate for microsatellite instability. P8-29 RADIATION EFFECTS ON THE CONFORMATION OF DNA MOLECULES IN SOLUTION Z. Paskalev1 , Tz. Marinova1 , D. Apostolova2 1 National

Center of Radiobiology and Radiation Protection, Sofia; 2 Clinic of Occupational Diseases, Medical University, Sofia, Bulgaria The biological activity of DNA molecule is directly related to DNA conformational change. A little information is available about conformation changes of the irradiated DNA in solution. The incorporation of the thymine analog 5-bromuracyl (5-BU) into DNA has been shown to result in sensitization of DNA to radiation in both cellular and extracellular systems. The function of a complex dielectric constant provides fundamental information on the interaction of DNA molecule with radiation. The present study investigates he applicability of the technique of measure the complex dielectric constant to determine small changes in the conformation of DNA molecule in solution after


irradiated with ␥-ray and 645-MeV-proton exposure. DNA, normal or sensitized, was isolated from prototrophic Bacillus subtilis 23, using Marmour’s technique. Gamma irradiation was carried out on a PXMGamma-20 60 Co. The 645 MeV-proton exposure was from the synchrocyclotron at the Joint Institute for Nuclear Studies, Dubna (Russia). Measurements of DNA electroconductivity and dielectric constant were made with a type SWM-2 bridge admittance meter coupled with a type M-2746 high-frequency generator. Our study indicated that exposure of DNA to small gamma dose produced in the molecular conformation change such as to convey conformational resistance to higher radiation doses. The exposed DNA molecules to 645 MeV-protons changed their conformation in higher degree compared to the DNA changes after gamma exposure in the same doses. The comparing radiosensitivities between irradiated normal DNA and BU-substituted DNA show the smaller conformation change in normal DNA. P9 Food Safety P9-01 CYTOTOXICITY AND ABSORPTION EVALUATION OF AFM1 ON HUMAN INTESTINAL CELL LINES F. Caloni1 , I. Foti1 , A. Stammati2 , I. De Angelis2 1 Department

of Veterinary Sciences and Technologies for Food Safety, University of Milan, Milan, Italy; 2 Department of Environment and Primary Prevention, Istituto Superiore di Sanit`a, Rome, Italy Aflatoxin M1 (AFM1 ), the principal hydroxylated metabolite of Aflatoxin B1, is a mycotoxin present in milk of mammals, classified as group 2B of the IARC. In order to reduce human risk, AFM1 limits in bovine milk (50 ppt by EU and 500 ppt by United States) and in milk based food for infants (25 ppt by EU), which represent the population more exposed to AFM1 , have been established in many countries. The aim of this study was to evaluate cytoxicity and absorption profile of AFM1 on Caco-2 cells, an intestinal cell line obtained from a human colon carcinoma, and on its derived clone TC7, both able to differentiate to small enterocyte in long-term culture but with slight differences in their metabolic activity. After treat-


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ment of undifferentiated and differentiated cells with AFM1 concentrations ranging from 10 to 10,000 ppt for 48 h, an high dose-dependent absorption was revealed only in Caco-2 and Caco-2/TC7 differentiated cells. Conversely, Caco-2 and Caco-2/TC7 viability was not affected by AFM1 treatments. In fact, about 90% of viable cells have been detected, in both phases of growth, at all the concentrations tested either with neutral red uptake (NRU) or with total protein content (TPC) assays. Indeed, LDH release data revealed a significant damage of undifferentiated cells at the end of treatment, for concentrations higher than 500 ppt. Present results can open new perspectives for the evaluation of human risk to AFM1 gastrointestinal exposure. P9-02 EFFECT OF FUSARIUM MYCOTOXINS ON ESTRADIOL PRODUCTION IN PIG GRANULOSA IN VITRO MODEL F. Caloni1 , G. Ranzenigo2 , F. Cremonesi2 , L.J. Spicer3 1 Department

of Veterinary Sciences and Technologies for Food Safety, University of Milan, Milan, Italy; 2 Department of Veterinary Clinical Science, University of Milan, Milan, Italy; 3 Department of Animal Science, Oklahoma State University, Stillwater, OK, USA Trichothecens, like deoxynivalenol (DON) and T2toxin (T-2), and zearalenone (ZEA), are mycotoxins produced by Fusarium, a common grain contaminant. The endocrine disruptor effect of ZEA is well documented in humans and in different species, but little information are reported on possible synergic effects with DON and T-2, already present together in food and feedstuffs. DON (0.01–16.0 ␮M), T-2 (0.0006–6.4 ␮M) and ␣-zearalenol (␣-ZEA) (0.009–31.2 ␮M) are tested on an in vitro model, pig granulosa cells, singularly or in combination at different concentrations in presence of insulin-like growth factor-1 (IGF-1; 0–30 ng/ml), FSH (0–30 ng/ml) and 0.5 ␮g/ml of testosterone. DON stimulated significantly estradiol production only at low concentrations until 0.03 ␮M after which doses were inhibitory, while ␣-ZEA did not show any effect with the exception of the inhibitory effects at high concentrations (9.4 and 31.2 ␮M), and T-2 had an inhibitory effect on estradiol production at all doses tested including the lowest concentration. Rank order potency for inhibition was

T-2 > DON > ZEA. No synergistic effects were shown during all the various combinations of the three mycotoxins on the production of estradiol by pig granulosa cells. P9-03 EVALUATION OF THE PRESENCE OF AFLATOXIN M1 IN GOAT MILK F. Caloni, L. Valnegri, G. Soncini Department of Veterinary Sciences and Technologies for Food Safety, University of Milan, Milan, Italy Aflatoxin M1 (AFM1 ), the principal hydrolyzed metabolite of Aflatoxin B1 excreted in milk, is classified in a group 2B of the IARC. Human exposure to AFM1 is due either for an endogenous production of AFM1 or from the consumption of contaminated dairy products. To reduce the human risk it was regulated the maximum levels of AFM1 in bovine milk on 50 ppt in EU and 500 ppt in United States. Little is known about the presence of AFM1 in goat milk and by-products. A preliminary investigation with ELISA test, specific for the determination of AFM1 in bovine milk, was carried out on 120 milk goat samples. The samples have been analysed after a preparative procedure, to avoid false positive. Results have shown a seasonal trend, with higher concentration during April and May with values over 60 ppt, and a decrease from June to September, where some samples showed a concentration over 50 ppt. Confirmation of the results by HPLC are in due course. P9-04 EFFECTS OF AFLATOXIN M1 IN XENOPUS LAEVIS DEVELOPMENT F. Caloni1 , A. Di Muzio2 , S. Panseri2 , I. Foti1 , M. Lucchino1 , C.Vismara2 1 Department

of Veterinary Sciences and Technologies for Food Safety, University of Milan, Milan, Italy; 2 Department of Biology, University of Milan, Milan, Italy The main hydrolyzed metabolite of Aflatoxin B1 excreted in milk is Aflatoxin M1 (AFM1 ), classified in group 2B by International Agency for Research on Cancer (IARC). It is important to underline that human exposure to AFM1 is due either for an endogenous pro-

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duction of AFM1 or from the consumption of contaminated dairy products. To reduce this risk, the maximum levels of AFM1 in bovine milk is 50 ppt in the EU and 500 ppt in the United States. Here, the research investigated the lethal and teratogenic potential of AFM1 using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX), a rapid, cost-effective and powerful bioassay for evaluating developmental toxicants, and a valid pre-screening to evaluate human embryotoxic compounds. Stage 9 blastulae were exposed to AFM1 at 4, 8, and 16 mg/L concentrations until stage 47, free swimming larva. No statistically significant differences were observed in mortality percents between control and exposed groups. A slight increase of malformed larva percents is observed in groups exposed to AFM1 but these differences were not statistically significant compared to the control and not in concentrationresponse. Therefore, AFM1 is to be considered a nonembryotoxic compound. P9-05 MYCOTOXIN CONTAMINATION OF FEEDSTUFFS: IN VITRO SYNERGISTIC EFFECTS OF OTA AND FB1 ON PROTEIN SYNTHESIS IN CACO-2 CELL LINE M.R. Carrat`u1 , A. Coluccia1 , P. Borracci1 , Th. Mobio2 , J. Kouadio2 , S. Mouka2 , E.E. Creppy2 1 Department

of Pharmacology and Human Physiology, Medical School, University of Bari, Italy; 2 Laboratory of Toxicology, University Victor Segalen Bordeaux 2, Bordeaux, France Ochratoxin A (OTA), a structural analogue of phenylalanine, inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminocylation reaction, constituting the main mechanism of OTA-induced cytotoxicity. Since it seems impossible to avoid pluricontamination of foodstuffs by toxigenic fungi, the present investigation was designed to study possible synergistic effects of OTA and Fumonisin B1 (FB1) on protein synthesis in Caco-2 cells. Protein synthesis was determined by incorporation of a radioactive precursor, [3 H]leucine (1). The results show that OTA and FB1 inhibit protein synthesis with IC50 of 7.9 and 1.02 ␮M, respectively. Moreover, the inhibition of protein synthesis was further increased by the combination of 5 ␮M OTA with different concentrations of FB1


(2.5 ␮M: p < 0.01; 5 and 10 ␮M: p < 0.001, Tukey’s test). Our findings suggest that the combined effects of OTA and FB1 observed in vitro are clearly of synergistic nature and underline the risk associated to the mycotoxin multicontamination, being of great concern for consumers safety. P9-06 TOXIC RISK’S CHARACTERIZATION CHILDREN EXPOSED TO TOXIC FOOD


G.F. Desogus Service of Prevention and Protection, Company USL 7 Carbonia, Italy The focus of this article is on the results of a toxic research on the majority of heavy metals in the diet of children exposed to occupational and environmental chemical exposures in a workplace or community setting (Sardinia, Italy). The working method is based on the alimentary residual risk depending on the concentration of specific metals such as lead and cadmium on food, the rate of food intake, the duration of exposure and the period on which the quantity is mediated. The intake’s parameters of lead and cadmium at which they affect children, are significantly higher in food such as fish, vegetable and fruit, compared to the provisional tolerable weekly intake (PTWI). The limit was recommended by the FAO/WHO group. This study demonstrates there is a good correlation between environmental pollution and contaminated food intake to which children are exposed. P9-07 OCHRATOXIN A INDUCES OXIDATIVE DNA DAMAGE IN VITRO 2 ¨ Undeger ¨ G.H. Degen1 , Y. Lektarau1 , U. 1 Institut

f¨ur Arbeitsphysiologie an der Universit¨at Dortmund, Ardeystr. 67, D-44139 Dortmund, Germany; 2 Hacettepe University, Faculty of Pharmacy, Ankara, Turkey Ochratoxin A (OTA), a common mycotoxin contaminant of human and animal foods, is nephrotoxic and a potent carcinogen in rats. Its mode of action is only partly understood, and the mechanism of OTA mediated genotoxicity (direct or indirect?) is still unclear


Abstracts / Toxicology Letters 158S (2005) S1–S258

and under debate. This calls for further characterization of the OTA-induced DNA damage. We investigated the concentration and timedependent effects of OTA on human bladder carcinoma (H5637) cells and V79 cells: Cytotoxicity, measured by Neutral red assay, was not observed with 3 h treatment up to 100 ␮M OTA, but apparent at 1 ␮M and more after 24 h exposure. DNA damage, assessed by alkaline single cell gel electrophoresis (Comet-assay) was detectable upon 3 h treatment at ≥1 ␮M OTA. The extent of DNA damage was clearly enhanced in the presence of fpg-glycosylase which is known to convert oxidative DNA damage into strand breaks. Significant increases in fpg-sensitive sites were already seen at 0.1 ␮M OTA, with the highest parameter values in H5637 and V79 cells at 3 h and 5 h, respectively. Our data indicate that genotoxicity of OTA is due to indirect effects such as formation of reactive oxygen species, and in line with the observation that OTAinduced damage is primarily clastogenic. This supports the existence of threshold concentrations for such adverse cellular effects of the mycotoxin. P9-08 NATURAL OCCURRENCE OF AFLATOXINS IN LOCAL AND IMPORTED WHEAT GRAINS (TRITICUM SATIVUM) IN EGYPT H.A. Amra1 , S.F. Masheal2 , M.M. Ebba2 1 Food

Toxicology and Contaminants Department, National Research Center, Dokki, Cairo; 2 Plant Pathology, Faculty of Agriculture at Kafr El-Sheikh, Tanta University, Tanta, Egypt Aflatoxins are the predominant mutagenic fungal metabolite isolated from serveal species of Aspergillus. These mycotoxins contaminate many food products and are consequently of worldwide heath concern. One hundred samples of imported and local were collected from El-Qualubia and Kafr El-Sheikh governorates, wheat granis (Triticum sativum) were examined for the natural occurrence of aflatoxins during 2000–2001. The results were indicated that that 7 samples out from 40 (17.5%) were positive for aflatoxin B1 in the local and 2 samples out from 10 (20%) were positive for aflatoxin B1 in imported wheat grains in same order. The concentration of aflatoxin B1 was ranged from 3 to 25 ␮g/kg in the imported and local wheat grains.

Only one sample of the white imported wheat grains was contented both aflatoxin B1 and B2. The highest concentration of aflatoxin B1 (20 and 25 ␮g/kg) were found in local wheat grains at Biella and Shobra El-Khema shops, while the lowest concentrations of aflatoxin B1 were 3 and 5 ␮g/kg which were found in wheat grain samples of white and the red imported wheat grain and samples of local wheat grain at Kaha shops, respectively in the summer season. The results also indicated that 4 out of 40 (10%) local wheat samples were positive for aflatoxin B1, with concentration ranged from 3 to 20 ␮g/kg wheat grains. The highest concentration of aflatoxin B1 (20 ␮g/kg wheat grain) was found in samples of Toukh, while the lowest concentration of aflatoxin B1 (3 ␮g/kg wheat grain) was found in wheat grain samples from Benha. On the other hand, no wheat grain sample was found positive in Kafr El-Sheikh Governorate. Two samples out of 10 of the imported wheat grain samples (20%) were positive for aflatoxin B1 with concentration at 4.2 and 7.8 ␮g/kg of white andred wheat grains, respectively, in winter season. From the present data, in aflatoxins wheat samples was depended on the area of collection as well as season (summer and winter). Generally, the consumers of wheat products in Egypt are exposed to aflatoxins health hazards. P9-09 PROBIOTIC BACTERIA AS A TOOL TO PREVENT FUNGAL GROWTH AND AFLATOXINS PRODUCTION BY ASPERGILLUS PARASITICUS E.A.M. AbdAlla1 , Y. Saleh2 , S. Mary2 , E. Aly Soher1 , A.S. Hathout1 1 Department

of Food Toxicology and Contaminants, National Research Centre, Cairo, Egypt; 2 Botany Department, Faculty of Science, Cairo University, Cairo, Egypt Lactic acid bacteria are used as probiotic bacteria in fermented dairy products and other fermented food products whereas they are known to prolong the shelf life of these products. The inhibitory effect of cell free supernatant (CFS) of several Lactobacillus species on the production of aflatoxin and mycelial growth by the aflatoxigenic Aspergillus parasiticus was studied. A complete inhibition (100%) of aflatoxin production

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and mycelial growth was recorded when cell free supernatant of Lactobacillus casei was placed in a dialysis sac or in the medium without a dialysis sac on the insertion of A. parasiticus after 16 h with L. casei. The obtained results also revealed that Lactobacillus reuteri also inhibited aflatoxin production and mycelial growth. Cell free supernatant (CFS) of Lactobacillus acidophilus and Lactobacillus bulgaricus showed the lowest effect on the aflatoxin production as well as on mycelial growth by A. parasiticus. Results also indicated that inoculation of bacterial strains before A. parasiticus inoculation by 16 h caused the higher inhibitory effect to aflatoxin production and mycelial growth. But these indications were not observed for the other treatments of L. acidophilus or L. bulgaricus. In conclusion probiotic bacteria produced active substances that can inhibit aflatoxins production by A. parasiticus in most treatments used and that the inhibitory effect depends on the type of lactic acid and/or the treatment used. P9-10 TOXICOLOGY STUDIES ON ACE-INHIBITING PEPTIDES DERIVED FROM CASEIN S. O’Hagan Safety and Environmental Assurance Centre, Unilever Colworth, Bedford, England Peptides derived from milk casein have been shown to lower blood pressure (BP) in individuals with an elevated BP and food products containing these peptides have been developed. Evidence suggests that the peptides isoleucine-proline-proline (IPP) and valineproline-proline (VPP) lower BP by having a mild inhibitory effect on angiotensin converting enzyme (ACE). ACE is a key enzyme in the renin–angiotensin system (RAS) and converts Angiotensin I to the potent vasoconstrictor Angiotensin II. Disruption of the RAS by ACE inhibiting drugs can produce adverse effects as well as the beneficial effect on blood pressure. When used in pregnancy the ACE inhibiting drugs can produce a fetopathy that is characterised by fetal hypotension, disruption of kidney development and reduction in amniotic fluid. Although the peptides have a much milder effect on the RAS, a number of toxicology studies were conducted to confirm their safety-in-use. A


hydrolysate containing IPP and VPP was tested in an embryo-fetal development study in rabbits at dose levels ranging from 335 to 1000 mg/kg bw/day. A pre and post-natal development study (including direct dosing of neonates) was conducted in rats to assess effects on kidney development and a 90-day subchronic study was also conducted in rats. The dose levels used in these studies ranged from 500 to 4000 mg/kg bw/day. No adverse effects were observed in any of the studies giving safety factors of 90–300 times the intended human intake level. P9-11 PREDICTIVE MODELS OF HEAVY METALS AND TOXIC ELEMENTS DISTRIBUTION DURING PARMIGIANO REGGIANO CHEESE PRODUCTION S. Ghidini, E. Zanardi, M. Conter, C. Malpeli, A. Ianieri, G. Campanini Department of Animal Production, Veterinary Biotechnology, Food Quality and Safety, University of Parma, Italy The concentration of aluminium, copper, iron and zinc were determined by ICP-AES on Parmigiano Reggiano cheese at several stages of production. On the same samples cadmium, lead, vanadium, cobalt, chromium, and molybdenum concentrations were determined by ICP-MS. These data together with quantitative data allowed to estimate the mass balance of the considered elements during the making of this Italian matured cheese. The affinity of elements for curd is variable: 90% of the zinc present in milk goes into the curd. This percentage is 68 for aluminium, 5 for vanadium, 15 for chromium, 93 for cobalt, 19 for iron and 50 for molybdenum. Both cadmium and lead seem to concentrate almost completely in curd, but their concentrations are very low in the starting milk (cadmium from non detectable to 0.23 ␮g/l; lead from non detectable to 4.91 ␮g/l) and even after concentration in cheese the final levels do not represent a risk for consumers. A cession of copper from the cheese vat (made of copper) to cheese was documented but, even in this case, the concentration of copper is quite low (lower than 12 mg/kg in curd) and does not represent a risk for human health.


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P9-12 OCHRATOXIN A AND FUMONISIN B1 INDUCE SYNERGISTIC INHIBITION OF DNA SYNTHESIS IN C6 GLIOMA CELLS M.R. Carrat`u1 , P. Borracci1 , A.Coluccia1 , Th. Mobio2 , J. Kouadio2 , S. Mouka2 , E.E. Creppy2 1 Department

of Pharmacology and Human Physiology, Medical School, University of Bari, Italy; 2 Laboratory of Toxicology, University Victor Segalen Bordeaux 2, Bordeaux, France Contamination of food by mycotoxins is a major problem of human health concern. Since multi-mycotoxin contamination of feed and foodstuffs occurs more frequently than a single mycotoxin contamination, the aim of the present study was to investigate possible synergistic effects of ochratoxin A (OTA) and Fumonisin B1 (FB1) on DNA synthesis in C6 rat glioma cells. DNA synthesis was determined by incorporation of [3 H]-thymidine (1). The results show that OTA and FB1 inhibit DNA synthesis (IC50 = 7.5 ␮M for OTA; IC50 = 33 ␮M for FB1, after 24 h incubation) in this cell line. The combination of OTA (5 ␮M) with different concentrations of FB1 (2.5, 5, 10 and 20 ␮M) significantly (p < 0.05, Tukey’s test) enhanced OTA-induced inhibition of DNA synthesis. FB1 at the dose of 2.5 ␮M inhibited the synthesis of DNA from <10% to >50% when combined to 5 ␮M OTA, while the same OTA concentration induced less than 20% of inhibition. The present findings underline that multi-mycotoxins contamination must be carefully considered when assessing the risk for humans exposed to mycotoxins. P9-13 ACCUMULATION OF CLENBUTEROL, A FOOD CONTAMINANT, IN PIG RETINAL TISSUE AFTER REPEATED EXPOSURE T. Gojmerac1 , J. Pleadin1 , I. Bratoˇs2 , A. GojmeracIvˇsi´c3 , M. Korˇsi´c4 1 Croatian

Veterinary Institute, Zagreb, Croatia; Institute, Zagreb, Croatia; 3 Faculty of Science, Department of Chemistry, University of Zagreb, Croatia; 4 School of Medicine, Department of Endocrinology, University of Zagreb, Croatia

2 Pliva-Research

Previous studies have documented adverse health effects in consumers of contaminated meat, associated with the illegal use of clenbuterol as a growth promotor in meat producing animals. As ␤2 -adrenergic agonists such as clenbuterol are bound to melanin and accumulate in pigmented tissues (choroid/pigmented retinal epithelium) over long periods, the aim of the study was to evaluate clenbuterol accumulation in pig retinal tissue after cessation of exposure, as a feasible target matrix of choice for detecting their abuse. The growth-promoting dose of clenbuterol (20 ␮g/kg body mass per day) was administered orally to cross-bred female pigs for 41 days. Upon cessation of administration, on days 0, 3, 7, 14 and 35, the pigs were randomly sacrificed and retina was dissected from the eye and stored at −25 ◦ C until analysis. The enzyme-linked immunosorbent assay (ELISA) was used as a “screening” method and reverse phase liquid chromatography (LC) and tandem mass spectrometry (MS/MS) as confirmation method for residual clenbuterol in pig retinal tissue. Analytical procedure showed a high correlation between ELISA and LC/MS/MS results (r(2) = 0.943) in quantification and confirmation of residual clenbuterol in pig retinal tissue. The results indicated >80% of clenbuterol depletion in pig retinal tissue to occur during the first 7 days after cessation of administration (21.5 ng/g). On day 35 of clenbuterol discontinuation, drug residues were detectable in pig retinal tissue at highly levels exceeding the maximal residue limit (MRL) of 0.5 ng/g. These findings suggest the pig retinal tissue to be useful in identifying residual clenbuterol at long withdrawal times after drug exposure. This is supported by the known facts about clenbuterol pharmacokinetics, with the ultimate goal of upgrading the control of food safety.

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B. Aspenstr¨om-Fagerlund1,2 , E. Netzel1 , J. Carlfors4 , J. Tallkvist2 , N.-G. Ilb¨ack1,3 , A.W. Glynn1,3

M. Bagchi1 , N. Maulik2 , D. Bagchi1,3 , L. Zhan2 , T. Yasmin3 , M.A. Shara3 , E. Altaf2

1 Toxicology

Research Center, Benicia, CA, USA; of Connecticut Health Center, Farmington, CT, USA; 3 Creighton University Medical Center, Omaha, NE, USA

Division, National Food Administration, P.O. Box 622, SE-75126 Uppsala, Sweden; 2 Department of Pathology, Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Uppsala, Sweden; 3 Department of Medical Sciences, Section for Infectious Diseases, Uppsala University, Uppsala, Sweden; 4 Department of Pharmacy, Uppsala University, Uppsala, Sweden We are investigating if high intake of fat might compromise the barrier function of the intestinal epithelium and thereby causing an increased passage of toxic substances and allergenic agents into the circulatory system. We addressed this hypothesis by testing free fatty acids such as oleic acid and docosahexanoic acid (DHA) in an experimental model of human absorptive enterocytes. Monolayers of human intestinal caco-2 cells were allowed to differentiate on permeable filters placed in bicameral chambers for 21–25 days followed by apical exposure to emulsions of oleic acid (15 or 30 mM) or DHA (5, 15 or 30 mM) for 90 min in medium with or in absence of calcium and magnesium ions. Paracellular transport of the extracellular marker 14 C-mannitol as well as the transepithelial electrical resistance (TEER) were monitored. Our results showed a dose dependent increase in 14 C-mannitol transport and decrease in TEER in all groups exposed to oleic acid or DHA. The effect of DHA was stronger than that of oleic acid. When monolayers were exposed to emulsions of oleic acid and DHA, in medium without Ca2+ and Mg2+ , effects were stronger and occurred at lower concentrations than in medium containing Ca2+ and Mg2+ . Our results indicate that fatty acids common in food appears to compromise the integrity of the intestinal epithelium but further studies are required to bring insight into the mechanisms involved and further testing of the hypothesis.

1 InterHealth

2 University

In this study, we assessed the bio-pathophysiological function and safety of a novel energy formula (NEF) containing niacin-bound chromium (NBC), standardized Ashwagandha extract, d-ribose and selected amines. NBC has been demonstrated for energy production as well as carbohydrate and fat metabolism. Male and female Sprague–Dawley rats were fed 25 ppm NEF for 90 consecutive days. Body weight, feed and water intake, and physical activities were monitored. NEF supplementation did not cause changes in hepatic lipid peroxidation and DNA fragmentation on 30, 60 or 90 days of treatment. Hematological, clinical chemistry and histopathological evaluations did not show any adverse effects in all organs. Efficacy of NEF was assessed by examining myocardial high-phosphate compounds, ATP and CP, by HPLC in hearts isolated at 30, 60 and 90 days post treatment. Myocardial ATP levels increased by 7%, 58% and 59%, and myocardial CP levels increased by 17%, 31% and 59% following supplementation of NEF for 30, 60 and 90 days, respectively. Expression of stress related proteins (HSP 70, HSP 32 and HSP 27) increased by approximately 20–30% at 30, 60 and 90 days of treatment. Incidence of myocardial infarction was significantly reduced and improved verntricular recovery in NEF-fed animals. These results suggest the broad spectrum safety and efficacy of NEF. P9-16 EVALUATION OF HEALTH HAZARDS BY EXPOSURE TO CONTAMINANTS IN TRADITIONAL GREENLAND DIET E. Nielsen, J.C. Larsen, O. Ladefoged Danish Institute for Food and Veterinary Research, Søborg, Denmark


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The traditional diet is a significant source of exposure to contaminants to people in Greenland. Some traditional food items contain very high levels of heavy metals as well as of persistent organic contaminants. A risk characterisation in order to evaluate the risk to Greenlanders of adverse health effects from intake of contaminants from traditional food items has been performed for the following contaminants: lead, cadmium, mercury, selenium, PCBs, DDT, chlordane and heptachlor, hexachlorocyclohexanes (HCHs), chlorobenzenes, dieldrin and toxaphene. The risk characterisation has been carried out by comparing the outcome of the effect assessment, i.e., a NOAEL or a LOAEL for the critical effect(s), to the outcome of the exposure assessment, i.e., the mean daily intake of the contaminant from traditional food items. The ratio resulting from this comparison, called Margin of Safety (MOS), has been compared with the so-called “minimal-MOS” (minMOS), which can be interpreted as an ‘overall’ uncertainty factor. A calculated MOS below the minMOS indicates a risk of adverse health effects and consequently, the exposure to the contaminant in question should be reduced. Based on the risk characterisation for the selected contaminants, it is recommended that the exposure to cadmium, lead, mercury, PCBs, chlordane and heptachlor, and toxaphene from the traditional Greenland diet as well as from other sources should be reduced. For the remaining contaminants, the exposure from the traditional Greenland diet is considered to be of low risk. P9-17 OCCURENCE OF OCHRATOXIN A IN BLOOD OF ITALIAN SLAUGHTERED PIGS V. Meucci, E. Costa, E. Razzuoli, G. Mengozzi, G. Soldani Department of Veterinary Clinics, Section of Pharmacology and Toxicology, University of Pisa, V.le delle Piagge 2, 56124 Pisa, Italy Ochratoxin A (OTA) is a secondary metabolite produced by several Penicillium and Aspergillus species with carcinogenic, hepatotoxic, nephrotoxic, teratogenic, and immunosuppressive properties. OTA has been implicated in the etiology of Balkan endemic nephropathy and has been associated with an increased incidence of tumours of the upper urinary tract. OTA

can occur in animal products as a result of either direct contamination with toxigenic moulds or indirect transmission (carryover) in animals fed with naturally contaminated feed. The aim of the present study was to evaluate the presence of OTA in serum samples of slaughtered pigs. Serum samples were analysed for OTA by an HPLC method. The limits of quantification (LOQ) and determination (LOD) of the method were 0.025 and 0.0125 ng/ml, respectively. The recovery of OTA was 91.0 ± 5%. Precision and accuracy were within 8.9%. Fifty-one Italian (Tuscany) pig serum samples (82% of 62 analysed) were found to be naturally contaminated with OTA in the range of 0.17–2.81 ng/ml. Similar OTA contamination was observed in the serum samples collected from the same farm. These results suggest that the amount of OTA in pig blood is expressive of the general exposure of the individuals to this mycotoxin and could be useful to give a general outlook over the contamination of an entire herd where the same diet was fed. P9-18 CHEMICAL CONTAMINATION OF FOOD, IN ROMANIA, 2004 C. Hura, I. Palamaru, B.A. Hura Food Toxicology Laboratory, Institute of Public Health, Iassy, Romania Ever since human have become aware that health is inseparably linked to an impact and healthy environment, the control and reduction of pollution have become the focus of worldwide concern. Investigation on possible health and environmental hazards involved have led many industrial countries to restrict or ban the use of chemicals (pesticides, heavy metals) and enforce the tolerance levels for the residues in food and feeds. The aim of the study was to investigate the variation of some chemical pollutants with cancer risk (nitrate/nitrite, heavy metals, pesticides residues) in some food (vegetables, meat, milk, daily diets) from the Romania area, in 2004. The concentration of the nitrates/nitrites and the heavy metals [Cu, Cd, Pb and Zn] were investigated in 5386 food samples (vegetables (1364 samples), meat and meat products (2743 samples), milk and dairy products (952 samples), total diets (327 samples)). The foods were harvested from the Romania area (37

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districts), in 2004. In the milk, vegetables, meat and total diets were analysed the metals (Cd, Pb, Zn and Cu) by absorption spectrophotometric method. The nitrate/nitrite was determined by colorimetric method and the pesticides residues by gas-chromatography. In all analysed samples these chemical pollutants were found. Generally, wide variations between in individual samples were observed. Nitrate/nitrite contents were, generally, in normal limits and the total diets contained quantities below acceptable daily intake. Analysis of the obtained results showed that concentrations of heavy metals in food were with the acceptable limits. The results gave emphasis that the pesticide residues are present in all food analyzed. The determinations of chemical pollutants in food are important in environmental monitoring for the prevention, control and reduction of pollution as well as for occupational health, legal, decisions and epidemiological studies. P9-19 OCHRATOXIN A CONCENTRATION AND SPHINGANINE AND SPINGOSINE RATIO IN URINE OF PERSONS FROM REGION WITH ENDEMIC NEPHROPATHY M. Peraica1 , M. Mileti´c-Medved2 , A.M. Domijan1 , R. Fuchs1 1 Unit of Toxicology, Institute for Medical Research and

Occupational Health, Ksaverska c. 2, 10000 Zagreb, Croatia; 2 Institute of Public Health of BrodskoPosavska County, Slavonski Brod, Croatia Endemic nephropathy (EN) is human fatal renal disease of unknown origin that occurs in geographically limited area of Croatia and some other countries. The most plausible theory of the aetiology of EN links it with the exposure to nephrotoxic mycotoxins through ingestion of contaminated food. In this study the concentration of mycotoxin ochratoxin A (OTA) and sphingosine/sphinganine (Sa/So) ratio, the biomarker of fumonisins exposure, were checked in human urine collected in endemic village (Kaniˇza) in Croatia and in a control village. The mean OTA concentration in urine of the group of subjects at risk (relatives of patients with EN, N = 31), group of suspected (with some signs of EN, N = 19), and in healthy persons from EN village, (N = 21) were 4.3, 1.8 and 0.9 ng/ml, respectively.


The mean concentration in control group (N = 33) was 2.1 ng OTA/ml, and was not statistically different to other groups. The mean Sa/So ratio in urine of subjects at risk, healthy subjects and in suspected was 0.82, 0.80 and 0.51, respectively. Although this ratio was the lowest in control group, (0.16) there was no statistical difference when compared to other groups. There was no correlation of OTA concentration and Sa/So ratio in urine. P9-20 ANALYTICAL OPTIONS IN THE IDENTIFICATION OF MYCOTOXINS ˇ J. Boˇsnir, D. Puntari´c, Z. Smit, A. Krivohlavek, L. ˇ Bariˇcevi´c, Z. Baklai´c, B. Jasna Department of Ecology, Zagreb Public Health Institute, Zagreb, Croatia Mycotoxins are metabolic products of various molds. In terms of diet, aflatoxins and ochratoxin A have come in the very focus of interest. These mycotoxins may occur in dry fruits and vegetables, spices and dairy products. Monitoring of mycotoxin levels in food is of great importance because of their adverse effects on human health. To present the procedure of identification and quantification of aflatoxins and ochratoxin A in particular foodstuffs by use of preliminary and confirmation techniques. A total of 350 food samples were analyzed by AOAC methods for the presence of mycotoxins. Identification of aflatoxins and ochratoxin A was performed by thin-layer chromatography (TLC) and enzymelinked immunosorbent assay (ELISA). Positive results were confirmed by the complex method of liquid chromatography–mass spectrometry (LC–MS). The maximal allowed concentration (MAC) of aflatoxins is from 0.05 ␮g/kg (infant formulas) through 30 ␮g/kg (spices). The presence of aflatoxins was suspected in 41 samples, whereas positive reaction to ochratoxin A was confirmed in 5 samples. The results obtained by screening techniques were confirmed by the complex LC–MS method. In positive samples, the concentration of aflatoxins ranged from 2 to 10 ␮g/kg. Total aflatoxin concentration exceeded MAC (>30 ␮g/kg) in two samples. The concentrations of ochratoxin A in various foods were within MAC limits (3–10 ␮g/kg).


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The reliability of analytical results for mycotoxins is directly related to the amount and representative quality of the food sample tested. P9-21 MONITORING OF PERSISTENT ORGANIC POLLUTANTS IN FOODSTUFFS J. Adamov, J. Hlpka University of Novi Sad, School of Sciences, Chemistry Department, Serbia and Montenegro In spite of bans and restrictions, organochlorine pesticides are still present in the environment and represent a serious threat to the human health and the state of the wildlife in general. Humans are exposed organochlorines mostly through their food. In this paper, monitoring of the food quality was assessed by determination of the content of organochlorine insecticides (OCI) in food samples of plant and animal origin. Lindan and its isomers were detected only in aquatic samples. Practically all investigated samples contained DDT and/or its metabolite DDE, except the sample of cheese. Almost threefold higher content of DDE in most samples is in accordance with its the metabolic pathway in living organisms It was shown that content of DDT and its metabolite DDE is the highest in the food of animal origin, due to the high lipid content. No samples exceeded the maximal residue limits for organochlorines set by FAO and national legislative. However, significant content of DDT in plants indicates more recent, probably atmospheric pollution. Relatively high content of this banned organochlorine insecticide calls for a strict policy in utilization and control of pesticides. This research was financially supported by the Ministry of Science and Environmental Protection of the Republic of Serbia, Project No. 1622. P9-22 ENVIRONMENTAL ENDOCRINE DISRUPTING CHEMICALS AND MALE REPRODUCTIVE SYSTEM A.M. Gharravi, R. Ghorbani, M. Khazaei, A. Pour Motabbad, M. Al Agha, J. Ghasemi, P. Sayadi Anatomy Department, School of Medicine, Kerman shah University of Medical Science, Kerman shah, Iran

There is presently considerable interest in exogen chemicals that cause adverse health effect in animals or humans. Consequent to change in reproductive male and female reproductive system function. Bisphenol A (BPA) a widely disturbed chemical used in making epoxy resin and polycarbonate plastics in packaging of canned food s and dental sealant. BPA has estrogenic potency. We studied effect of BPA on spermatogenesis in rats. Adult male wistar rats (30 N) exposed to several doses of BPA, 10, 50 and 100 ␮g/kg bw/day for 6 and 12 days. Moreover, sperm count determined by haemocytometre. All data were expressed as mean ± S.E. two-way ANOVA was performed. Analysis of data showed sperm counts of all examined groups in compare with control group decreased. In conclusion, these finding suggests that environmental endocrine disrupting chemical (BPA) decreased sperm production subsequently affect spermatogenesis in rats. P9-23 CORIANDER SATIVUM AND ITS MAIN OIL INGREDIENTS: IN VITRO EFFECT ON HEPG2 CELLS AND MITOCHONDRIAL RESPIRATORY CHAIN COMPLEXES J. Usta1 , K. Knio2 , S. Dagher2 , P. Barnabe1 , Y. Boumouglabay1 , S. Kreydiyyeh2 1 Department

of Biochemistry, Faculty of Medicine, American University of Beirut, Beirut, Lebanon; 2 Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon Coriander is used as an appetizer and a common food seasoning in Mediterranean dishes as food seasoning because of its flavored leaves and seeds. Extracts of this plant were claimed to be beneficial as a remedy for many ailments. This study investigates the in vitro effect of an oil extract of coriander seeds and its main components on HepG2 cell viability and rat liver mitochondrial functions. GC–MS of the oil extract identified linalool and trans-anethole as main components. The crude extract oil reduced HepG2 viability and inhibited complexes 1 and 2 of rat liver submitochondrial particles. Linalool and trans-anethole the main oil ingredients were tested as well. Whereas linalool resulted in 52% cell death at 0.4 ␮M, anet-

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hole had no effect. A 50% inhibition of both complex I (4.5 ␮M) and complex II (4.2 ␮M) by linalool was obtained. A 50% inhibition of complex I (8 ␮M) and complex II (45 ␮M) by anethole was obtained. HepG2 cell death by linalool was partially protected by: 30% GSH (1 mM); 5 3% PMSF (100 ␮M); 3 8% FK019 a caspase-3 inhibitor (20 ␮M); 2 4% FK022 a caspase-9 inhibitor (20 ␮M); and 56% FK009 a general caspase inhibitor (20 ␮M). FK030 a caspase-13 inhibitor had no protective effect. The results obtained suggest that coriander oil effect on HepG2 cells may in part be attributed to: (a) a direct effect of linalool and anethole on a proton pumping site complex-I, (b) effect on GSH level; and (c) effect on proteases and caspases 3 and 9. P9-24 FLUORIDE LEVELS IN NATURAL JUICES CONSUMED IN TENERIFE M.I. Rodr´ıguez1 , C. Rubio1 , C. Revert1 , A. Burgos2 , A. Hardisson1 1 Toxicology

Department, University of La Laguna, School of Medicine, 38071 La Laguna, S/C de Tenerife, Spain; 2 Preventive Medicine and Public Health Department. University of La Laguna, School of Medicine, 38071 La Laguna, S/C de Tenerife, Spain Fluorine is the most electronegative of all the known chemical elements, and the most reactive. The body’s main source of fluorine is undoubtedly drinking water, as this is the cheapest and most efficient way of providing it and fluoride plays an important role in human health. There are also traces of fluorine in almost all food products. The fluoride content in food is of great importance, as it can have both beneficial and prejudicial effects on our health when added to the quantities supplied by fluoride treated water and tooth-paste. The present study concentrates on fluoride provided by the intake of fruit juice. We have determined the fluoride content of the most widely consumed natural juice in Tenerife. The analytical technique used for the determination of fluoride in fruit juice is based on the direct potentiometry method. We have analysed 31 fruit juice samples. The mean concentration obtained was 0.39 mg/L (S.D. = 0.30). Only two natural juice samples presented a fluoride concentration over 1 mg/L, the reason for


these high levels is that the water used in their manufacturing process presented a fluoride concentration between 1 and 1.5 mg/L. P9-25 DETERMINATION OF FLUORIDE IN TENERIFE TEA M.I. Rodr´ıguez1 , C. Rubio1 , C. Revert1 , A. Burgos2 , A. Hardisson1 1 Toxicology

Department. University of La Laguna, School of Medicine, 38071 La Laguna, S/C de Tenerife, Spain; 2 Preventive Medicine and Public Health Department, University of La Laguna, School of Medicine, 38071 La Laguna, S/C de Tenerife, Spain Fluoride content in food is increasing by contamination from: impure phosphate fertilizers, irrigation water with high fluoride levels, fluoridation of drinking water and from excess gas and dust of polluting industries. For this reason, we also know that tea leaves have a high fluoride content. These values are above 400 mg/kg (dry weight), but tea infusions (teabags) have concentrations of around 0.5–1.5 mg/L. The amount of F− present in a cup of tea will depend not only on its volume, but also on the brand of tea, the amount of tea used, how long it is brewed for, whether or not it is a dilution of a previously prepared cup of tea and whether or not it has been made with fluoride enriched water. The F− intake of tea drinkers can vary between 0.04 and 2.7 mg per day. The analytical technique used for the determination of fluoride in natural juice is based on the direct potentiometry method. 46 tea samples were analyzed, the mean concentration obtained was 2.19 mg/L (S.D. = 1.45). P9-26 A RAPID METHOD FOR SCREENING FOR CEREULIDE, THE EMETIC TOXIN OF BACILLUS CEREUS IN FOODS M. Andersson, E.L. J¨aa¨ skel¨ainen, T. Johansson, M.S. Salonen Department of Applied Chemistry and Microbiology, POB 56, Fi-00014, Helsinki University, Finland Cereulide, the emetic toxin of Bacillus cereus is a heat stable hydrophobic depsipeptide and a serious foodpoi-


Abstracts / Toxicology Letters 158S (2005) S1–S258

soning agent. Cereulide intoxications may be underdiagnosed as a causative agent because a rapid assay for detecting the toxin in heated or pasteurized food. We developed a screening assay for cereulide and used it to resolve five cases of foodpoisonings cases where the causative agent had remained unclear in routing laboratory investigation. The test procedure is as follows: heat 2 g of food (meat stew, pasta cassarole, boiled rice, fried meat and fish stew) at 80 ◦ C in one volume (2 ml) of ethanol for 10 min. Expose 200 ␮l of boar spermatozoa, obtained as commercial extended boar semen, to 5 ␮l of the ethanolic food suspension. After exposure of 30 min investigate the motility of the sperm by phase contrast microscopy. Results: Suspensions prepared from the boiled rice, pasta cassarole and meat stew inhibited sperm motility. Suspensions prepared from the fish or the fried meat as well as two control foods did not inhibit. The sperm motility inibiting agent in the three foods was analysed by LC–MS and identified as cereulide, based on four molecular adduct ions. The meat stew contained 1.35 ␮g cereulide g−1 , the pasta casserole 0.3 ␮g g−1 and in the boiled rice approximately 0.1 ␮g g−1 . This shows that the rapid screening assay described here detected the food poisoning agent in three out of five unsolved food poisonings, and that it gave the same result as did the more laborious LC–MS assay.

Turkey, Ankara. For this purpose, 39 samples of cheese and 40 samples of yogurt were randomly collected from supermarkets in Ankara. Aflatoxin M1 levels were determined by competitive enzyme-linked immunosorbent assay (ELISA) kit. The limits of detection were 50 ng/l for cheese, and 60 ng/kg for yogurt. Levels of aflatoxin M1 in 11 cheese samples were detected. These were ranging from 32.18 to 188.44 ng/l. Thirty-two of the 40 yogurt samples had aflatoxin M1 levels between 61 and 365 ng/kg of yogurt. According to EC Regulation 2174/2003, actual limits of aflatoxin M1 contamination both in milk and dairy products including cheese and yogurt is 50 ng/kg. Therefore, 10 samples of cheese and 34 samples of yogurt were over this limit. The results of this study indicated the necessity of frequent monitoring surveys in dairy products.


The increased use of organophosphorus insecticides in agriculture, inside homes and schools, and widespread existence in the environment poses a potential health hazard. As the use of these agents expands, acute and chronic exposure has become more common. Like other organophosphates, chlorpyrifos kills insects and other animals, including human being itself because of its toxicity to the nervous system. Exposure of organophosphates in pregnant women is an important clinical entity because of its effects on two organisms, both mother and baby. There are limited reports about fetal toxicity of organophosphates in the literature because of relatively less reported cases. In this paper we reported a case with chlorpyrifos intoxication, an organophosphorus compound, during pregnancy, causing fetal death.

A. G¨urbay, A.B. Engin, A. C¸a˘glayan, G. S¸ahin Hacettepe University, Faculty of Pharmacy, Department of Toxicology, 06100 Sıhhiye, Ankara, Turkey Milk products such as cheese and yogurt are main nutrients of Turkish population for centuries. On the other hand, contamination of foods including dairy products with aflatoxins is considered as a potential risk for human health. Since there is a limited data about aflatoxin M1 levels in dairy products in Turkey, determination of its level in these type of food samples is important. Therefore, in the present study, the aim was to investigate the presence of this toxin in various types of cheese, and yogurt samples consumed in capital city of

P10 Development Toxicity P10-01 ORGANOPHOSPHATE POISONING ASSOCIATED WITH FETAL DEATH A. Sebe, S. Satar, R. Alpay, N. Kozacı, A. Hılal, A. Avcı 1 Cukurova

University, School of Medicine, Department of Emergency Medicine, Balcali, Adana, Turkey; 2 Cukurova University, School of Medicine, Department of Forensic Sciences, Balcali, Adana, Turkey

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P10-02 EFFECTS OF IN UTERO AND LACTATIONAL EXPOSURE TO LOW DOSES OF PCB126 AND PCB 153 ON BONE TISSUE IN FEMALE GOAT OFFSPRING R. Lundberg1 , P.M. Lind1 , M. Wikstr¨om2 , J.U. 2 , J.L. Lyche4 ¨ Skaare3,4 , E. Ropstad3 , J. Orberg 1 Institute of Environmental Medicine, Karolinska Insti-

tutet, Stockholm, Sweden; 2 Department of Environmental Toxicology, Uppsala University, Uppsala, Sweden; 3 The Norwegian School of Veterinary Science, Oslo, Norway; 4 National Veterinary Institute, Oslo, Norway Results presented previously have shown that bone tissue is a target for endocrine disrupting chemicals. The aim of this study was to investigate whether environmental levels of the putative estrogenic non-dioxin like PCB 153 and the dioxin-like PCB 126 cause changes in bone composition and dimensions in female goat offspring. Goat dams were dosed orally with PCB 153 in corn oil (98 ␮g/kg body wt/day) and PCB 126 (49 ng/kg body wt/day) during gestation. Bone composition and dimensions were determined in the offspring by peripheral quantitative computed tomography (pQCT) on excised metacarpus. PCB153 exposure significantly decreased the total cross-sectional area of the diaphysis (125 ± 4 mm2 (n = 7) versus nonexposed (142 ± 5 mm2 (n = 7)) and increased trabecular bone mineral density of the metaphysis (121 ± 5 mg/cm3 (n = 7) versus nonexposed (111 ± 3 mg/cm3 (n = 7)). PCB126 did not produce any observable effects in this study. In conclusion, in utero and lactational exposure to PCB 153 may result in altered bone composition in female goat offspring. P10-03 RAT BRAIN MONOAMINERGIC NEUROTRANSMISSION PARAMETERS AFTER PERINATAL EXPOSURE TO METHYLMERCURY AND PCB153 T. Coccini1 , F. Blandini2 , L. Manzo1,3 , A.F. Castoldi1 1 Toxicology

Division, IRCCS Maugeri Foundation, Italy; 2 Functional Neurochemistry Laboratory, IRCCS Mondino, Pavia, Italy; 3 University of Pavia, Pavia, Italy


The central monoaminergic system is a target for the common seafood contaminants methylmercury (MeHg) and polychlorinated biphenyls. This study explored, in 21-day-old rat pup brain, the individual and joint effects of MeHg (1 mg/kg/day, GD7-PND7) and PCB153 (20 mg/kg/day, GD10GD16), given orally to rat dams, on the activity of monoamine oxidase B (MAO-B; determined radiochemically) and on dopamine (DA), serotonin (5-HT), 5-hydroxy-indolacetic acid (5-HIAA) and homovanillic acid (HVA) levels measured by HPLC. Neither treatment elicited changes in MAO-B in striatum, hippocampus, cerebellum and cerebral cortex of female pups. At variance, in males the cerebellum displayed a significantly reduced enzyme activity (25–45%) following all treatments. In the cerebellum of all pups, 5-HIAA and HVA levels (+40%) were enhanced by MeHg, while 5-HT levels (+133%) by PCB153. In the striatum, PCB153 alone and in mixture diminished to a similar extent the content of DA, HVA and 5-HIAA (20–40%). In the hippocampus, MeHg and PCB153 alone reduced the concentration of both monoaminergic metabolites (30–55%), without synergistic effects after combined exposure. No changes were observed in cortex. Sub-cortical dopaminergic and serotoninergic systems are affected by developmental exposure to MeHg and/or PCB153 in a different manner according to the brain area considered. The combined exposure does not seem to exacerbate the effects of the individual compounds (EU Grants: QLK4-CT-2001-00186; FOODCT-2003-506543). P10-04 BRAIN DOPAMINERGIC AND CHOLINERGIC RECEPTORS IN PUBERTAL RATS PERINATALLY EXPOSED TO METHYLMERCURY AND PCB153 A.F. Castoldi1 , T. Coccini1 , E. Roda2 , L. Marastoni2 , L. Manzo1,2 1 Toxicology

Division, IRCCS Maugeri Foundation, Pavia, Italy; 2 University of Pavia, Pavia, Italy

The dopamine and muscarinic receptors (MRs) represent targets for the neurodevelopmental contaminants methylmercury (MeHg) and PCBs. The effects of the oral administration to rat dams of MeHg


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(0.5 mg/kg/day) and/or PCB153 (5 mg/kg/day) from GD7 to PND21 were investigated on the density (Bmax ) of cortical and striatal dopamine D2-like receptors (D2R) and MRs in the rat offspring on PND36 by saturation binding studies using 3 H-YM-09151-2 and 3 H-QNB as specific ligands, respectively. In striatum a common finding to males and females (control Bmax = 200 ± 54 and 189 ± 42 fmol/mg protein) was the MeHg-induced 10% decrease in D2-R density, in the absence of any effect elicited by PCB153, either alone or combined. In the cerebral cortex of both genders (control Bmax = 48–57 fmol/mg protein), PCB153 augmented D2-Rs (+50%), MeHg was uneffective and the co-exposure mimicked the PCB-like effect. Concerning striatal MRs, females but not males displayed a 20% decrease in Bmax (controls 1250 ± 350 and 1360 ± 380 fmol/mg protein) following MeHg exposure. The opposite gender-susceptibility was observed after PCB153 exposure. Cortical MRs of all pups were decreased to a similar extent (10–20%) by MeHg and PCB153, administered either alone or combined. Low doses of MeHg and PCB153 administered prenatally and lactationally affect both the dopaminergic and muscarinic systems in a gender- and brain area-dependent manner without additivity (EU Grants: QLK4-CT-2001-00186; FOOD-CT-2003-506543).

animals prenatally exposed to MeHg showed approach latencies that did not differ significantly with respect to controls. However, when the trial was repeated 24 h later, the avoidance latencies of animals exposed on GD8 were significantly shorter than those of controls (p < 0.005, Kruskal–Wallis test). Furthermore, during the active avoidance task, MeHg-treated rats exhibited a significant impairment in the acquisition of an active avoidance schedule (GD 8: p < 0.001; GD 15: p < 0.05, Tukey’s test), suggesting an impairment of learning ability. The animal’s sensitivity to footshock in the hot plate test and motor ability in the open field test were not affected by prenatal MeHg exposure, thus indicating that the impairment in cognitive processes could be attributed to an altered hippocampal function.


Nofer Institute of Occupational Medicine, 8 Teresy St, 90-950 Lodz, Poland

A. Coluccia1 , P. Borracci1 , M.R. Carrat`u1 , V. Cuomo2 1 Department

of Pharmacology and Human Physiology, Medical School, University of Bari, Italy; 2 Department of Human Physiology and Pharmacology “V. Erspamer”, University “La Sapienza”, Rome, Italy Developmental exposure to methylmercury (MeHg) increases the risk of learning and memory deficits in adulthood. In the present study, we report the effects of a single oral dose of MeHg administered to Sprague–Dawley rats on gestational days (GD) 8 and 15. The behavior of 90-day-old offspring was tested in both passive (1) and active avoidance apparatus (2). In passive avoidance test, during the acquisition trial

References: Kopf et al., 2001. Brain Res. 894, 141–144. Thompson and Rosen, 2000. Neuropeptides 34, 38–44.


Mercury is a common environmental pollutant. In microbial soil and aquatic systems it is being biotransformed into methylmercury (MeHg) and as such it enters the food chain. MeHg is highly neurotoxic particularly at the early developmental stages. The purpose of the present work was to study the effect of MeHg exposure during gestation and lactation on the postnatal offspring’s development. Virgin female rats were mated and, starting from day 7 of pregnancy to day 21 post partum they were exposed orally to MeHg at doses 0.5 or 2.0 mg/kg/day. A battery of tests was used for assessment of the morphological and functional offspring development. The reproductive performance of dams was not overtly affected by the exposure. In the offspring of the high-dose group, the growth rate was significantly retarded, the pinna detachment was delayed and the incisor eruption was significantly hastened. In the negative geotaxis test the pups of the low-

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dose group performed better than their counterparts in the remaining groups. In the forepaw suspension test no significant differences between groups were noted, and in the free-fall righting test a greater proportion of pups of the high-dose group was delayed in developing the skill of 4 ft. landing. In rats, like in humans, a high-dose maternal MeHg exposure affects negatively morphological and functional development of the offspring. The low-dose maternal exposure has no overt adverse effect on the morphological and functional development of the offspring at the early stage of life. This study was performed within the frame of the “DEVNERTOX” project supported by the European Commission: Contract 6PR/03/506143. P10-07 GLYCINE REDUCES CADMIUM-INDUCED TERATOGENIC DAMAGE IN MICE N. Paniagua, G. Escalona, G. Chamorro Pre-clinical Toxicology Laboratory, National School of Biological Sciences, National Polytechnic Institute Mexico City, Mexico Pregnant animals exposed to cadmium (Cd) show a broad spectrum of adverse effects on both embryos and fetuses. On the other hand, glycine has been reported to protect against Cd toxicity by reducing oxidative stress. Accordingly, the purpose of this study was to determine whether glycine protects from Cd-induced teratogenicity in mice. Cd was administered as cadmium chloride subcutaneously at 1, 2 or 4 mg/kg doses on gestation days (GD) 8, 9 and 10. Glycine was given ad libitum (in the drinking water) from GD1 through GD19 (the day when animals were killed), as a 1% and 2% drinking water solution. Cadmium and nucleic acid concentrations in embryos were determined. The most common finding seen after cadmium 4 mg/kg exposure was exencephaly. The incidence of this malformation was significantly reduced in mice receiving 2% glycine while fetal cadmium significantly decreased as compared to control animals. Increased nucleic acid levels were seen in the same embryos. In non-supplemented mice given Cd 4 mg/kg, embryonic lipid peroxidation proved to be increased. In conclusion, lipid peroxidation appears to play a major role in cadmium-induced teratogenicity and glycine has a protective effect from it due to its oxidative process inhibition. However, further


detailed studies are needed to establish the mechanism(s) of action. P10-08 PYRAZINAMIDE EFFECTS ON MALE RAT’S REPRODUCTIVE FUNCTION V.M. Kovalenko, L.B. Bondarenko, T.F. Byshovetz, G.M. Shayakhmetova, A.K. Voronina, O.S. Voloshina, N.A. Saprykina Institute of Pharmacology and Toxicology, Academy of Medical Sciences of Ukraine, Kiev, Ukraine In spite of broad utilization of pyrazinamide in tuberculosis and AIDS treatment schemes its effects on reproductive function and posterity remains insufficiently investigated and present results are discrepant. Effects of pyrazinamide (500, 1000 and 2000 mg/kg bw) on liver and testis biochemical parameters and reproductive toxicity indices have been studied in experiments on Wistar rats. It was shown that pyrazinamide caused dose dependent increasing of p-nitrophenol hydroxylase activity (from 1.4 to 2.3 times), cytochrome P-450 contents (from 1.3 to 1.8 times) and NADPH-dependent lipid peroxidation (to 1.5 times) in liver microsomal fraction with simultaneous decrease of DNA (to 30%), RNA (to 34%), total histones (to 49%), phospholipids (to 31%), esterified cholesterol contents (to 47%) in testis. Ratio DNA/histones was increased from 1.2 to 1.4 times. Pyrazinamide introduction produced spermatozoid number decreasing in epididymis in dependence of dose. Decrease of spermatogonia number and compensatory increase of 12th meiosis stages with synchronous dystrophic changes of spermatogenic epithelia also were registered at these conditions. Pyrazinamide caused dose dependent increase of preimplantational embryos death and postnatal newborn death in different terms. P10-09 INHIBITORY EFFECT OF ETHYLENE GLYCOL MONOETHYL ETHER ON SPERM MOTION IN RAT R.S. Wang, K. Ohtani, M. Suda, T. Honma National Institute of Industrial Health, Kawasaki 2148585, Japan


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Ethylene glycol monoethyl ether (EGEE) can cause testicular toxicity, as demonstrated by testicular atrophy and decreased sperm count. To elucidate whether EGEE has any effect on sperm motion, especially in the case of short time exposure, we conducted a series of in vivo experiments with rats, as well as an in vitro study with rat sperm. Sperm from cauda epididymis and spermaduct was analyzed for the change in motion with Hamilton-Thorne Sperm analyzer. Administration of EGEE at 600 mg/kg/day for 5 weeks significantly decreased total and progressive motility of sperm to 15–30% of controls, in both the cauda epididymis and the spermaduct. Decreased curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL) were also observed in epididymal sperm. In the acute experiment, abnormal motion of sperm was detected by decreased total and progressive motility, and straightness. Percentage of sperm with rapid velocity significantly declined, but that of static sperm rose. The time-course experiment showed that damage to sperm motion began 6 h after EGEE administration and reached its most remarkable level at 12–24 h. Addition of EGEE to the medium of sperm had no effect on its motion, but EAA showed inhibitory effect at moderate degree. These results suggest that besides its toxicity to spermatogenesis, EGEE may also directly affect the motion of mature sperm. P10-10 REPRODUCTIVE/DEVELOPMENTAL TOXICITY SCREENING TEST OF 1,3-DIO-TOLYLGUANIDINE IN RATS M. Ema1 , E. Kimura2 , A. Hirose1 , E. Kamata1 1 Division

of Risk Assessment, Biological Safety Research Center, National Institute of Health Sciences, Tokyo, Japan; 2 Panapharm Laboratories Co., Ltd., Kumamoto, Japan 1,3-Di-o-tolylguanidine (DTG) is used as a basic rubber accelerator. The reproductive and developmental toxicity of DTG was determined according to an OECD 421 reproduction/developmental toxicity screening test protocol. SD rats were given DTG by gavage at 0, 8, 20 or 50 mg/kg/day. Males were dosed for 49 days from day 14 before mating. Females were dosed from day 14 before mating to day 3 of lactation throughout the

mating and pregnancy period. Deaths were found in two males and three females at 50 mg/kg/day. Lower body weight accompanied by reduced food consumption was also noted at 50 mg/kg/day. Salivation, mydriasis, hypoactivity, bradypnea, prone position and/or tremor were observed in males and females at 20 and 50 mg/kg/day. No effects of DTG were found in estrous cyclicity, precoital interval, copulation, fertility and gestation indexes, numbers of corpora lutea and implantations, and gestation length. Number, body weight and viability of offspring were decreased at 50 mg/kg/day. At this dose, incidence of fetuses with external malformations was increased. Oligodactily, anal atresia and tail anomalies were observed. The data show that DTG is reproductive toxic at general toxic dose. DTG is suggested to be teratogenic at maternal toxic dose. The NOAELs of DTG for general and reproductive/developmental toxicity were 8 and 20 mg/kg/day, respectively, in rats. P10-11 THE EFFECTS ON SKELETAL DEVELOPMENT OF PRE-INCUBATION OF FORMALDEHYDE FUMIGATION AT CHICK EMBRYOS S. Hayretda˘g, D. Kolankaya Hacettepe University, Department of Biology, Beytepe Campus, 06532, Ankara, Turkey In this study, the possible effects of formaldehyde fumigation on the hatching eggs from fumigated chicken eggs were aimed. Pre-incubation fumigation was applied once and was done at two different concentration (3X and 4X per m3 ) and two different periods (20 and 40 min). At the end of incubation, embryo mortality in early (0–5 days), middle (6–17 days) and last developmental period (18–21 days) and developmental anomalies occurred in death embryo were established. At the end of the investigation, increase in embryo mortality in the early and last developmental period, decrease in hatchability and skeletal malformations such as crooked in vertebra, delay of chondrogenesis and osteogenesis, cross bill and abnormal eye development in death embryos were detected depending on the increase in fumigation concentration and periods.

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Research Institute, Chonbuk National University, Jeonju, South Korea; 2 College of Veterinary Medicine, Chonbuk National University, Jeonju, South Korea Embryonic movements (EM) are considered to be the first sign of life sensed by mothers and cigarette smoking during pregnancy has been linked to affect the EM. Exposure to sidestream smoke, produced from the emissions of a smoldering cigarette, may result in poor pregnancy outcome. In this study, the chicken embryo bioassay was used to systematically assess the effects of short-term exposure to sidestream whole smoke solutions (SSWSS) of 6-day-old chicken embryos. EM was recorded in real time by a video camera for 60 min and each motion was counted for every 3 min interval. Application of SSWSS, obtained from five commercial cigarettes of different nicotine concentration; 0.2, 0.5, 0.6, 0.7 and 1 mg to the embryos resulted to significant changes in all four types of EM of the head, tail, whole body and swing-like movements. Significant reduction (P < 0.01) in swing-like movements and whole body movements were observed in all SSWSStreated embryos. Highly significant (P < 0.001) decline with incomplete recovery of all four types of EM were observed by application of SSWSS from cigarette containing 1 mg nicotine. This study has established a link between exposure of SSWSS and substantial decrease in EM. P10-13 EFFECTS OF PRENATAL EXPOSURE TO DAIDZEIN OR 17-ALPHA ETHINYLESTRADIOL ON OVARIAN SURFACE EPITHELIAL CELL HEIGHT Ch.E. Talsness, J. Nowak, K. Grote, S.N. Kuriyama, I. Chahoud Institute of Clinical Pharmacology and Toxicology, Department of Toxicology, Charit´e Universit¨atsmedizin-Berlin, Germany


The ovarian surface epithelium (OSE) must proliferate to restore the ovarian surface after ovulation. The majority of ovarian cancer (85–90%) originates from this site. Epidemiological studies suggest that reduction in ovulations through oral contraceptive use or pregnancy is protective against the development of epithelial ovarian carcinoma. One of the factors to place women at increased risk for ovarian malignancy is long-term use of estrogen-only replacement therapy. We evaluated whether prenatal exposure to the phytoestrogen, daidzein, and the synthetic estrogen, 17-alpha ethinylestradiol (EE), alters the height of the OSE. Pregnant Sprague–Dawley rats were treated with vehicle, 5 or 60 mg daidzein/kg/day or 0.002 mg EE/kg/day on gestation days 6–21. Females were sacrificed in estrous at approximately 4.5 months of age and the OSE was measured. We observed a significant 12% reduction in OSE following exposure to 0.002 mg EE (N = 9 ovaries) or 5 mg daidzein (N = 8) compared to their respective controls (N = 4–5). A 27% reduction occurred following exposure to 60 mg daidzein (N = 8) compared to control (N = 5). Prenatal exposure to a synthetic or phyto-estrogen affects the OSE in a persistent manner. This decrease in epithelial cell height is in contrast to the proliferative responses reported for postnatal exposures to 17␤-estradiol (Stewart et al., 2004) or methoxychlor (Borgeest et al., 2002). Funding: 07HOR01/7-BMBF. P10-14 IMPACT OF A NEW ANTI-TUMOROUS ESTROGENE COMPOUND ONTO THE RATS’ REPRODUCTIVE SYSTEM M. Kudrya, I. Palagina, N. Melnikovskaya Institute of Endocrine Pathology Problems, Kharkov, Ukraine One of the new effective compounds created for treatment of prostate cancer and adenoma is 3-oxyestra1,3,5-trien-17-hydrazone (OETH). This drug possesses the prolonged anti-tumorous action as well as estrogenic and anti-androgenic properties. Aim of this study was to analyze OETH’s impact on generative function of rats. Experiments were conducted over 52 female and 60 male Wistar rats. Female rats were exposed to OETH by its placement on skin in doses of 20 and 100 mg/kg during 20 days. Male rats inhalated OETH


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one-fold in concentrations of 0.83, 3.6 and 11.9 mg/m3 . We found that OETH’s impact on female rats’ reproductive system was expressed in changes of the estrous cycle duration and phase structure and in inbition of hypophysis’ gonadotropic function. Tentative animals had lower progesteron level, and ratio between estradiol/progesteron shifted toward estradiol. Inhalation of OETH by male rats notably worsened the spermatozoa functional status (we fixed reduction of their mobility time and quantity) and decreased spermaries’ relative weight. Study concluded in forecasting of OETH’s possible adverse effects on human reproductive system and in elaboration of preventive measures.

made to increase to over 50% by dibromoacetic acid, trichloroacetonitrile, bromoacetonitrile, dichloroacetonitrile, propiconazole, azoxystrobin were decreased. These results indicate that many kinds of environmental pollution chemicals cause adverse effect during neutrophilic differentiation.


Reproductive and Developmental Toxicology Division, National Institute of Toxicological Research, Korea FDA 5, Nokbun-dong, Eunpyung-gu, Seoul 122-704, South Korea

T. Nishimura, K. Shimizu, R. Kubota, M. Tahara, H. Tokunaga

Alcohol consumption during pregnancy results in morphological abnormalities in the fetuses of humans and experimental animals, and is referred to as fetal alcohol syndrome (FAS). However, the molecular mechanism underlying FAS has not been completely elucidated. The aim of the present study was to investigate the potential molecular mechanisms of ethanol-induced FAS in the developing embryo and fetus. cDNA microarray analysis was used to screen for altered gene profiles. Ethanol at a teratogenic dosage (3.8 g/kg, twice a day) was administered intraperitoneally to pregnant C57BL/6J mice from gestation days (GD) 6 to 8. Morphologic observations showed excessive malformations of the craniofacial regions (reduction of the face, the absence of eyes, nose, jaw and mandible, underdevelopment of vibrissae areas, cleft lip, and palate) in ethanol-exposed embryos and fetusus (GD 10–15). cDNA microarray analysis showed alterations in several genes profiles, including the “palate, lung, and nasal epithelium clone (plunc)”, “neurofilament (nfl)”, and “pale ear”. Of these genes, the expressions of plunc were confirmed by RT-PCR and whole mount in situ hybridization (WISH). The plunc was highly expressed in the craniofacial region, specifically in upper airways and nasopharyngeal epithelium. RT-PCR analysis revealed that normal plunc mRNA expression levels were present in GD 14 and 15 fetuses, but not GD 10–13 embryos. Interestingly, ethanol significantly down-regulated the plunc expres-

Division of Environmental Chemistry, National Institute of Health Sciences, Tokyo, Japan Environmental pollution chemicals are thought to cause adverse effect for the development or maturation of reproductive organs during ontogeny. The immune, endocrine and nervous systems are interlinked and many of the same cytokines and their receptors are expressed by three systems. Therefore, it is important to investigate the effect of environmental pollution chemicals on maturation process of immunocompetent cells in order to understand the effect of the chemical during ontogeny. We investigated the effect of environmental pollution chemicals on the neutrophilic differentiation of human promyelocytic HL60 cells induced by dimethyl sulfoxide and granulocyte colony-stimulating factor. Fifty chemicals, including endocrine disrupting chemicals, chlorophenols, haloacetic acids, halo-acetonitriles and pesticides, were examined at the concentration of 20% or less in the lethality of cells. CD18, which was the specific and inducible expression protein in neurtrophic cells, was evaluated the effect as an index. The expression levels of CD18 were made to decrease to over 25% by toriclosan, 4-chlorophenol, bromoacetic acid, chlorpyrifos, metalaxyl were decreased at the concentration of 20% or less. The expression levels of CD18 were


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sion in GD 14 and 15 fetuses. Our results suggest that ethanol-induced FAS is due in part to the downregulation of plunc expression in the fetus and this gene may be a candidate biological marker for FAS. Also, we observed stage-specific proteome alterations of developing embryo and fetus during organogenesis. Proteome alterations were observed using twodimensional polyacrylamide gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). The proteins also revealed in this experiment might be considered novel FAS biological marker candidate. P10-17 SUSCEPTIBILITY OF NEWBORN RATS TO SIX CHEMICALS, COMPARED WITH YOUNG RATS R. Hasegawa, M. Hirata-Koizumi, M. Takahashi, E. Kamata, M. Ema National Institute of Health Sciences, Tokyo, Japan To elucidate the susceptibility of newborn rats to chemicals, newborn and young rats were administered six industrial chemicals by gavage from postnatal days 4 to 21 and for 28 days starting at 5–6 weeks of age, respectively, under the same experimental conditions as possible. For the appropriate comparison, presumed NOAEL (pNOAEL) was estimated as an expected no adverse effect dose based on both main and dose-finding studies and presumed unequivocally toxic level (pUETL) was speculated as the equivalently toxic intensity dose between newborn and young rats. Ratios of pNOAELs for young/newborn rats were 5.0 for 2-chlorophenol, 1.0 for 4-chlorophenol, 3.3 for p-(␣,␣-dimethylbenzyl) phenol, 0.4 for (hydroxyphenyl)methyl phenol, 0.2 for trityl chloride, and 3.0 for 1,3,5-trihydroxybenezene. To speculate the most likely equivalent pUETLs between newborn and young rats for specific purpose in this analysis, the range of dose was adopted in several cases because of limited experimental doses. Ratios of pUETLs for young/newborn rats were 4–5 for 2chlorophenol, 1.7 for 4-chlorophenol, 2.3–2.7 for p(␣,␣-dimethylbenzyl) phenol, 6.3–7.1 for (hydroxyphenyl)methyl phenol, 0.6–0.8 for trityl chloride, and 2.0 for 1,3,5-trihydroxybenezene. In most cases, newborn rats were two to five times more susceptible than


young rats in term of both pNOAEL and pUETL. Exceptionally, young rats were clearly more susceptible than newborn rats for trityl chloride. P10-18 ROLE OF JUVENILE ANIMALS IN ASSESSMENTS OF PEDIATRIC SAFETY D.J. Sandler ILSI Health and Environmental Sciences Institute, One Thomas Circle, NW, Ninth Floor, Washington, DC 20009, USA The ILSI Health and Environmental Sciences Institute’s Developmental and Reproductive Toxicology Technical Committee have undertaken a project to provide information and data for developing sound science pertaining to juvenile animal studies. Reviews on clinically relevant endpoints of comparative postnatal development of several organ systems, including skeletal, renal, pulmonary, female reproductive, male reproductive, cardiovascular, immune, and behavioral aspects of the central nervous system, have been published as part of Phase I of the project, in Birth Defects Research, Part B—Developmental and Reproductive Toxicology. A review of postnatal development of the gastro-intestinal system is in press, and a review of postnatal anatomical development of the central nervous system is under development, and will be submitted for publication. A review of common clinical manifestations of pediatric toxicity and their relationship to juvenile animal testing is also planned. A workshop was held in Washington, DC, as part of Phase II of the project, to provide a forum for discussion of the processes to determine when juvenile animal studies are necessary, and to propose appropriate study designs and testing strategies for these studies. More than 100 scientists from industry, academia, and government agencies from the U.S., the E.U., and Canada participated. Proceedings of the workshop were also published in Birth Defects Research, Part B—Developmental and Reproductive Toxicology, in 2004. This poster presentation will include conclusions reached at the HESI workshop, and information on the reviews of comparative postnatal development and relevance of clinical manifestations of pediatric toxicity to juvenile animal testing.


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P10-19 COMPARATIVE STUDY OF ABORTION BETWEEN OPERATION ROOM WORKERS AND THE WORKERS OF OTHER WARDS OF HOSPITALS IN AHVAZ, IRAN R. Kalantarhormozi, S. Simin Ahvaz Jondishapour Medical Sciences University, Iran Several factors including chromosomal disorders, in creasing maternal age, multi parity and other factors might be causes of abortion. One of the important factors of abortion is environmental toxin. The effect of some compounds such as nitrous oxide on spontaneous abortion has been reported. Material and method: It was a case control study conducted on 40 workers in each of operating room and the other wards. Confounding variables such as age, sex and some of the other risk factors of abortion were matched and the prepared questionnaires were filled by all participants. The data were analyses by using t-student test and Mantel-Haenzel test. The mean age of workers in operation room was 31 ± 2 and in the other wards was 32 years. Both of groups worked in their ward at least for 1 year and the mean of it was 8. The rate of abortion in case group was 24.2% and in control it was 10.2%, statistical tests showed significant difference between two groups (p < 0.05). In this study the rate of abortion between case and control group was significant. It seems that to be exposed by anesthesia gases could be a risk factor for abortion among the workers of operation room. Further investigations regarding this hypothesis strongly recommended. P10-20 SEMEN QUALITY IN POLAND E. Tyrkiel1 , A. Hernik1 , K. G´oralczyk1 , P. Milewczyk2 , J.K. Ludwicki1 , Inuendo3 1 National

Institute of Hygiene, Warsaw, Poland; and Obstetric Hospital of the Warsaw School of Medicine, Warsaw, Poland; 3 2 Gynecological

Data on semen collected from reports published worldwide indicate clearly that sperm quality has declined

appreciably during 50 last year. Such remarkable changes over a relatively short period are more probably due to environmental rather than genetic factors. It has become evident that chemical and physical agents can impair the male reproductive capability by action at several sites—in the testis, or in organs which have a role in the proper testicular function. The recent hypothesis suggested that male infertility, testicular cancer, cryptorchidism and hypospadias share a common etiology as a part of the so-called testicular dysgenesis syndrome (TDS). TDS was suggested to be caused by “endocrine disrupters”. The semen quality in the polish population was studied in preliminary investigate. Semen samples were analyses for motility and sperm concentration according to a manual for the project inuendo based on the whom manual for basic semen analysis. Total 193 men participated in the semen study (from January 2003 to March 2004). Participants were divided to four age groups. Additionally, semen samples from 13 man claiming infertility have been analyses. The main results of the present study indicate the relationship between semen quality and age of the participants. These results show, that when the role environmental factors in semen quality is analyses the age of donors should be taken into account. P10-21 COMPARATIVE INVESTIGATION OF SEVERAL SPERM ANALYSIS METHODS FOR EVALUATION OF SPERMATOTOXICITY OF 2BROMOPROPANE K. Ohtani, H. Kubota, J. Saegusa National Institute of Industrial Health, Independent Administrative Institution, 6-21-1 Nagao, Tama-ku, Kawasaki, Kanagawa 214-8585, Japan Reproductive toxicity of 2-bromopropane (2BP) was reported to be found among the workers in an electronics factory in 1995. Therefore the importance of spermatotoxicity has been realized in the industrial toxicology. However, classical manual methods must rely on subjective assessment. Recently, computer-assisted sperm analysis (CASA) was proposed but this system requires vast investment. We then investigated the applicability of the MTT method with a microplate and sperm quality analyzer (SQA) as simple, rapid,

Abstracts / Toxicology Letters 158S (2005) S1–S258

and economic instrumental methods for the examination of sperm quality in rats, comparing it with the manual microscopic method and CASA. Epididymal fluid derived from male F344/N Slc (Fischer) rats intraperitoneally injected with 2BP in the dose range of 125–1000 mg/kg/day twice a week (total eight times) were examined by these methods as a model experiment. Sperm count measured by the manual method and CASA in the epididymal fluid, absorbance by the MTT method and sperm motility index value by the SQA method were significantly lower in the 2BP 1000 mg/kg administered group than in the control group. Sperm analyses by the MTT method with the microplate reader and the SQA method are available for reproductive toxicity study in rats. P10-22 EFFECT AND TOXICITY OF 14F7 MONOCLONAL ANTIBODY LABELED WITH REIUM 188 IN BALB/C MICE WITH PX63 MYELOMA CELLS B.O. Gonz´alez1 , A. Casaco, R. Leiva, N. Subiros, A. Perera, M. Leon, O. Hernandez 1 Expositor

address, National for Breeding of Laboratory Animals Center, CENPALAB, Finca Tirabeque, Km 2½ Carretera a Cacahual, Bejucal, La Habana, Cuba The 14F7 murine monoclonal antibody (MAb) is an IgG1 that was generated by immunizing Balb/c mice with GM3 (NeuGc) ganglioside hydrophobically conjugated with human very-low-density lipoproteins and in the presence of Freund’s adjuvant and recognized human melanoma and breast tumors. The antibody was developed at the Center of Molecular Immmunology, Havana Cuba. In this study we evaluated the toxicity and effectivity of the anti-tumoral treatment; by administration iv a single dose of the monoclonal antibody 14F7 labeled with 188 Re in BALB/c mice; with PX63 murine myeloma cells, to obtain ascitic tumor. At day zero of treatment: using a small dose of protein (25 ␮g of 14F7) and eight dose levels of radionuclide (175–700 ␮Ci of 188 Re) The treatment effect was noted in the decrease of body weight at day seventh in all groups treated with RAIT and its repercussions upon the tumoral growing at 14 days. The doses 550, 625 and 700 ␮Ci showed a higher survival and a raising tumoral


effect. The Hb decreased in all groups; and reached the lower values in the higher doses at 14th days. The total leucocytes decreased in a same time but starting from the dose of 325 ␮Ci. These results shed light on the efficiency of the treatment against murine tumors, and the 14F7-188 Re toxicity, contributing to the development of the RAIT clinical for solid tumors. P10-23 OECD VALIDATION OF THE HERSHBERGER ASSAY IN JAPAN: BLIND STUDY USING CODED CHEMICALS K. Yamasaki1 , R. Ohta2 , H. Okuda3 1 Chemicals

Evaluation and Research Institute, Oita, Japan; 2 Food Drug Safety Center, Kanagawa, Japan; 3 Japan Bioassay Research Center, Kanagawa, Japan The OECD is developing new guidelines for the screening and testing of potential endocrine disrupters. The Hershberger assay was selected for validation based on the need for an in vivo screening method to detect androgen agonists or antagonists. The assay measures the response of five sex accessory organs to the test chemical in castrated juvenile male rats. Phases 1 and 2 of the Hershberger validation program have been successfully completed. Phase 3, the final stage of the validation program, has also been performed, using coded androgen agonists and antagonists. Three Japanese laboratories contributed to the Phase 3 validation studies of the Hershberger assay using coded chemicals. All chemicals were orally administered for 10 consecutive days. In the agonist version of the assay, all sex accessory organs consistently responded with significant changes in weight within a narrow window among the laboratories. In the antagonist version of the assay, 0.2 mg/kg/day of testosterone propionate was coadministered by subcutaneous injection, and the results showed that all sex accessory organ weights consistently responded within a narrow window among the laboratories. Therefore, the Japanese studies support the use of the Hershberger assay as a suitable screening assay for the detection of androgen agonistic and antagonistic effects.


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P10-24 PERSPECTIVES OF COMBINED RADIOIMMUNOTHERAPY AND ANTI-EGFR MONOCLONAL ANTIBODY, H-R3, THERAPY FOR THE TREATMENT OF SOLID TUMORS OF NEUROEPITHELIAL ORIGEN B. Gonzalez-Navarro1 , A. Casaco2 , N. Subiros1 , R. Leyva3 , M. Leon3 , O. Hernandez1 , E. Hernandez1 , I. Bosulei2 , A. Perera4 , M. Legro1 1 National

for Breeding of Laboratory Animals Center, CENPALAB, Finca Tirabeque, Km 2½ Carretera a Cacahual, Bejucal, La Habana, Cuba; 2 Center of Molecular Immunology, Cuba; 3 Isotopes Center, Cuba; 4 Clinical Research Center, Cuba The humanized anti-epidermal growth factor receptor (EGF-R) monoclonal antibody (MAb), h-R3, labeled with Rhenium 188 administered intratumorally may have potential for the treatment of patients bearing high grade tumors of neuroephitelial origin in CNS. In an effort to enhance the efficacy of radioimmunotherapy (RAIT), we evaluate the combined treatment of 188-Re-h-R3 and the naked h-R3 in nude mice bearing subcutaneously the human squamous cell carcinoma A431 cells. Group 1 was treated with a single intravenous (i.v.) administration of 150 ␮Ci of 188 Relabeled 1 mg h-R3 and six (i.v.) administration of 1 mg of h-R3 every 48 h. Group 2 was treated with seven i.v. administration of 1 mg of h-R3 each 48 h and group 3 was treated with seven i.v. administration of PBS. Animals were weighted and tumors were measured with a vernier caliper. Hematological, biochemical and anatomo-pathological study was carrying out to all animals. The combined treatment and the unlabeled monoclonal antibody did not show any toxic effects on mice corporal weights and elicited a significantly reduction of tumor size regarding to the control group. Platelets, leukocytes and hemoglobin peripheral values as well as the bone marrow studies did not show toxicological effects. Hepatic and renal function did not show any alteration according to the creatinine, aspartate aminotransferase, alanino aminotransferase values. A similar

reduction of the overall microvascular density and an elevated apoptotic index in the remaining tumors were observed in the treated groups with RAIT and h-R3 and the group treated with h-R3 alone. h-R3 MAb proved to be effective in mice with a xenotransplanted squamous cell carcinoma, the combinate h-R3 + RAIT treatment at the administered doses did not improve the results. P10-25 POSTNATAL DEVELOPMENT IN RAT OFFSPRING FOLLOWING IN UTERO AND LACTATIONAL EXPOSURE TO DI (2-ETHYLHEXYL) PHTHALATE K. Kobayashi, M. Miyagawa, R.S. Wang, M. Suda, S. Sekiguchi, T. Honma National Institute of Industrial Health, Kawasaki, Japan Di (2-ethylhexyl) phthalate (DEHP) has been widely used as a plasticizer for a large number of plastic products. In the present study, we investigated the effect of in utero and lactational exposure to DEHP on postnatal development in rat offspring. Pregnant Sprague–Dawley rats (Crj: CD (SD) IGS) were administered DEHP in corn oil orally at dosage levels of 0, 25, 100 or 400 mg/kg per day from gestation day (GD) 6 through postnatal day (PND) 20. Gestational length and maternal body weight gains during gestation in the DEHP groups remained the same as those in the control group. The number of live births did not differ between the control group and the DEHP groups. There were no significant changes from the controls in any of the somatic or organ growth, including body weight, body length, tail length or organ weights (liver, kidneys, testes, prostate, seminal vesicles, ovaries and uterus) in the offspring. In addition, there were no doserelated differences between the controls and DEHP groups in somatic developmental parameters, including several plasma hormone concentrations. These results suggest that in utero and lactational exposure to DEHP (GD6-PND20) does affect postnatal development in rat offspring for either sex under the experimental design of the present study.

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P10-26 GLYCINE DECREASES DEVELOPMENTAL DAMAGE INDUCED BY HYPERGLYCEMIA IN MOUSE EMBRYOS E. Mart´ınez-Galero1 , R. P´erez-Past´en2 , L. Gardu˜no1 , G. Chamorro1 1 Preclinical

Toxicology Laboratory, National School of Biological Sciences, National Polytechnic Institute, Mexico; 2 Pharmacy Academic Area, Health Sciences Institute, Autonomous University of the State of Hidalgo, Mexico It has been shown that glycine reduces the embryolethality of streptozotocin-induced diabetes in mice. On the other hand, hyperglycemia has been confirmed as inducing neural tube defects and growth retardation in cultured mouse and rat embryos. In this study the possibility of glycine in preventing hyperglycemia-induced embryopathy was researched. Early somite mouse embryos were cultured in normal, hyperglycemic (50 mmol/l glucose), glycine 1 mmol/l supplementation of normal and glycine-supplemented hyperglycemic rat serum for 48 h followed by embryo growth and differentiation determinations to estimate development and congenital malformations. The addition of glycine to control culture media did not affect embryonic development. Embryos cultured in hyperglycemic medium showed significant growth retardation and malformations affecting mainly central nervous and vesicular systems. Nevertheless, the amino acid prevented dysmorphogenesis induced by hyperglycemic media for telencephalon affectation, decreased DNA content, number of somites and morphological score, but retarded differentiation of the otic system. Partial protection from hyperglycemiainduced teratogenesis due to glycine in this study could be related to its antiglycation, antioxidative or other protective effects. Thus, the mechanism(s) by which glycine acted as an antiteratogenic agent needs to be clarified.


P11 Pesticide Toxicology P11-01 MECHANISMS OF NEUROTOXIC ACTION OF ORGANOPHOSPHATES V. Tonkopii Institute of Limnology, Russian Academy of Sciences, Russia The present study was undertaken to elucidate the relations between lipid peroxidation, organophosphates (OPs) toxicity and delayed, long lasting, noncholinergic changes. In the experiments on the rats we studied the influence of OPs intoxication by paraoxon, sarin, malathion, soman on lipid peroxidation in rat cerebral hemispheres. The level of lipid peroxidation was measured as the amount of common phospholipids, peroxidate lipids and malondialdehyde (MDA) in reaction with thiobarbituric acid. Results were compared to those with pre-treatment with atropine and reversible cholinesterase inhibitor—galanthamine alone or together with different antioxidants (␣tocopherol, oxymetacyl and ionole). The rate of reaction of conditioned reflex of active avoidance was measured. OPs caused a rapid, dose-dependent increase of peroxidate lipids and MDA 15–30 days after intoxication. With paraoxon and sarin pre-treatment with atropine and galanthamine totally prevents the all symptoms of intoxication and changes in lipid peroxidation. Comparatively such type of prophylaxis in malathion and soman poisoned rats did not normalize the biochemical and physiological parameters. The protective effect of antioxidants against somanand malathion-induced lipid peroxidation was shown. Therefore, malathion- and soman-associated lipid peroxidation is likely to arise mainly as a primary change which may, however, play a significant role in delayed neurotoxicity and conditioned reflex activity.


Abstracts / Toxicology Letters 158S (2005) S1–S258


Davila” University of Medicine and Pharmacy, Toxicology Department, Bucharest, Romania; 2 “Victor Babes” National Institute, Bucharest, Romania Membrane potential controls several functions of both excitable and non-excitable cells. Evidence exists that, in the case of immune cells, membrane potential regulates receptor-mediated intracellular signaling. Membrane potential measurement is performed either by electrodes techniques or fluorescent voltagesensitive dyes (molecules that interact with cell membranes and their fluorescence spectrum is correlated with the membrane potential changes). We investigated the effects of organophosphates pesticides chlorpyrifos, diazinon and malathion (1–1000 ng/mL), on the membrane potential of human lymphocytes and K562 lymphoblastic cells. To evaluate membrane potential changes, several slow-response fluorescent dyes were used comparatively. The experimental procedure has been set up by verifying microscopically the cell loading with dyes. The optimal concentrations of the dyes and the time required for cells loading have been set. The obtained results indicate the depolarizing effect exerted by organophosphates, mainly at high doses of organophosphate compounds, but, in some cases, lower concentrations seem to be active. Low-proliferating tumor cells are more sensitive to the depolarizing effect of organophosphates. The results show that the effect of pesticides on lymphocytes is more obvious, K562 cells giving a smaller response. Membrane potential alteration induced by organophosphates could result in negative consequences related to apoptosis, ion homeostasis, altered activation, proliferation capacity, etc.

P11-03 DNA DAMAGING EFFECTS OF PESTICIDES ON HUMAN PERIPHERAL LYMPHOCYTES ¨ Unde˘ ¨ U. ger, N. Bas¸aran Department of Toxicology, Faculty of Pharmacy, University of Hacettepe, Ankara, Turkey Despite the beneficial effects associated with the use of pesticides for domestic and industrial applications many of these chemicals may pose potential hazards to humans and to nature. And the evaluation of their genotoxic effects is of major concern to public health. Although various experimental data have provided evidence that pesticides can possess genotoxic properties in animals and in vitro test systems after acute and chronic exposure, the information on the genotoxic effects of some of pesticides is limited and inconsistent. In the present study, the genotoxic potential of commonly used pesticides (i.e., dimethoate and methyl parathion from the organophosphates, propoxur and pirimicarb from carbamates, and cypermethrin and permethrin from pyrethroids) have been evaluated. The genotoxic effects of these substances were examined using the single cell gel electrophoresis (comet) assay in freshly isolated human peripheral lymphocytes. The cells were incubated with 10, 50, 100 and 200 ␮g/ml concentrations of the test substances for 0.5 h at 37 ◦ C and DNA damage was compared with that obtained in lymphocytes from the same donor not treated with substances. Hydrogen peroxide, 100 ␮M, was used as a positive control. Within the concentration ranges studied, no significant cytotoxic effects were observed. Dimethoate and methyl parathion at 100 and 200 ␮g/ml; propoxur at 50, 100 and 200 ␮g/ml, and pirimicarb, cypermethrin and permethrin at 200 ␮g/ml significantly increased DNA damage in human lymphocytes. This study was supported by a grant from Research Foundation of Hacettepe University, Turkey, 01-02301-005.

Abstracts / Toxicology Letters 158S (2005) S1–S258

P11-04 ANTIDOTAL TREATMENT OF SOMAN INTOXICATION IN MICE WITH BISPYRIDINIUM OXIMES ˇ A. Luci´c-Vrdoljak, B. Radi´c, V. Zlender, M. Peraica Institute for Medical Research and Occupational Health, Ksaverska c. 2, Zagreb, Croatia Like organophosphosphate insecticides, nerve agents phosphorylate and inactivate acetylcholinesterase (AChE), leading to accumulation of acetylcholine at nicotinic and muscarinic receptors, and other receptors in the central nervous system (CNS). Together with atropine, pyridinium oximes are known to be successfully used to treat poisoning with many OPs. The aim of this study was to evalute the efficacy of three new bis-pyridinium compounds: [1-(4-hydroxyiminomethyl pyridinium)3-(4-carbamoyl pyridinium)] propane dibromide (K 027), [1-(4-hydroxyiminomethyl pyridinium)-4-(4carbamoylpyridinium)] butane dibromide (K 048) and [1,4-bis (2-hydroxyiminomethyl pyridinium)] butane dibromide (K 033), in combination with atropine in the therapy of soman intoxication in mice in vivo. Their acute intraperitoneal (i.p.) toxicity (LD50 with 95% confidence limits) was tested and observed for 24 h. The therapeutic effect was expressed as the therapeutic factor (TF) with 95% confidence limits and as the therapeutic dose (TD). In vivo results show that the tested compounds are relatively toxic (their LD50 was from 33.4 to 672.8 mg/kg body weight). Generally, in vivo toxicity of these compounds and their antidotal efficacy expressed as the TF, TD and the ratio between dead and injected experimental animals, were depended of type of the substituent in the pyridinium ring, and chains between pyridinium moiety. P11-05 ANTIDOTAL EFFECT OF ADAMANTYL DERIVATIVE TAMORF AND CARBAMATE PHYSOSTIGMINE IN SOMAN INTOXICATION ˇ B. Radi´c, A. Luci´c Vrdoljak, V. Zlender, M. Peraica, R. Fuchs Institute for Medical Research and Occupational Health, Zagreb, Croatia


Acethylcholinesterase (AChE) is an extremely active enzyme. Irreversible inhibition of AChE by organophosphorus compounds (OPs) results in the accumulation of endogenous acetylcholine in synaptic cleft and paralysis of nerve impulse transmission in the central and peripheral nervous system. In order to find the best treatment of organophosphate poisoning new compounds have been synthesized and tested. The aim of this study was to evalute the pretreatment efficacy of adamantyl tenocyclidine derivative TAMORF which antagonize the NMDA receptors and might protect AChE. Its antidotal effect was compared with physostigmine which also has a good prophylactic efficacy in soman poisoning. The therapeutic efficacy of TAMORF and physostigmine was tested on rats poisoned with two different sub-lethal dose of soman (1/4 and 1/2 of LD50 ). Our results indicate that TAMORF when administered 5 min before intoxication clearly stopped soman-induced seizures and markedly improved respiration of animals. In contrast to TAMORF, physostigmine seems to be slightly effective in the elimination of soman-induced toxicity in rats. Catalytic activities of AChE in brain, especially after administration of higher dose of soman (1/2 of LD50 ) were additionally lower throughout of the experiment, except after 24 h. Similar results with physostigmine in contrast to TAMORF were obtained in plasma also. In conclusion, treatment with TAMORF seems to be a good alternative for current pretreatment in soman intoxication. P11-06 THE REACTIVATION EFFECT OF PRALIDOXIME IN HUMAN BLOOD ON PARATHION AND PARAOXON-INDUCED CHOLINESTERASE INHIBITION M. Jafari1 , G. Pourheidari2 1 Department

of Biochemistry, Baqiyatallah University, Tehran, Iran; 2 Department of PharmacologyChemical Research Center, Faculty of MedicineMilitary Medicine Institute, Baqiyatallah University, Tehran, Iran Organophosphates (OP) are widely used throughout the world as agricultural pesticides. Two OP compounds, parathion and its active metabolite, paraoxon, are potent inhibitors of acetylcholinesterase (AChE) and


Abstracts / Toxicology Letters 158S (2005) S1–S258

butylcholinesterase (BChE). AChE is found primarily in nervous tissue and erythrocytes and BChE is found in liver and plasma. Pralidoxime is one of the oximes used for treatment of organophosphate poisoning. In this study the reactivation effect of pralidoxime on parathion and paraoxon-induced cholinesterase inhibition was studied in plasma and erythrocytes. For this purpose, human plasma and erythrocytes were incubated with various concentrations of parathion (0.1–10 ␮M) and paraoxon (0.03–0.3 ␮M) for 10 min at 37 ◦ C. After this time, pralidoxime (10–300 ␮M) was added to the samples and incubated for 10 min prior to assay of cholinesterases. The results show that the effect of parathion and paraoxon were dose dependent. Parathion and paraoxon inhibited greater than 85% of BChE and AChE activity, but the inhibitory effect of paraoxon was 10 times higher than parathion. In the presence of pralidoxime (100 ␮M) alone, BChE activity was significant higher than the control. Also, it removed less than 50% of parathion and 42% of paraoxon inhibitory effects. When pralidoxime (10 ␮M) was added to erythrocytes, the inhibitory effects of two organophosphates removed less than 15%. P11-07 SYNTHESIS OF THE NOVEL SERIES OF BISPYRIDINIUM COMPOUNDS AND EVALUATION OF THEIR REACTIVATION ACTIVITY AGAINST CHLORPYRIFOS-INHIBITED ACETYLCHOLINESTERASE K. Musilek1 , K. Kuca2 , D. Jun2 , V. Dohnal3 , M. Dolezal1 1 Department

of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic; 2 Department of Toxicology, Faculty of Military Health Sciences University of Defence, Hradec Kralove, Czech Republic; 3 Department of Food Technology, Mendel University of Agriculture and Forestry Brno, Czech Republic The reactivators of acetylcholinesterase (AChE, EC are very important components in the treatment of intoxications caused by organophosphate inhibitors such as nerve agents and insecticides. These inhibitors covalently bind on active site of mentioned enzyme and

irreversibly inhibit its activity. The reactivator breaks the inhibitor-enzyme covalent bond and restores its activity. Unfortunately, there is no reactivator applicable for every type of inhibitor; it means that every structural change in the molecule of inhibitor needs a specific structure of the reactivator. Therefore, development of more active AChE reactivators with broader spectrum is a major challenge actual from the point of view of war operations, accidents or terroristic attacks. The novel potential AChE reactivators were synthesized using modification of currently known synthetic pathways. Their potency to reactivate acetylcholinesterase inhibited by insecticide chlorpyrifos was tested in vitro. Promising results were obtained. The reactivation potency of these compounds depends on structural factors such as presence of quaternary nitrogen atoms, length of the linking chain between both pyridinium rings and position of the oxime moiety at the pyridinium ring. P11-08 THE CYTOCHROME P450 SYSTEM IN THE LIVER OF RATS INTOXICATED WITH DIMETHOATE AND PYRANTEL A. Wiaderkiewicz1 , P. Czekaj1 , B. Czajkowska1 , A. Pałasz1 , R. Wiaderkiewicz1 , A. Zasadowski2 1 Department

of Histology and Embryology, Medical University of Silesia, Katowice, Poland; 2 Department of Veterinary Toxicology and Environmental Protection, University of Warmia and Mazury, Olsztyn, Poland Antyparasitic drugs (pyrantel) affecting cytochrome P450 system may modify the metabolism of many xenobiotics, including pesticides (dimethoate). In the case of simultaneous exposure possible interactions between xenobiotics may lead to adverse health effects which are unpredictable on the base of their individual toxicity. In the presented study we analyzed the cytochrome P450 system in rats intoxicated with dimethoate (5 days, 1/10 DL50), pyrantel embonate (3 days, 1/5 DL50) or both xenobiotics simultaneously. Both compounds were delivered directly to the stomach by gavage and the components of cytochrome P450 system were analyzed in the microsomal fraction of the liver up to 14 days after the last applied dose.

Abstracts / Toxicology Letters 158S (2005) S1–S258


Intoxication with dimethoate and pyrantel resulted in the increase in the total amount of cytochrome b5 and activities of NADH:cyt.b5 and NADPH:cyt.P450 reductases, mostly during the period of several hours following the intoxication. On the other hand, the increase in the total content of cytochrome P450 was evident as late as 7 days after the last applied dose. In animals receiving dimethoate as well as both xenobiotics simultaneously we observed significant increase in the level of CYP2B1/2 protein. The increase in CYP1A2 protein expression corresponded with the changes observed for the total amount of cytochrome P450. However, stimulatory effect of dimethoate on the expression of CYP1A2 was abolished by simultaneous intoxication with pyrantel.

group 3 treated with antidotes after poisoning. Acute organophosphate poisoning and antidotal treatment in this model are not associated with histopathological changes in the rat kidney but the models with different orgonophosphate compounds, by administrating the different dosages, may be more illuminative to explain the effects of these chemicals in kidney. This project was funded by C ¸ ukurova University Research Project Foundation (Project No: TF 2002 BAP 90).


1 Cukurova

University, School of Medicine, Department of Emergency Medicine, Balcali-Adana, Turkey; 2 Cukurova University, Faculty of Agriculture, Department of Plant Protection, Balcali-Adana, Turkey

S. Satar1 , D. Satar2 , U.O. Mete3 , J.R. Suchard4 , M. Topal1 , M. Kaya3

Accidental or intentional ingestion of carbofuran can produce a life-threatening syndrome that requires prompt diagnosis and treatment. This paper investigates the unintentional carbofuran poisoning in farm workers. We reviewed the patients’ medical charts retrospectively and detailed demographic data, intoxication route, clinical and laboratory presentations, and outcome. We made the diagnosis according to a compatible exposure history and clinical findings. Thirteen patients were admitted to emergency department with carbofuran poisoning between January 2002 and August 31, 2004 (2 female, 11 male). The patients were poisoned while mixing the liquid form of carbofuran with seeds and their hands were coloured in red on admission. Complaints, most commonly reported by the patients on admission were: nausea, vomiting, headache, weakness, dizziness, and blurred vision. The most commonly observed signs were: tachycardia, tachypnoea, salivation, miosis, elevated blood pressure, and fasciculation. Three patients were agitated and patient 9 was lethargic on admission. The most commonly observed laboratory finding was hyperglycaemia that was found in six patients. Serum pseudocholinesterase level was decreased only in one patient. All the patients were cured and discharged from the hospital in good physical conditions.

1 Cukurova

University, School of Medicine, Department of Emergency Medicine, Balcali/Adana, Turkey; 2 Adana Numune Education and Research Hospital, Pathology Department, Balcali/Adana, Turkey; 3 Cukurova University, School of Medicine, Department of Histology and Embryology, Balcali/Adana, Turkey; 4 University of California Irvine Medical Center, Department of Emergency Medicine Orange, CA, USA We investigated the ultrastructural effects of the organophosphate compound methamidophos and treatment with atropine and pralidoxime (2-PAM) on rat kidneys. Male Wistar albino rats were assigned to four groups. Group 1 received 30 mg/kg methamidophos, the LD50 for this compound in rats, via oral gavage. Group 2 received only physiologic saline. Group 3 rats received 30 mg/kg methamidophos and were treated with 2-PAM and atropine via intraperitoneal injection when cholinergic symptoms were noted. Group 4 served as a control, and received physiologic saline in equivalent volumes and routes to Group 3. Kidney tissues were prepared for electron microscopic studies. No ultrastructural changes were detected in group 1 after acute poisoning with methamidophos and in

P11-10 CARBOFURAN POISONING AMONG FARM WORKERS S. Satar1 , S. Satar2 , A. Sebe1 , H. Yesilagac1


Abstracts / Toxicology Letters 158S (2005) S1–S258

Rapid onset, mild illness and quick recovery are typical characteristics of occupational acute carbofuran poisoning. We conclude that public health efforts should be made to educate the farm workers about the dangers of pesticide application so that their threat may be diminished. P11-11 ANTICHOLINESTERASE EFFECT OF GALANTHAMINE INFLUENCED BY l-CARNITINE ˇ y, J. Kvˇetina, Z. Svoboda, J. Bajgar, J. Herink, J. Zivn´ V. Paliˇcka Institute of Experimental Biopharmaceutics, Joint Research Centre of the Academy of Sciences of the Czech Republic and PRO.MED.CS Praha a.s., Hradec Kr´alov´e, Czech Republic Most of Alzheimer disease treatments have been focused on the inhibition of acetylcholinesterase (AChE). Galanthamine (GAL) is a cholinesterase inhibitor and allosteric modulation ligand at cholinergic receptors. l-Carnitine (CAR), a natural component of mammalian tissue, is known to increase penetration of some substances through biological barriers. Male Wistar Han II rats were divided into three groups with seven in each. The control group was administered with water p.o. during three consecutive days, then was injected i.m. saline and 30 min later the animals were killed and blood, liver, hypophysis (Hyp), frontal cortex (FC), basal ganglia (BG), septum (S), and hippocampus (Hipp) were collected. In the second group GAL was administered (i.m.) in different doses (from 2.5 to 10 mg/kg). In the third group, CAR was administered daily during three consecutive days 250 mg/kg (p.o.) and after last CAR dose GAL was injected (i.m.) 10 mg/kg. In tissue homogenates and plasma, activities of AChE were determined. Cholinesterase activities were decreased significantly following higher GAL dose in plasma, Hyp, FC, Hipp. Pretreatment with CAR followed by GAL administration showed higher AChE inhibition in comparison with single GAL administration in FC and Hipp only, but statistical nonsignificantly.

P11-12 NEW RUSSIAN TOXICOLOGICAL-HYGIENIC CLASSIFICATION OF PESTICIDES A. Potapov, V. Rakitski, N. Nikolaeva The Federal Research Center of Hygiene named after F.F. Erisman, Russia Classification of pesticides regarding their danger for man is the main regulating tool for determining a class of hazard for certain preparations, their active ingredients and, if necessary, dangerous metabolites and that in its turn is the basis for regulation of their safe circulation. The Classification has been in force in the territory of the RF since 1996, its new version has been recently developed, expanded and made more precise. The pesticides Classification has been supplemented with the data of assessing pesticides irritation effects on skin and eye mucous membranes. Evaluation criteria of pesticides stability in soil for the third and the fourth hazard classes have been revised. Recommendations for experts engaged in classifying pesticides, their active ingredients and dangerous metabolites have been made more accurate and specific. Thus in the Classification data on toxicity at peroral, skin and inhalation effects, criteria of irritating, allergic, teratogenic, embryotoxic reproduction, mutagenic and carcinogenic effects, stability in soil for four hazard classes (extremely dangerous, dangerous, moderately dangerous and low dangerous) are included. A class of hazard is being determined for use and storage conditions. For the conditions of pesticides production a Classification of chemical substances is being used. P11-13 CHARACTER OF PESTICIDES AND HEAVY METALS COMBINED EFFECT ON ORGANISMS OF WARM-BLOODED ANIMALS V. Rakitski The Federal Research Center of Hygiene named after F.F. Erisman, Russia Pesticides and heavy metals are widely spread and hazardous contaminations of the environment. High biological activity, stability and migration ability of these xenobiotics in the environment create a real threat of their simultaneous intake into human organisms.

Abstracts / Toxicology Letters 158S (2005) S1–S258

At the same time data on a character of combined effect on an organism of pesticides and heavy metals in literature is extremely limited. In this connection toxicological experiments were conducted for determining the character of combined effect on organisms of warm-blooded animals (rats) of primary contaminations for the environment: pesticides (2.4-D, copper sulphate, deltamethrin and dimethoate) and heavy metals (methylmercury and lead chloride). At that our own method of mathematical planning of the experiment was used (Rakitski, 1985). Each combination of a pesticide and a heavy metal was studied in nine series of essays with different components correlation at levels of LD16, LD33 and LD50 or LD2.5, LD9.25 and LD16 (in case of effect intensification). As the result corresponding mathematical models of the studied processes were developed for the combined effects of methylmercury and 2.4-D, methylmercury and copper sulphate, methylmercury and deltamethrin, methylmercury and dimethoate and also lead chloride and 2.4-D, lead chloride and deltamethrin, lead chloride and dimethoate. It was founded out that the combined effect of lead chloride and pesticides was characterized as close to additive effect (Kn = 0.8–1.06) and methylmercury and pesticides as more than additive effect (intensification) (Kn = 1.41–3.2). In all cases, for the exclusion of combination with dimethoate, heavy metals were the primary components of the combinations which 1.5–2 times exceeded the pesticides action and 1.3–12 times exceeded their combined effect. P11-14 EFFECTS OF DELTAMETHRIN ON HEMATOLOGICAL AND BIOCHEMICAL PROFILE ON COMMON CARP (CYPRINUS CARPIO) J. Vel´ısˇek1 , Z. Dobˇs´ıkov´a3

Svobodov´a2,3 ,


Modr´a3 ,


1 University of South Bohemia Ceske Budejovice, Czech

Republic; 2 Research Institute of Fish Culture and Hydrobiology Vodnany, University of South Bohemia Ceske Budejovice, Czech Republic; 3 University of Veterinary and Pharmaceutical Science Brno, Czech Republic The aim of this study was to assess the effect of deltamethrin [(S)-a-cyano-3-phenoxybenzyl-(1R,3R)3-(2,2-dibromvinyl)-2,2-dimethylcyclo-propancarbox-


ylate] on common carp (Cyprinus carpio). The effect was assessed on the basis of the results of haematological and biochemical examination of a control and an experimental group exposed to Decis flow 2.5 pesticide preparation (active substance 25 g l−1 of deltamethrin) in a concentration of 0.13 mg l−1 . The experimental group showed significantly lower values (p < 0.01) of erythrocyte count (RBC), haemoglobin content (Hb), haematocrit (PCV) and significantly higher values (p < 0.01) of ammonia (NH3 ), aspartate aminotransferase (AST) and alanin aminotransferase (ALT) compared to the control group. Values of mean erythrocyte volume (MCV), mean colour concentration (MCHC), erythrocyte haemoglobin (MCH), glucose (GLU), total protein (TP), albumins (ALB), total globulins (GLOB), triacylglycerols (TRIG), lactate dehydrogenase (LDH), creatinkinase (CK), calcium (Ca2+ ), inorganic phosphate (PHOS) alkalic phosphatase (ALP), lactate and cholinesterase (ChE) were comparable in the two groups during the study. Also, the leukocyte count (Leuko) and relative and absolute count of lymphocytes, monocytes, neutrophil granulocytes and their developmental forms were comparable in the two groups. Changes in the erythrocyte and biochemical profile after exposure to deltamethrin-based preparation may be referred to possible disruption of haematopoiesis and mild damage of liver. This research was supported by the MSM Project No. 621571240, FRVSˇ Project No. 744/2005/G3, GACR Project No. 523/03/H076. P11-15 EFFECTS OF PYRETHROIDS ON DOPAMINE RELEASE AND UPTAKE IN THE RAT STRIATUM M.M. Hossain1,3,4 , T. Suzuki1,3 , I. Sato1,3 , T. Takewaki3 , K. Suzuki2 , E. Tachikawa4 , H. Kobayashi1,3 1 Department

of Veterinary Medicine, Faculty of Agriculture, Iwate University, Japan; 2 Department of Agrobioscience, Faculty of Agriculture, Iwate University, Japan; 3 United Graduate School of Veterinary Sciences, Gifu University, Japan; 4 Department of Pharmacology, School of Medicine, Iwate Medical University, Japan


Abstracts / Toxicology Letters 158S (2005) S1–S258

The effects of allethrin (type I), cyhalothrin (type II) and deltamethrin (type II) on changes in outflow of extracellular level of dopamine (DA) and its metabolites in the striatum of conscious rats were studied by microdialysis techniques. Rats were treated i.p. with pyrethroids or vehicle. The increase in the extracellular level of DA by 10 mg/kg allethrin in the striatum reached a maximum of 178% of baseline but 20 and 60 mg/kg inhibited DA release to 63% and 52% of baseline with a peak time of 60–80 min after injection. Cyhalothrin 10, 20 and 60 mg/kg inhibited DA release to 65%, 56% and 45% of basal release, respectively with a peak time of 40–80 min. Deltamethrin 10 and 20 mg/kg increased DA release to maximum of 187% and 252% of basal release while 60 mg/kg first reduced the efflux for 60 min to 50% of basal release and then increased the efflux to a maximum of 344% of basal release with a peak time of 120 min. Local infusion of 1 ␮M tetrodotoxin through the dialysis probe completely prevented the effect of allethrin (10 and 60 mg/kg), cyhalothrin (60 mg/kg) and deltamethrin (20 mg/kg) on DA release but not deltamethrin 60 mg/kg (partly abolished). Three pyrethroids did not alter the release of DOPAC, 3-MT and HVA except 20 and 60 mg/kg of allethrin and also cyhalothrin increased the release of 3-MT. Our results show that striatal DA release is differently affected by type I and two type II pyrethroids. P11-16 THE INFLUENCE OF CYPERMETHRIN (PYRETHROID) ON FISH SENSITIVITY TO DIFFERENT INFECTIOUS FACTORS H. Lutnicka Subdepartment of Fish Diseases and Biology, Faculty of Veterinary Medicine, Agricultural University, Poland Pyrethroids are very toxic to fish. They influence immunological system. The aim of the study was to find out if the exposure of fish to cypermethrin influence their response to the infection of Aeromonas hydrophila bacteria and infection of Aeromonas bestiarum bacteria complicated by latent invasion of Chilodonella sp. parasite. The experiments were made in aquaria conditions, at spring season, on carp, weighing 70–80 g. Fish were

exposed for three days to cypermethrin in low concentration: 0.02 ␮g/l, added to water only once. The fish were infected after the third day of exposure by pathogenic bacteria—A. hydrophila (5 × 107 ) (group I) and pathogenic bacteria—A. bestiarum (5 × 105 ) (group II), which was complicated by invasion of Chilodonella sp. parasite. Group III was control fish. The fish sensitivity to bacterial infections was lower than in control. The symptoms of bacterial diseases lasted longer and were suppressed in the experimental groups than in control fish. The mortality of experimental fish was higher than in control (40% and 100%, respectively). Fish exposure to cypermethrin caused the manifestation of latent invasion of Chilodonella sp. as a first disease and the next, after disappearance of this ill the bacteria disease appeared. The symptoms were shorter and lower in exposed groups than in control fish. P11-17 ORAL TOXICITY OF ALFA-CYPERMETHRIN AND ITS DISTRIBUTION IN MICE ORGANS EVALUATED BY HPLC METHOD S. To´s-Luty, A. Haratym-Maj, M. Tokarska-Rodak, J. Latuszy´nska, T. Saran Department of Pathology, Institute of Agricultural Medicine, Lublin, Poland The aim of this study was to evaluate the amount of alpha-cypermethrin in internal organs of mice after oral intoxication by HPLC method with ultraviolet detection, and to asses the toxicity of this insecticide based on histological and ultrastructural examination of selected internal organ. A synthetic pyrethroid—alpha-cypermethrin, applied per os in following doses: 25 and 5 mg/kg b.m. for 28 days, was used. The studies were conducted on male and female Swiss mice. After 28 days of the experiment, the following organs were taken: lung, heart, liver, kidney, brain. Slices of tissue were placed in 5 ml of acetonitrile and homogenized. Homogenates were subjected to the HPLC system. The HPLC analysis was performed with Dionex system: a gradient pump P580 with internal vacuum degasser, a diode array detector UVD 340S

Abstracts / Toxicology Letters 158S (2005) S1–S258

and a manual injector (20 ␮l loop). The retention time for cypermethrin was 2.95 min. Surprisingly, among the two concentrations used, higher amounts of residues were detected in mice poisoned at lower concentration of alpha–cypermethrin compared to the higher dose of pyrethroid tested. Oral application of alfa-cypermethrin resulted in slight histological changes in heart, spleen, lung and brain. Focal inflammation in liver and kidneys and additionally parenchymatous degeneration in kidneys were observed. Ultrastructural changes were observed only in kidney. P11-18 MAJOR SPIRODICLOFEN METABOLITE INTERFERES WITH STEROIDOGENESIS BY DECREASING NADPH LEVELS A. Freyberger1 , W. Bomann2 , H.J. Ahr1 , B. Stahl3 1 Bayer HealthCare AG, Wuppertal (DE), USA; 2 Bayer

CropScience AG, Kansas City, USA; 3 Sophia-Antipolis (F), USA Findings in adrenocortical and Leydig cells of spirodiclofen(SDF)-treated animals suggested interference of SDF with steroidogenesis. As SDF was rapidly and completely hydrolyzed, investigations focused on the resultant BAJ-2510. This metabolite inhibited testosterone synthesis in human chorionic gonadotropin or dibutyryl-cAMP stimulated rat testicular explants. The inhibition could not be ameliorated by addition of 25-OH-cholesterol. Whereas cytochrome-P-450 dependent and other microsomal enzymes of testosterone synthesis were not affected, BAJ-2510 inhibited malate-dependent cholesterol side chain cleavage (CSCC) in rat testicular mitochondria. This effect could be ameliorated by increasing malate concentrations and by replacing malate with citrate suggesting an indirect mode of interaction. Further studies revealed reduction of intramitochondrial NADH and NADPH levels by BAJ-2510 resulting from decreased utilization of malate due to strong inhibition of mitochondrial malate dehydrogenase (MDH). Investigation of CSCC in testicular explants using progesterone formation as a marker showed that testosterone synthesis was much more affected by BAJ2510 compared to progesterone formation providing evidence of an additional target site located in the


cytosol. Additional investigations characterized BAJ2510 as an inhibitor of cytosolic MDH, which as part of the citrate/pyruvate shuttle is involved in cytosolic NADPH generation for steroidogenic reactions. Accordingly, BAJ-2510 interferes with Krebs cycle and pyruvate/citrate shuttle thereby decreasing mitochondrial and cytosolic NADPH levels and unspecifically affecting steroidogenesis, thus representing a novel mechanism of steroidogenesis inhibition. P11-19 STUDY OF THE EFFECTS OF SODIUM CROMOLYN ON LEVELS OF TNF␣, IL1-␤ AND COLLAGEN IN THE PARAQUAT INDUCED PULMONARY FIBROSIS IN RAT A.A. Hemmati1 , Z. Nazari1 , M. Ghafurian2 , N. Ranjbari2 , B. Darabi1 1 Department

of Pharmacology and Toxicology, Pharmacy School, Ahvaz Jundishapour University of Medical Sciences, Ahvaz, Iran; 2 Department of Immunology, Ahvaz Jundishapour University of Medical Sciences, Ahvaz, Iran Female rats weighting 130–180 g were divided into five groups. Lung fibrosis was induced by single oral dose of paraquat (40 mg/kg). Treatment groups (1 and 2) were nebulised with 6 or 8 mg cromolyn 7-day prior and 2 weeks after paraquat treatment. One of the control groups was given distilled water at the same volume of paraquat and the other nebulised with 8 mg cromolyn for 2 weeks. Fibrotic group received just paraqut. Histological examination of fibrotic group showed marked infiltration of inflammatory cells in the alveolar spaces, septal thickening and fibrosis. Collagen content of lung tissue were 709, 755, 1660, 1129 and 819 ␮g/g in controls, fibrotic and treatment groups (1 and 2), respectively. Serum level of IL 1␤ in controls, fibrotic and treatment groups (1 and 2) were 67, 57, 170, 83 and 77 ␳g/ml, respectively. Serum level of TNF-␣ in controls, fibrotic, treatment groups (1 and 2) were 124, 138, 438, 153 and 143 ␳g/ml, respectively. In cromolyntreated groups such factors were near to controls and were significantly lower than those in fibrotic group (P < 0.01). Results indicate that sodium cromolyn can decrease the fibrogenic effects of paraquat on lung.


Abstracts / Toxicology Letters 158S (2005) S1–S258



A.K. W´ojtowicz1 , J. Falandysz2 , E.Ł. Gregoraszczuk1

M. Tutudaki1 , I. Leukidou2 , M.N. Tzatzarakis1 , T. Nzeim3 , G. Dolapsakis1 , A.M. Tsatsakis1

1 Laboratory

of Physiology and Toxicology of Reproduction, Department of Animal Physiology, Institute of Zoology, Jagiellonian University, Krak´ow, Poland; 2 University of Gdansk, Faculty of Chemistry, Department of Environmental Chemistry and Ecotoxicology, Gda´nsk, Poland Dichlorodiphenyltrichloroethane (DDT) is persistent environmental pollutant, which exert a variety of toxic effects in animals. In mammals DDT is converted by reductive dechlorination to DDD and then by dehydrochlorination to DDE. Therefore it must be assumed that not only DDT themselves, but also their metabolites can participate in the toxic effects of DDT. The aim of the present study was threefold: (i) to examine the effects of p,p -DDT, o,p -DDT as well as their metabolite o,p -DDE or p,p -DDE on estradiol secretion, (ii) to analyze the involvement of estrogen receptor (ER), and (iii) to confirm our hypothesis that the endocrine disruptive effects of PCBs on ovarian functions is due to modulation of aromatase activity in follicular cells. Co-cultured granulosa and theca cells was used as a model resembling in vivo follicles. Cells were initially cultured for 24 h to allow their attachment to the plates. Than, the media were changed for fresh ones and reagents were added at a dose of 4 ␮g/ml. Down-regulation of ER was noted under the influence of o,p -DDT and o,p -DDE while up-regulation of ER was noted in the presence of p,p -DDT and p,p DDE. The metabolites of both isomers showed higher estrogenic activity than parent compounds. Fact that estrogenic effect of the metabolites was abolished by an aromatase inhibitor CGS 16949A indicates an action on aromatase activity. We hypothesize that: (1) o,p-DDT and its metabolite o,p-DDE preferentially bind to ER in contrast to p,p-DDT isomers, (2) huge estrogenic action of the metabolites is due to activation of aromatase activity in ovarian follicular cells. This work was supported by BW/IZ/25b/2004.

1 Center

of Toxicology, Medical School, University of Crete, Heraklion 710 03 Crete, Greece; 2 Laboratory of Toxicology, Department of Forensic Medicine, Ministry of Justice, Athens, Greece; 3 School of Food Technology and Dietetics, Department of Human Nutrition and Dietetics, TEI of Crete, Sitia, Greece Most of the organochlorine pesticides have been banned from use in most countries. Very few of them are still used due to their low cost and high effectiveness. Their main characteristic is their long half life in the environment and their accumulation in lipid tissues. The use of DDT has been banned in Crete and Lebanon since the 70 s. Lindane is still used in very few cultivations in both countries. In the present study hair samples coming from inhabitants of Heraklion and Beirut were analyzed for the presence of lindane, DDT and its metabolites. The analytes were extracted from the hair matrix by acidic hydrolysis, cleaned by liquid–liquid extraction with hexane:dichloromethane 4:1, multilayer column chromatography and analyzed by GC-ECD. Although the studied populations had no occupational exposure to pesticides and lived in big cities, traces of DDT and metabolites were detected in all hair samples. Lindane concentration was considerably higher than that of DDT in all hair samples. The findings suggest that the main route of exposure of the studied population was the diet. P11-22 FUNCTIONAL NEUROTOXIC EFFECTS IN RATS AFTER SUBCHRONIC EXPOSURE TO AMITRAZ, COMBINED WTH TWO OTHER INSECTICIDES Z. Lengyel, A. Papp, L. Instit´oris, L. Nagymajt´enyi Department of Public Health, University of Szeged, Hungary The application of various pesticides likely results in combined human exposure, the effects of which are not

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fully understood. Most modern insecticides attack the nervous system, in the target species but also in others including humans. In the present study, the LOEL dose (26.5 mg/kg b.w.) of amitraz (A), a formamidinetype insecticide, was combined with NOEL doses of the organophosphate dimethoate (D; 7 .04 mg/kg) and the pyrethroid cypermethrin (C; 11.1 mg/kg), and was given to male Wistar rats for 12 weeks (4th to 16th week of life) five times a week by gavage. Then, the rats were prepared for electrophysiology in urethane anaesthesia. Spontaneous cortical and stimulus-evoked cortical and peripheral activity was recorded. From the records, frequency distribution of the cortical activity, latency and duration of the evoked responses, as well as tail nerve conduction velocity and refractory period were measured. In the spontaneous activity, amitraz alone caused no alteration, the combinations AD and ADC shifted the activity to higher frequencies. ADC, and AC, also decreased the latency and increased the duration of the somatosensory evoked response significantly, AD and A only influenced duration. The visual evoked potential had a similar but non-significant trend, the alterations of the auditory response were inconsistent. In the tail nerve, the relative refractory period was significantly lengthened by AD. The combinations of the insecticides studied had generally a stronger effect on the parameters examined, indicating possible positive interactions. Consequently, occupational and environmental limit values of individual agents may be inadequate in combined exposures. Supported by the Hungarian OTKA grant no. T 042955. P11-23 CARBENDAZIM ADMINISTRATION TO THE BLOOD SYSTEM OF RATS V. Lisovska, V. Shulyak, N. Nedoputanska L.I. Medved’s Institute of Ecohygiene and Toxicology, 6 Heroiv Oborony Street, 03680, Kyiv, Ukraine The base of administration carbamates (in particular carbendazim) blood index of the laboratory animals and human in the result of acute poisoning and the appearance, of different diseases are not completely described.


The aim of this work was to research the influence of carbendazim to the morphofunction blood index of the laboratory animals as a result of acute intoxication per os. The study was held using 24 male albino rats of Wistar strain. The testing group and control group consisted of six animals per group. Carbendazim was administered once at the dose of 750 mg/kg per os. The peripheral blood was investigated with the routine clinical-laboratory methods and cytochemical one, which allow to detect the early changes of blood cells functional state before the appearance of morphological alterations and indicate the maturity of blood cells (neutrophilous and lymphocytous). The obtained results shown acute intoxication with carbendazim decreases the quantities of erythrocytes and hemoglobin, accordingly, the number of reticulocytes. Also we observed morphological changes which characterized of increasing the quantity of acanthocytes and atypical lymphocytes. On the terminal stage of experiment we detected of the leukopenia which development of light of the monocitosis and decreasing the quantities of the thrombocytes. P11-24 EVALUATION OF HEALTH HAZARDS BY EXPOSURE TO TRIAZINES AND DEGRADATION PRODUCTS P. Nørhede, E. Nielsen, O. Ladefoged, O. Meyer Danish Institute for Food and Veterinary Research, Søborg, Denmark Atrazine, simazine, terbutylazine and cyanazine are triazines, which either have been used or still are in use in agriculture as herbicides in Denmark. We have recently published a review on the health hazards by exposure to these compounds as well as to their degradation products. The general population can be exposed to the triazines and their degradation products through their widespread occurrence in the environment, especially in drinking water, whereas no significant exposure is expected to occur via foodstuffs. In humans, the available data on health effects are mainly focused on the possible carcinogenic effects. However, data from many of these studies, are of limited value for the evaluation of a carcinogenic potential to humans due to simultaneous exposure to other chemicals. A


Abstracts / Toxicology Letters 158S (2005) S1–S258

few studies have focused on toxicity to reproduction without significant findings. In laboratory animals, the main effects observed are neuroendocrine effects (hormonal disturbances, and reproductive and developmental effects caused by the disruption of the hypothalamicpituitary-gonadal axis), haematological changes, and cardiac and kidney effects. In conclusion, the critical effects in humans following exposure to these triazines and their degradation products are considered to be the neuroendocrine effects, and a NOAEL of 0.20 mg/kg b.w./day has been evaluated for the human risk characterisation for exposure via drinking water. The ratio ‘NOAEL to the exposure’, Margin of Safety (MOS), has been estimated for children and adults at different concentrations in the drinking water. The MOS is higher than 250 for children and 650 for adults for concentrations in drinking water of 10 ␮g/l or below implicating a low risk of adverse health effects from exposure via drinking water to the selected triazines and their degradation products at concentrations below 10 ␮g/l. P11-25 PRELIMINARY TOXICOKINETIC EVALUATION IN CHICKENS OF THE INSECT GROWTH REGULATOR HYDROPRENE M. Giorgi1 , S. Cerri1 , F. Attuali1 , E. Cacciuttolo2 , Soldani1 , G.P. Mani 2 1 Pharmacology

and Toxicology Section, Department of Veterinary Clinics, University of Pisa, Pisa, Italy; 2 Department of Animal Pathology, University of Pisa, Pisa, Italy Hydroprene (HYD) is an insecticide belonging to the insect growth regulator (IGR) class. An IGR insecticide is a juvenile hormone mimic that inhibits growth/maturation of insect pests. An IGR is an important pest management tool: it helps reducing, eliminating or preventing infestations of targeted pests without the use of conventional contact insecticides. As a consequence, the need for pesticides in homes, hospitals, restaurants, animal treatments is reduced. Due to its toxicity, data of HYD are not sufficiently investigated in several animal species. The aim of the present work was to develop and validate, according to the EMEA guidelines on validation of analytical procedures a new HPLC method to determine

HYD plasmatic concentrations in chickens. The mobile phase consisted in CH3 CN/H2 O 95:5 (v/v), wavelength 266 nm, flow 2 ml/min, attenuation 0.4 a.u.f.s., column was Xterra C18 (3.5 ␮m). This study was carried out in 15 chickens, 0.9–1.2 kg, divided in three groups (n = 5). Two groups were treated with HYD 140 mg/kg single dose, via IP or OS, respectively, while a group was held as control. Plasma was collected at different times. Chicken microsomes were incubated with HYD according to Hogemann et al. (1990). The LOQ and LOD for HYD were 0.04 ␮g/ml and 0.01 ␮g/ml, respectively. Recovery resulted 77 ± 8% (S.E.). Precision and accuracy were within 7%. Reproducibility was assessed by means of a inter-laboratory trials. HYD plasmatic concentration resulted under LOQ in all the withdrawal times after IP and OS administration, whereas a new peak was identified, as HYD acid, a metabolite, in both treatments. The metabolite concentration had a Tmax of 1.88 and 1.2 h after IP and OS administration, respectively. Cmax was 1.93 and 3.04 ␮g/ml and T1/2 was 0.77 and 0.70 h after IP and OS administration, respectively. Moreover, HYD metabolism in chicken microsomes was maximum after 40 min. Pharmacokinetics data and rapidity of metabolism development, suggest a rapid and extensive esterification of the parental compound. P11-26 SCREENING ANALYTICAL METHOD FOR PESTICIDES DETECTION IN ANIMAL TISSUES A.L. Rodrigues, M.I. Correia, A. Belas, A.I. Santos, B. S˜ao Braz, B. Carrapic¸o, A. Moreira, A.J. Silva Nucleus of Pharmacology and Toxicology, CIISA, Faculty of Veterinary Medicine, Lisboa, Portugal Pesticides group includes a wide range of chemicals, with great diversity of applications, both in agriculture and animal health, and are often implicated in acute intoxications in humans and animals. The development of screening methods, with a quick detection and eventual quantification of these xenobiotics in animal tissues, will allow a more efficient intervention in pesticides poisoning. Based on analytical methods of thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) in silica gel G 60

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F254 , already developed, combinations of eluent (nhexane/acetone; 8 0:20) with several spraying reagents have been tested retention factor value and visualization with UV-light at 254 nm, allowing pesticide group and, in some cases, the compound identification. This improved methodology had been tested in animal tissues sampled from live animals (blood) and animal corpses (liver, cardiac blood cloth and contents from stomach and intestine) with the advantage of using a small amount of sample, a simultaneous running for three major groups of pesticides: organophosphates (yellow spot), organochlorines (brown spot) and carbamates (blue-green spot) and a limit of detection suitable for a screening test (≥0.25 ␮g). P11-27 STUDY ON THE NON-SPINNING SYNDROME OF SPRING SILKWORM IN KOREA K.H. Park, Y.K. Park, J.B. Joo, K.S. Kyung, J.S. Shin, B.S. Kim, G.H. Ryu, B.H. Sohn, C.H. Bae National Institute of Agricultural Science and Technology, Republic of Korea Pupation ratios of silkworm in Korea two representative farming areas, Yecheon and Sangju, are below 10% in spring season, but up to 90% in autumn season. In the survey area, 12 insect growth regulator (IGR) pesticides are sold, 8 of them having a possibility of being sprayed on and drifting to the mulberry leaf and silkworm. To investigate the influence of IGR pesticides, diflubenzuron wp was sprayed at recommended application dose (300 l per 10a). It affected silkworms that eat the mulberry leaves to a 50 m distance in orchard. Also, this current study was performed to clarify the pupation disorder symptoms in silkworm which herbicide, molinate has been regarded as a causal origin. Water diluted solution of molinate at the concentration of 1, 10, and 100 mg/l gave no abnormal symptoms in silkworm irrespective of spraying to silkworm or spiking to diet. Exposing the silkworm to the air containing 250, 2500, and 10,000 ␮g/m3 also revealed no abnormal symptom. These results illustrated that the abnormal growth of silkworm encountered in the field was not caused by pesticide contamination.


P11-28 THE POISONING OF SILKWORM INFLUENCED BY INSECT GROWTH REGULATORS WHICH ARE REGISTERED AND ON THE MARKET IN KOREA B.H. Sohn, K.H. Park, N.H. An, P.D. Kang, S.W. Kang, S.W. Lee, Y.S. Kim, J.B. Ju, I.S. Jeong National Institute of Agricultural Science and Technology, Republic of Korea Since the spring of 1995, non-spinning silkworms occurred in Korea, and many silkworm raising farmhouses were economically damaged by wide occurrence of this phenomenon in 1998, 1999 and 2002. It seemed that IGR insecticides sprayed around the rearing farmhouse in the spring rearing season were a cause of this phenomenon. In Korea, 13 IGR insecticides were registered and are on the market. Thus the effects of these insecticides on silkworms were investigated. The results are as follows. Among the registered IGR insecticides, two ingredients of phenyl ether insecticides and carbamate insecticides induced non-spinning silkworms. The characteristics of nonspinning silkworms induced by these insecticides were the same as those of silkworms occurred in the rearing farmhouses. Normally, sound silkworms pupate at 7th or 8th day of fifth instar, but non-spinning silkworms remain as larvae after 20th day of fifth instar, and show shape characteristics such as enlargement of body and silk gland. Two ingredients of benzohydrazide insecticides chemical showed poisoning syndromes such as decreasing and yellowing of body, thinning of annulus and becoming transparent. Each ingredient of thiadiazine and triazin insecticides showed poisoning characteristics of little body color change similar to control. Seven ingredients of benzoyl urea insecticides showed distinct poisoning syndrome by agricultural insecticides such as burst of body caused by thinning of skin, browning, prolapse of the anus.


Abstracts / Toxicology Letters 158S (2005) S1–S258


against a background of elevated thromboxane A2 ). The regularities revealed allowed the treatment-andrehabilitation measures to be optimized.

G.M. Balan, A.I. Ivanova


L.I. Medved’s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine

L. Ivanova, G. Zvarich, N. Kolontaeva, I. Leposhkin, A. Kravchuk, L. Yermolova

The prevalence of acute and chronic intoxications with pesticides was studied for the last decade among agricultural workers under the conditions of implementation of new management forms with altered ownership forms. In the structure of acute intoxications with pesticides among agricultural workers, intoxications with herbicides based on 2,4-dichlorophenoxyacetic acid predominated (47.4%). Noted more rarely were intoxications with herbicides based on sulfonyl urea (7.7%), organophosphorous compounds (3.5%), pyrethroids (1.7%), aminoformic preparations (1.3%), metalcontaining pesticides (0.9%), derivatives of phenyl pyrosols (0.4%), and other compounds. Chronic intoxications resulted from prolonged exposures to a combination of pesticides (OPC, COC, herbicides, pyrethroids, cuprum-containing compounds, etc.) were revealed in agricultural workers with work experiences of above 15 years: more often among workers at storehouses for pesticides, tobacco-growers, fruit-growers and wine-growers; not so frequently, among machine-operators and agronomists. Neurological impairments (toxic encephalopathy, asthenovegetative syndrome, vegetosensory polyneuropathy) predominated in the clinical picture of acute and chronic intoxications with pesticides. Not so frequently, there were noted toxic hepatitis, cardiomyopathy, hypochromic (erythronormoblastic) anemia, toxicodermia, exogenous allergic alveolitis and bronchoobstructive syndrome: in 28.8% of acute cases and in 34.6% of chronic cases, being a cause of workers’ invalidization. The criteria were substantiated for prediction of the progressive course of intoxications with pesticides. Most informative among them are indices of pronounced cytolytic syndrome of hepatic impairment, endotoxicosis, non-compensated activation of lipid peroxidation, elevation in small pathogenic circulating complexes, and the development of endothelial dysfunction (a decrease in blood prostacyclin level

L.I. Medved’s Institute of Ecohygiene and Toxicology, Kiev, Ukraine Fenhexamid, a derivative of cyclohexancarboxamid, is the perspective new fungicide for use in agriculture. Fenhexamid is a highly effective protective fungicide for the control of Botrytis diseases on grapes, strawberries and peaches. The fate of fenhexamid and safety of grown products have been investigated in different agroclimatic zones of Ukraine. The toxicological assessment of formulations and their active ingredients have been studied in Ukraine during State trials. Residual quantities of fenhexamid in the products have been studied by high-performance liquid chromatography (HPLC). Assessment of acute toxicity of fenhexamid formulation has shown that it may be referred to the pesticide of 3 class of hazard accordance Ukrainian Classification of Pesticides. On the basis of toxicological assessment of active substance the ADI were recommended and approved in Ukraine. Field trails were conducted in Ukraine during 1999–2004. Dynamics of fungicide residues were studied in different agroclimatic zones of Ukraine. As a result of conducted experiment we establish that the rate of degradation of active ingredient is not stable. In some cases character of decrease of pesticide residues may be described as a wavy type. Fenhexamid is more stable in plants than other fungicides. Residues of fenhexamid were found in plant products during the harvest time at the level of 0.1–0.2 mg/kg in grapes. Simulated industrial preparation of juice allowed us to study the fate of residue during processing. On the basis of conducted experiments, an MRL and post-harvest interval has been recommended, providing safe use of new fungicides in Ukraine. Theoretical maximum daily intake of new fungicides with food products did not exceed 2% of acceptable daily intake.

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P11-31 ACCUMULATION OF CHLOROPHENOXY HERBICIDE RESIDUES WITHIN WEED TISSUES R. Krzyzanowski, B. Leszczynski Department of Biochemistry, University of Podlasie, Siedlce, Poland In agricultural practice, commercial formulation of (4chloro-2-methylphenoxy)acetic acid (MCPA) is very often applied in cereal protection towards weeds. However, there is little information on accumulation of these herbicides within the weed tissues. In the present paper we report on accumulation of active chlorophenoxy herbicides from the Chwastox 300SL on very sensitive weeds (scentless mayweed, shepherd’s purse), less sensitive species (field horsetail, garden cornflower) and resistant weeds (corn cockle, wireweed). Dichloromethane extract of the weed seedlings was performed. The extracts were evaporated to dryness and redissolved in a small volume of methanol. Then the MCPA were analyzed using gas chromatography combined with mass spectrometry. The chlorophenoxy herbicide showed different accumulation within tissues of the studied weeds. The highest concentration of MCPA was found within tissues of the sensitive weed species (scentless mayweed, shepherd’s purse) and the lowest within tissues of the resistant weeds (corn cockle, wireweed). The MCPA accumulation within the weed tissues was clearly related to applied doses of the studied herbicide. Various reaction of the studied weeds to the applied herbicides is discussed. P11-32 RESIDUES OF PLANT PROTECTION PRODUCTS IN PEACHES AFTER LONG-TERM IMPLEMENTATION OF ICM T.G. Danis1 , I.N. Tsakiris2 , A.M. Tsatsakis2 , A.K. Alegakis2 , M.N. Tzatzarakis2 1 DELCOF

SA, Post Office Box 1393, Pleuroma Skydra 58500, Greece; 2 Center of Toxicological Sciences and Research, Department of Medicine, University of Crete, Heraklion, Greece Based on recent market research, during the last years a specific demand of consumers related with the produce of healthy and safe agricultural products is developed.


Part of consumers speculation consists the issue of plant protection products residues on and in the agricultural products. One of the most important cultivation systems, characterized from the capability to secure the quality of produce is the Integrated Crop Management (I.C.M). The collection period of peach samples originate from ICM was from 15 June to 30 August. The first year of sampling was 2000 and the last one was 2003. The number of samples was 85, 95, 156 and 66 for each year, respectively. The monitoring was performed for the following products: methamidophos, dimethoate, chlorothalonil, chlorpyriphosmethyl, parathion-methyl, malathion, chlorpyriphos, fenthion, endosulfan, ethion, biffethrin, phosmet, phosalone, azinphos-methyl, ␭-cyalothrin, deltamethrin chlorothalonil, captan, tebuconazole dicofol. The levels of plant protection products for the entire sample were lower than the MRLs. Thus all the samples were safe for the consumers. Chlorpyriphos had the highest frequency of detection especially during the years 2001–2002 due to withdrawal of methamidophos. It was detected in 36, 67, 62 and 31 samples with average concentration 0.04, 0.03, 0.03 and 0.02 ppm for the years 2000–2003, respectively. P11-33 EFFICACY AND TOXICITY STUDY OF A NEW PIROXICAM TOPICAL GEL FORMULATION A.H. Moghbel, H. Aghel, Z. Nazari, F. Faghiri Pharmacy school, Ahvaz Jundishaour University of Medical Sciences, Ahvaz, Iran Piroxicam is a non steroidal anti-inflammatory agent which has an extend use in rheumatic disorders. Adverse effects coming from oral piroxicam lead to development of piroxicam gel using carbomer 934 as a way to decrease its side effects. In this study two types of 0.5% piroxicam gel, so called Red gel and green gel have been prepared by using ethanol, propylene glycol and hydroxypropyl methycellulose solution. In vitro release of piroxicam from Red gel, Green gel and commercial available gel bases were measured and data showed that there were no meaningful differences among these three types of gels in release of piroxicam. Piroxicam serum levels after application of 1 gram of these three products on deltoid muscle twice daily for two weeks were measured in three groups of


Abstracts / Toxicology Letters 158S (2005) S1–S258

healthy volunteers. In this determination red gel had the highest serum levels and there was significant difference between piroxicam serum levels due to Red gel with those of the other gels (P < 0.005) but Green gel and commercial gel had no statistical difference. The results indicated that Red gel and green gel in comparison to commercial gel are in parallel quality even though Red gel has some superiority in vivo. Toxicity evaluation of the subject showed neither soft tissue injuries or skin diseases producing rashes, swelling, inflammation, redness, irritation, itching nor any other toxic symptoms. P11-34 INFLUENCE OF DIFFERENT ADSORBENTS ON SURVIVING OF RATS POISONED BY ABSOLUTE P.O. LETHAL DOSE OF LAMBDACYHALOTHRIN Jezdimirovic1 ,

Ivanovic1 ,

M. S. M. TomasevicCanovic2 , S. Trailovic1 , D. Stojanovic3 1 Faculty

of Veterinary Medicine, Belgrade; 2 Institute for Technology of Nuclear and Other Raw Materials, Belgrade; 3 Scientific Veterinary Institute “Novi Sad”, Serbia and Montenegro

Lambda-cyhalothrin is an insecticide. Animals can be poisoned after dermal intoxication, inhalation or after ingestion. Efficiency of adsorbents is not reliable enough, but also not examined enough in this group of poisons. For that reason, we wanted to examine protective effects of medicinal charcoal, organically modified natural zeolithe and mixture of medicinal charcoal (20%) + caoline (5%) + magnesium oxide (10%), as function of time after poisoning. Investigation was performed on 88 adult, male, Wistar rats (220–260 g bw). Commercial product OXYFLY 10CS (1 ml = 100 mg ␭-cyhalothrin, Novartis) was applied by a gastric tube, 15 g/kg bw, which corresponded to LD90 . It was determined that LD100 was 17 g/kg. 0.5, 1 and 2 h after administration of LD100 of the examined product, mixture, MINAZEL Plus® (ITNMS, Belgrade, SCG) or medicinal charcoal was administrated (2 ml/kg). Percentage of lethality was observed in function of time, during 24, 48, 72 and 96 h.

By correlation analysis it was determined that only product Minazel Plus® administrated 30 min after administration of LD100 , significantly (** p = 0.0087) decreased lethality of ␭-cyhalothrin in function of time. Therefore MINAZEL Plus® has important protective effect and increases percentage of survival for even 33.33% during the period of 96 h. Tuesday, September 13, 2005-06-14 P12 Environment and Health P12-01 MICRONUCLEI AND EXPOSURE TO LEAD IN CHILDREN E. Siwi´nska1 , L. Kapka1 , J. Kwapuli´nski2 , D. Miel˙zy´nska1 1 Institute

of Occupational Medicine and Environmental Health, Sosnowiec, Poland; 2 Department of Toxicology, Silesian Medical University, Sosnowiec, Poland Environmental exposure to lead in childhood is still an important health problem in urban and industrial regions of Poland. The objective of the study was to confirm lead’s contribution in the development of cytogenetic damage detected at the level of micronuclei by examining lead blood level (PbB) and micronuclei (MN) in peripheral blood lymphocytes (PBL) in 4–14-year-old children living in four different regions of the south of Poland: Katowice (n = 46), Sosnowiec (n = 25), Ustro´n (n = 49) and Bukowno (n = 87). Exposure to lead was assessed by PbB determination (AAS) and the frequency of micronuclei (MN) by cytokinesisblock micronucleus assay. MN were scored as the number of micronuclei in 1000 binucleated cells. The results showed significantly increased levels of both: PbB and MN in children living in the industrialized and densely populated centre of Katowice Agglomeration as compared to two other populations. Geometric mean of PbP in children from Sosnowiec was 7.9 and from Katowice 6.1 versus Bukowno: 4.9 and Ustro´n: 3.2 ␮g/dL. The same pattern referred to the level of MN. Mean level of micronuclei in children from Sosnowiec was 5.2 and from Katowice 4.0, versus Bukowno 1.4 and Ustro´n 1.3. Pooled data showed low but significant correlation between PbB and MN (Spearman R = 0.284, p < 0.05) due to the

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relationship between these parameters in Sosnowiec population. In conclusion, the study indicated higher PbB levels in children living in the most industrialized part of Katowice province compared to two other groups resulting in higher cytogenetic damage measured as MN in PBL. P12-02 ASSESSMENT OF SOME HEAVY METALS IN HAIR AND URINE OF CHILDREN FROM IASSY AREA, RISK IN CANCER DISEASE C. Hura, I. Palamaru, B.A. Hura Institute of Public Health, Iassy, Romania Human hair can be used effectively for biological monitoring of the highest priority toxic trace metals. The hair, nail and teeth are tissues in which trace minerals are sequestered and stored. Toxic elements (Pb, Cd, Cu and Zn) concentration in hair may be up to several hundred times higher than in the blood and urine, but there is a strong correlation between their concentrations in hair and concentrations in internal organs. Due to that, trace metals determination in hair is easier to study. The aim of this study was the evaluation of the heavy metals contents in the hair and the urine collected from children (15–18 years old) living in the Iassy area, from Romania. The study presents the results obtained in 2004 period of some metals [Pb, Cd, Zn, Cu and Mn] in the hair and the urine of children, in Iassy district, from Romania. A total of 126 children (15–18 years old) participated in the study voluntarily. Trace elements concentrations were analyzed by atomic absorption spectrophotometry, using a Carl Zeiss Jena, Model AAS-1N, with flame air-acetylene. In all analysed samples these metals were found. Generally, a wide variation between individual samples was observed. Children 15–18 years old—girls (61 samples): The mean metals levels in the hair varied between 3.02 ␮g/kg Cd and 9.49 ␮g/kg Pb. Children 15–18 years old—boys (65 samples): The mean metals levels in the hair varied between 3.6 ␮g/kg Cd and 17.6 ␮g/kg Pb. Determinations of these pollutants in human body (hair, urine) are important in environmental monitoring


for the prevention, control and reduction of pollution as well as for occupational health and epidemiological studies. P12-03 RISK ASSESSMENT OF FORMALDEHYDE FOR GENERAL POPULATION IN JAPAN M. Naya, J. Nakanishi National Institute of Advanced Industrial Science and Technology, Japan Formaldehyde is used in the production of resins, molding compounds, photographic film, bactericide and tissue preservative. The purpose of this study is to provide an up to date critical review of the information to the toxicological profile of formaldehyde, and assess the risk of formaldehyde for the general population in Japan. Inhaled formaldehyde is an effective sensory irritant at a dosage of 0.5 ppm in mice. Following inhalation in laboratory animals, more than 6 ppm of formaldehyde causes degenerative non-neoplastic effects in mice and monkeys, and nasal tumors in rats. It is considered that formaldehyde induce genotoxic effects directly in vitro, and secondary in vivo. The sensory irritation of eyes and respiratory tract of inhalation exposure to formaldehyde were reported at 0.08 ppm and above in human study. Formaldehyde is carcinogenic at site of contact, as a consequence of epithelial cell regenerative proliferation resulting from cytotoxicity and mutation, based on studies in both animals and humans. Levels of formaldehyde in air detected in rural, suburban and urban areas in Japan were 2.5–3.2 ppb from 1998 to 2003. The majority of the population is exposed to airborne concentration of formaldehyde less than those associated with sensory irritation. The reference concentration of formaldehyde in air for Japanese general population is recommended to be 0.01 ppm. P12-04 THE LYSOSOMAL ENZYMES IN URINE IN THE ASSESMENT OF KIDNEYS FUNCTION IN WORKERS EXPOSED TO GASOLINE Z. Marchewka, A. Stokłosa, A. Długosz Department of Toxicology, Wroclaw Medical University, Poland


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The possibility of early detection subclinical kidneys damages among persons occupationally exposed to chronic influence of chemical toxins is still the object of the investigations. The present study attempts at the assessment of kidneys damage in the chronic inhalation exposure to gasoline vapors. The examinations have been carried out with the participation workers of Polish fuel consortium employed directly in the bottling plant department and reference group. The enzymes plentiful in kidney proximal tubular cells lysosomes have been evaluated in urine: NAG, NAG-B, ␤-Gr, GAL. The activity of nephron brush border enzyme GGT has been marked additionally (the cytoplasmic and microsomal fraction). In the examined group NAG activity was statistically significant higher than in the control group (p ≤ 0.006), NAG-B (p ≤ 0.03). In spite of low GGT activity the significantly increase (p ≤ 0.04) of the cytoplasmic fraction of this enzyme has been confirmed. Higher NAG, NAG-B and ␤-Gr activity levels have been identified among persons employed longer. The observed increase of NAG, NAG-B and GGT cytoplasmic fraction activity can indicate the extended permeability of cellular membranes in the exposure to gasoline vapors. The proved correlation of NAG and ␤Gr with NAG-B in the lysosomes membrane indicates the damage of lysosomal membranes of renal cells. The obtained results enable to detect slight changes in kidneys even in the reversible phase, before appearance clinical symptoms. P12-05 CHILDREN’S ENVIRONMENTAL HEALTH IN SWEDEN K. Victorin Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden On behalf of the government and the national board of health and welfare, the institute of environmental medicine at the karolinska institute, together with the work and environmental medicine unit at Stockholm county council has prepared an environmental health report with focus on children. Many experts participated in the preparation of the report. Important information came from a national survey of environmentrelated health in 30,000 children aged 8 months, 4

years and 12 years old. (Sweden has a population of about 9 million inhabitants and 1.5 million children). The questionnaire contained questions about health status, family and building conditions, parental smoking, asthma and allergy, exposure to noise and exhausts, etc. Children’s health in Sweden is generally good. Some effects on health can be linked to environmental factors. Children with allergic diseases are a particularly vulnerable group. Parental smoking and damp problems in the home are estimated to result in more than 500 and 1000 cases of infant asthma each year, respectively. Air pollutants can induce airway symptoms in children and increase the risk of impaired pulmonary function. Exposure to certain metals and persistent organic substances are near the level that may influence foetal development and small children. Perhaps the most widespread environmental problem for children is noise. The children themselves indicate that they are disturbed most by noise from other children and loud music. Impaired hearing, buzzing in the ears (tinnitus), sleeping problems and reduced concentration are serious effects of noise. P12-06 HANDBOOK OF EXPOSURE FACTORS IN EUROPEAN CHILDREN AND ADULTS CONSUMERS WITH AN INTEGRATED ENVIRONMENT AND HEALTH PROSPECTIVE A. Steenhout Group for Ecotoxicological Evaluation of Pollutants, Safety and Impacts on Humans (GEEPSIH), IGEAT, Free University of Brussels, Belgium A handbook has been built on determinants of exposure, with a particular emphasis to sensitive groups such as children. It develops a database for European populations with relevance to inhalation, ingestion and dermal exposure. An assessment of the resulting database is presented here. This demanding long-range project included identification and review of existing data covering parameters involved prior to absorption. The parameters describe lifestyles and behavior, activity patterns, time budget, dietary habits, living conditions, housing characteristics, population and sociological factors, consumption habits, anthropometric and physiological factors, etc. Variability and data distributions were

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gathered in terms of gender, age and socio-economic differences, when possible. The extent of variations among the European Member States or regions has been stated as well. Scopes of work assessed gaps in data sources and availability with reference to the various bodies of information, addressed comparability and uncertainty issues, standardization, etc. A built-in, flexible, data acquisition system allows further development of fields in functional tree structure and users search (including within documents) for information of interest to assessing generic or specific exposure. The data are then discussed with reference to European and US activities and needs in the field, in particular, to sensitive groups. Research has been funded by the CEFIC Long Range Research Initiative (LRI) program. P12-07 HEART RATE VARIABILITY IN PAINT MANUFACTURE WORKERS A. Siela´nczyk1 , L. Jagodzi´nski1 , J. Gmyrek1 , A. Bortkiewicz2 , E. Gadzicka2 1 Departament

and Clinic of Internal Diseases, Angiology and Physical Medicine, Medical University of Silesia, Bytom, 15 Batory St., Poland; 2 Department of Work Physiology and Ergonomics, Nofer Institute of Occupational Medicine, Lodz, 8 Teresy St., Poland Thirty-five workers of the Factory of Paints and Varnishes were examined. 13 of them were exposed to organic solvents at the admissible exposure limits (subgroup I); 10 workers were exposed to paint dust (subgroup II); 12 administration workers were the control group. Using the standard computer system, a minimum 10 min recording of heart rate was carried out. The recording was done after five-day work at the same time of the day. From among the parameters heart rate variability (HRV), LF/HF index was analyzed as a reliable description of autonomic nervous system balance. There was no statistical difference in the workers age and work period. Index LF/HF in subgroup I was 70.517 S.D. ±41.336; in subgroup II 51.436 S.D. ±28.214 and in control group 68.5 S.D. ±48.354. Comparison of LF/HF index values in the examined subgroup and in the control group did not show any statistical differences.


The study suggests no influence of organic solvents within admissible occupational exposure limits on LF/HF index. However, this is preliminary observation and needs further study. P12-08 THE INFLUENCE OF A MIXTURE OF ORGANIC SOLVENTS ON THE LEFT VENTRICULAR EJECTION FRACTION IN EXPOSED WORKERS A. Siela´nczyk1 , L. Jagodzi´nski1 , J. Gmyrek1 , A. Bortkiewicz2 , E. Gadzicka2 1 Department and Clinic of Internal Diseases, Angiology and Physical Medicine, Medical University of Silesia, Bytom, 15 Batory St., Poland; 2 Department of Work Physiology and Ergonomics, Nofer Institute of Occupational Medicine, Lodz, 8 Teresy St., Poland

We examined 23 male workers, at the same age and work tenure at the Factory of Paints and Varnishes, exposed to organic solvents. The control group consisted of 12 non-exposed workers from the same factory. The exposed group was then divided into two subgroups. Subgroup A: 13 workers exposed to organic solvents at admissible occupational exposure limits. Subgroup B: 10 non-exposed physical workers recruited from the same manufacture, working in the paint production line. Ultrasonocardiographic examination estimating ejection fraction (EF), the diastolic and systolic dimension of intraventricular septum (IVSd and IVSs) was carried out on the fifth day of their work at the same time of the day. In subgroup A, mean EF was 58.230% S.D. ±2.117, IVSs dimension was 13.230 S.D. ±1.120 mm and IVSd dimension was 9.169 S.D. ±1.100 mm. In subgroup B: EF −55.7% S.D. ±5.19, IVSs 12.580 mm S.D. ±1.774, IVSd 8.75 mm S.D. ±1.284. In control group: EF −52.916, IVSs −14.0 mm S.D. ±1.154, IVSd 10.025 mm S.D. ±2.102. Statistically significant difference in IVSs dimension was found between Subgroup B and the control group, p = 0.0437. Comparison of the remaining parameters did not show any statistically significant differences. Our study was performed in a small group of workers and further investigations have been undertaken to explain these results.


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P12-09 EUROPEAN NETWORK ON CHILDREN’S SUSCEPTIBILITY AND EXPOSURE TO ENVIRONMENTAL GENOTOXICANTS QLK4-CT-200202198 (CHILDRENGENONETWORK) L.E. Knudsen Department of Environmental and Occupational Health, University of Copenhagen, Blegdamsvej 3, DK 2200 Copenhagen N, Denmark The Concerted Action, Children Genotoxicity Network, explores gene-environment interactions during the foetal, neonatal and infancy developmental periods, concentrating on genotoxic exposures and environmental factors with focus on air pollution (traffic and tobacco). Work-package 1 has evaluated several studies analysing the effects of children’s exposure to chemical environmental pollutants with biomarkers of genetic damage. A number of biomarkers resulted to be consistently increased in children (MN, CA, DNA and protein adducts). MN show increasing frequencies with age even in children. Work-package 2 identified several mother/child cohorts within Europe with biobanks enabling follow up studies with biomonitoring. A pilot family study has been performed in work package 4 with samples from 24 families (12 mothers with 2 children in a polluted and 12 in a less polluted area) in Czech Republic as well as studies in Belgium, The Netherlands, Hungary, UK, Norway, Spain and Greece to explore adult versus child/newborn gene expression, DNA repair and susceptibility. Also work-package 3 has initiated pilot studies of placental transfer. In work package 6 (Ethics) state of the art in studies with focus on ethical issues in pre- and post-natal studies on children have been summarized. These issues are related to incentives to participate, data protection, data storage and dissemination. Discussions of maturity age and assent/consent from children are also important. The network is described at website and a number of publications have been issued.

P12-10 WORKING NOXAE AND NEUROPHYSIOLOGIC CHANGES IN THE MEDICAL STAFF G.F. Desogus Service of Prevention and Protection, Company USL 7 Carbonia, Italy The purpose of this study is to compare the epidemiological effects of non-degenerative neuropathies with neurobehavioral dysfunctions in the medical and nursing staff. The assessment of some neurophysiologic parameters—altered by working exposures (anaestetic gas, microclimate, psychophysical stress, overwork, biological agents, use of medical devices) or working organizations (which are: sleep disorders, heart rate troubles, muscular tremors, giddiness, cephalalgia, asthenia, depression and bad digestion) enable to detect the risk of neurophysiologic alterations. Constantly the toxicological data underline the neurotoxic risks linked to the physicophysical stress to which is exposed the medical staff is in its own workplace. P12-11 BIOLOGICAL MONITORING OF GENOTOXIC HAZARDS IN CIGARETTE MANUFACTURING INDUSTRY ˇ V. Kaˇsuba, R. Rozgaj, N. Kopjar, D. Zeljeˇ zi´c, M. Mili´c Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia Biological monitoring of genotoxic hazard was performed in 45 workers manufacturing cigarettes and 45 control subjects. The exposed workers were additionally exposed to a source of strontium 90, a strong beta-ray generator that measures or gauges the density of the moving tobacco-containing rod. The control subjects were not exposed to ionizing radiation or chemicals at their workplaces. Peripheral blood lymphocytes were analyzed for cytogenetic parameters (chromosomal aberrations and the micronuclei frequency). An analysis of variance (ANOVA) was performed to model the relationship between the confounding factors such as sex, age, years of exposure, smoking habits and X-ray diagnostics and chromosomal aberrations and

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micronuclei frequency. The exposed subjects significantly differed from controls in double chromosome breaks, acentrics and chromosomal exchanges (triradials and tetra-radials), as well as in the total micronucleus count. Total micronucleus count was significantly higher in the group exposed from 21 to 30 years of exposure than in the control group. In the exposed women smokers, only the total micronucleus count was significantly different from controls. The frequency of chromatid breaks was significantly higher in women. The detected increase in cytogenetic endpoints in the exposed workers might be mediated by a possible additive or synergistic action of different chemical agents used in cigarette manufacturing, and not by the genotoxic effect of a single cigarette component. P12-12 PROFESSIONAL DIFFICULTIES AMONG MIDWIFERY STAFF IN TEACHING HOSPITAL IN AHVAZ, IRAN F. Athari Ahvaz Jondishapour Medical Sciences University, Iran In Iran both governmental and private institutions are responsible for occupational health and occupational hygiene. The activity of the Ministry of Health consist in the protection against occupational diseases and improvement of the working environment. The aim of the study was to evaluate the professional problems of midwives. Materials and methods: For data gathering we used a questioner. We gave the questioners to 64 of midwives who were working in the Ahvas teaching hospitals. After a while we received 54 questioners. We used descriptive statistic for data analyzing. Results: In respect of treatment facilities, 98% of subjects had a negative issue. Seventy-six percent of them claimed that, they have not labor welfare facilities. Eighty-one percent suggested, there are no labor health facilities. Fifty-two percent of them had not educational facilities. 48.15% were not satisfied with ethical issues with their coworkers. Conclusion: According to results, we found that, midwives have a lot of professional problems, such as lack of health facilities. This can cause spreading of diseases such as Hepatitis and AIDS. So the authorities should pay attention to midwives professional problems.


P12-13 PLUTOCRACY PROJECT M. Saunders1 , L. Palkovicova2 , R.Van Den Heuvel4 , K. Desager5


Stoian3 ,

1 University of Bristol, UK; 2 Slovak Medical University,

Slovakia; 3 University Carol Davila, Romania; 4 VITO, Belgium; 5 University of Antwerp, Belgium This EU project investigates the epidemiologic relationship between exposures to pollutants which occur in utero with the incidence of early childhood allergy and alterations in immune responses. To achieve the objectives, pregnant women were recruited in five different regions across Europe (Bratislava (Br) and Stara Lubovna (SL) in Slovakia; Bucharest (Bu) and Giurgiu (G) in Romania; Mol (M) in Belgium. Placenta, cord blood, maternal peripheral blood and breast milk samples were collected. Serum IgE levels were determined. Samples were analysed for xenobiotics. Preliminary results indicated a wide variation across the regions. The recruited subjects were defined as atopic, nonatopic or indeterminate based on specific serum IgE levels and questionnaire data. The percent defined as atopic ranged from 10% in SL/Bu to 38% in M, percent non-atopic from 17% in M to 35% in SL. The percent of cord blood samples with positive IgE levels varied from 5% in SL to 42% in Bu. Maternal blood Pb and Cd concentrations ranged between 17–45 ␮g/L and 0.20–0.48 ␮g/L, respectively, with highest concentrations in Romania (p < 0.01). Concentrations of Cd in placenta varied between 5.1 and 8.9 ng/g with highest values in Romania (p < 0.05). Levels of organochlorine insecticides in placenta, maternal blood and breast milk were 3–10× higher in Romanian samples (p < 0.01). Chlorinated benzenes in placenta (0.49 ng/g) and breast milk (63 ng/g) were 2× lower in Belgian samples (p < 0.01). P12-14 EFFECT OF HYROALCOHOLIC EXTRACT OF LAVANDULLA OFFICINALIS (L.O.) ON NICOTINE-INDUCED CONVULSION IN MOUSE A. Arzi1 , M. Ahameh2 1 Department of Pharmacology and Toxicology, School

of Pharmacy, Ahvaz, Iran; 2 Jundishapour University of Medical Sciences, Ahvaz, Iran


Abstracts / Toxicology Letters 158S (2005) S1–S258

In this study the preventive effect of hydroalcoholic extract of Lavandulla offlcinalis on nicotine—induced convulsion in mouse has been investigated. In this study animals were divided into seven groups of eight mice each. Different doses (100, 200, 400, 600 and 800 mg/kg) of extract were administered intraperitoneally (IP). The negative and positive control groups received normal saline (10 ml/kg IP) and diazepam (0.15 mg/kg IP), respectively. After 30 min, nicotine (5 mg/kg IP) was given to all groups and the time of onset, duration and intensity of the convulsion and percentage of death were recorded. The results showed that 200, 400, 600 and 800 mg/kg of extract, increased onset of convulsion and decreased duration and intensity of the convulsion, significantly (P < 0.05). The best response observed with 600 mg/kg dose of extract. The time schedule for the best extract dose (600 mg/kg) administration and nicotine injection was 0, 15, 30, 45 and 60 min. The results showed that, the time of onset, duration and intensity of convulsion, increased, decreased and decreased significantly (P < 0.05), respectively for all the times. The best response observed in 30, 45 and 60 min. The results of this study showed that L.O. has anticonvulsant effect. In order to know the mechanism effect of extract, it needs more study on different animal models.

centration ranging from 1 to 50 mg/m3 . The results obtained show that the Bulgarian TWA 1 mg/m3 was unreasonably low. It is too difficult and very costly to be supported. The outline of the “Dose-effect” and “Dose-response” relationships gives reasons to accept temporary TLV-TWA of 10 mg/m3 . After 1999 investments were done and a decrease in concentrations was achieve. The results of the follow-up study show the improvement of the health status of workers exposed to CS2 . It was the reason to recommend 30 mg/m3 as a temporary TLV-TWA. Though the health status of workers with high sensitivity and low resistance should be monitored.


Lead is one of the dangerous and widespread poisons in the environment, especially for children. Erythrocyte and isolated erythrocyte membrane are suitable models to investigate membrane infringements at the lead intoxication in vitro: (1) getting into blood erythrocytes accumulate more than 90% of lead; (2) human erythrocyte has not nucleus, it excludes genotoxical effects; (3) erythrocyte membrane is less specialized, but has highly organized structure. Therefore the aim of our work was to study in vitro membrane-acting effects of lead at concentrations comparable to maximum available limit (0.5 mcM). Lead intoxication was modeled by incubation of erythrocytes and isolated erythrocyte membranes to 2–10 mcM of lead acetate for 1–3 h at 37 ◦ C. It was shown by methods of atomic absorption spectrometry that erythrocyte and isolated erythrocyte membranes accumulated lead ions effectively at these conditions. Using fluorescent probes and spin labels it was shown that under action of lead at subhemolytic concentrations on erythrocytes membranes

K. Lyubomirova, T. Popov, Tz. Georgieva, T. Panev, V. Nikolova, A. Mihailova, Hr. Copcheva Department of Toxicology, National Centre of Public Health Protection, Sofia, Bulgaria The TLV-TWA of carbon disulfide (CS2 ) accepted in the majority of European countries is 31 mg/m3 . At this exposure levels health effects remain contradictory. Five years ago the TLV-TWA of CS2 in Bulgaria was 1 mg/m3 , which was very low and technically hardly achievable. That was the reason for special planned studies to investigate the health status of workers exposed to 10 mg/m3 in 1999. A follow-up study of the same cohort of workers was performed in 2005. Examinations were performed on 110 workers exposed to CS2 in the production of synthetic fibres in con-

P13 Metals Toxicology P13 Metals Toxicology P13-01 LEAD AS A MEMBRANE-ACTING TOXIC METAL O.B. Oleksiuk, L.M. Lukyanenko, E.I. Slobozhanina The Belarus Satellite Centre of Trace Element Institute of UNESCO Academicheskaja 27, Minsk, 220072, Belarus

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in vitro microviscosity of lipid bilayer changes. Character of these changes depends not only on concentration of the toxic agent, but also from its distribution in lipid bilayer of membrane. It was found that erythrocytes of children are more sensitive to effect of lead at subhemolytic concentrations than erythrocytes of adult donors. For the first time it was demonstrated that level and functional activity of the integrated membrane protein multidrug resistance protein (MRP1) associated with resistance of cells to xenobiotics depend on concentration of lead in a blood and its presence in the incubation medium of erythrocytes in vitro. The data obtained may be concluded that lead has membraneacting toxic metal and erythrocytes and erythrocyte membranes can be used by models of lead intoxication. P13-02 NEUROBEHAVIOURAL PERFORMANCE IN ADULT RATS EXPOSED PERINATALLY TO METHYLMERCURY D. Wiaderna, P. Lutz, S. Gralewicz Nofer Institute of Occupational Medicine, Department of Toxicology and Carcinogenesis, Lodz, Poland The aim of the study was to investigate neurobehavioural effects of perinatal methylmercury exposure in adult rats. MeHg was administered orally to females during pregnancy and lactation at two dosing levels: 0.5 mg/kg/day or 2.0 mg/kg/day. The following functions were assessed at adulthood: locomotor activity in open field, spatial short-term memory in radial maze, long-term memory (passive avoidance learning), sensitivity to pain and vulnerability to stress in hot plate test and efficiency of the sensorimotor gating in startle response test. In the female offspring the level of open field locomotor activity was decreased. Radial maze test revealed no significant differences between groups (male and female). Males exposed to MeHg were worse in the passive avoidance performance. Testing on the hot plate


have shown disappearance of the effect of stress in males of the high exposure group. In both exposed groups of males, the prepulse inhibition values were reduced. In the rat perinatal exposure to MeHg results in some behavioural alterations detectable in adulthood rats. Daily dose of MeHg as low as 0.5 mg/kg is effective in inducing some alterations. Alterations developing in males differ qualitatively from those developing in females. This study was performed within the frame of the “DEVNERTOX” project supported by the European Commission, contract no: 6PR/03/506143. P13-03 BEHAVIORAL SENSITIVITY TO AMPHETAMINE IN ADULT RATS BORN TO MOTHERS EXPOSED TO METHYLMERCURY P. Lutz, S. Gralewicz, D. Wiaderna Nofer Institute of Occupational Medicine, 8 Teresy St., 90-950 Lodz, Poland Mercury is a common environmental pollutant. In soil and aquatic systems mercury is being biotransformed into methylmercury (MeHg) and as such it enters the food chain. There is evidence that even lowlevel maternal dietary exposure to MeHg may result in altered functioning of the dopaminergic system during maturity. The present experiment was performed in order to find out whether and how maternal exposure to MeHg affects the offspring’s sensitivity to amphetamine (AMPH) at adulthood. Female rats, Wistars, were exposed orally to MeHg from day7 of pregnancy to day 21 post partum, at doses 0.5 mg/kg/day) or 2.0 mg/kg/day. The testing started at about PND90. It consisted in measuring the behavioural response to a test dose of AMPH (0.5 mg/kg, i.p.): (a) before an acute treatment with a high dose (5.0 mg/kg) of AMPH in order to assess the rat’s “normal” sensitivity to the psychostimulant, and (b) three weeks after an acute treatment with AMPH at the dose of 5.0 mg/kg (sensitization induction). Data from step1 have shown no group


Abstracts / Toxicology Letters 158S (2005) S1–S258

differences in the locomotor behaviour induced by the AMPH test dose. In step 2, in males of both MeHg exposed groups, the locomotor response to AMPH challenge was stronger than in the control group. (i) Sensitivity to amphetamine in adulthood is apparently not affected by maternal exposure to MeHg; (ii) in males, maternal exposure to MeHg may result in an increased susceptibility to behavioral sensitization by an acute AMPH treatment. This study was performed within the frame of the “DEVNERTOX” project supported by the European Commission: Contract no:6PR/03/506143). P13-04 SELENIUM-INDUCED DECREASE OF CADMIUM IN TISSUES OF SUCKLING RATS M. Lazarus, M. Blanuˇsa, K. Kostial Institute for Medical Research and Occupational Health, Zagreb, Croatia Cadmium (Cd) as a toxic metal is of special concern regarding exposure and effects in neonates. Selenium (Se) prevents the toxicity of Cd through undefined mechanisms causing Cd redistribution in tissues. This protective effect has not yet been studied in the early postnatal period. Suckling Wistar rats were orally exposed to CdCl2 ·xH2 O and/or Na2 SeO3 in equimolar amounts from day 6 to day 14 after birth. Two experiments were carried out. In the first experiment Se and Cd were given concurrently over nine days (0.004 mmol/kg/day, each). Pups of the same age in the second experiment were pre-treated with Se for four days and then concurrently treated for five days with Cd (0.008 mmol/kg/day, each). The concentrations of Cd and essential metals in kidneys, liver and brain at day 15 after birth were determined by electrothermal atomic absorption spectrometry. A decreasing effect on Cd concentrations in pups receiving Cd and Se in the first experiment was less pronounced, as indicated by a 26%, 68% and 54% reduction in kidneys, liver and brain, respectively, than in those receiving comparable dosages of Cd but pretreated with Se, indicated by a 72%, 87% and 67% decrease in the same organs. The present study demonstrated that Se significantly decreased organ Cd concentrations in pups in both experimental designs.

P13-05 INTERFERENCE OF SELENOMETHIONINE ON MERCURY ACCUMULATION IN TARGET ORGANS IN RATS EXPOSED TO METHYLMERCURY M.C.C. Bator´eu, M.L. Mateus, C.M.L. Carvalho, A.P.M. Santos CECF, Laboratory of Toxicology, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal Repeated exposure of rats to low doses of methylmercury (MeHg) induces the accumulation of mercury in target organs, namely in kidney and brain. Previous work in our laboratory demonstrated that the increase of urinary porphyrins and Hg accumulation in brain are correlated with the increase of neurobehavioural dysfunctions in rats exposed to mehg. On the other hand, it is well known, that the alterations in urinary porphyrins excretion patterns are due to Hg accumulation in the kidney and to consequent effects of Hg2+ on renal porphyrin metabolism. In this work, the protective effects of selenomethionine (SeMet) against MeHg toxicity was investigated by the quantification of Hg in kidney and brain and by the determination of porphyrins concentration in urine of rats repeatedly i.p. exposed to two and four doses (1.5 mg/kg/day) of MeHg and in rats co-exposed to MeHg (1.5 mg/kg/day) and semet (1.0 mg/kg/day). Urine of 24 h was weekly (5 weeks) analysed for total porphyrins using a routine colorimetric assay and at the end of the experiment brain and kidney total hg concentration was determined by cvafs. The interference of semet on the toxicokinetics of MeHg was further investigated by the evaluation of porphyrins profile and specific porphyrins were selected as potential biomarkers of mercury internal dose. The urinary porphyrins were identified and quantified by hplc/fld and the concentration of pentacarboxyl and coproporphyrins in rats exposed to mehg were significantly higher compared with the ones detected in rats exposed to the mixture (SeMet + MeHg). Finally, the correlation between the changes on hg contents in target organs and porphyrins excretion is under discussion in order to clarify the mechanism of selenium protection against methylmercury toxicity.

Abstracts / Toxicology Letters 158S (2005) S1–S258


Supported by Project POCTI/41741/ESP/2001 (FEDER, OE) Risk Assessment of Methylmercury Exposure: integrated actions through the food chain.

the HPLC method is the right choice to characterize the urinary porphyrins and to identify a specific metal exposure.


Supported by Project POCTI/41741/ESP/2001 (FEDER, OE) Risk Assessment of Methylmercury Exposure: Integrated actions through the food chain.

M.L. Mateus, R.I.L. Ferreira, C.M.L. Carvalho, A.P.M. Santos, M.C.C. Bator´eu CECF, Laboratory of Toxicology, Faculty of Pharmacy of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal Urinary porphyrins are a sensitive but unspecific biomarker of animal and human exposure to metals. In this study, a simple and rapid spectrophotometric method was compared to the HPLC/FLD method using two different sample pre-treatments to evaluate porphyrins in urine of rats exposed and not exposed to methylmercury (MeHg) (1.5 mg/kg/day). The results obtained in the colorimetric assay confirm a significant increase of porphyrins in rats exposed to MeHg and the subsequent identification of porphyrins in the same extract of urine by HPLC show coproporphyrins as the main porphyrins (96%) detected in the colorimetric assay. Comparison between the different sample treatments (Wang et al., 2002; Woods and Simmonds, 2001) for HPLC analysis demonstrated that the previous urine extraction proposed by Woods decreases the matrix interferences but is time consuming and didn’t increase the sensitivity and resolution of chromatographic peaks. Thus, no significant changes in porphyrins profile between the two different sample treatments were observed. The characterization of urinary porphyrins by HPLC show a significant increase of pentacarboxylporphyrin and coprophorphyrins in urine of rats exposed compared with control samples. However, the ratio Copro/Penta is lower in exposed than in control urine, which indicates a higher relative increase of pentacarboxyl (4-fold) as compared to coproporphyrins (2.5fold). In conclusion, pentacarboxylporphyrins seem to be a more sensitive biomarker of MeHg exposure, and

References: Wang, J.P., et al., 2002. Cellular and Molecular Biology 48 (8), 835–843. Woods, J.S., Simmonds, P.L., 2001. In: Current Protocols in Toxicology, Unit 8.9.

P13-07 ORALLY ADMINISTERED MELATONIN DECREASES TISSUE ACCUMULATION AND TOXICITY OF CADMIUM IN MICE E. Chwelatiuk, T. Wlostowski, E. Bonda, A. Krasowska University of Bialystok, Institute of Biology, Bialystok, Poland The aim of study was to determine whether a long-term oral administration of melatonin, a known antioxidant, free radical scavenger and metal chelator, would protect against cadmium (Cd)-induced injury in the kidneys and liver of mice exposed subchronically to the metal. The animals received drinking water containing 50 ␮g Cd/ml and 2, 4 and 6 ␮g/ml of melatonin for 8 weeks. Histopathological changes occurred only in the kidneys (glomerular swelling and focal tubular degeneration) in all mice from a Cd alone group. In mice co-treated with melatonin only slight (2 ␮g/ml) or no damage (4 and 6 ␮g/ml) was seen. Cd and melatonin treatments did not affect renal lipid peroxidation and iron concentration; in the liver the two parameters were significantly reduced upon the exposure. Melatonin brought about a dose-dependent decrease in the renal, hepatic and intestinal Cd concentrations, and the renal and hepatic metallothionein levels followed a pattern similar to that of the Cd accumulation. These data suggest that orally administered melatonin protects the kidneys against Cd-induced injury by reducing Cd accumulation rather than suppressing oxidative stress.


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P13-08 GENE EXPRESSION MODULATION IN THE LIVER OF MICE FOLLOWING ACUTE OR CHRONICAL EXPOSURE TO ARSENIC B. Casati, A. Collotta, L. Clerici, E. Marafante Physical and Chemical Exposure Unit, IHCP, Joint Research Centre, 21020 Ispra, Italy DNA macroarrays was applied to evaluate gene expression response in the liver of mice exposed to a single acute dose of arsenic or chronically exposed to arsenate in drinking water. Adult male mice were exposed to sodium arsenate in drinking water (1 mg As/l) for 4 months and the modulation of gene expression in the liver compared to that induced by exposure to a single acute dose at 4 and 24 h after treatment. After 4-month of exposure to arsenate significant up modulated genes were found, mostly belonging to families of metabolism, DNA-synthesis and repair, protein turnover, receptors, transcription and post translation modification. At 24 h following the exposure of the acute dose, some of these genes were already up-modulated while at 4 h after treatment only down modulated genes were see. Major differences between the acute and chronic doses were observed in the down-modulated genes at 4 and 24 h following the acute dose, involving gene families of extra-cellular transport, metabolism and protein turnover. These results suggest that the length of exposure affects the biological response to arsenic exposure and the modulation of specific gene expression. P13-09 EVALUATION OF CADMIUM CHLORIDE EFFECTS ON MOUSE SPERMATOGENESIS BY FLOW CYTOMETRY H. Oliveira, L. Filipe, J. Loureiro, C. Santos, M.L. Pereira Department of Biology, University of Aveiro, 3810 Aveiro, Portugal Cadmium is a well known testicular toxicant and several studies on testis histology are available. However, as far as we know, quantification of testicular cell ratios

was not yet performed. The aim of the present work was to quantify the different testicular cell types in mice after a cadmium chloride exposure, by flow cytometry using paraffin embedded material. Seven weeks old male mice were subcutaneously injected with 1, 2 or 3 mg of CdCl2 /kg body weight. Control animals were injected with saline. After 24 h animals were sacrificed and testis were removed, fixed in neutral 10% buffered formalin and embedded in paraffin. Sections were deparaffinized, rehydrated and digested with pepsin. The nuclear suspension was incubated with RNAse and PI and analysed by flow cytometry. Cadmium chloride (particularly the dose of 3 mg of CdCl2 ) induced significant changes in number and ratios of testicular cells. A decrease in the number of haploid (1C) cells and an increase in diploid (2C), S phase and tetraploid (4C) cells was also observed. These changes may be due to the effects of CdCl2 on both blood testis barrier and vascular endothelium. Cadmium disrupts inter Sertoli tight junctions and alters Sertoli-germ cell adhesion with consequent exfoliation of spermatids within the seminiferous tubules. On the other hand a decrease in blood supply to germ cells leads to testicular necrosis. The results obtained by FCM are in accordance with the histopathological evaluation. The use of embedded material for FCM analysis allows long time storage and the possibility of making some replicas, which increase the reliability of the results. P13-10 PLASMA METALLOTHIONEIN LEVELS IN LEAD POISONED CHILD R. Prusa, O. Blastik, J. Kukacka, R. Kizek, H. Stuchlikova 1 Department

of Clinical Biochemistry and Pathobiochemistry, 2nd Faculty of Medicine Charles University, V Uvalu 84, 150 06 Praha 5, Czech Republic; 2 Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zemedelska 1, 611 37 Brno, Czech Republic A 11-year-old girl was referred to the hospital by her general practitioner because of abdominal pain, vomiting, dark colour of tongue, low intake of fluid and food, and abnormal laboratory results (bilirubin 55 ␮mol/l,

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ast 2.94 ␮kat/l, alt 3.46 ␮kat/l, haemoglobin 86 g/l). Blood film exhibited basophilic stippling, prompting for investigation of lead poisoning. Blood lead levels were measured by automated graphite furnace aas (varian). Plasma metallothionein levels were measured by electroanalytical technique—voltametric Brdicka reaction. In time of admission, the blood lead was 648 ␮g/l, and metallothionein 153 ␮mol/l (normal values below 10 ␮mol/l). Chelation therapy by EDTA was administred for five consecutive days. During the course of therapy the b-Pb decreased to 360 ␮g/l, u-Pb increased to 6019 ␮g/24 h, p-metallothionein increased to 276 ␮mol/l. After 5 days of chelation treatment, the b-Pb increased (535 ␮g/l), p-metallothionen decreased (147 ␮mol/l). The source of 6 months lead exposure was identified as tea from a ceramic tea pot with insufficient glazing (lead concentration in tea after 30 min was 45332 ␮g/l). Both this child’s mother and grandparents were also poisoned by lead. In conclusion, there is need to be aware of lead exposure risk from commonly used materials. This work was supported by grants: gacr 525/04/p132, raso 2005. P13-11 PRO-INFLAMMATORY CHANGES FOUNDED IN AORTIC WALL OF CADMIUM TREATED RATS V. Mlynek1 , M. Rzeszutko2 , A. Skoczynska1 1 Department

of Internal Medicine, Medical University of Wroclaw, Poland; 2 Department of Pathological Anatomy, Medical University of Wroclaw, Poland The histopathological changes caused by cadmium in blood vessels wall were determined. Male Buffalo rats were treated by solution of cadmium chloride in concentration of 50 ppm (n = 12), 5 ppm (n = 12) and distillated water (n = 12). After three months, in the deep anesthesia, the thoracic aortas were prepared. Mean blood cadmium level in poisoned rats (36.5 ± 3.6 ␮g/l and 2.95 ± 1.07 ␮g/l, respectively, in animals given 50 ppm or 5 ppm of metal) was significantly higher (p < 0.001) than in controls (0.016 ± 0.01 ␮g/l). Mean thickness of aortic wall in the rats treated with 50 ppm of cadmium was significantly higher in comparison to controls (646 ± 85 ␮m versus 498 ± 86.2 ␮m; p < 0.01) and aortic wall was asymmetric.


Histopathological changes were observed especially in rats treated with cadmium in a dose of 5 ppm. There were lymphocytes linked to endothelium, subendothelial infiltration of lymphocytes, lack of the endothelium and different endothelium thickness. Moreover, aortas of these rats displayed excavations which formed pseudocrypts. Collagen fibers distribution was nonparallel and irregular. Also lymphocytic infiltration in the periaortic soft tissue was founded more often in rats treated with cadmium in a dose of 5 ppm. It was concluded that proinflammatory effect of cadmium in vessels wall was dose-dependent and it was more evident in rats poisoned with cadmium in the smaller dose. P13-12 HOMEOPATHIC MEDICATION CURY’S CHELATING AGENT



L.A. Beringhs-Bueno1 , A.E. Paschalicchio2 1 Brazilian

Institute Louis Blanc of Studies and Research, S˜ao Paulo, Brazil; 2 Health Institute, S˜ao Paulo State Heath Department, Epidemiological Division, S˜ao Paulo, Brazil Mercury (Hg) is one of the toxic metals presenting the most environmental and occupational hazards. For many years specialists have been searching for ways of reducing heavy metal toxicity in vivo by using chelating agents or other competing substances. Another effective method is the use of ultradiluted quantities of the toxic metals themselves, either in pre or posttreatment of poisoning. Homeopathy can control metal poisoning levels in human beings. This paper evaluates the results of homeopathic medication in the treatment of Hg-contaminated patients. Clinical and laboratory data on 52 patients with a history of occupational exposure to mercury were collected. The patients were randomly selected and blindly distributed into two different groups: placebo and Mercurius solubilis (7 CH and 12 CH). The choice of homeopathic medication was based on the principle of similitude to the toxic metal. Those patients had been submitted to the Hg blood, urine and in the hair analyses before the beginning of the treatment, in 30 and 60 days. By the end of the treatment it had a significant Hg hair level reduction in those individuals treated with homeopathic medication, beyond the presence of indications of the increase Hg urinary elimination. Mercury symptoms had also


Abstracts / Toxicology Letters 158S (2005) S1–S258

significant improvement during the homeopathic treatment. This study showed that Hg highdilution was effective in treating mercury contamination. Further studies with a bigger population are necessary to a greater comprehension about the effectiveness of the homeopathic treatment. P13-13 INTERACTION OF DNA WITH LEAD NITRATE IN SOLUTION M. Jafari1,2 , A. Rabbani1 , S. Mohmadian1 1 Institute

of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; 2 Department of Biochemistry, Faculty of Medicine, Baghiytollah Medical Sciences University, Tehran, Iran Lead is one of the most common heavy metals in the environment that the genome of the cell has been introduced as a target molecule. In this study, interaction of lead nitrate with calf thymus DNA was investigated employing UV–vis spectroscopy, thermal denaturation and equilibrium dialysis techniques. The results show that when lead nitrate was added to DNA solution hypochromicity was occurred at 210 nm accompanied by red shift. Thermal denaturation profiles showed that Tm of DNA was reduced in the presence of lead nitrate in a dose dependent manner. Equilibrium dialyses indicated that the binding is cooperative with a positive slope at low r values. The results suggest that binding of lead nitrate to DNA is concentration dependent, at low values has no considerable effect on DNA but at higher concentrations destabilizes DNA and produces structural changes on DNA. The interaction is mainly with the negatively charged residues of DNA. P13-14 GENOTOXIC RESPONSES IN RATS EXPOSED TO INHALATIONS OF URANIUM COMPOUNDS M. Monleau1 , M. De M´eo2 , V. Chazel1 , F. Paquet1 , G. Dum´enil2 , M. Claraz1 1 Laboratoire

de radiotoxicologie exp´erimentale, IRSN/DRPH/SRBE, BP166, 26702 Pierrelatte, France; 2 Laboratoire de Biog´ enotoxicologie et mutagen`ese environnementale (EA 1784), Facult´e de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille, France

Inhalation of airborne uranium compounds is a major consideration for the health protection of nuclear workers. Different situations of contamination by inhalation occur at the workplace. The aims of this study were to determine the genotoxic effects in function of (i) dose with several acute inhalations of uranium dioxide (UO2 ), (ii) solubility with acute inhalations of insoluble UO2 and moderately soluble uranium peroxide UO4 and (iii) mode of inhalation with acute and repeated UO2 inhalations. Male Sprague-Dawley adult rats were exposed to uranium aerosols in the different described conditions and were sacrificed 1, 3, 8, 14 and 30 days postexposure. The general health conditions of rats were assessed by the follow-up of weight, food and water consumptions and biochemical analyses in serum. The genotoxicity study was assessed by comet assay on nasal epithelial cells, broncho-alveolar lavage (BAL) cells and kidney cells. The general physiological effects of uranium were dependent on the dose, the solubility and the mode of inhalation (acute or repeated). Repeated exposure did not alter the general conditions of rats. No genotoxic effect was detected in epithelial nasal cells with the different tested conditions. In BAL cells, the genotoxic effect appeared to be linked to a threshold dose because it was limited to the highest tested dose of UO2 (600 ␮g g−1 Lung). No solubility effect was observed at the tested dose (40 ␮g g−1 Lung). Repeated inhalations induced higher and longer responses than acute inhalation. These results raise questions on the long-term effects of repeated exposure in lungs. P13-15 SMOKING IS ASSOCIATED WITH HIGHER CADMIUM LEVELS IN BLOOD BUT NOT WITH LEAD J. Kukacka, J. Cepova, R. Prusa, K. Kotaska Department of Clinical Biochemistry and Pathobiochemistry, 2nd Faculty of Medicine, Charles University, V Uvalu 84, 150 06 Prague 5, Czech Republic A significant flux of heavy metals such as cadmium (Cd) and lead (Pb) reaches the lungs through smoking. The objective of the present study was to evaluate the association between blood levels of lead and cadmium

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in 50 adult smokers (25 males and 25 females) and 50 non-smokers. Lead and cadmium levels were measured by graphite furnace atomic absorption. Smoking was assessed by self-reported cigarette consumption and serum cotinine levels. The mean serum cotinine concentration was 303 ± 29 ␮g/L in male, 316 ± 26 (mean ± S.D.) in female smokers. The both male and female smokers had significantly higher concentration of Cd (1.4 ± 0.55 ␮g/L and 1.5 ± 0.74 ␮g/L) than nonsmokers (0.5 ± 0.27 ␮g/L for male and 0.5 ± 0.25 ␮g/L for female). There was a significant positive association between the blood Cd and serum cotinine levels (r = 0.71; P < 0.001). In contrast, Pb concentrations did not show any significant correlation nether with serum cotinine nor with Cd. Blood Pb levels were 53 ± 51.1 ␮g/L in male smokers and 42 ± 39.6 ␮g/L in female smokers. In conclusion, smoking is associated with increased cadmium levels in blood but not with lead. The main source of cadmium is cigarette smoking whereas Pb intake comes from food and air in roughly equal proportions. P13-16 TOXICITY OF CADMIUM AND CELLULAR STRESS: BILIRUBIN A POTENTIAL ENDOGENOUS SUBSTRATE OF CYTOCHROME P450 2A5 A.A. Bakar1 , M.R. Moore1 , M.A. Lang2


induction of firstly HO-1 then CYP2A5 mRNAs, which was supported by Western blot analysis of the respective proteins. BR degradation was measured in liver microsomes of Cd-treated and control mice. Eighteen hours after treatment in vivo, a 46% reduction in CYP was observed. In contrast, COH activity increased 7fold and there was a 10-fold increase in the rate of microsomal BR degradation. Microsomal COH activity in the presence of various concentrations of BR showed that inhibition of activity by BR is 100%. The double-reciprocal plot of microsomal COH activity in the absence and presence of BR showed that Km increased and Vmax fell in the presence of bilirubin. BR-degrading activity was found to be inhibited by an antibody raised against CYP2A5. Coumarin, a specific substrate for CYP2A5, was also shown to inhibit the rate of BR degradation. The observation that BR degradation by CYP2A5 was induced by Cd treatment in vivo indicates its role in the regulation of cellular haem levels. P13-17 EVALUATION OF NEUROTOXIC EFFECTS OF LEAD ACETAT ON RAT RADIAL NERVE M. Mehdizadeh1 , F. Kermanian2 , G. Farjah3 , P. N.Tabatabaei1 1 Iran University of Medical Sciences, Iran; 2 Army Uni-

1 EnTox,

versity of Medical Sciences, Iran; 3 Oromieih University of Medical Sciences, Iran

Oxidative stress is an important component of cadmium (Cd) toxicity. To understand how the cell protects itself from Cd-induced oxidative stress we studied in vivo effects of Cd on concurrent upregulation of cytochrome P450 2A5 (CYP2A5) and haem oxygenase-1 (HO-1). With the role of bilirubin (BR) in the CYP2A5-dependent coumarin metabolism. CdCl2 treated male mice had a significant decrease of total hepatic cytochrome P450 (CYP) and significant increase of HO activity. Treatment caused a progressive increase in Coumarin 7-hydroxylase (COH) activity (CYP2A5). Northern blot analysis showed concurrent

Lead toxicity is a common popular problem. Many researches was performed about this toxicity both in vivo and in vitro from 100 years ago. Those studies showed that lead have toxic effects such as behavioral disorders (9, 11), decrease of IQ (3) and decrease of learning and memory (2). Also lead have neurotoxic effects such as decrease of neuronal density in visual cortex of monkey (6), cell death in hippocampus (10) and decrease of acetylcolin in rat’s hippocampus (1). In this study we examine neurotoxic effects of lead on rat’s radial nerve because radial nerve is a mix nerve. 24 adult male rats was divided in six groups. Groups I and II received lead acetate 4% and 2%, groups III and IV received distilled water and normal water for one month. After this time, we killed rats and exposed radial

The University of Queensland, Queensland Health Scientific Services, Brisbane Australia; 2 Department of Biochemistry, Uppsala University, Sweden


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nerve from behind of arm. Then studied them with light and electron microscopy. In experimental groups we saw decrease of myelin sheath diameter and decrease of nuclear density in schwann cell. Also we saw many granules in mitochondrial matrix, active macrophage, edema and disarrangement of myelin sheath layers. We suggest that lead neurophaty is due to schwann cell injury and this injury lead to decrease of myelin sheath. P13-18 PROTECTIVE EFFECTS OF VITAMINS AND ZINC AGAINST HEMATOLOGICAL CHANGES ON LEAD EXPOSURE IN RATS DURING PREGNANCY AND LACTATION E. Mass´o, I. Corpas, M.T. Antonio Garcia Department of Physiology, Faculty of Biology, Complutense University, 28040 Madrid, Spain The assumption of oxidative stress as a mechanism in lead (Pb) toxicity, suggests that some antioxidants, as vitamins and others minerals, e.g. Zinc, might play a role in the treatment of lead poisoning. This study aimed to investigate the effects of gestational and lactational exposure to 300 mg/L of lead, administered in drinking water, on haematological parameters of pups. It also explored the possibility that combined treatment with lead and antioxidants, Vitamins A (50.000 U.I./L), E (500 mg/L), C (2000 mg/L), B6 (500 mg/L) and Zn (20 mg/L), was capable of reversing the alterations caused by lead. In our protocol of lead exposure during gestation and lactation, at 21 days of age, pups showed a normochromic and microcytic anaemia. Lead combined with antioxidants treatment restored the mean corpuscular volume and increased the hematocrit value towards normal. There were significant differences on erythrocyte osmotic fragility that was four-fold greater in lead exposed pups than in control pups. Lead combined with antioxidants treatment reduced the osmotic fragility of red blood cells to control values. Finally, blood ␦-aminolevulinic acid dehydratase (ALA-D) activity, was significantly reduced (90%) in both lead-treated animals, when compared to control values. Lead combined with antioxidants treatment produced a significant increase of this activity, but it could not reach control values.

P13-19 ULTRASTRUCTURAL AND IMMUNOCYTOCHEMICAL ANALYSIS AFTER THE INHALATION OF VANADIUM, MANGANESE AND ITS MIXTURE ON MICE CORPUS STRIATUM AND SUBSTANTIA NIGRA M.R. Avila-Costa1 , L. Colin-Barenque1 , V. AnayaMart´ınez1 , A. Reyes1 , P. Aley1 , J.L. Ordo˜nezLibrado1 , A.L. Gutierrez-Valdez1 , G. Pi˜no´ n-Z´arate2 , V. Rodr´ıguez-Lara2 , M. Rojas-Lemus2 , P. MussaliGalante2 , O.L. Saldivar3 , M.G. Espejel3 , V. Delgado2 , T.I. Fortoul2 1 FES Iztacala, National University of Mexico (UNAM),

Mexico; 2 Facultad de Medicina, National University of Mexico (UNAM), Mexico; 3 Facultad de Qu´ımica, National University of Mexico (UNAM), Mexico Human population is constantly exposed to chemical mixtures of pollutants such as metals; information about the consequences of the interactions of these compounds on the nervous system is scarce. It has been reported that vanadium (V) and manganese (Mn) alters brain catecholamine concentrations, thus the current study explores the effects of the inhalation of V, Mn and its mixture (V–Mn) in a mice model, analyzing brain metal concentrations, tyroxine hydroxylase (TH) immunocytochemistry in the Substantia Nigra and ultrastructural modifications in the Corpus Striatum after eight weeks of inhalation. Our results showed that brain concentrations of both metals alone were higher compared to controls; however V concentrations drastically decrease with the mixture. These data correlate with the morphological alterations observed, which consisted in decreased number of TH-positive cells, neruopil changes and neuronal necrosis. These modifications were mainly given by Mn, alone or in combination with V. The additive effect of V–Mn is suggested, since the extensive damage observed was more evident when mice were exposed to the mixture, and the results endured more research in the area of inhaled mixtures.

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P13-20 GENDER DIFFERENCES IN SPLEEN MEGAKARYOCYTE FEATURES AFTER CHRONIC INHALATION OF VANADIUM G. Pi˜no´ n-Z´arate1 , V. Rodr´ıguez-Lara1 , M. RojasLemus1 , M.R. Avila-Costa2 , P. Mussali-Galante1 , L. Colin-Barenque2 , V. Delgado1 , F. Pasos1 , A. Rondan1 , S. Antuna1 , T.I. Fortoul1 1 Facultad de Medicina, National University of Mexico

(UNAM), Mexico; 2 FES Iztacala, National University of Mexico (UNAM), Mexico A previous report from our group informed the presence of thrombocytosis as a consequence of vanadium inhalation. This time, we decided to evaluate the effects of this element on megakaryocytes and to identify its morphological modifications and to compare the changes between males with females. CD-1 male and female mice inhaled V2 O5 0.02 M 1 h, twice a week during 10 weeks. Spleens were extracted, weighed, and processed by the usual histological technique. The main changes observed were: an increase in the amount of megakaryocytes in those mice exposed to V, mainly in females. These findings explain in part, the thrombocytosis reported previously. The questions to answer are: why this gender difference takes place, and what is its effect on platelets function. P13-21 INHIBITION OF ERYTHROCYTE PHOSPHORIBOSYLTRANSFERASES (APRT AND HGPRT) BY LEAD: A POTENTIAL MECHANISM FOR HEMOLYSIS IN LEAD POISONING I. Baranowska-Bosiacka1 , V. Dziedziejko1 , B. Dol˛egowska1 , K. Safranow1 , M. Marchlewicz2 , D. Chlubek1 1 Department

of Biochemistry and Chemistry, Pomeranian Medical University, 72 Powsta´nc´ow Wlkp. St., 70-111 Szczecin, Poland; 2 Department of Histology and Embryology, Pomeranian Medical University, 72 Powsta´nc´ow Wlkp. St., 70-111 Szczecin, Poland Adenine and hypoxanthine phosphoribosyltransferases, APRT and HGPRT, enable the salvage of purine bases (adenine, guanine and hypoxanthine) and transform them to nucleotides (AMP, GMP and IMP, respectively). Recent studies show that red


blood cells in people exposed to lead have lower ATP concentrations and adenylate energy charge values. The aim of this study was to find if lead ions inhibit the activities of erythrocyte APRT and HGPRT. Red blood cell hemolysate from a healthy person was incubated (for 30 min, at 37 ◦ C) in the presence of lead acetate at concentrations 100 ␮M, 10 ␮M and 0 ␮M (control). The HGPRT and APRT activities were measured by checking the respective IMP and AMP levels (HPLC) after 20 min hemolysate incubation with PRPP, hypoxanthine and adenine, in the presence of the aforementioned concentrations of lead ions. Without lead the mean activity was 0.914 ± 0.067 nmol/mg hemoglobin/min for APRT and 3.610 ± 0.244 nmol/mg hemoglobin/min for HGPRT. Lead acetate at concentrations 100 ␮M and 10 ␮M inhibited the activity of APRT by 36.4% (p < 0.035) and 34.4% (p < 0.009), and that of HGPRT by 30.5% (p < 0.035) and 22.2% (p < 0.009), respectively, versus control. Lead ions moderately inhibit both phosphoribosyltransferases even at the very low lead ion concentrations. It seems important that the inhibitory influence of lead should be further examined at the physiological concentrations of substrates and in intact erythrocytes. Our results suggest that the short life of erythrocytes exposed to lead may be due to the energy metabolism inhibition caused by lower salvage pathway enzyme activity. P13-22 INVOLVEMENT OF MAP-KINASES AND PKC IN CADMIUM-INDUCED CYTOKINE RELEASE FROM LUNG CELLS M. L˚ag, E. Lilleaas, P.E. Schwarze, R. Hetland, M. Refsnes Norwegian Institute of Public Health, Oslo, Norway Lung is one of the main target organs for cadmium toxicity. We have established an in vitro system of type 2 cells and Clara cells isolated from rat lung, which are used for studying signal transduction mechanisms involved in cadmium-induced apoptosis and cytokine release. In this study, we studied Cd-induced release of interleukin-6 (IL-6) from epithelial lung cells and involvement of MAP-kinases and PKC. The expres-


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sion of the different MAP-kinases and PKC isoforms, both total and activated (phosphorylated) were studied by Western blotting technique. The results show that the levels of phosphorylated p38, -JNK and -ERK were increased 2 h after cadmium exposure in both cell types. However, the levels of phosphorylated PKC␣ and PKC␦ did not change after cadmium exposure. The cell cultures were preincubated with inhibitors of p38 (SB202190), JNK (SP600125), ERK (PD98059), PKC (GF109203X and rottlerin) prior to cadmium exposure. SB202190, GF109203X and rottlerin reduced the cadmium-induced IL-6 release in both Clara cells and type 2 cells. However, PD98059 did only reduce the IL-6 release in type 2 cells, whereas in Clara cells the IL-6 release was not affected. In conclusion, p38 and PKC seem to be involved in the cadmium-induced IL-6 release in both epithelial cell types; however, activation of ERK seems only to be involved in IL-6 release from type 2 cells. P13-23 ZINC CONTENT IN THE KIDNEY OF SUBACUTE CADMIUM INTOXICATED MICE: INFLUENCE OF MAGNESIUM D. Djuki´c, V. Matovi´c Institute of Toxicological Chemistry, Faculty of Pharmacy, Belgrade, SCG Cadmium–zinc interactions are considered to be of special importance in cadmium toxicity. Cadmium causes a redistribution of Zn and replaces Zn in zinc-dependent enzymes, but these interaction can also be confounded by interactions between the essential ions themselves. On the other hand, our and some other findings confirm beneficial effect of magnesium in heavy metals intoxication. The goal of this work was to investigate the effect of increased oral magnesium pretreatment on zinc metabolism in kidney of mice exposed to subacute cadmium intoxication. The experiment was performed on male Swiss mice divided into three groups: a control group; a Cd group—mice intoxicated orally, every day for 1 or 2 weeks with 10 mg Cd/kg; a Mg + Cd group—mice given cadmium the same dose and for the same period of time, but pretreated with 20 mg Mg/kg b.w. Zinc kidney content was determined after wet mineralization by AAS.

Subacute Cd intoxication induced high enhancement of Zn content in kidney after 1 and 2 weeks. On the other hand, 2 weeks co-administration of Mg and Cd did not alter the metabolism of Zn in kidneys of mice compared with controls. These data confirm negative effect of cadmium on zinc metabolism and suggest beneficial effects of magnesium in subacute cadmium intoxication. P13-24 DETERMINATION OF CADMIUM CHLORIDEINDUCED MICRONUCLEI IN V79 CELLS USING GIEMSA, DAPI AND SILVER STAINING TECHNIQUES M. Mili´c, R. Rozgaj, V. Kaˇsuba Mutagenesis Unit, Institute for Medical Research and Occupational Health Zagreb, Croatia Soluble compounds of highly toxic heavy metals are well known as inductors of genotoxic damage in humans and animals. We evaluated the toxic effects of cadmium chloride as one of them. The induced DNA damage was observed through the formation of micronuclei (MN) in Chinese hamster cells. V 79 cells were simultaneously treated with different doses of cadmium chloride (10−4 , 10−5 and 10−6 M) for 24 h. MMC-treated cells served as positive control. Cytochalasin-B was added to arrest cytokinesis. Three separate cultures per concentration were analyzed. Slides were stained using conventional Giemsa, DAPI (4 ,6-diamidino-2-phenylindole-dihydrochloride) and silver staining techniques. For each staining technique, 1000 binucleated cells per slide were analyzed. All techniques revealed an increased number of MN when compared to control. DAPI staining gave more information about the origin of MN and allowed us to distinguish between MN+ and MN−. In respect to the patterns of nucleolar organizer (NOR) activity in MN we distinguish Ag-NOR+ and Ag-NOR− MN. P13-25 THE EFFECT OF BISPHOSPHONATES ON TRACE ELEMENTS DISTRIBUTION IN MICE—INTERACTION WITH CADMIUM V. Eybl, D. Kotyzova Charles University School of Medicine in Pilsen, Czech Republic

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Bisphosphonates, which are used for treating of some bone disorders, form metal complexes. However, their influence on trace elements (TE) distribution in the body was not systematically studied. In this study the effect of bisphosphonates – clodronate, etidronate and risedronate – (administered at the dose of 100 mg kg−1 b.w., p.o., once daily for 10 days) on the level of TE was investigated in standard male mice (CD-1, Anlab) and also in mice pre-loaded with cadmium (5 mg CdCl2 ·2.5H2 O kg−1 , s.c., once daily for 3 days before bisphosphonates treatment). Twenty-four hours after the last dose of bisphosphonates TE level was estimated in target organs of cadmium toxicity, i.e. in the liver, kidneys and femurs. All bisphosphonates caused an increase of Fe level in the liver (to 142–210%) and in kidneys (to 120–128%) and risedronate treatment increased Zn level in the liver (to 134%). In femurs, clodronate increased Ca, Mg, Zn and Cu concentration and risedronate increased Ca and Zn level (always p < 0.01). In cadmium preloaded mice bisphosphonates did not influence any changes of TE distribution caused by cadmium. In previous experiments etidronate influenced lead distribution (Toxicol. Lett. 109 (Suppl. 1) (1999) 54), however in this study cadmium distribution was not affected by bisphosphonates treatment. Further experiments are supposed especially for explanation of bisphosphonates effect on zinc and iron distribution. ˇ 305/05/0344. Supported by the grant GACR

complex mixtures, to define endpoints that can be easily employed to assess the risk associated to the exposure, as well as to assess the effectiveness of the remediation technologies. The in vitro BALB/c 3T3 model resembles the in vivo multistep tumor process and it is a valid procedure to assess the cancer potential of chemicals. It shows good predictability, high sensitivity and specificity. The assay is also useful to analyze the role of the tested agents in the initiation and promotion stages of carcinogenesis. Extracts from contaminated soils were chemically characterized and then analyzed in order to evaluate the associated cytotoxicity and to select the effective dose range to be used in further testing. A modified BALB/c 3T3 transformation assay was performed to verify the ability of the samples to increase the occurrence of transformed foci. Positive controls were represented by cells treated with the well-known chemical carcinogen, 3-methylcholanthrene (2.5 ␮g/ml). A cytotoxicity test was also performed in parallel, in order to calculate the number of cells survived to the chemical treatment. The results gave evidence that the proposed endpoints were affected by the tested mixtures. The test battery allows the assessment of the dose-related toxicity and transforming activity of environmental samples and their role in the multistep carcinogenesis.



Vaccari1 ,

Silingardi1 ,

Chiozzotto1 ,

M. P. D. M.G. Mascolo2 , C. Severini1 , W. Horn1 , A. Colacci1 1 Excellence

Environmental Carcinogenesis and Laboratory Mechanisms of Carcinogenesis and Anticarcinogenesis, EPA-Emilia-Romagna Region (ER-EPA), Bologna, Italy; 2 Department Experimental Pathology, Cancer Research Section, Bologna, Italy The toxicological evaluation of environment-occurring mixtures plays a key role in the risk assessment of soils contaminated by heavy metals. The present study aims at verifying the suitability of in vitro cell models as useful tools to profile the toxicological behavior of

This work was supported by the National Research Program “Environment”, Item 15, Ministry of Education, University and Research, Italy.

A. Rafaell , I. Parreira1 , M.C. Alpoim2 , A.M. Silv´erio Cabrita1 , R. Alves3 l Instituto

de Patologia Experimental da FMUniversidade de Coimbra Portugal; 2 Departamento de Bioqu´ımica da FCTUniversidade de Coimbra, Portugal; 3 Servi¸co de Nefrologia dos Hospitais da Universidade de Coimbra, Portugal Lead is the most abundant toxic heavy metal, which is found naturally in the environment as well as in manufactured products, such as waste oil and solid waste incineration, iron and steel production, battery and lead alkyl manufacturing. Exposure occurs from lead’s presence in air, food, water, soil, dust fall, paint, and


Abstracts / Toxicology Letters 158S (2005) S1–S258

other materials. Lead rarely occurs naturally in drinking water. Instead, lead contamination usually occurs at some point in the water delivery system. It is most commonly caused by the corrosion of lead service connections, pipes, or lead solder used to join copper pipes in the home. Homes that are less than 10 years old and have lead solder or that are connected to the water main by a lead service line are more likely to have higher levels of lead in the water. Lead is readily absorbed by the body via the primary routes of entry, inhalation and ingestion. The aim of our study is to evaluate the effects of chronic lead intoxication on kidney. To perform the study we used 20 Wistar rats housed in groups of ten, they were fed with ad libitum with standard feed and had free access to water. They also maintained under standard conditions of humidity, temperature and 12 h light/darkness cycle. The animals were acclimatized for a week before the commencement of the study. The principles of laboratory animal care were also followed in this study. Group one was the control group, and group two was exposed to drinking water contaminated with 25 ppb of lead acetate. After two months the animals were sacrificed and the kidney collected to histology studies. The kidney show tubular necrosis and a discrete interstitial fibrosis. We believe that more studies should be performed but that chronically exposure to drinking water contaminated with lead will cause kidney damage.

motivational state of the rats was tested in open field, and their psychomotor performance, by evoking the acoustic startle reflex. Finally, spontaneous and evoked cortical activity from the primary sensory areas was recorded. The treated rats had, compared to the controls, worse performance in acquiring a memory task (finding food pellets in a maze). With increasing summed dose, the performance in the working memory test was significantly decreased in the high dose group, then in both treated groups. In the open field, treated rats had lower horizontal and vertical motility and spent longer time in the corners. The number of noisepositive responses in the acoustic startle test, and the peak force exerted by the muscle contraction, was dosedependently decreased. In the spontaneous activity of the cortex, lower frequencies increased in the treated rats, and the latency of the evoked responses increased. Our results indicate that lead effects on the higher nervous functions constitute an important segment of the neurotoxicity of this metal, and deserve further mechanistic investigations.


Department of Biochemistry and Biophysics, Saratov State University, Saratov, Russia

L. Nagymajt´enyi, A. Papp, T. Vez´er Department of Public Health, University of Szeged, Hungary Lead is one of the metals known and used for over thousand years. Mining, smelting, processing and reprocessing of lead, and the use of lead-based products, resulted in considerable environmental pollution and human exposure. Tetraethyl lead used to be a commonly used additive in petrol. In the present study, young adult male Wistar rats were treated, by gavage, with 0.123 and 0.492 mg/kg b.w. tetraethyl lead for 10 weeks. During the 10 weeks, memory tests were performed. At the end of the treatment period, the


The research viewed the changes of bioimpedance in rat pups, born from the mothers, exposed to the cadmium treatment during pregnancy. The effects of cadmium exposure during pregnancy on the postnatal development were investigated. Pregnant rats in control group received 0.9% NaCl, while the Cd group received Cd(NO3 )2 per os during 10 days (from 6 to 15 days gestation) in dosage of 2 mg/kg. On the 60 and the 120-postnatal days the male and female offspring were decapitated. For measuring blood, erythrocytes and tissues impedance (IMP) there has been used the frequency diapason from 10 Hz up to 10 MHz. It has been detected that in blood of the experimental males the IMP values decrease is observed only on the 120 day of postnatal development as contrasted to

Abstracts / Toxicology Letters 158S (2005) S1–S258

females, where the IMP changes are registered already on the 60th day. In erythrocytes the IMP values in the experimental female group significantly decrease only at low frequencies on the 60-day and do not differ from the control values on the 120-day. While in male groups the IMP decrease in erythrocytes take place on the 60th day as well as on the 120th day. In the impedancemetric measurements in liver, brain and heart there has been detected on the 60th day a decrease of the cell electroconductivity in the male and female groups. On the 120th day in males groups there take place even more significant changes in the IMP decrease at the high frequencies. Impedancemetry thus helped to register the tissue homeostasis changes in offspring that was born from mothers exposed to the Cd ions during pregnancy. P13-30 EFEECTS OF PRENATAL EXPOSURE OF CADMIUM IONS ON FREE RADICAL PROCESSES AND MITOCHONDRIAL ENZYMES ACTIVITY IN ADULT RAT OFFSPRING R.A. Kireev, E.S. Solovijeva, E.A. Konychenko, E.A. Rozgnova Department of Biochemistry and Biophysics, Saratov State University, Saratov, Russia The research involved the lipid peroxidation processes and activity of mitochondrial enzymes in rat pups, born from the mothers, exposed to the cadmium treatment during pregnancy. Pregnant rats in control group received 0.9% NaCl, while the Cd group received Cd(NO3 )2 per os during 10 days (from 6 to 15 days gestation) in dosage of 2 mg/kg. On the 60th day the male and female offspring were decapitated. The measured parameters were tissue MDA and Na+ -K+ -ATP-ase and succinate dehydrogenase (SDG) activity in mitochondrias. As compared to the control indexes, the test results showed an increase of SDG in the experimental female group in heart (p < 0.04) and in brain (p < 0.04) mitochondrias. A corresponding increase of SDG activity in experimental male group displayed the indexes in liver (p < 0.04) and in heart (p < 0.01) as compared with the control male group. There have been discovered in the Na+ -K+ -ATP-ase activity in 2-months old offspring changes: a decrease of mitochondrias activity in liver


in the experimental female group and in heart of experimental males, compared to control animals. The MDA level was higher in liver, brain and heart in the experimental male and female groups on the 60th day of age, compared to the control groups. However, the MDA level in kidney was higher in the experimental male group as compared to a female one. The data indicate that the exposure of pregnant mothers to Cd produced changes in the lipid peroxidation processes and activity of the mitochondrial enzymes at critical periods of development, which may have serious implications in the following period of life. P13-31 Hg, Pb AND Cd LEVELS IN CANNED VARIEGATED SCALLOPS (CHLAMYS VARIA) NORMALLY CONSUMED IN TENERIFE (CANARY ISLANDS, SPAIN) ´ Guti´errez2 , G. Lozano2 , A. Hardisson1 C. Rubio1 , A.J. 1 Toxicology Department, University of La Laguna, S/C

de Tenerife, Spain; 2 Animal Biology (Marine Sciences Section) Department, University of La Laguna, S/C de Tenerife, Spain A study of the metal content (Hg, Pb and Cd) of 190 samples of canned variegated scallops (Chlamys varia) has been carried out. The studied brands have been those most consumed and distributed in the island of Tenerife and correspond to seven different manufacturers located in Galicia, north-west coast of Iberian Peninsule. Only one commercial presentation of the variegated scallops has been considered: Pickled sauce. Graphite furnace absorption spectrometry was used in the determination of Pb and Cd and cold vapour atomic absorption spectrometry in the study of Hg (His et al., 2000; Pokorny et al., 2000). The obtained Hg levels are underneath the maximum level fixed for human consumption (0.5 mg/kg, w/w). The concentration levels of mercury were 8.6 ± 6.5 to 48.33 ± 5.97 ␮g/kg. The obtained Pb and Cd (187.15 ± 95.08– 301.64 ± 118.12 ␮g/kg and 693.86 ± 176.88– 1087.45 ± 434.58 ␮g/kg, respectively) have resulted to be in the same range of those values referred by other authors and the obtained levels are underneath the maximum level fixed for human consumption (1 mg/kg, w/w).


Abstracts / Toxicology Letters 158S (2005) S1–S258

P13-32 Fe, Mn, Ni AND Cu LEVELS IN CANNED VARIEGATED SCALLOPS (CHLAMYS VARIA) NORMALLY CONSUMED IN TENERIFE (CANARY ISLANDS, SPAIN) ´ Guti´errez2 , G. Lozano2 , A. Hardisson1 C. Rubio1 , A.J. 1 Toxicology Department, University of La Laguna, S/C

de Tenerife, Spain; 2 Animal Biology (Marine Sciences Section) Department, University of La Laguna, S/C de Tenerife, Spain A study of the metal content (Fe, Mn, Ni and Cu) of 190 samples of canned variegated scallops (Chlamys varia) has been carried out. The studied brands have been those most consumed and distributed in the island of Tenerife and correspond to seven different manufacturers located in Galicia, north-west coast of Iberian Peninsule. One different commercial presentation of the mussels has been considered: Pickled sauce. Inductively coupled plasma atomic emition spectrometry was used in the determination of Fe, Mn, Ni and Cu. The obtained Fe, Mn, Ni and Cu contents (19.00 ± 5.76–42.89 ± 29.59, 7.57 ± 6.70– 33.09 ± 26.81, 0.08 ± 0.10–0.91 ± 0.08 and 1.33 ± 0.41–2.93 ± 1.90 mg/kg, respectively) have resulted to be in the same range of those values referred by other authors. P13-33 INFLUENCE OF CHELATING AGENTS ON THE TOXICITY, DISTRIBUTION AND EXCRETION OF THORIUM IN RATS P. Sharma, A. Kumar, K.P. Mishra Radiation Biology and Health Sciences Division, Bhabha Atomic Research Center, Mumbai-85, India Thorium (Th) is ubiquitous in our environment. Releases of Th to the atmosphere can occur both from natural and anthropogenic sources. The primary anthropogenic sources of Th exposure to workers is through mining, milling and processing of uranium and thorium, phosphate fertilizer production, coal fired utilities, welding with thoriated tungsten electrodes and industrial boilers. The conventional management of Th is grossly inadequate. However, DTPA has been shown to remove the Th from the system but this

chelating agent has several drawbacks. So the present study was planed to investigate the therapeutic effectiveness of five different chelating agents viz. Tiron, dpenicillamine, gallic acid, deferioxamine, free and liposome encapsulated DTPA against Th induced toxicity in rats. Th was given as thorium nitrate (12 mg/kg/day i.p.) for 3 days. Chelating agents were given after 24 h of Th administration for 3 days. The administration of Th caused marked elevation in the level of serum creatinine, lactate dehydrogenase, bilirubin, transaminases (AST and ALT) and catalase however decrease in reduced glutathione. Concentration of Th was estimated in the brain, liver, kidney, lungs, heart, blood, femur and urine from each animal by inductively coupled plasma (ICP). Significantly high concentration of Th was found in the liver and bone. Treatment with chelating agents reduces the accumulation of the thorium in almost all the organs studied. P13-34 IRON INDICES: FERRITIN, TRANSFERRIN, IRON IN SERUM AND TIBC OF SMOKING AND NON-SMOKING WORKERS CHRONICALLY EXPOSED TO MERCURY VAPOURS ˙ nski2 , P. Moszczy´nski2 J. Rutowski1 , Z. Zabi´ 1 E. Szczeklik’s Specjalistic Hospital in Tarn´ ow, 13 Szpi-

talna St., 33-100 Tarn´ow, Poland; 2 Regional Hospital in Brzesko, 68 Ko´sciuszki St., 32-800 Brzesko, Poland The study involved 46 men, aged 21–56, exposed to mercury vapours from 7 months to 32 years (14.7 ± 10.8), working in a three shift system for 8 h a day, smokers constituted 50% of the studied group. Urine mercury concentrations of workers exposed to mercury vapours were in range 10–215 ␮g/dm3 (81.4 ± 72.9) and in blood 4–72 ␮g/dm3 (16.3 ± 15.0). Controls were 46 men, aged 20–54, workers never exposed to mercury vapours or toxic agents, smokers was 16 men. The serum concentration of transferrin and ferritin was determined by nephelometry (Behring Nephelometer Type 100). The serum concentration of iron and TIBC were determined by colorimetry (Beckman Synchron CX System). The TWA mercury concentration in the air, measured before tests was 0.028 mg m−3 . Toxicological studies of the showed that the highest excess NDS indices for mean concentrations of mercury occurred in 1968–1969, and were

Abstracts / Toxicology Letters 158S (2005) S1–S258

1.5–1.8, whereas in the 70-ties, 80-ties and the 90ties—around 1.1. There were no statistically significant differences in the mean serum iron concentrations between all the studied groups. The mean ferritin concentration in the serum of the control group amounted to 61.45 ␮g l−1 , while in groups exposed to mercury vapours of 141.87 ␮g l−1 (statistically significant increase occurred in ferritin by 130.9%, p < 0.001). Both these values are inside the normal distribution range (15–200 ␮g l−1 ). The mean transferrin concentration in the serum of the control group was 1.47 ␮g l−1 , while in groups exposed to mercury vapours of 3.21 ␮g l−1 (p < 0.001). The accepted range of TIBC is 45–70 ␮g l−1 . The mean value of TIBC in control group was 65.45 ␮g l−1 , while in groups of men exposed to mercury vapours was 72.78 ␮g l−1 (p < 0.05). Moreover, no statistically significant differences in the mean values of all determined parameters between groups of smokers occupationally exposed to mercury vapours and non-smokers occupationally exposed to mercury vapours also were observed. P13-35 BASIC HAEMATOLOGICAL PARAMETERS OF THE PERIPHERAL BLOOD OF SMOKING AND NON-SMOKING WORKERS CHRONICALLY EXPOSED TO MERCURY VAPOURS ˙ nski2 , P. Moszczy´nski2 J. Rutowski1 , Z. Zabi´ 1 E.Szczeklik’s Specjalistic Hospital in Tarn´ ow, 13 Szpi-

talna Str., 33-100 Tarn´ow, Poland; 2 Regional Hospital in Brzesko, 68 Ko´sciuszki Str., 32-800 Brzesko, Poland The study involved 46 men, aged 21–56, exposed to mercury vapours from 7 months to 32 years, working in a three shift system for 8 h a day, smokers constituted 50%. Urine mercury concentrations of workers exposed to mercury vapours were in range 10–215 ␮g/dm3 (81.4 ± 72.9) and in blood 4–72 ␮g/dm3 (16.3 ± 15.0). Controls were 46 men aged 20–54 workers never exposed to mercury vapours, other toxic agents, smokers was 16 men. RBC, HCT, Hb and MCHC of the peripheral blood were determined using AVL 808 haematological counter. The TWA mercury concentration in the air, measured before tests was 0.028 mg m−3 . Toxicological studies of the showed that the highest excess NDS indices for mean concentrations of mercury occurred in


1968–1969, and were 1.5–1.8, whereas in the 70-ties, 80-ties and the 90-ties—around 1.1. The RBC number in mm3 of peripheral blood was higher in the workers group than in controls. In the subgroup of smoking and non-smoking workers, RBC numbers were higher than in the control subgroup. In all subgroups separated in respect to duration of exposure as well as in respect to mercury concentration in urine, the number of RBC in the peripheral blood was higher than in the control group. In workers exposed to mercury vapours, the HCT value was higher than in the controls. In the subgroup of smokers exposed to mercury vapours HCT was lower than in controls, in the subgroup of non-smoking workers it was however higher than in the control subgroup. In all subgroups separated in respect to mercury concentrations in the urine, HCT values were higher than in the control group. Hb concentrations did not reveal any statistically significant differences among the investigated groups/subgroups. The value of MCHC in the total group exposed to mercury vapours was lower than in the controls, whereas in the subgroups separated in respect to mercury concentration in the urine, it was lower only in workers showing the highest concentration of this metal. In workers exposed to mercury vapours, MCV values were lower than in the controls. In the subgroups of workers who smoked and those who did not smoke, they were also lower than in the controls. P13-36 ENZYMATIC ACTIVITY OF GLUCOSE-6PHOSPHATE DEHYDROGENASE (G-6PD), HEXOKINASE (Hx) AND ACETYLCHOLINESTERASE (AchE) IN PERIPHERAL BLOOD ERYTHROCYTES OF SMOKING AND NON-SMOKING WORKERS CHRONICALLY EXPOSED TO MERCURY VAPOURS ˙ nski2 , P. Moszczy´nski2 , Z. J. Rutowski1 , Z. Zabi´ 3 D˛abrowski 1 E.Szczeklik’s Specjalistic Hospital in Tarn´ ow, 13 Szpi-

talna St., 33-100 Tarn´ow, Poland; 2 Regional Hospital in Brzesko, 68 Ko´sciuszki St., 32-800 Brzesko, Poland; 3 Laboratory of Experimental Haematology, Institute of Zoology, Jagiellonian University, 6 Ingardena St., 30060 Krak´ow, Poland The study involved 46 men, aged 21–56, exposed to mercury vapours from 7 months to 32 years


Abstracts / Toxicology Letters 158S (2005) S1–S258

(14.7 ± 10.8), working in a three shift system for 8 h a day, smokers constituted 50% of the studied group. Urine mercury concentrations of workers exposed to mercury vapours were in range 10–215 ␮g/dm3 (81.4 ± 72.9) and in blood 4–72 ␮g/dm3 (16.3 ± 15.0). Controls were 46 men, aged 20–54, workers never exposed to mercury vapours or toxic agents, smokers was 16 men. Activity of the studied enzymes was estimated by the Beutler’s spectrophotometric method. The TWA mercury concentration in the air, measured before tests was 0.028 mg m−3 . Toxicological studies of the showed that the highest excess NDS indices for mean concentrations of mercury occurred in 1968–1969, and were 1.5–1.8, whereas in the 70ties, 80-ties and the 90-ties—around 1.1. The activity of G6PD was lower in the group of subjects occupationally exposed to mercury vapours. When comparing the subgroups of smokers and non-smokers with the controls, workers showed lower G6PD activity than in the matching control subgroups in the subgroups of smokers as well as in non-smokers, respectively. Erythrocyte G6PD activity was lower in all studied groups separated in respect to duration of exposure or mercury concentration in the urine. The lowest enzyme activity was found in the subgroups showing the highest mercury concentration in the urine. Hx activity levels were lower than in the control group. The AchE activity was higher in the group exposed to mercury vapours compared to the controls and the respective values were 50.22 ± 14.44 versus 36.87 ± 2.92 IU/gHb. A similar trend was observed when comparing AchE activity between the subgroups of smokers and non-smokers. In the subgroups separated in respect to length of exposure or in respect to the mercury urine concentration, the activity of AchE was statistically significantly higher than in the control group. P13-37 THE EXPRESSION OF MRP-TRANSPORTERS AND UMAT IN TUMOR CELLS AND PRIMARY CULTURES OF HUMAN LUNG CELLS EXPOSED TO HEAVY METALS F. Glahn1 , A.W. Torky1 , E. Stehfest1 , S. Hofmann2 , H. Foth1 1 Institute

of Environmental Toxicology, Germany; Surgery, Martin Luther University, D-06097 Halle/Saale, Germany 2 Cardio-Thoracic

The multidrug resistance associated proteins (MRP) transport a wide range of substrates including metal complexes, e.g. glutathione–metal complexes. The ubiquitously expressed mammalian ABChalftransporter (UMAT) has high sequence similarity to the fission yeast heavy metal tolerance protein hmt1, thus it is assumed to be involved in metal ion homeostasis. We incubated primary cultures of normal human bronchial epithelial cells (NHBEC), peripheral lung cells (PLC) and lung tumor cell lines H322 and H358 with sub-toxic concentrations of inorganic As(III), Cu(II) and Hg(II). Expression of different MRP-isoforms and UMAT was determined by realtime RT-PCR: As(III) led to a slight up-regulation of MRP1 in H358 (1.2-fold) (5 ␮M; 2 4 h), and of MRP5 in PLC (1.1-fold) (5 ␮M; 2 4 h). Incubation with Cu(II) (25 ␮M; 2 4 h) induced MRP3 and 5 expression 1.5and 1.3-fold in NHBEC whereas H322 cells were not affected. Prolonged incubation with Cu(II) (72 h) increased expression of MRP3 in H322 (2.3-fold) and PLC (1.9-fold) and expression of UMAT in H322 (1.5-fold), but not in PLC. Also Hg(II) (5 ␮M; 2 4 h) increased MRP3 expression in NHBEC (1.2–2-fold), while it had no effect on MRP3 and decreased MRP4 (81% of control) in H322. Supported by Philip Morris External Research 2002 Program. P13-38 MATRIX METALLOPROTEINASE-9 ACTIVITY IN PLASMA CORRELATES WITH PLASMA AND WHOLE BLOOD LEAD LEVELS F. Barbosa Jr.1 , R.F. Gerlach2 , J.A. Uzuelli1 , J.E. Tanus-Santos1 1 Department

of Pharmacology, Faculty of Medicine of Ribeirao Preto-USP, Ribeirao Preto-Brazil; 2 Department of Morphology, Estomatology and Physiology, FORP-USP, Ribeirao Preto-Brazil Matrix metalloproteinases (MMPs) are a family of structurally related, zinc-dependent enzymes involved in the degradation of many components of the extracellular matrix during both physiological and pathological processes. Specifically, MMP-2 and MMP-9 (gelatinase A and gelatinase B, respectively) play a role in many physiological processes, and increased expression and activity of these enzymes have been

Abstracts / Toxicology Letters 158S (2005) S1–S258

reported in a variety of pathological conditions including cardiovascular. Lead (Pb) exposure is widely recognized as a serious environmental health problem. In this regard, a causal association between lead exposure and cancer/increased cardiovascular risk has been strongly suggested. To investigate whether there is an association between plasma MMP-2 and MMP-9 activities and the concentrations of Pb in whole blood (B-Pb) or in plasma (P-Pb) from lead-exposed subjects, we evaluated the relationship between plasma MMP-2 and MMP-9 activities measured by gelatin zymography. It was found a significant correlation between pro-MMP-9 activity in plasma and B-Pb (r = 0.454, p = 0.003). Moreover, pro-MMP-9 activity in plasma was also positively correlated with P-Pb levels (r = 0.312; p = 0.049). No significant correlations were found between MMP-9 or pro-MMP-2 with BPb or P-Pb levels. P13-39 VOLUNTARY FATAL INTOXICATION WITH INORGANIC MERCURY: EVALUATION OF THE DISTRIBUTION OF THE METAL IN SEVERAL AUTOPSY SAMPLES P. Triunfante1 , M.E. Soares2 , M. de Lourdes Bastos2 1 Master

Student of the Course of Forensic Sciences, University of Porto, Portugal; 2 REQUIMTE, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, Rua An´ıbal Cunha, 164, 4099-030 Porto, Portugal A 39-year-old man tried the suicide by ingestion of a massive amount of inorganic mercury (mercury chloride salt). He arrived at the hospital unconscious and remained in the intensive care unit for 49 days. After admission he was immediately submitted to the removal of stomach contents. Chelanting therapy and haemodialysis were subsequently performed in an attempt to remove the metal from the organism. At the end of that period he died mainly by general organ failure and, at the autopsy, samples of blood, liver, brain and lung were collected to quantify mercury levels and to evaluate its distribution after this long period of treatment. The samples were wet digested and mercury was determined by cold vapour atomic absorption spectrometry.The levels found were 11.7 ␮g/mL in blood


and 166.2, 1.11 and 10.9 ␮g/g dry weight in liver, brain and lung, respectively. In spite of the long period to tentatively remove mercury from the organism, the levels found in organs were similar to those referred in the literature for acute fatal poisoning with this metal. P13-40 METALS FROM AMALGAMS IN SALIVA: ASSOCIATION WITH LICHENOID LESIONS, LEUKOPLAKIA AND BURNING MOUTH SYNDROME P.D. Pigatto1 , L. Arancio1 , G. Guzzi2 , G. Severi3 1 Department

of Dermatological Sciences, University of Milan, Maggiore Hospital IRCCS, Milan, Italy; 2 Italian Association for Metals and Biocompatibility Research—AIRMEB, Milan, Italy; 3 Cancer Epidemiology Centre, The Cancer Council Victoria, Melbourne, Australia Exposure to mercury vapor from amalgams could be related to oral lichenoid lesions (OLL), burning mouth syndrome (BMS), and oral leukoplakia (OL). Mercury concentrations were measured in saliva samples – after chewing-gum stimulation – by spectrometry methods. We also measured other metals released in saliva from amalgams (silver, copper, tin, nickel). We investigated the relation of sensitization to metal allergens, which were patch-tested, and the corresponding metal levels in saliva. Of the 21 patients with OLL, 86% had at least one allergic sensitization to metals. Ten had allergy to nickel (47.6%); had gold (38%); palladium (23.8%); cobalt (23.8%); mercury (9.5%), 1 thimerosal (as organic mercury) (4.7%); 1 chromium allergy (4.7%). Three had no allergy to metals (14.3). Of the 35 patients with BMS, 22 (62.8%) were associated with at least one allergy to metal. Ten had allergy to nickel (28.6%); had gold (17.1%); had mercury (11.4%); chromium (11.4%); palladium (8.5%); one had allergy to amalgam (2.8%); one had cadmium (2.8%); one had cobalt allergy (2.8%). 13 had no evidence of allergy to metals (37.1%). Of the four patients with oral leukoplakia, three were associated with allergy to metals (gold, mercury, and thimerosal, respectively).


Abstracts / Toxicology Letters 158S (2005) S1–S258

Gold and nickel were substantially elevated and highly correlated with the presence of metal restorations. Mercury was the most frequent metal in saliva in both conditions: at baseline and in post-chewing gum test. The resulting leakage of metal into the oral tissues causes higher levels of mercury – which were measured after oral biopsy – in subjects with dental metal restorations compared with controls (6 versus 5). In all cases, the level of metal in saliva was reduced to undetectable level by dental restoration removal. These analyses directly implicate a metals dissolution in saliva from dental restorations. These results strongly support that metals in saliva and in close contact to tissues may elicit allergic sensitization and cytotoxicity, implying a role in oral pathologies.

longer. These of H358 revealed a length increase of 30% (0.02 mM) and 74% (0.1 mM) compared to the control samples. These experiments show clearly that cadmium has a dose-dependent genotoxic effect on lung tumor cells. To image the human lung in a model as physiological as possible cultivation of other cell types (endothelial cells) obtained from the human lung were cultivated and investigated on their possible involvement in the model. Beyond the “dry/wet”-membrane cell culture was tested for their expression of the transport proteins MRP1-5 in comparison to the submerge culture.


H.P. Tran1 , A.S. Prakash1,2 , J.C. Ng1

E. Stehfest, A.W. Torky, F. Huang, H. Foth Institute of Environmental Toxicology, Martin-LutherUniversity Halle-Wittenberg, 06114 Halle (Saale), Germany The lung is exposed to a great multitude of environmental chemicals because of its function and tissue structure. It works partly as a filter and is therefore affected by its own process of filtering chemicals like heavy metals which appear very often in respirable particles of nano size. In the present work the genotoxic potential of cadmium, was tested in three human tumor lung cells by comet assay. The tail lengths of A549 cells under exposure to cadmium (0.05 and 0.1 mM) are about 50% longer compared to the control samples. The tail lengths of H322 cells under cadmium exposure are about 70% (0.05 mM) and about 80% (0.1 mM)


1 National

Research Centre for Environmental Toxicology, The University of Queensland, Brisbane, Qld, Australia; 2 Reckitt Benckiser, Sydney, NSW, Australia Female Sprague–Dawley rats were dosed with sodium arsenite (10 mg/kg body weight) with or without benzo(a)pyrene (Bp, 800 ␮g) by intramammilary injection. Mammary gland tissues were collected on days 1, 3, 5, 10 and 27 for DNA-adduct measurement using a 32 P post-labeling technique. Rats given arsenic alone did not show any DNA-adducts. Rats given Bp alone, DNA-adducts reached a maximum level by day 5 then reduced to 13% of this level by day 27. Rats given arsenic and Bp (As + Bp), DNA-adducts also reached a maximum by day 5 but yielded only 80% of the level compared to the Bp group; 8 4% of this amount still remained by day 27. Rats given sodium selenite (10 mg Se/kg b.w.) alone yielded no adducts. Se + Bp rats had an adduct profile similar to that of the Bp group. If Se was given 5 days after As + Bp, it had no effect on both, the level of, and persistency of DNA adducts as observed in the As + Bp group. However, Se co-injected with As + Bp, DNA-adducts decreased

Abstracts / Toxicology Letters 158S (2005) S1–S258


significantly over 27 days. This indicated a protective mechanism of sodium selenite against the inhibitory effect of arsenic on the repair of DNA-adducts induced by Bp. However, for this protection to be operating, it appears that Se must be present at the very early stage of arsenic exposure. Further, it is plausible to conclude that a potential way for inorganic arsenic to cause carcinogenicity is via the inhibition of DNA repair mechanism(s).

compared with controls, p < 0.05. Our findings suggest Pb probably affects MMP-2 activity in rat aorta. It could be a possible mechanism wherein lead impairs vascular function inducing hypertension.


F. Barbosa Jr.1 , T. Saint’Pierre2 , A. Curtius2 , M. Buzalaf3 , R. Gerlach4 , J.E. Tanus-Santos1

M.M. Castro1 , E. Rizzi1 , L. Figueiredo-Lopes1 , M.L. Martinez1 , R.F. Gerlach2 , F. Barbosa Jr.1 , J.E. TanusSantos1 1 Department

of Pharmacology, FMRP-USP, Av. Bandeirantes, 3900, Monte Alegre, 14049-900, Ribeir˜ao Preto-SP, Brazil; 2 Departments of Morphology, Estomatology and Physiology, FORP-USP, Ribeir˜ao PretoSP, Brazil Lead (Pb) exposure may promotes endothelium dysfunction, probably through oxidative stress, which is a key factor regulating the expression and activity of MMPs. Specifically, MMP-2 has recently been described as a modulator of vascular tonus. In this study, we hypothesized that MMP-2 activity would be increased in aortas from Pb exposed rats. Male Wistar rats (250 g) were randomly assigned to one of eight experimental groups as follows: two control groups (n = 5, each), one treated with sodium acetate in water for two weeks and the other for two months, three groups (n = 5; each) treated with Pb in water (30, 90 or 600 mg/L Pb, as lead acetate for two weeks), and three additional groups (n = 5; each) treated with Pb as the former, but for two months. MMP-2 from aorta samples was performed by gelatin zymography and enzyme activity was assayed by densitometry. Significant differences in MMP-2 activity were seen in aortas from rats treated with 600 mg/L Pb


1 Department

of Pharmacology-FMRP-USP, Av. Bandeirantes, 3900, Monte Alegre, Ribeir˜ao Preto-SP, Brazil; 2 UFSC, Florian´opolis-SC, Brazil; 3 FOB-USP, Bauru-SP, Brazil; 4 FORP-USP, Ribeir˜ao Preto-SP, Brazil Much attention has been given to the fraction of lead (Pb) in plasma, since it might better reflect lead toxicological effects. We examined the influence of age and gender on the relationship between the concentrations of Pb in blood (Pb-B) and in plasma (Pb-P) in an adult exposed population. We studied 154 adults (56 men and 98 women) from 18 to 60 years old. Pb-B levels varied from 10.0 to 428.0 ␮g/L in men (mean, 98.3 ␮g/L) and from 10.0 to 263.0 ␮g/L (mean, 62.8 ␮g/L) in women. Corresponding Pb-Ps were 0.02–2.9 ␮g/L (mean, 0.66 ␮g/L) and 0.02–1.5 ␮g/L (mean, 0.42 ␮g/L) in men and women, respectively. The relationship between Pb-B and Pb-P was found curvilinear (r = 0.90, P < 0.05). When data were separated by gender, the correlation between %Pb-P/Pb-B ratio and Pb-B levels was significant and stronger for men (r = 0.36, p = 0.005) compared to women (r = 0.17, p < 0.05). Moreover, we found an interesting positive correlation between %Pb-P/Pb-B and age for women (r = 0.38, p = 0.007) and a negative correlation for men (r = −0.34, p = 0.01). Taken together, these results suggest contrasting effects of age on the plasma/whole blood lead ratio in men and women with a history of lead exposure.


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of Pharmacology-FMRP-USP, Av. Bandeirantes, 3900, Monte Alegre, Ribeir˜ao PretoSP, Brazil; 2 FOB, Bauru-SP, Brazil; 3 Cena-USP, Piracicaba-SP, Brazil; 4 FORP-USP, Ribeir˜ao PretoSP, Brazil We conducted a study to evaluate the use of parotid salivary lead (Pb-saliva) levels as a surrogate of the blood lead (Pb-B) or plasma lead levels (Pb-P) to diagnose lead exposure. Therefore, the relationship between these biomarkers was assessed in a lead exposed population. We studied 88 adults (31 men and 57 women) from 18 to 60 years old. Pb-saliva levels varied from 0.05 to 4.4 ␮g/L, with a mean of 0.85 ␮g/L. Blood lead levels varied from 32.0 to 428.0 ␮g/L in men (mean, 112.3 ␮g/L) and from 25.0 to 263.0 ␮g/L (mean, 63.5 ␮g/L) in women. Corresponding Pb-Ps were 0.02–2.50 ␮g/L (mean, 0.77 ␮g/L) and 0.03–1.6 ␮g/L (mean, 0.42 ␮g/L) in men and women, respectively. Significant correlation was found between log Pbsaliva and log Pb-B (r = 0.277, p < 0.008), and between log Pb-saliva and log Pb-P (r = 0.280, p = 0.006). The Pb-saliva/Pb-P ratio ranged from 0.20 to 18.0. Age or gender does not affect Pb-saliva levels or Pb-saliva/PbP ratio. Taken together, these results suggest that salivary lead may not be used as a biomarker to diagnose lead exposure nor as a surrogate of plasma lead levels. P14 Oxidative Stress P14-01 THE EFFECTS OF NEUROTOXIC INSECTICIDES ON THE LIPID PEROXYDATION ON VARIOUS TISSUES J. Stankovic-Ciric1 , V. Davidovic1 , V.M. Varagic2 1 Department

of Physiology and Biochemistry, Faculty of Biology, Belgrade; 2 Department of Pharmacology, Faculty of Medicine, Belgrade, Serbia and Montenegro

The neurotoxic insecticides lindan, malathion and permethrine have been already known to affect many physiological functions. In the present experiments all the three insecticides have been found to affect the activity of enzymes of the antioxidative protection. The level of the lipid peroxides was determined by measuring the concentration of the products obtained after reaction between thiobarbituratic acid and degradation products of polyunsaturated lipid acids after peroxidation in vitro. The increasing doses of malathion (1, 3 and 5 mg/kg) in the brown fat tissue produce a statistically significant increase in the level of lipid peroxidation. Quite contrary, in the homogenates of heart of animals treated with the same doses of malathion, a significant decrease of lipid peroxidation was observed. In the hypothalamic tissue only the highest dose produced an increase in peroxidation. In animals treated with lindan (the same dose levels) a statisticaly significant decrease of lipid peroxidation was found in the brown fat tissue. In the heart tissue only higher doses of lindan produced a highly significant increase in lipid peroxidation. The same type of response was observed in the hypothalamic tissue. The same range of doses of permethrine produced a dose-dependent decrease in lipid peroxidation in the brown fat tissue. In the homogenates of heart and hypothalamus an increase in lipid peroxdation was observed. It is concluded that all the three neurotoxic insecticides produce a modification in the function of enzymes of antioxidative protection. P14-02 FLOW CYTOMETRIC DETERMINATION OF GRANULOCYTE RESPIRATORY BURST ACTIVITY IN MICE AFTER ␣-CYPERMETHRIN POISONING A. Haratym-Maj1 , S. Luty1 , M. Skrzypczak2 1 Department of Pathology, Institute of Agricultural Medicine, Lublin, Poland; 2 Second Department of Gynecology, Medical Academy, Lublin, Poland

Synthetic pyrethroids possess immunomodulatory properties manifested by a decreased immunological response, both of cellular and humoral type. Growing evidence also suggest that generation of reactive oxygen species (ROS) has been implicated in the toxicity of various pesticides. Flow cytometry was used to study oxidative burst after ␣-cypermethrin poison-

Abstracts / Toxicology Letters 158S (2005) S1–S258


ing in Swiss mice. Chemically pure (min. 97.7%) ␣cypermethrin was administered per os in two concentrations: 2 mg/kg b.m. and 10 mg/kg b.m., once daily for 28 days. Peripheral blood granulocytes were stimulated by phorbol-12-myristate-13-acetate (PMA), Nformyl-methionyl-leucyl-phenylalanine (FMLP), and Escherichia coli. The oxidative burst was estimated by the amount of nonfluorescent dihydrorhodamine 123 converted to green fluorescent rhodamine 123. The results showed that E. coli-, FMLP-induced oxidative burst decreased in mouse poisoned with ␣-cypermethrin compared to control group. This data demonstrate the modulating effect of synthetic pyrethroids on the oxidative burst.

into the nascent RNA. BrUridine (BrU) or BrUridinetri-phosphate (BrUTP) incorporation into the nascent RNA was quantified. We also evaluated transcription repression by diamide which is a glutathione depletor and a positive control for ROS production. Results from confocal analyses clearly showed that sodium arsenate induces the production of ROS and is able to repress general transcription in the ECV 304 bladder carcinoma cell line. BrU and BrUTP incorporation was clearly affected in arsenate-exposed cells when compared to untreated groups. In addition, we found that general transcription level was also repressed by diamide. This result suggests that oxidative stress may be involved in repression of transcription.



R. Pires-Das-Neves1 , M.L. Pereira1 , F. Carvalho2 , M.L. Bastos2 , F.J. Iborra3 1 Biology

Department, University of Aveiro, Portugal; of Pharmacy, University of Porto, Portugal; 3 Institute of Molecular Medicine, University of Oxford, England 2 Faculty

Arsenic compounds are known to display both chemotherapeutical and/or carcinogenic effects, depending on the chemical species and dose. The molecular mechanisms responsible for these different responses are still not fully understood. It is known that arsenic compounds may induce oxidative stress through production of reactive oxygen species (ROS). ROS can induce specific molecular alterations which mediate cancer cell death or activate/inactivate transcription factors that alter gene expression and can induce uncontrolled cellular proliferation, differentiation and carcinogenesis. The aim of the present study was to evaluate sodium arsenate-induced production of ROS and its overall effect on general transcription in ECV 304 bladder carcinoma cell line. The fluorescent probe DCFH-DA was used to study the production of ROS. To evaluate the effect on transcription, we have adopted the approach of incorporation of halogenated nucleotides

M. Curcic, M. Djukic Institute of Toxicological Chemistry, Faculty of Pharmacy, Belgrade, Serbia and Montenegro Paraquat (PQ) is often used as substance to induce oxidative stress in experimental models. PQ neurotoxicity was explored in vulnerable brain regions of Wistar rats by nitrate concentration assessment, taking in consideration that nitrates are stabile and final reactive nitrogen species (RNS) metabolism product. According to regional brain distribution of enzyme nitric oxide synthaze (NOS) and other enzymes of antioxidative defense it might be that PQ, as a redox-cycling compound, interferes with RNS metabolism. The aim of the study was to examine the role of NO compounds in PQ neurotoxicity. Single dose of PQ (2.5 ␮g/10 ␮L) was intrastriatally (i.s.) applied to Wistar rats and nitrates were measured in homogenates of hyppocampus, striatum and cortex, in both ipsilateral and contralateral side. After 30 min, the highest nitrate concentration was found in hyppocampus and cortex, slightly less in striatum, and all of them were about 1.5 times higher than physiological (18.78 ± 6.09 nmol/mg proteins). 24 h after the poisoning, nitrate levels were about 20 times less than physiological in the selected brain structures, unlike after 7 days when physiological level was reached.


Abstracts / Toxicology Letters 158S (2005) S1–S258

Nitrate concentration was higher in contralateral side. Nitrate contents showed positive correlation with regional distribution of NOS. Higher values in ipsilateral side were expected due to damage caused by i.s. administration. Changes in nitrate contents through the time contribute the fact that RNS are involved in mechanisms of PQ neurotoxicity. P14-05 STATE OF FREE RADICAL HOMEOSTASIS IN RAT LIVER AFTER INTERRUPTED AND CHRONIC ALCOHOL INTOXICATION D. Miskevich, A. Borodinsky, Konovalenko, N.E. Petushok, V.V. Lelevich Regulation of Metabolism Department, Institute of Biochemistry, Grodno, Belarus Reactive oxygen species (ROS), nitric oxide (NO) and their interaction are crucial factor in alcohol intoxication. We aimed to evaluate free radical homeostasis under chronic and interrupted alcohol administration and investigate possible mechanisms of adaptation of the ROS to interrupted alcohol intake. The experiments were performed on male Wistar rats (180 g). Five groups of the animals n = 6) were used. Ethanol was administered in a dose of 3.5 g/kg (a 25% solution) intragastrically, two times a day. Rats from Group I were treated with ethanol during 28 days. Group II was injected during 7 days. The animals from these groups were sacrificed 1 day after the last ethanol injection. Group III was injected during 7 days and sacrificed after 7 day following the last ethanol injection. In Groups IV and V the cycle of alcoholization/withdrawal was repeated twice, and the animals were sacrificed as follows: Group IV—after 1 day following withdrawal and Group V—after 7 days of withdrawal. The control animals were injected with NaCl solution 0.95%. The activities of superoxide dismutase (SOD), catalase (CAT), state of gluthatione system, level of thiobarbituric acid-reactive substances (TBARS) were measured in liver homogenates. Serum activity of gammaglutamyl transpeptidase (GGTP) and levels of NO, Vitamin A (Vit A), E (Vit E), were also determined. It was shown that the activity of GGTP in Groups I, II and III was increased (200%, 214% and 164%),

which indicates significant liver injury. Levels of Vit A, Vit E in all Groups and NO in Groups II, III and IV were diminished. At the same time it was shown that the TBARS level was elevated in the Groups I, II and III which indicates oxidative stress. The activity of glutathione peroxidase (GSHPO) was increased in Groups I, III, IV and V. The results showed that both interrupted and chronic alcohol treatment caused in liver damage and oxidative stress. However, interrupted alcohol intake turn on certain mechanisms of adaptation to ethanol. In our opinion it seems that the GSHPO can play important role in adaptation to toxic ROS action. P14-06 OXIDATIVE STRESS AND ANTIOXIDANT STATUS IN PROSTATE CANCER AND BENIGN PROSTATIC HYPERPLASIA Z. Arsova-Sarafinovska1 , A. Aydın2 , A. Sayal2 , O. ¨ ok4 Erdem2 , A. Eken2 , A. Dimovski3 , Y. Ozg¨ 1 Department

for Drug Quality Control, Republic Institute for Health Protection, 50 Divizija 6, 1000 Skopje, Republic of Makedonia; 2 Department of Toxicology, G¨ulhane Military Medical Academy, 06018 Etlik, Ankara, Turkey; 3 Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, Vodnjansk 17, 1000 Skopje, Republic of Makedonia; 4 Department of Urology, G¨ulhane Military Medical Academy, 06018 Etlik, Ankara, Turkey Prostate cancer and benign prostatic hyperplasia are diseases associated with aging. Also commonly associated with increasing age is a shift in the prooxidant–antioxidant balance toward a more oxidative state, i.e., increased oxidative stress. The present study has been undertaken to investigate the possible alteration of oxidant/antioxidant status in the circulation of patients with prostate cancer and benign prostatic hyperplasia. Samples were obtained from 22 prostate cancer patients, 39 benign prostatic hyperplasia patients and 24 age- and sex-matched healthy subjects (controls). Thiobarbituric acid reactive substances (TBARS), the enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and copper (Cu) and zinc (Zn) levels were estimated in the erythrocytes of the patients and controls. The enhanced lipid peroxidation with

Abstracts / Toxicology Letters 158S (2005) S1–S258

concomitant decrease in the activities of erythrocyte GSH-Px and SOD were found in the prostate cancer patients as compared to control (p < 0.001) and benign prostatic hyperplasia patients (p < 0.05). The zinc levels were found to be decreased in prostate cancer patients compared to controls (p < 0.01) with no significant changes between hyperplasia and cancer patients. Similarly, the lipid peroxidation was found to be increased (p < 0.05) with decreased SOD activity (p < 0.05) and zinc level (p < 0.01) in benign hyperplasia patients as compared to controls. These results reveal an altered prooxidant–antioxidant status in the circulation of prostate cancer and either benign hyperplasia patients. The further researches should be planned in order to find out whether the oxidative stress related parameters could be used as differential diagnostic and prognostic markers regarding these diseases. Moreover, the use of antioxidant supplementation should be encouraged in the prevention and therapy of the prostate cancer and benign prostatic hyperplasia. P14-07 NITRIC OXIDE LEVELS AND LIPID PEROXIDATION IN THE CIRCULATION OF PATIENTS WITH BENIGN PROSTATIC HYPERPLASIA NAD PROSTATE CANCER Z. Arsova-Sarafinovska1 , A. Aydın2 , A. Sayal2 , O. Erdem2 , A. Eken2 , A. Dimovski3 , K. Erten4 1 Department

for Drug Quality Control, Republic Institute for Health Protection, 50 Divizija 6, 1000 Skopje, Republic of Makedonia; 2 Department of Toxicology, G¨ulhane Military Medical Academy, 06018 Etlik, Ankara, Turkey; 3 Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, Vodnjansk 17, 1000 Skopje, Republic of Makedonia; 4 Department of Urology, G¨ulhane Military Medical Academy, 06018 Etlik, Ankara, Turkey The alteration in prooxidant–antioxidant balance has been linked with the human cancer development. It is also believed that nitric oxide synthase overexpression and the increased production of nitric oxide may be involved in human cancer etiology. On the basis of these findings, we undertook the present study to evaluate the plasma levels of nitrite/nitrate (the end products of nitric oxide), plasma and erythrocyte malondialdehyde levels (a non-invasive biomarker of oxidative


stress) in patients with prostate cancer and benign prostatic hyperplasia. Samples were obtained from 22 prostate cancer patients, 39 benign prostatic hyperplasia patients and 24 age- and sex-matched healthy subjects (controls). Both increased plasma nitrite/nitrate levels and erythrocyte MDA levels were found in the prostate cancer group as compared to healthy control group (p < 0.001) and benign prostatic hyperplasia group (p < 0.05). Either plasma MDA concentrations were found to be elevated in prostate cancer group (p < 0.05) as compared to the control group and to the benign hyperplasia group (p < 0.05). Only erythrocyte MDA levels were significantly increased in the benign prostatic hyperplasia group (p < 0.05), with no significant changes in the plasma nitrite/nitrate and MDA levels as compared to the controls. In conclusion, we have found that increased NO production and MDA levels were present in the plasma of the patients with prostate cancer, which can be related to an alteration in oxidant–antioxidant status. Since NO seems to have a dual role in tumor progression, high concentration of NO for a long period could result in damage to DNA, leading to mutation and cancer. P14-08 INVESTIGATION OF CHANGES IN AMOUNTS OF ANTIOXIDANTS IN SALIVIA OF PATIENTS WHO HAVE HEAD AND NECK CANCER 2 , Y. G¨ ¨ A. Sayal1 , A. Aydın1 , V.M. Ozkurt unaydın2 , 2 1 Y.S. Aydıntu˘g , O. Erdem 1 G¨ ulhane

Military Medical Academy, Department of Toxicology, 06018 Etlik, Ankara, Turkey; 2 G¨ulhane Military Medical Academy, Department of Oral and Maxillo Facial Surgery, 06018 Etlik, Ankara, Turkey Free radicals can be form via many diverse mechanisms in the body. The reactive oxygen derivatives, which belong to the free radical group forming through intra- and extra-cellular mechanisms, can be extremely toxic and mutagenic and carcinogenic for the cells. In this study, we attempted to evaluate the relation between salivary selenium (Se), selenium-dependent glutathione peroxidase (Se-GSH-Px), and superoxide dismutase (SOD). We also examined the level of malondialdehyde, which is one of the most important indicators of lipid peroxidation in saliva. We did not see SOD activity in any of saliva samples. No difference


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was found between MDA level in the samples of the control group and the MDA level in the preoperative samples of the group with head and neck carcinomas. The activities of Se-GSH-Px in the preoperative samples of the patient group with head and neck carcinomas were found to be lower than the activity of Se-GSH-Px in the samples of the control group. No difference were found between the amounts of Se-GSH-Px in preoperative samples of the patient group with head carcinoma and neck carcinoma. The level of Se in the preoperative samples of the patient group with head and neck carcinomas were found to be lower than the level of Se in the samples of the control group. The results of our study indicate that there exists a general reduction in the levels of some antioxidants in saliva in patients with head and neck carcinoma, and/or the reduction in the level of antioxidants increase predisposition to malignancies. P14-09 ALTERATIONS OF ANTIOXIDANT ENZYMES IN LIVER AND ERYTHROCYTES IN EXPERIMENTALLY-INDUCED LIVER FIBROSIS S. Theocharis1 , F. Apostolakou1 , G. Gribilas1 , G. Kouraklis2 , C. Spiliopoulou1 , C. Giaginis1,2 , C. Liapi3 1 Department

of Forensic Medicine and Toxicology, School of Medicine University of Athens, Greece; 2 Second Department of Surgery, School of Medicine University of Athens, Greece; 3 Department of Experimental Pharmacology, School of Medicine University of Athens, Greece Thioacetamide (TAA) administration in rats represents an established model for generating liver fibrosis and cirrhosis. Oxidative stress is involved in the fibrotic process induction. Aim of this study was to investigate the alterations of enzymes related to antioxidant potential in liver tissue and erythrocytes membranes during liver fibrotic process induction with TAA treatment. Liver fibrosis was induced on adult male Wistar rats by TAA administration in drinking water (300 mg/L) for 3 months. The animals were sacrificed at 0 (control group), 1, 2 and 3 months later on. Malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CT), glutathione peroxidase (GSH-Px) activities were determined enzymatically in rat liver. SOD, CT

and GSH-Px activities were also determined in preparations of erythrocytes membranes. TAA administration resulted in progressively increased liver fibrosis, leading finally to the formation of cirrhotic nodules. MDA levels in liver tissue were progressively, statistically significantly, increased during fibrosis induction. The activities of antioxidant enzymes SOD, CT and GSHPx in the liver were statistically significantly decreased during the experimental time. Similar decrease was also noted in the activities of the same enzymes in preparations of erythrocytes membranes. The alterations of antioxidant enzyme activities observed in liver and erythrocytes could participate in the liver fibrotic process. Additionally, the administration of substances able to enhance antioxidant status in liver and erythrocytes could eliminate the induction of liver fibrosis. P14-10 MARKERS OF OXIDATIVE STRESS IN THE BLOOD OF WORKERS OCCUPATIONALLY EXPOSED TO FINE PARTICLES (PM 2.5) E. Pytlinska, J. Gromadzi´nska, I. SzadkowskaSta´nczyk, W. W˛asowicz Nofer Institute of Occupational Medicine, Lodz, Poland It is supposed that exposition to fine particles is associated with increased risk of cardiovascular and pulmonary diseases. The aim of study was assessment of oxidative stress markers in the blood of workers occupationally exposed to PM 2.5. The study was conducted on 56 men in the age between 23 and 43, who were workers of the same pottery production company. The blood was collected from workers three times: before first shift (after weekend), after first shift and after fourth shift. The control group consisted of 34 healthy, non-exposed men. The final results showed differences in the activities of antioxidant enzymes between PM 2.5 exposed and non-exposed workers. The activity of superoxide dismutase (SOD) in erythrocytes was higher compared to control subjects (p < 0.05) in contradiction to the activity of glutathione peroxidase (GSH-Px) in erythrocytes, which occurred to be lower (p < 0.0001). Lipid peroxidation measured by TBARS concentration was higher compared to control subjects (p < 0.0001) and was increased after fourth shift. The total antioxidant status was negatively correlated with the concentration of particles in the air (r = −0.226;

Abstracts / Toxicology Letters 158S (2005) S1–S258

p < 0.05). In conclusion, exposition to PM 2.5 affects the antioxidant system, which may be involved in the ethiology of cardiovascular and pulmonary system diseases. P14-11 THE EFFECTS OF SULFAQIUNOXALINE ON ˙ ˙ OXIDAT IVE DAMAGE AND PLASMA MALONDIALDEHYDE LEVEL IN QUAILS B.C. Liman, A. Alan University of Erciyes, Faculty of Veterinary Medicine, Department of Pharmacology-Toxicology, Kayseri, Turkey Sulfaqiunoxaline alone or in combination with a diaminopyrimidine has been used extensively for many years to control diseases in animals. The objective of the study was to evaluate the effect of sulfaqiunoxaline on oxidative damage according to plasma malondialdehyde levels. Two hundred forty, 10-day-old Japanese male quail chicks (Coturnix coturnix Japonica) were used in this study. The birds were divided in 5 groups with first group being as control (Group 1), Group 2, 3, 4 and 5 received 144 mg/L, 288 mg/L, 576 mg/L and 1152 mg/L sulfaqiunoxaline, respectively as control group received only water for 3 days. Blood samples were taken on days 1, 3, 5 and 7 from all groups. Plasma malondialdehyde (MDA) was measured. MDA levels increased in groups received sulfaqiunoxaline compared with control. The magnitude of the increase was greatest on day 3 after dosing followed by a decrease after day 3. In addition, there was a positive correlation between MDA levels and the dose of sulfaqiunoxaline. In conclusion, sulfaqiunoxaline caused on oxidative damage, but it was reversible based on the fact that MDA level returned to the normal upon discontinuing the drug application. P14-12 SAFETY ASSESSMENT OF POTENTIAL ANTIDIABETIC DRUGS CONSIDERING PARAMETERS OF LIPOPEROXIDATION AND ANTIOXIDANT SYSTEMS IN VITRO I. Palagina, M. Kudrya, F. Kolodub, V. Lipson Institute of Endocrine Pathology Problems, Kharkov, Ukraine


Dicarboxilic acids amides (DAAs) are the promising compounds with anti-diabetic and antioxidant activities. An important aspect in their safety assessment covers study of processes of lipoperoxidation (PLP), systems of anti-radical (ARP) and antioxidant protection (AOP). Aim of our study was to reveal nature of changes in PLP, ARP and AOP parameters in rat liver microsomes under DAAs in vitro exposure. The indicated parameters were measured by chemiluminescence method. DAAs were added to suspension of liver microsomes in concentrations 1.2 × 10−5 to 9 × 10−3 M, and incubated for 15 min under 26 ◦ C. Within control the microsomes were incubated with solvents (96◦ ethanol or dimethylsulphoxide). We found that methylamide of 2-hydroxy- and benzylamide of 2-methoxysuccinanilic acids, even in very high concentrations (9 × 10−3 and 3 × 10−4 M), did not affect PLP, ARP and AOP parameters, as opposed to di-(␤-phenylethyl)succinamide which activated ARP and AOP systems even in concentration 1.2 × 10−5 M. ␤-Phenylethylamide of 2-methoxysuccinanilic acid caused similar changes in concentration 3 × 10−4 M. ␤-Phenylethylamide of 2-hydroxysuccinanilic acid and dibenzyl-succinamide decelerated PLP and activated ARP in concentrations 3.2 × 10−5 M and 1.9 × 10−4 M. Benzylamide of 2-hydroxysuccinanilic acid in concentration 8 × 10−4 M depressed ARP microsomal system. Cis-3-(2 -benzimidazolyl)-1,2,2trimethylcyclopentancarboxilic acid at maximum concentration 6 × 10−4 M accelerated lipids hydroperoxides oxidation. We concluded in establishing the DAAs potential safety concentrations ranged 1.2 × 10−5 to 8 × 10−4 M. Nature and extent of changes are dependent on DAAs structure and concentration. Toxic properties were expressed by benzylamide of 2hydroxysuccinanilic acid. P14-13 THE EFFECT OF OCHRATOXIN A ON THE CONCENTRATION OF PROTEIN CARBONYLS IN RATS A.M. Domijan1 , K. Rudeˇs2 , M. Peraica1 1 Unit of Toxicology, Institute for Medical Research and

Occupational Health, Ksaverska c 2, Zagreb, Croatia; 2 Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia


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It was found that the mechanism of the toxicity of ochratoxin A (OTA) involves the production of free radicals and consequently oxidative stress. Until now the only studied pathway of OTA-caused oxidative damage was peroxidation of lipids. The aim of this study was to check whether OTA induces the production of protein carbonyls, markers of protein oxidation. Rats (5 per group) were given daily OTA (0.5 mg/kg b.w., i.p.) for 7, 14, and 21 days, respectively or solvent only (TRIS) as controls and sacrificed 24 h after the last treatment. The concentration of OTA in plasma, kidney and liver homogenates increased gradually during the whole length of experiment. The concentration of protein carbonyls in kidney homogenates of OTA treated rats was significantly higher after 14th and 21st treatment than in controls (P < 0.05). In liver the concentration of protein carbonyls was significantly higher in OTA treated animals only after 21st treatment (P < 0.05). These results confirm that oxidative stress is involved in the mechanism of OTA toxicity, and that it causes the oxidation of proteins. P14-14 THE EFFECT OF CADMIUM ON LIPID PEROXIDATION AND THE ANTIOXIDANT SYSTEM IN THE HF2FF CELL LINE A.B. Zaree Mahmoodabady Biochemical Department, Faculty of Medicine, Baqyattalah University, Tehran Iran We investigated the effects of chronic exposure to cadmium (Cd) on the levels of lipid peroxidation (LPO) and reduced glutathione (GSH) associated with activities of glutathione peroxidase (GSH-Px) and the catalase (CAT) enzyme in the human skin fibroblast (HF2FF) cell line. The control group was grown in DMEM media, while the experimental group was exposure to 2.5 mM CdCl2 in media culture and incubated for 24 h. Lipid peroxidation contents increased significantly in the (p < 0.001) Cd-treated cell line. Glutathione levels decreased significantly (p < 0.005) in the experimental group. Cadmium treatment caused a significant decrease in the activities of GSH-Px and CAT (p < 0.005, p < 0.1) of the HF2FF cell line. These results indicated that a Cd-induced increase in LPO was associated with reductions in GSH-Px and CAT enzyme activities, and in GSH levels in the human skin fibroblast cell line.

P14-15 IMPACT OF CHLORPYRIFOS AND ALPHACYPERMETHRIN ON OXIDATIVE STRESS PARAMETERS IN RATS B. Wielgomas, J. Krechniak Department of Toxicology, Medical University of Gda´nsk, Hallera 107, 80-416 Gda´nsk, Poland Development of civilization intensified food production and consumption. Therefore the use of chemicals to protect animals and crops from insects and parasites is needed to raise the production efficiency. Organophosphates and synthetic pyrethroids are the most common groups of insecticides, widely used in agriculture, households and veterinary medicine. They exhibit relatively low toxicity to humans, and high efficiency in control common pests. Main mechanisms of action are known for both groups: organophosphates and synthetic pyrethroids, although other possible mechanisms involved in toxicity are still subjects of interest. Studied chemicals are available on the market in formulations containing single compounds or combination of two, what increases the risk of interaction between them. The study was conducted to investigate, whether the free radical mechanism is involved in organophosphates and pyrethroids toxicity in rats and what kind of interaction may occur. Male Wistar rats were treated orally for 14 and 28 days with 10 mg/kg b.w. of chlorpyrifos or alpha-cypermethrin separately or as a mixture of both compounds with 5 mg/kg b.w. of each. Such doses were low enough to avoid visible signs of toxicity. Estimation of free radical-mediated damage was performed by measuring total sulphydryls, lipid peroxidation products by means of TBARS concentration and the activity of catalase and glutathione-S-transferase in blood, liver and brain homogenates. Also blood and tissues were examined by GC-ECD for residue levels of investigated pesticides and their metabolites. P14-16 ANTIOXIDANT DEFENCES IN PREGNANT ´ WOMEN FROM THE GDANSK REGION M. Szyszko, J. Krechniak Department of Toxicology, Medical University of Gda´nsk, Poland

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Selenium and glutathione (GSH) system play key functions in defense against pro-oxidants. Major roles of GSH include direct interception of pro-oxidants as well as a reduction of other antioxidants from their oxidized forms. It plays a role in the detoxification of hydrogen peroxide, lipid peroxides and free radicals. Variety of xenobiotics and endogenous metabolites also interact with GSH to form less toxic glutathione derivatives that are easily excreted. Protection by selenium in the mammalian cell is mediated by proteins and enzymes, which contain selenoaminoacids. The family of glutathione peroxidase (GSH-Px) isoenzymes, selenoprotein P (Se-P) and thioredoxin reductase are the most important selenocompounds. Glutathione peroxidases and the glutathione system are closely linked in the antioxidant defense reactions. There is much evidence that placenta is a major source of lipid hydroperoxides in maternal circulation during pregnancy. We have found a weak positive correlation [r = 0.4, p < 0.01] between Se concentration in the placenta and GSH/GSSG ratio in umbilical cord blood (CB), but not in maternal blood (MB). In the resting cell, the GSH/GSSG ratio exceeds 100, whereas in oxidative stress this ratio decreases to values between 10 and 1. We have also found that in maternal blood this ratio was 13.5 ± 10.8, whereas in umbilical cord blood 21.9 ± 21.4 [p < 0.01]. Our study indicated that the concentration of TBARS, as the index of lipid peroxidation was higher in maternal blood than in cord blood [8.2 ± 3.0 versus 4.9 ± 1.9 mM l−1 , p < 0.001]. P14-17 OXIDATIVE IMPAIRMENT IN POISONING WITH HEAVY METALS F. Bronet, L. Hontecillas, D. Hormigo, M.T. Antonio, I. Corpas Department of Physiology (Animal Physiology II), Faculty Biology, U.C.M., Madrid, Spain Lead and cadmium were enviromental toxins. This metals have an oxidative effect in liver causing an accumulation of reactive oxygen species in this organ that takes to hepatotoxity. The activity of some physiologic parameters was evaluated in four groups of Wistar rats. I, received only distilled water during treat-


ment (control); II, were treated with PbAc (300 ppm) in drinking water from pregnant period to dead day; III, were exposed to CdAc (10 ppm); IV, received PbAc and CdAc (150 ppm–5 ppm) in drinking water for the same period of time. All animals were sacrificed to the age of 100 days. After that, we extracted the liver and measured some blood parameters (hematocrite, haemoglobine, glucose, ALP, ACP, red and white blood cells count). Furthermore, we evaluated the oxidative damage in liver, in this way total lipid, lipid peroxidation, catalase activity and GSSG/GSH were measured. The result about this study show that lead–cadmium intoxication cause a minor increase that the originated for the cadmium intoxication, in lipid peroxidation, catalase activity and GSSG/GSH index in liver, while lead produce a decrease in catalase activity. We also observed a highly significant decrease in alkaline phosphatase activity in lead and cadmium coadministration that does not exist in the poisoning of only a metal. Lead and cadmium produce an interaction and attenuate the oxidation effect in cotreatment. P14-18 PREVENTIVE EFFECTS OF ␣-KETOGLUTARATE AND OXALOACETATE AGAINST BRAIN MITOCHONDRIAL DNA DAMAGE, LIPID PEROXIDATION AND SEIZURES INDUCED BY CYANIDE IN MICE H. Yamamoto Institute of Community Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan The effects of ␣-ketoglutarate (␣-KG) and oxaloacetate (OXA) in brain mitochondrial DNA (mtDNA) damage, lipid peroxidation and seizures induced by KCN were investigated in mice. A subcutaneous injection of KCN (7 mg/kg) produced broad-spectrum limbic and severe sustained seizures in all of the treated mice. The seizures were abolished when ␣-KG (1 g/kg) or OXA (1 g/kg) was injected intraperitoneally in the animals 1 min before KCN administration. In addition, the administration of KCN caused damage to mtDNA in brain frontal and middle cortex of mice. These effects were completely abolished by the intraperitoneal preinjection of ␣-KG (1 g/kg) or OXA (1 g/kg). Furthermore, the subcutaneous injection of KCN (7 mg/kg) elicited an increase in lipid peroxidation in brain of


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mice. However, the increased lipid peroxidation was completely inhibited by preinjection of ␣-KG (1 g/kg) or OXA (1 g/kg). These results suggest that ␣-keto acids, such as ␣-KG or OXA play a role in the inhibition of seizures and subsequent mtDNA damage induced by the neurotoxic agent, KCN. P14-19 THE INFLUENCE OF COMBINED ANTINEOPLASTIC AGENTS CONTAINING ANTHRACYCLINES ON METABOLIC LIVER FUNCTION IN MICE M. Popovic1 , J. Kolarovic2 , B. Kaurinovic1 , V. Jakovljevic2 1 Department

of Chemistry, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia and Montenegro; 2 School of Medicine, Hajduk Veljkova 3, 21000 Novi Sad, Serbia and Montenegro The anthracycline antibiotics are cytotoxic drugs used in treatment of the malignant diseases. In spite of great efficiency, their clinical use is limited by dose-related cardiotoxicity. The aim of this study was to investigate on an animal model (mice), if and how, the use of combined antineoplastic therapy containing anthracyclines, used in phase la of the induction therapy of protocol YU ALL 95 for treatment of non-B ALL of childhood, administrated to animals in the adjusted dose and adequate interval, influences function of myocardium and metabolic liver function. White laboratory BALBc mice were divided into 10 groups of 15 animals. Dosage and intervals of application of cytotoxic drugs were corrected according to mice metabolism. The following biochemical parameters were determined: extent of lipid peroxidation, glutathione peroxidase, and the content of reduced glutathione. Intensity of LPx was significantly increased by agents SOD, MKH, DVPSE and DVPMKH. It is interesting to mention that selenium ion itself does not increase LPx, but in combination with anthracycline drugs cause statistically significant increase. Due to large standard deviation, there is no statistical significance of effects of applied substances on GSHPx. Treatment with doxorubicin increased content of GSH.

P14-20 STUDIES ON ANTIOXIDATIVE POTENTIAL IN RATS EXPOSED TO DIMETHOATE AND PYRANTEL EMBONATE A. Spodniewska, A. Zasadowski, D. Barski Division of Veterinary and Environmental Toxicology, Department of Pathology and Pharmacology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland The aim of the present paper was determination of glutathione peroxidase (GSH-Px) activity in blood as well glutathione reductase (GR) in serum of rats exposed to dimethoate and pyrantel embonate. Investigations were conducted in 2 stages. In the first stage of the experiment, animals in group I were given pyrantel embonate at a dose of 1/5 DL50 for 3 consecutive days, animals in group II received dimethoate (technical concentrate) at a dose of 1/10 DL50 for 5 consecutive days and in group III both of mentioned above substances at the same doses and regime administration, but pyrantel embonate was given during the last 3 days of dimethoate intoxication. In the second stage the scheme of experiment was similar but administered dimethoate was in the commercially available form Bi-58 Nowy preparation. All studied substances were delivered directly to the stomach by gavage. Blood samples were taken from animals after 3, 6, 12 h and 2, 7, and 14 days after the end of intoxication with examined compounds. The activity of glutathione peroxidase in blood was determined using analytical kit RANSEL (RANDOX) and glutathione reductase activity in serum with GLUTATHIONE REDUCTASE analytical kit (RANDOX). After both individual as well combined intoxication with pyrantel and dimethoate, activity of GSH-Px decreased in comparison to control group till day 2 of the experiment, whereas in case of GR the opposite effect was observed. The observed changes in GSH-Px activity were more evident after Bi-58 Nowy application, while in GR activity after administration of dimethoate technical concentrate.

Abstracts / Toxicology Letters 158S (2005) S1–S258




D. Barski, A. Zasadowski, A. Spodniewska

B. Kaurinovic1 , M. Popovic1 , I. Jezdimirovic2 , M. Vojinovic-Miloradov1

Division of Veterinary and Environmental Toxicology, Department of Pathology and Pharmacology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland Estimation of chosen markers of oxidative stress (SOD, CAT, MDA) in rats exposed to dimethoate and pyrantel was the aim of this study. Investigations were carried out on Wistar rat. The experiment was performed in 2 stages. In the first stage dimethoate (concentrate) was administered to rats in water solution at a dose of 1/10 DL50 for 5 consecutive days by stomach-tube, pyrantel embonate at a dose of 1/5 DL50 for 3 consecutive days as well both of these substances in a mixed way, applying pyrantel in last 3 days of dimethoate intoxication. The second stage assumed the same way of dosage and application as in the first one, but dimethoate was given in the commercially available form of the preparation Bi-58 Nowy. The biological samples were taken from each group of rats in selected periods, i.e., 3, 6, 12 h and 2, 7 and 14 days after the last intoxication with examined compounds. Activity of superoxide dismutase (SOD) in blood was determined with kinetic method using analytical kit RANSOD (RANDOX), catalase (CAT) with kinetic Aebi’s method and malonodialdehyde (MDA) concentration in the liver with BIOXYTECH® MDA (OxisResearchTM ) analytical kit. It was shown that compounds used in the experiment differently influence activities and concentration of chosen parameters of antioxidative barrier. The observed changes occurred at different time points and were more significant in combined intoxication. The results obtained also indicate that dimethoate used in form of Bi-58 Nowy preparation induces more pronounced changes in analyzed parameters compared to the same dose of technical concentrate of dimethoate.

1 Faculty

of Sciences, Department of Chemistry, Trg Dositeja Obradovi´ca 3, 21000 Novi Sad, Serbia and Montenegro; 2 Faculty of Technical Sciences, Trg Dositeja Obradovi´ca 1, 21000 Novi Sad, Serbia and Montenegro

The aim of this research was to investigate effects of bastard balm leaves and their combination with ascarel and pyralene, on the production of hydroxyl radicals (which cause oxidative stress) and to prove the potential antioxidative effect of this secondary medicament. Crude methanol extracts of macerated leaves were obtained in extraction of leaves with 70% methanol. After evaporation to dryness, dry matter was dissolved in water and extracted with ether, chloroform, ethylacetate, and n-butanol, thus leaving water solution as well. The effects of these extracts on production of OH• radicals was determined by monitoring the chemical degradation of deoxyribose. Butanol and water extract increased the production of hydroxyl radicals, while ethylacetate extract showed statistically significant decrease of the production of hydroxyl radicals. Ascarel shows dose-dependant increase of the production of hydroxyl radicals. The combination of this extract with ascarel has statistically significantly increased the production of hydroxyl radicals, which means that higher concentrations of ascarel act as better radical promoter. Effects of pyralene on the production of hydroxyl radicals is not so pronounced and there is no dose-dependant relationship. On the basis of these results, it can be concluded that pyralene has no prooxidative characteristics.


Abstracts / Toxicology Letters 158S (2005) S1–S258

P14-23 IN VITRO EVALUATION OF THE ANTIOXIDANT EFFECT OF YOHIMBINE L. Todorov1 , M. Traykova2 , T. Traykov3 1 Faculty

of Pharmacy, Medical University, Sofia, Bulgaria; 2 Department of Pharmacology and Toxicology, Medical Faculty, Medical University, Sofia, Bulgaria; 3 Department of Physics and Biophysics, Medical Faculty, Medical University, Sofia, Bulgaria The main therapeutic effects of yohimbine have been related with its activities as ␣-2-adrenergic blocker and MAO inhibitor. The yohimbine molecule contains both OH and NH groups, which might potentate some antioxidant activity. This could explain the use of the Yohimbe bark against fever in the traditional African medicine. The aim of the present investigation was to monitor in vitro the antioxidant properties of yohimbine. The effect yohimbine hydrochloride on the MDA was monitored by measuring TBARS in yolk-derived liposomes and rat’s brain homogenate. Some other assays for analyzing the scavenging properties against superoxide radical, hydroxyl radical and hypochlorous anion were used as well. In presence of Yohimbine, the MDA level decreased in both yolk-derived liposomes and brain tissue homogenate. Within concentrations of 10−6 ÷ 10−4 M, ROS-scavenging effects of yohimbine were concentration-dependent. All effects observed were weaker in presence of rat’s brain homogenate in comparison with the other model solutions. Yohimbine hydrochloride exhibited a significant in vitro antioxidant effect. It was proposed that yohimbine hydrochloride might be involved in some extent in the control over the oxidative stress. This participation in EUROTOX 2005 is financially supported by the Faculty of Pharmacy, Medical University of Sofia, Bulgaria.

P14-24 THE EFFECT OF RESVERATROL ON CADMIUM INDUCED OXIDATIVE DAMAGE AND ESSENTIAL ELEMENTS CHANGES IN MICE D. Kotyzova, V. Eybl Charles University School of Medicine in Pilsen, Czech Republic Cadmium (Cd) is a carcinogenic metal and a serious environmental pollutant. Resveratrol, a naturally occurring polyphenol, has been referred to exert protective effects in cardiovascular diseases and cancer, possibly partly due to antioxidant properties. In this study the influence of resveratrol on Cd-induced oxidative stress was investigated relating to lipid peroxidation, antioxidant defense system and essential element level in male mice injected with CdCl2 ·2 21 H2 O (7 mg/kg, sc) with/without resveratrol pre-treatment (20 mg/kg, po) 48 h, 24 h and 1 h before Cd intoxication. The increase in lipid peroxidation (122% of controls, p < 0.01) and decrease of GSH level (75% of controls, p < 0.01) 24 h after Cd administration was prevented in Cd+ resveratrol treated group. The decreased catalase activity (73% of controls, p < 0.05) remained unaffected by resveratrol pretreatment. GSH-Px activity increased in Cd+resveratrol group (129% of controls, p < 0.05). Resveratrol administered alone did not influence measured parameters. The Cd distribution in the tissues was not affected by resveratrol pre-treatment. The alteration in essential element concentration caused by Cd intoxication (increased Zn, Cu, Ca, Mg level in the liver; increased Zn level in the kidneys; increased Ca, Fe level and decreased Mg, Zn, Cu level in the testes) remained unaffected by resveratrol pretreatment. Resveratrol administered alone did not influence essential elements level in the tissues with the exception of decreased Cu level in the brain. Considering the role of copper in various pathological conditions in CNS, this finding deserves further study. ˇ 305/05/0344. Supported by the grant GACR

Abstracts / Toxicology Letters 158S (2005) S1–S258


of Pharmacy, Medical University, Sofia, Bulgaria; 2 Department of Pharmacology and Toxicology, Medical Faculty, Medical University, Sofia, Bulgaria The prolonged NaCl overload is among the highest risk factors for developing Alzhimer’s Disease (AD) pathology. The aim of this investigation was to monitor the effect of high NaCl intake on the working memory and oxidative status of the brain cortex. Twelve male Wistar rats (groups “Control” and “NaCl”) were used as model animals. For two weeks, they received 10 g/100 g food and water ab libitum. The group “NaCl” was administered per os. every day with 14 g/kg NaCl (dissolved in 0.5–1 ml water). The 8 arm Radial maze task performances of groups “Control” and “NaCl” were compared. All treatments were in agreement with the standards for animal treatment in the Medical University-Sofia. In the end of the experiment, the increased oxidative stress in the rat’s brain cortex (according spectrofotometric MTT and NBT-tests) of the “NaCl” rats was accompanied with decline of their performance in 8 arm Radial maze task. The kidney function remained intact in both groups. Thus, the NaCl overload might alter the performance of the brain, even before altering the kidney function. This work is financially supported by the Scientific Board of the MU-Sofia, Bulgaria (grant no. 44/2005). The authors thank the Faculty of Pharmacy, MU-Sofia for supporting financially their participation in EUROTOX 2005. P14-26 CURCUMIN HAS A PALLIATIVE ACTION ON GENTAMICIN-INDUCED NEPHROTOXICITY IN RATS Naser Al-Wabel Al-Qassim University, College of Agriculture and Veterinary Medicine, Saudi Arabia


Generation of free radicals in kidney cortex plays an important role in the pathogenesis of gentamicin (GM) nephrotoxicity, and curcumin, the yellow curry pigment isolated from turmeric, has been confirmed to have a strong anti-oxidant action. Therefore, in the present work, we aimed at testing the possible protective or palliative effect of curcumin on GM nephrotoxicity. Curcumin was given to rats at an oral dose of 200 mg/kg/day for 10 days, and in some of these rats GM was also injected intramuscularly at a dose of 80 mg/kg/day during the last 6 days of the treatment. Nephrotoxicity was evaluated histopathologically by light microscopy, and biochemically by measuring the concentrations of creatinine and urea in serum, and reduced glutathione concentration and superoxide dismutase (SOD) activity in renal cortex. The concentration of GM in renal cortex was measured microbiologically. GM significantly increased the concentrations of urea and creatinine (P < 0.05) by about 111% and 97%, respectively. GM treatment reduced cortical GSH concentration by about 31% (P < 0.05), and the activity of SOD by about 27% (P < 0.05). Curcumin significantly mitigated these effects. Sections from saline and curcumin-treated rats showed apparently normal proximal tubules. However, kidneys of GM-treated rats had a moderate degree of necrosis. The degree of necrosis appeared lessened when GM was given simultaneously with curcumin. The concentration of GM in the renal cortex of the rats given GM + curcumin was significantly (P < 0.05) lower than that found in rats treated with GM alone by about 39%. The results suggested that curcumin had ameliorated the histopathological and biochemical indices of nephrotoxicity in rats. Pending further studies, curcumin may potentially be useful as a nephroprotectant agent. P14-27 VARIOUS BIOLOGICAL EFFECTS OF COMBINED LOW DOSES OF HYDROCARBONS AND ALCOHOLS A. Dlugosz, E. Sawicka, D. Piotrowska Department of Toxicology, Wroclaw Medical University, Poland Previous study showed differentiated influence of hydrocarbons and alcohols on free radicals processes measured as lipids peroxidation level, hydroxyl radi-


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cal generation or proteins sulfhydryl groups level. The investigation of combined hydrocarbons and alcohols exposure on free radicals processes pointed on synergetic as well antagonistic interaction. It was observed that low doses of styrene or ethylene glycol did not change lipids peroxidation level or hydroxyl radical generation but the effect of combined low doses exposure statistically significant increased free radicals processes measured as MDA production or • OH generation. So in the case of combined exposure for low doses of styrene and ethylene glycol synergistic effect was observed. Different effect was observed in combined methanol/xylene or methanol/Solvesso 100 exposure. In contrary the mixture of these hydrocarbons gives antagonistic effect. It seems that the differences in final biological effect are caused by various mechanism of interaction: in the case of styrene/ethylene glycol-biotransformation; in the case of aromatic hydrocarbons/methanol-distribution. Styrene is bioactivated to styrene oxide by cytochrome P-450. Short chain aliphatic alcohols can increase xenobiotics toxicity through induction of cytochrome P-450 izoenzymes. On the other hand the detoxication of styrene oxide to phenylglycol might be inhibited by ethylene glycol. In the case of combined aromatic hydrocarbons/methanol exposure the decrease in the lipophylic properties of mixture could reduce its penetration into cells membrane and cause antagonistic effect. P14-28 SELENIUM SUPPLEMENTATION PROTECTS AGAINST CADMIUM INDUCED OXIDATIVE DNA DAMAGE – IN VITRO STUDY J. Gromadzi´nska, J. Palus, M. Sta´nczyk, E. Reszka, W. W˛asowicz Nofer Institute of Occupational Medicine, Lodz, Poland Toxic effects of heavy metals on cells involve among others generation of reactive oxygen species, disturbance of antioxidative processes and damage of lipids, proteins and DNA. Cadmium shows affinity to thiol groups, and the decreased concentration of intracellular glutathione is most likely the underlying cause of oxidative stress. Selenium is trace element involves in antioxidative processes and heavy metal detoxication, through activity of glutathione peroxidases, tioredoxin reductases, selenoprotein P, etc. The aim

of this study was to investigate the protective effect of selenium on cadmium induced DNA damage. The mice fibroblasts (WEHI 164) was cultured in medium with low (0.15 ng/ml), standard (1.5 ng/ml) and high selenium (50 ng/ml) concentration and stressed with 1.5 ␮g CdCl2 /ml medium for 24 h. Oxidative DNA damage measured by the percent of DNA in comet tail and by fpg sites, was lower in cells with standard and high selenium medium as compared with cells growing in low selenium medium (p < 0.05). Incubation cells with cadmium alone or Cd in presence of H2 O2 induce statistically significant oxidative DNA damage (p < 0.05). Percent DNA in comet tail in cadmium treated cells were inversely correlated with selenium concentration in medium. It was not found differences in DNA damage in high Se cells treated with Cd and controls (without Cd) – Se supplementation protect against cadmium-induced DNA damage. This project was support by Polish Committee of Scientific Research grant no 0548/P05/2003/25. P14-29 EFFECTS OF ACUTE DIMETHOATE ADMINISTRATION ON ANTIOXIDANT STATUS OF LIVER AND BRAIN OF EXPERIMENTAL RATS Y. Sharma1 , S. Bashir1 , M. Irshad2 , S. Datta Gupta3 , T.D. Dogra1 1 Department

of Forensic Medicine and Toxicology, India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India; 2 Department of Laboratory Medicine, India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India; 3 Department of Pathology, India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India Organophosphorus compounds may induce oxidative stress leading to generation of free radicals and alterations in antioxidant and scavengers of oxygen free radicals. The present study demonstrates effect of acute exposure of dimethoate in causation of oxidative stress in male Wistar rats. Dimethoate was administered orally at doses 45 mg/kg, 75 mg/kg and 90 mg/kg of body weight on the basis of LD50 for 24 h. After administration of doses, the liver and brain homogenates were analyzed for various parameters of oxidative stress. The results indicated an increase in hepatic Cytochrome P450, lipid peroxidation, catalase, super-

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oxide dismutase, glutathione peroxidase, glutathione reductase in liver and brain at 90 mg/kg and 75 mg/kg doses. There were no significant changes in the levels of glucose-6-phosphate dehydrogenase activity in both liver and brain. Similarly, there was no significant changes in hepatic glutathione and glutathione-Stransferase activities. However, there was a significant increase in glutathione and glutathione-S-transferase in brain at 90 mg/kg dose only. Erythrocyte acetylcholinesterase was inhibited at all doses used. Dosedependent histopathological changes observed in both liver and brain is also described. P14-30 PROTECTIVE EFFECT OF SAFRANAL AGAINST HEXACHLOROBUTADIENE NEPHROTOXICITY IN RAT M.T. Boroushaki, B. Naghizadeh, H.R. Sadeghnia Department of Pharmacology and Pharmacological Research Center on Medicinal Plants, Ghaem Hospital, Mashhad, Iran Hexachlorobutadiene (HCBD) is a potent nephrotoxine in rodents, which can cause degeneration, necrosis and regeneration in renal tubular epithelial cells. There are some studies that safranal, the active ingredient of saffron with antioxidant properties, has a protective effect against ischemic injuries. Therefore, in this study the protective effect of safranal against HCBD-induced nephrotoxicity in rat was examined. In this study W/A rats were used. After acclimatization, animals divided in five groups. On day 1, each animal was separately put in a metabolic cage for collecting 24-h urine samples. On day 2, after collecting urine samples for measuring glucose and protein, group one received corn oil 1 ml/kg, group two received HCBD 50 mg/kg as a single dose, and groups 3–5 received 0.5, 0.25 and 0.1 mg/kg safranal as a single dose, 1 h before HCBD 50 mg/kg, respectively. All injections were carried out intraperitoneally. After 24 h, urine samples were collected, animals were killed; blood samples were taken by cardiac puncture for measuring urea and creatinine. Both kidneys were removed. The right kidney was fixed in formalin for histopathologic examinations and the left one were homogenized for measuring malodialdehyde (MDA).


Data showed that in groups 2 and 5, there was substantial necrosis to straight portion of the proximal tubules. In groups 3 and 4 the renal proximal tubules showed a normal appearance compare to control. Concentration of urea, creatinine, glucose, protein and MDA in different treated groups was as follows, Group 1: Urea (59 ± 3.8), creatinine (0.63 ± 0.03), glucose (7 ± 0.7), protein (1.68 ± 0.9) mg/dl and MDA (0.24 ± 0.03 nmol/g). Group 2: Urea (115.75 ± 0.4), creatinine (1 ± 0.19), glucose (38.25 ± 0.63), protein (9.85 ± 0.78) mg/dl and MDA (0.26 ± 0.05 nmol/g). Group 3: Urea (76.5 ± 6.5), creatinine (1.07 ± 0.14), glucose (21.24 ± 1.18), protein (1.3 ± 0.28) mg/dl and MDA (0.2 ± 0.006 nmol/g). Group 4: Urea (68.25 ± 2.75), creatinine (0.68 ± 0.28), glucose (24.25 ± 1.11), protein (1.75 ± 0.43) mg/dl and MDA (0.31 ± 0.03 nmol/g). Group 5: Urea (122.75 ± 8.7), creatinine (1.15 ± 0.11), glucose (39.25 ± 1.25), protein (3.36 ± 0.87) mg/dl and MDA (0.27 ± 0.01 nmol/g). In conclusion, safranal, with doses 0.25 and 0.5 mg/kg is able to protect the kidney against toxic effect of HCBD in rat. P14-31 ASPECTS REGARDING HYPEGLYCEMIC TOXICITY EVALUATED AS REDOX STRESS PARAMETERS D. Margin˘a1 , D. Gr˘adinaru1 , N. Mitrea1 , M. Vl˘adic˘a2 , A. Nit¸ulescu-Arsene1 1 UMF

Carol Davila, Faculty of Pharmacy, Department of Biochemistry, Traian Vuia 6, Bucharest, Romania; 2 N. Paulescu National Institute of Diabetes and Metabolic Diseases, Bucharest, Romania Hypeglycemic patients must be closely biochemically and clinically monitored a proper diet and medication being also essential. We have designed a 3-month prospective study on 60 newly discovered type II diabetes patients, 28 women (46.6%) and 32 men (53.4%). All patients received standard lifestyle changes recommendation. The following data were recorded: age, body mass index, fasting plasma glucose and insulin, glycated hemoglobin, plasma total cholesterol, LDL-


Abstracts / Toxicology Letters 158S (2005) S1–S258

cholesterol, HDL-cholesterol, triglycerides, serum apparent redox potential, and susceptibility of low density lipoproteins to lipid peroxidation. After 3 month of follow-up, patients showed a statistically significant increase of the insulin level (10.52 ± 5.62 ␮U/mL versus 14.07 ± 6.21 ␮U/mL, p < 0.05) and of apparent redox potential (−29.4 ± 11.96 mV versus −13.09 ± 8.76 mV, p < 0.001) and a decrease of the glycated hemoglobin level (6.86 ± 1.12% versus 5.91 ± 1.10%, p < 0.0001). There was no significant change of the body mass index. Our study emphasized that diet and physical exercise has a beneficial effect on the activity of pancreatic ␤ cells of hyperglycemic patients, leading to a decrease of the glycated hemoglobin. Although there was a significant increase in the glycemic control, the hyperglycemic oxidative stress lead to a deterioration of the redox homeostasis during the study.

put screening (96-well format) within 1 day. Using two application examples, we demonstrate that the “hmox1 reporter-gene-assay” can determine the oxidative stress-inducing potential of CS fractions and CS constituents in both quantitative and qualitative terms. Our results suggest that this assay may be a useful routine assay for the high through-put, short-term determination of the oxidative stress-inducing potential of CS as well as other xenobiotic compounds and mixtures.


College of Pharmacy, Pusan National University, Busan 609-735, Korea

C. Kn¨orr-Wittmann, S. Gebel, T. M¨uller Philip Morris Research Laboratories GmbH, Cologne, Germany Exposure to cigarette smoke (CS) provokes a strong antioxidant cellular response in vitro and in vivo. A central feature of this cell protective response is reflected by the paramount up-regulation of the gene encoding heme oxygenase-1, which catalyzes the initial and rate-limiting steps in heme catabolism. Due to its reaction products, i.e., biliverdin and, in the presence of biliverdin reductase, eventually bilirubin, which represent a kind of SOS redox buffer against an imbalanced redox homeostasis, heme oxygenase-1 (hmox1) expression is considered a reliable marker of (oxidative) stress. Here we show that NIH3T3 cells, stably transfected with an hmox1 promoter/enhancer reporter (luciferase) gene construct, express endogenous hmox1 and the reporter gene in a co-linear manner upon exposure to aqueous fractions of CS, thus rendering this system a potential experimental tool for routine determination of cellular (oxidative) stress. Optimization of the assay protocol now allows high through-


The aim of present study is to investigate the effects of di(n-butyl) phthalate (DBP) on the oxidative DNA damage and antioxidant enzymes activity in the testes of hyper- or hypothyroid rats. Hypothyroidism was induced in Sprague-Dawley pubertal male rats (4 weeks of age) by adding 0.1% propylthiouracil (PTU) to their drinking water for 30 days and hyperthyroidism was induced by treatment with T3 (10 ug/kg/day, i.p.). DBP (750 mg/kg/day) was administered to the normal, hypo- and hyperthyroid rats via oral gavages over a 30-day period. DBP (750 mg/kg/day) significantly reduced the weights of the testes in both the hyperand hypothyroid rats. The 8-OHdG levels were significantly increased in the testes of DBP-treated hyperthyroid rats, but there was no significant differences were observed in DBP-treated hypothyroid and normal rats. Catalase activity was reduced in hypothyroid status, whereas DBP significantly increased catalase activity in all treatment groups. Glutathione peroxidase (GPx) activity was decreased in the testes of hypothyroid rats, whereas superoxide dismutase (SOD) activity was significantly increased in hypothyroid rat. These results suggest that hypothyroidism may cause changes in the metabolism of DBP in the testis, thus may protect against oxidative damages induced by metabolic activation of DBP.

Abstracts / Toxicology Letters 158S (2005) S1–S258

P14-34 COMPARATIVE STUDY ON PARAMETERS OF EXPERIMENTAL DEPRESSION IN MALE AND FEMALE RATS M. Varadinova, D. Drenska, T. Shumkova, N. Boyadjieva Department of Pharmacology and Toxicology, Medical University, Sofia, Bulgaria The rapid development of psychopharmacology in recent years has resulted in numerous biochemical hypotheses with respect to depression and its treatment. There appears to be a growing demand of experimental models for studying the different mechanisms of depression. The models of oxidative stress and ovariectomy are proposed. The aim of this study is to compare depressive symptoms in male rats exposed to oxidative stress for 14 days with depressive symptoms in ovariectomised female rats. We determined the weight gain, food intake, sleep rhythm and behaviour in male and female, experimental and control rats. We used forced-swimming test and 8-arm radial maze test to estimate the differences in rats’ parameters. Both male and female animals demonstrated depressive symptoms. However, we observed certain differences between female and male experimental rats. Our data suggest that these two models can be used for further investigation of the mechanisms of depression and antidepressants. Moreover, these results are an opportunity for improvement the pharmacotherapy of depression. P14-35 DETERMINATION OF PARAMETERS INDICATIVE FOR OXIDATIVE STRESS IN CARBON TETRACHLORIDE TREATED RATS P. Sagelsdorff1 , E. Sommer1 , Y. Balduchelly2 1 RCC Ltd., 4452 Itingen, Switzerland; 2 Filiˇ cre Chimie, HEV, 1950 Sion, Switzerland

A role of oxidative stress has been proposed in the toxicity of numerous chemicals, in the process of ageing as well as in the pathogenesis of many diseases and carcinogenesis. Oxidative stress is known to be followed by cellular and tissue damage whereby lipids and proteins are the major targets.


The aim of this study was to compare the sensitivity of different markers for oxidative stress in male Wistar rats treated with carbon tetrachloride (1 ml/kg body weight). As a measure for lipid peroxidation hepatic isoprostane F2a was determined by ELISA and LC-MS/MS following solid phase extraction. Hepatic malondialdehyde was analysed following derivatisation with thiobarbituric acid by HPLC with fluorescence detection. As a measure for cellular antioxidants in the liver α-tocopherol and ascorbic acid were determined by HPLC, and reduced as well as oxidized glutathione by spectrophotometry following derivatisation with dithio-bis-nitrobenzoic acid. Two and six hours after treatment, hepatic 8-isoprostane F2a concentrations were maximally increased by a factor of about 40 and 13, respectively. Malondialdehyde was increased only by a factor of 1.5. α-Tocopherol and ascorbic acid concentrations in the liver were increased to 160% and decreased to 40%, respectively. The glutathione status in the liver was not influenced following treatment. This study demonstrates the sensitivity of hepatic 8isoprostane F2a concentration as marker for oxidative stress induced by carbon tetrachloride. P14-36 EFFECTS OF HYPERBARIC OXYGEN TREATMENT ON OXIDATIVE STRESS AND GENOTOXICITY IN HUMANS. IS IT A RISK FACTOR FOR GENOTOXICITY? ¨ unda˘g2 , Y. A. Aydın1 , A. Eken1 , A. Sayal1 , A. Ust¨ 2 3 Duydu , K. D¨undar 1 Department of Toxicology, G¨ ulhane Military Medical Academy, 06018 Etlik, Ankara, Turkey; 2 Department of Toxicology, Faculty of Pharmacy, Ankara University, 06100 Tando˘gan, Ankara, Turkey; 3 Department of Underwater Medicine, G¨ulhane Military Medical Academy, 06018 Etlik, Ankara, Turkey

Hyperbaric oxygen (HBO) therapy is a useful method for treatment of various clinical conditions but it can also cause an increased production of free radicals and oxidative DNA base damage. In this study, our aim was to investigate the effects of hyperbaric oxygen on oxidative stress and genetic toxicity. Fifteen patients exposed to HBO treatment (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total of


Abstracts / Toxicology Letters 158S (2005) S1–S258

three 20-min periods, interspersed with 5 min of air breathing) for various pathologies related to hypoxia. Blood samples were taken before HBO therapy, at the end of the 1st, 10th and 20th HBO sessions. Antioxidant parameters and genetic toxicity were studied. We have observed that an increment of lymphocyte sister chromatid exchange (SCE) frequency was observed at the end of the 1st HBO session, the 10th HBO session and the 20th HBO session compared to before HBO treatment (P < 0.05). No significant difference in erythrocyte copper-zinc superoxide dismutase (CuZnSOD), selenium dependent glutathione peroxidase (SeGSHPx) and malondialdeyde (MDA) levels were observed at the end of the 1st HBO therapy and the prolonged HBO exposure as compared to before HBO treatment (P > 0.05). Our results indicate that HBO treatment could not cause significant changes on erythrocyte antioxidant capacity and lipid peroxidation, however it could induce genotoxicity due to different mechanisms. P14-37 MALONDIALDEHYDE AND CARBONYL CONTENT IN THE ERYTHROCYTES OF STREPTOZOTOCIN-INDUCED DIABETIC RATS D. Qujeq, M. Habibinudeh, H. Daylmkatoli Department of Biochemistry and Biophysics, Faculty of Medicine, Babol University of Medical Sciences, Babol, IR. Iran Oxidative stress appears to be involved in degenerative disease, including atherosclerosis and diabetes mellitus. Impaired antioxidant defense mechanism may be an important factor in the pathogenesis of diabetes. The oxidative stress, as measured by the change in blood malondialdehyde and carbonyl content. Determination of carbonyl level in proteins is used an index of the extent of protein oxidative damage. Malondialdehyde level is as marker of lipid oxidation. We investigated lipid peroxidation and protein oxidative damage status in erythrocytes of streptozotocin-induced diabetes rats by measuring erythrocyte malodialdehyde and carbonyl level. Malondialdehyde and carbonyl content were measured by using spectrophotometer. Carbonyl level in erythrocytes of streptozotocin-induced diabetes rats group was increased compared to control

group [2.21 ± 0.38 (mean ± S.D.) versus 1.12 ± 0.16] mmol/mg of protein, P < 0.05. Malondialdehyde content in erythrocytes of streptozotocin-induced diabetes rats group was increased compared to control group [1.85 ± 0.23 (mean ± S.D.) versus 0.82 ± 0.14] mmol/mg of protein, P < 0.05. We found that carbonyl and malondialdehyde levels were significantly increased in erythrocytes of streptozotocin-induced diabetes. These indirectly suggest an increased production of oxygen free radicals-mediated damage of the membrane lipid and protein. P14-38 BETAINE PREVENTS LOSS OF SIALIC ACID RESIDUE AND PEROXIDATIVE INJURY ON ERYTROCYTE MEMBRANE IN ETHANOLGIVEN RATS 1 , F. C ¨ G. Kanbak1 , F. Ozdemir ¸ alıs¸kan2 , M. ˙Inal1 1 Osmangazi

University, The Medical School, Department of Biochemistry, Eski¸sehir, Turkey; 2 Osmangazi University, The Science and Art Faculty, Department of Biology, Eski¸sehir, Turkey To evaluate chronic ethanol toxicity on erythrocyte membrane and preventive effect of betaine, 24 male Wistar albino rats were divided into three groups: control, ethanol and ethanol plus betaine group. Animals were fed 60 ml diet per day for 2 months. Rats in the ethanol group were fed ethanol 8 g/kg/day. The ethanol + betaine group were fed ethanol plus betaine (0.5% w/v). After 2 months, all animal were killed. Malondialdehyde and sialic acid levels were determined in plasma samples. Osmotic fragility test were measured in whole blood and erythrocyte membrane thiol contents were determined in membrane suspension. MDA levels in ethanol-given rats were increased as compared to control group (p < 0.05). MDA in the betaine group was lower than in the ethanol group (p < 0.05). Erythrocyte membrane thiol contents in ethanol group were decreased compared with the control group (p < 0.05). Thiol contents were increased slightly after betain therapy. But, this increase was not statistically significant (p > 0.05). Plasma sialic acid levels in ethanol group were significantly higher than in the control group (p < 0.01). Sialic acid was decreased in betaine group as compared to ethanol group

Abstracts / Toxicology Letters 158S (2005) S1–S258


(p < 0.01). In the osmotic fragility test, we observed that chronic ethanol consumption was increased erythrocyte hemolysis. Betaine was protected ethanol-induced hemolysis. Our findings show that the toxicity of chronic ethanol administration affects erythrocyte membrane structure and this may be related to oxidative stress. Betaine protects erythrocyte membrane alterations against chronic ethanol toxicity. Therefore, betaine may protect ethanol induced clinical problems subject to membrane abnormalities as a nutritional agent.

fonic acid (0.46 mM) adjusted to pH 3.0 at 1 mL/min were used. The GSH mono- and bi-conjugates were characterized according to their UV spectra. Further NMR analysis of these adducts confirmed their identity.


1 Department

R. Silva1,2 , V. Costa1 , S. Boldt1 , H. Carmo1 , F. Carvalho1 , M. Carvalho1,3 , R. Carvalho4 , M. Lourdes Bastos1 , F. Remi˜ao1 1 Requimte,

Servi¸co de Toxicologia, Faculdade de Farm´acia, Universidade do Porto, Portugal; 2 Departamento de Biologia, Universidade de Aveiro, Aveiro, Portugal; 3 Universidade Fernando Pessoa, Porto, Portugal; 4 Centro de Neurociˆencias de Coimbra, Universidade de Coimbra, Coimbra, Portugal Sustained high levels of circulating catecholamines can lead to cardiotoxicity and neurotoxicity. There is increasing evidence that these processes can result from the oxidation of catecholamines. We have previously shown that the catecholamines oxidation products can further conjugate with GSH in isolated rat cardiomyocytes and hepatocytes. The aim of this study was to develop an HPLC methodology to detect GSH adducts of the catecholamines epinephrine, norepinephrine and dopamine in biological samples. Human serum samples were incubated with a reaction mixture of each catecholamine (160 nM to 4 ␮M), tyrosinase and GSH. After 10 min of reaction, the adducts were extracted after adsorption to alumina. Aliquots of the extract were injected into an HPLC equipped with a photodiode array detector and a coulochem detector. An Spherisorb S5 ODS2 column and an isocratic mobile phase consisting of methanol (7% or 10%, depending on the catecholamine) with citric acid (50 mM) and octanesul-

P14-40 INVOLVEMENT OF OXIDATIVE STRESS AND CYCLOOXYGENASE IN FLUORO-EDENITEINDUCED MESOTHELIOMA V. Cardile1 , A.Panico2 , L. Lombardo1 of Physiological Sciences, V.le A. Doria 6, University of Catania, Catania, Italy; 2 Department of Pharmaceutical Sciences, V.le A. Doria 6, University of Catania, Italy In this study, we investigated the involvement of cyclooxygenase-2 (COX-2) and prostaglandin (PG)E2 in the probable carcinogenic effect caused by fluoroedenite in monocyte-macrophage cell line J774. Alveolar macrophages occupy a key position in mediating the interaction between inhaled particulates and various cell types. Fluoro-edenite is a new asbestos-like amphibole recently extracted from stone quarries in Biancavilla, a village located in the Etna Volcanic Complex (Catania, Italy) of eastern Sicily, in which an epidemiological survey evidenced a cluster of cases of the mortality due to pleural mesothelioma. The aim of this research was to understand whether fluoroedenite present in Biancavilla was in relation to the development of the high incidence of cancer of the respiratory tract among the population. The expression of COX-2, enzyme, which participates in the processes of inflammation and cell proliferation and differentiation, was evaluated by Western blot analysis. The concentration of PGE2 , implicated in the pathogenesis of solid tumours, was measured in the culture media by enzyme-linked immunosorbent assay (ELISA). Fluoro-edenite at 50 ␮g/ml for 24, 48, 72 and 96 h has been demonstrated significantly to increase COX-2 and PGE2 productions in time-dependent manner. The overall data provides convincing evidence that oxidative stress and inflammatory factors, such as COX-2 and PGE2 , mediate the fluoro-edenite induced carcinogenesis.


Abstracts / Toxicology Letters 158S (2005) S1–S258

P14-41 THE EFFECT OF WOOD DUST ON OXIDATIVE STRESS AND CELL VIABILITY OF HUMAN BRONCHAIL EPITHELIAL CELLS (BEAS-2B) L. Pylkk¨anen, H. Stockmann-Juvala, H. Alenius, K. Husgafvel-Pursiainen, K. Savolainen Finnish Institute of Occupational Health, Helsinki, Finland Regular occupational exposure to wood dust has been shown to be associated to a variety of respiratory diseases. Human bronchial epithelial cell-line cells were exposed to three different wood dusts (pine, birch and oak) at final concentrations of 10–500 ␮g/ml for 0–24 h. Untreated control cultures were included in all time points. After exposure the production of reactive oxygen species (ROS), and frequency of both necrotic and apoptotic cell death were measured. Increased production (1.5–1.7 fold induction) of ROS was observed after 2 and 6 h exposure to each of the wood dusts studied. After exposure to 12–24 h the levels of ROS returned back to the control levels. The wood dusts also induced both necrotic and apoptotic cell death. The caspase-3 enzyme activity, measured as a marker of apoptosis, increased significantly compared to the control levels after 2 and 6 h exposure to each of the wood dusts. After 24 h, the level of caspase-3 activity of the untreated cultures was also increased, and only pine dust caused a clearly elevated effect. In addition, necrotic cell death increased after 2 and 6 h exposure, especially to the highest concentrations of wood dusts, but after 12 h the cell viability was recovered. Activation of different repair mechanisms, the formation of a new generation of cells, and the antioxidants contained in the wood dusts may play a role in the transient oxidative stress as observed in these human bronchial epithelial cells. Supported by the Academy of Finland, project no. 53631 and by the European Union, Wood-Risk project No QLK4-2000-00573.

P14-42 CYTOTOXICITY STUDIES OF NOVEL EMPODERIVED SPIN TRAPS N. Rohr-Udilova1 , K. Stolze1 , B. Marian2 , R. SchulteHermann2 , H. Nohl1 1 Research Institute of Biochemical Pharmacology and

Toxicology, University of Veterinary Medicine Vienna, A-1210 Vienna, Austria; 2 Institute of Cancer Research, Medical University of Vienna, A-1090 Vienna, Austria Free radicals are playing a major role in different regulatory and pathological processes. Especially detection of superoxide in living cells or whole organisms using the ESR spin trapping technique is of interest. Therefore, the design of adequate spin traps with low toxicity and high selectivity is necessary. Our study investigates the toxicity of novel derivatives of the spin trap 5-ethoxycarbonyl-5-methyl-pyrroline Noxide (EMPO) to cultured human lung carcinoma cells (A549), breast carcinoma cells (SKBR3), colon carcinoma cells (SW480) as well as to human fibroblasts (F2000). A dose-dependent decrease of the cell number was observed for all spin traps. At 100 mM the n-, sec- and tert-butoxycarbonyl derivatives of EMPO (BuMPO, sBuMPO, tBuMPO) caused pronounced cell loss (>90%) and increased LDH-release. The use of 5-diethoxyphosphoryl-5-methyl-pyrroline N-oxide (DEPMPO), EMPO and its 1- and 2-propoxycarbonyl derivatives (PrMPO, iPrMPO) caused only moderate cell loss (<60%) without effect on LDH-release after 24 h. At 10 mM and 50 mM the latter agents even decreased LDH-release acting like antioxidants. Staining for apoptotic nuclei did not reveal any differences between controls and treated cultures indicating necrotic cell death possibly due to membrane toxicity. The following toxicity ranking was obtained: tBuMPO > BuMPO > sBuMPO > PrMPO > iPrMPO ∼ DEPMPO ∼ EMPO. The least toxic compounds were DEPMPO (LD50 = 143 mM for SW480, 117 mM for A549 or 277 mM for F2000) and iPrMPO

Abstracts / Toxicology Letters 158S (2005) S1–S258

(LD50 = 114 mM for SKBR3), the most toxic one was tBuMPO (LD50 = 5 to 6 mM for all cell types). In conclusion, up to 100 mM iPrMPO (t1/2 = 18.8 min) and up to 50 mM sBuMPO (t1/2 = 26.3 min) can be recommended for further investigation of superoxide in biological systems. P14-43 INDUCTION OF OXIDATIVE STRESS BY ACRYLAMIDE IS RELATED TO THE DIFFERENTIATION GRADE OF CACO-2 CELLS B. Z¨odl1 , A. Bielik1 , S. Humpeler1 , B. Schweiger1 , T. Thalhammer2 , W. Marktl1 , C. Ekmekcioglu1 1 Medical

University of Vienna, Center for Physiology and Pathophysiology, Institute of Physiology, Schwarzspanierstr. 17, 1090 Vienna, Austria; 2 Medical University of Vienna, Center for Physiology and Pathophysiology, Institute of Pathophysiology, W¨ahringer G¨urtel 18-20, 1090 Vienna, Austria Objectives: Acrylamide is metabolized via cytochrome P450 2E1 (CYP2E1) to glycidamide (phase I), a chemically reactive epoxide that forms DNA adducts. This metabolite is detoxified via glutathione-S-transferase (GST) to N-acetyl-S-(3-amino-3-oxopropyl) cysteine (phase II). Caco-2 cells express both, CYP2E1 and GST, but expression of the latter is dependent on the degree of differentiation, for which apical alkaline phosphatase expression is a marker. Methods: In the presented study Caco-2 cells were grown until different stages of confluency (day 3 and 7) and differentiation (day 3, 7, 14 and 21). Cells were incubated for 24 h with 0.1, 0.5 and 1 mM of acrylamide dissolved in deionized water added directly into the culture medium. Thereafter activity of GST and alkaline phosphatase as well as reduced/oxidized glutathione GSH/GSSG ratio and lipid peroxidation were analyzed. Results and discussion: GST and alkaline phosphatase activities increased during differentiation of the cells while addition of acrylamide showed no effects. Glutathione levels decreased until confluency was reached (day 7). At day 14 and 21 (day 21: cells are fully differentiated) acrylamide significantly decreased total glutathione levels and at day 14, also the GSH/GSSG ratio


decreased in a concentration dependent manner. This indicates oxidative stress burden. However, regarding lipid peroxidation all samples analyzed were below the detection limit. Conclusion: The present data suggest that, by reducing glutathione levels, acrylamide or its metabolite might affect phase II detoxification in differentiating Caco-2 cells. Additional measurements of CYP2E1 expression are currently performed to assess the role of this CYP in acrylamide induced oxidative stress. P14-44 EFFECT OF MELATONIN ON POLY(ADPRIBOSE) POLYMERASE-1 (PARP-1) ACTIVITY IN HUMAN TUMOR LUNG CELLS TREATED WITH COPPER, MERCURY M.H. Ahmad1 , Abdel-Rahman Raemisch1 , F. Glahn1 , H. Foth1


Torky1 ,


1 Institute of Environmental Toxicology, Martin-Luther-

University Halle, Germany; 2 Institute of Cardiothracic Surgery, Martin-Luther-University Halle, Germany Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein and DNA binding enzyme. It plays a primary role in the process of poly(ADP-ribosyl)ation and this post-translational modification of nuclear proteins is activated in response to DNA damage. Melatonin, the chief secretory product of the pineal gland is a free radical scavenger and antioxidant that protects DNA from damage induced by heavy metals and oxidative stress. Induction of DNA damage by H2 O2 (100 ␮M), leads to formation of ADP-ribose-polymers, which were detected immunhistocemically by means of fluorescence detection. Pre-incubation of human tumor lung cells A549 with melatonin (0.1 mM) added to the inhibitory effect of copper sulfate (0.05 mM) on H2 O2 induced PARP1 activity. Mercury (II) chloride (0.03 mM) exerted no effect on PARP-1 activity but decreased the activity of PARP-1 initiated upon co-incubation with H2 O2 . Incubation of A549-cells with both H2 O2 and mercury showed more fluorescence signals of PARP1-activity compared to melatonin pre-treated A549-cells. All


Abstracts / Toxicology Letters 158S (2005) S1–S258

used concentrations of metals were within the subtoxic ranges determined by MTT-assay. Modification of PARP1-activity in normal human lung cells using different antioxidants, e.g. melatonin, Vit C and Vit E remains an interesting point to be studied in the next step. P14-45 AMIFOSTINE REDUCES MORTALITY, BONE MARROW AND CARDIO TOXICITY IN RATS SUBJECTED TO CHRONIC DOSE REGIMEN OF DOXORUBICIN V. Dragojevic-Simic1 , S. Dobri´c1 , D. Bokonjic1 , Z. Tatomirovic2 , V. Jacevic1 1 National

Poison Control Centre, Military Medical Academy, Belgrade, Serbia and Montenegro; 2 Insitute of Pathology and Forensic Medicine, Military Medical Academy, Belgrade, Serbia and Montenegro Therapeutic success of doxorubicin (DOX) is limited by its dose-dependant bone marrow and cardiotoxicity. Although its toxic effects on the heart is probably mediated initially by highly reactive oxygen free radical action, we suggest that final irreversible changes are the result of inflammatory reactions involving cells of bone marrow origin. Amifostine (AMI) is strong free radical scavenger, although its protective effect on bone marrow is more complex. Our aim was to investigate AMI effects on DOX-induced changes in bone marrow and peripheral blood, as well as in the heart of the animals treated by DOX. AMI (75 mg/kg i.p.) was given 30 min before each dose of DOX (cumulatively 20 mg/kg i.p., during 28 days). Microscopic examination of bone marrow, peripheral blood and heart was done 28 days after the last dose of drugs. AMI significantly attenuated reduction of both immature and mature forms of neutrophil granulocytes, as well as increase of eosinophil leukocyte number in bone marrow caused by DOX. In AMI-pretreated rats number of peripheral blood leukocytes was significantly higher comparing to DOX-only treated animals, preferentially protecting neutrophils and monocytes. Heart polymor-

phonuclear cell infiltrates were prominently reduced in AMI-pretreated rats, especially when mast cell number was concerned. Observed protective effects of AMI highly correlated with better survival of DOX-treated rats compared with that of unprotected ones. P14-46 ANTIOXIDANT POTENTIAL AND FREE RADICAL DAMAGE IN RATS SUBCHRONICALLY EXPOSED TO SODIUM FLUORIDE I. Inkielewicz, J. Krechniak Department of Toxicology, Medical University in Gda´nsk, Poland Fluoride effects on structure and functions of organs may suggest that fluoride produces free radicals and negatively influences the defence mechanisms of living cells. Authors investigating the influence of fluoride on free radical parameters received different, even contradictory results. The aim of this study was to investigate if fluoride exerts an impact on free radical parameters in rats. Male Wistar rats weighing 180 g were given sodium fluoride in drinking water in a concentration of 5 and 25 mg F− /L. Control animals received tap water containing 0,3 mg F− /L. At the beginning of the experiment and after 2, 4 and 12 weeks of exposure 6 animals of each group were sacrificed and samples of blood, liver, brain and testis were collected. In this material the activity of glutathione peroxidase and concentrations of GSH, TBARS and carbonyl groups were determined. In exposed animals the activity of glutathione peroxidase and concentration of GSH decreased significantly in all investigated tissues in a dose and exposure – time dependent manner. A positive correlation between GPx and GSH was ascertained. The concentration o MDA and carbonyl groups increased significantly in all investigated tissues in a dose and exposure – time dependent manner. The results of this study indicate that fluoride exerts its toxic action through free-radical mediated mechanisms.

Abstracts / Toxicology Letters 158S (2005) S1–S258

P15 Exposure Monitoring P15-01 EARLY GENOTOXIC EFFECTS BY PAH EXPOSURE IN PAVING WORKERS C.L. Ursini1 , D. Cavallo1 , P. Bavazzano2 , C. Cassinelli2 , A. Frattini3 , B. Perniconi1 , A. Di Francesco1 , A. Ciervo1 , R. Maiello1 , B. Rondinone1 , S. Iavicoli1 1 Department of Occupational Medicine, ISPESL Mon-

teporzio Catone, Rome; 2 Laboratory of Public health, ASL-Florence; 3 Department of Prevention ASL RMG, Guidonia, Rome Paving workers are exposed during road paving to several PAHs contained in asphalt fumes. We aimed to evaluate early genotoxic effects in 19 paving workers and 22 controls. We measured the concentration of 14 PAHs and urinary OH-pyrene following 3 working days. We assessed oxidative DNA damage by Fpgmodified comet assay calculating tail moments from fpg-enzyme treated cells (TMenz) and from untreated cells (TM). For each subject the TMenz/TM ratio higher than 2.0 was used to indicate the presence of oxidative damage. We also evaluated DNA damage analysing comet percentage. Moreover, we analysed sister chromatide exchange frequency. Personal air sampling showed low level of total PAHs (2.79 ␮g/m3 ) with prevalence of 2–3 ring PAHs (2.67 ␮g/m3 ). Urinary OH-pyrene after 3 days were significantly higher than that found at the beginning of working week. We found oxidative DNA damage in 37% of exposed in respect to the absence in controls. Comet percentage was significantly higher (P = 0.000 ANOVA) in the exposed than in controls. SCE analysis did not show any difference between two groups. The results demonstrate the high sensitivity of comet assay to assess early oxidative effects induced by exposure to PAH mixtures at low doses and confirm the suitability of urinary OH-pyrene as biomarker of PAH exposure.


P15-02 DIETARY EXPOSURE TO POLYCYCLIC AROMATIC HYDROCARBONS IN BRAZIL M.C.R. Camargo, M.C.F. Toledo Department of Food Science, Faculty of food Engineering, UNICAMP, Campinas, SP, Brazil A study was carried out in Brazil in order to estimate the intake of 10 PAHs by the the general population. Food consumption data were inferred from a national household economic survey, accomplished by the Brazilian Institute of Geography and Statistics (IBGE). Samples of food groups regularly consumed by the Brazilian population were analysed for the following PAHs: fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(e)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(ah)anthracene and benzo(ghi)perylene. The analytical methodology involved liquid–liquid extraction, clean up on silica gel column and determination by high performance liquid chromatography using fluorescence detection. Different PAHs were detected in all food groups. The most frequently occurring PAHs were fluoranthene, benzo(k)fluoranthene and benzo(a)pyrene, which were detected in 91, 84 and 81% of the analysed samples, respectively. The levels of the individual PAHs ranged from not detected to 42.90 ␮g/kg. The estimated mean daily intake of the total and the carcinogenic PAH fraction by the general population was, respectively, 6.15 and 2.07 ␮g/day/person. Oils and fats were the major contributors to the total PAHs dietary intake (21.5%), followed by sugar (15.4%) and smoked meat (14.6%). These data may be helpful in assessing the risks to public health arising from the presence of PAHs in the Brazilian diet. P15-03 OPTIMIZATION OF SPE FOR 1-HYDROXYPYRENE AS A BIOMARKER OF OCCUPATIONAL EXPOSURE TO PAHS PRIOR TO HPLC S.J. Shahtaheri, L. Ibrahimi, F. Golbabaei, M. Hosseini, B. Fouladi Department of Occupational Health, Tehran University of Medical Sciences, Tehran, Iran


Abstracts / Toxicology Letters 158S (2005) S1–S258

Urinary 1-hydroxypyrene (1-OHP) is commonly used as a major metabolite and biological indicator of the overall exposure to polycyclic aromatic hydrocarbons (PAHs). For evaluation of occupational exposure, biological monitoring is an essential process, in which, preparation of samples is one of the most timeconsuming and error-prone aspects prior to chromatography. SPE was optimized with regard to sample pH, sample concentration, loading flow rate, elution solvent, washing solvent, sample volume, elution volume, and sorbent type and mass. 1-OHP was retained on C18 sorbent based on specific interactions. Further study employed methanol to extract the analyte from spiked urine and gave a clean sample for HPLC-fluorescent system. Extraction recovery exceeded 99.96%, achieving detection limit of 0.02 ␮g/L. The procedure was validated with three different pools of spiked urine samples and showed a good reproducibility over six consecutive days as well as six within-day experiments as suitable results were obtained for CV% (less than 3.1%) for both day-to-day and within-day reproducibility. In HPLC, C18 column was used with mobile phase of methanol/water run at constant flow rate of 0.8 ml/min, using a fluorescence detection system, setting at 242 and 388 nm. It is concluded that, this optimized method can be successful in simplifying sample preparation for trace residue analysis of PAHs metabolites. P15-04 EXPOSURE TO PAH IN ASPHALT ROAD PAVERS BY ENVIRONMENTAL AND BIOLOGICAL MONITORING S. Fustinoni1 , M. Buratti1 , P.E. Cirla1 , I. Martinotti1 , O. Longhi1 , L. Campo1 , D. Cavallo2 , V. Fo`a1 1 Dipartimento

di Medicina del Lavoro, Universit`a degli Studi e Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy; 2 Scienze Chimiche ed Ambientali, Universit`a dell’Insubria, Como, Italy In the present study, we assessed exposure to PAH in 100 road pavers exposed to bitumen fumes and diesel exhausts in Italy, by means of environmental and biological monitoring. 1-Hydroxypyrene (HO-Py) was determined in 3 urine spot samples collected, respectively after 2 days of vacation (baseline, BL), at the beginning (BS) and at the end (ES) of the monitored workshift, in the second part of the workweek. Median

airborne levels during the workshift of sixteen PAH (both vapour and particulate phases), from naphtalene to indeno(1,2,3-cd)pyrene, ranged from 426 to below 0.03 ng/m3 . Excretion of HO-Py was 228, 402 and 690 ng/l at BL, BS and ES, with higher values in smokers compared to non-smokers (e.g. 565 and 781 versus 252 and 506 ng/l in BS and ES, respectively). In all subjects a very weak correlation between personal exposure to airborne 16PAH and OH-Py was observed by the exclusion of smokers, a higher correlation coefficient was obtained. The results of this study show that asphalt road pavers experienced a moderate occupational exposure to airborne PAH, resulting in a significant increase of urinary OH-Py during the workweek. The contribution of working activities to internal dose seems to be in the same order of magnitude of the contribution of cigarette smoking. P15-05 PAH DERMAL CONTAMINATION IN ASPHALT ROAD PAVERS S. Fustinoni, O. Longhi, L. Campo, P.E. Cirla, I. Martinotti, M. Buratti, V. Fo`a Dipartimento di Medicina del Lavoro, Universit`a degli Studi e Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy Dermal exposure has been suggested as a major determinant of the total PAH dose absorbed by road pavers from bitumen fumes. In the present research we assessed dermal contamination by sixteen PAH [from naphtalene to indeno(1,2,3-cd)pyrene] in 24 asphalt road pavers in Italy. To each subject six pads (round polypropylene membrane) were applied in different parts of the body: neck, shoulder, upper arm, wrist, upper leg and ankle, during the workshift. Personal exposure to airborne 16PAH was in the range 323–1365 ng/m3 . Phenantrene (PhA), pyrene (Py), and benzo(a)pyrene (BaP) were detected in 100, 98 and 75% of pads. Their relative abundances in the different positions were similar. Wrist and neck had the highest PAH contamination with median values of 1.82, 0.52 and 0.06 and 1.19, 0.24 and 0.10 ng/cm2 for PhA, Py and BaP, respectively. In these positions PhA and Py were correlated with almost all 2- to 4-ring volatile PAH (0.41 < r < 0.91), but not with BaP; BaP was correlated with almost all 4- to 5-ring PAH (0.43 < r < 0.78).

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The results of this study suggest that, for a proper estimation of dermal contamination, at least one light (e.g. PhA and Py) and one heavy (e.g. BaP) polycyclic aromatic hydrocarbon, selected as the most representative compounds because of their abundance and/or toxicological relevance, should be determined; wrist and neck may be suggested as the most representative and convenient positions to perform sample collection. P15-06 URINARY LEVELS OF 1-HYDROXYPYRENE, 1-, 2-, 3-, AND 4-HYDROXYPHENANTHRENE AFTER DERMAL EXPOSURE TO COAL TAR Z. Fiala1 , L. Borska1 , V. Krajak2 , K. Hamakova3 1 Charles

University, Faculty of Medicine, Hradec Kralove, Czech Republic; 2 Institute of Public Health, Pardubice, Czech Republic; 3 University Hospital, Hradec Kralove, Czech Republic Objective: Excretion of selected urinary metabolites after dermal exposure to therapeutic coal tar ointment containing polycyclic aromatic hydrocarbons (PAH). Methods: Patients with psoriasis cured by therapeutic coal tar ointment created observed group. The ointment contained 5% of therapeutic coal tar. The range of ointment applications varied from 32 to 77% of patient’s body surface. Urine samples were collected before the therapy, after the first application of ointment, in the middle, after the last application and first day after the last application of ointment. Urinary levels of 1hydroxypyrene, 1-,2-,3-, and 4-hydroxyphenanthrene were analyzed by GC/MS after extraction (Solid Phase Extraction) and derivatization. In parallel, urinary levels of creatinine were analyzed by spectrophotometric method. Results: The therapeutic coal tar contains 29–36% of PAH. The excretion characteristics of 1hydroxypyrene, 1-,2-,3-, and 4-hydroxyphenanthrene varied markedly in dependence with the area and the time of application. Conclusions: The findings indicate that the determination of described metabolites could be useful method for monitoring of dermal exposure to coal tar. Supported by the grant no. NR/8154-3, IGA, Ministry of Health, Czech Republic.


P15-07 OCCUPATIONAL EXPOSURE TO XYLENE IN PATHOLOGISTS EMPLOYING AT HOSPITALS, QAZVIN CITY, IRAN S.J. Shahtaheri1 , M. Afshar2 , M. Majedi2 1 School of Public Health, Tehran University of Medical

Sciences (TUMS), Tehran, Iran; 2 School of Medicine, TUMS, Tehran, Iran Aromatic hydrocarbons, such as benzene, toluene, and xylene are extensively used in different environments and industries, causing adverse effects on individuals. Occupational exposure to xylene in pathologists at different hospitals belonging to the Qazvin University of Medical Sciences has been investigated. Methyl hippuric acid (MHA) as a main metabolite of xylene in urine was used to evaluate the workers exposure to this chemical compound at hospitals. The urine samples were taken from all 30 pathologists from 4 main hospitals. Thirty administrative employees were also selected as control group. The direct DBA colorimetric method was used to measure MHA in the pathologists’ urine. Significant differences between MHA of workers’ urine and working days, type of jobs, and length of exposure time (p < 0.05) were seen. This study also showed that, there were no significant difference between urinary MHA concentration and sex, age, and smoking habit (p > 0.05). It was found that, xylene exposure cannot affect to the total and direct serum bilirubine in the workers’ blood. Although this study showed no acute exposure to xylene in hospitals pathology wards, chronic exposure to such compounds cannot be ignored due to their adverse effects, therefore, protecting pathologists against like these organic solvents are strongly recommended as their TLVs have been considerably reduced during recent years. P15-08 DETERMINATION OF METHYLENE DIPHENYL DIISOCYANATE FOR EVALUATION OF OCCUPATIONAL EXPOSURE IN AN AUTOMOBIL MANUFACTURING COMPANY, IRAN S.J. Shahtaheri, H. Kakooei, H. Karbasi School of Public Health, Tehran University of Medical Sciences, Tehran, Iran


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A cross-sectional evaluation of exposures to Methylene diphenyl diisocyanate (MDI) among 39 employees, working in the window fixation and window glue in an automobile manufacture was performed. Exposure assessment was conducted for both case and control groups. Personal inhalation exposure, was evaluated by means of UV–vis spectrophotometry, at 593 nm as well as spirometric assessment of lung function was performed. The average concentration of MDI in the window fixation, and window glue were 34.53 mg/m3 and 27.37 mg/m3 , respectively, which was lower than the TLV recommended by ACGIH (51 mg/m3 ). Respiratory symptoms, such as sputum, cough, wheezing in the exposed group were significantly different compare to the unexposed group (p < 0.05). FVC, FEV1 , FEV1 /FVC, and PEF in case group were lower than the control group (p < 0.05). Exposed workers who were in higher and lower values than the average concentration of MDI (31.22 mg/m3 ) had a significant difference with the symptoms of difficulty in respiratory system, eyes, mental, FVC, and FEV. It can be concluded that, after sampling and a simple sample preparation processes, MDI can be easily measured making possible to evaluate the risk of adverse effects of occupational exposure to such common and hazardous compound in relevant workplaces. P15-09 PRELIMINARY RESULTS OF AN INVESTIGATION ON SMOKING HISTORY AND NICOTINE ADDICTION IN A SAMPLE OF ITALIAN SMOKERS Bassi1 ,

Picciolo1 ,

Andreoli1 ,

Calapai2 ,

A. M. C. G. A. Nunziata1 , S. Saltini1 , A. D’Alessio1 , V. Arcoraci2 1 Research

Centre, BAT-Italia S.p.A., Naples, Italy; and Exp. Dep. of Medicine and Pharmacology, University of Messina, Italy 2 Clinical

Nicotine is one of the major constituents of tobacco smoke and is considered responsible for the establishment of smoking dependence. In recent years, diagnostic tools, such as ad hoc questionnaires, to evaluate psycho-behavioural components of the addiction have been formulated. An explorative study using an ad hoc questionnaireinterview built up by the integration of three addiction evaluation tests, FTND (Fagerstr¨om test for Nicotine

Dependence), SDS (Severity Dependence Scale) and VAS (Visual Analogical Scales), has been carried out, in a limited sample of smokers and ex-smokers, to the aim to evaluate the relationships among life amount of tobacco smoked, daily nicotine intake and nicotine addiction. Preliminary results show that years of smoking and daily nicotine intake might be directly correlated to nicotine addiction. In fact, most of people smoking from less than 20 years show low FTND score, while high nicotine intake and long time smokers exhibit higher FTND score. The nicotine intake of smoked cigarettes appears to be not associated to the nicotine dependence degree. Total daily nicotine intake seems to be related with nicotine addiction evaluated with SDS test. Because of this study has been conducted on a limited sample, no definitive conclusion can be reached for the smokers and any association can be explored for ex-smokers. However, results suggest that nicotine addiction in smokers appears to be associated both to the daily nicotine intake and smoking years. On this basis, a transversal study on representative sample of the smoker population of the city of Messina will be planned. P15-10 EXPOSURE AND RISK ASSESSMENT OF APPLICATOR TO FUNGICIDE THIOPHANATE-METHYL DURING TREATMENT ON PEPPER H. Choi1 , K.H. Liu2 , J.K. Moon1 , H.W. Park1 , Y.B Ihm3 , J.H. Kim1 1 School of Agricultural Biotechnology, Seoul National

University, Shillim-dong, Gwanak-gu, Seoul 151-742, Korea; 2 Department of Pharmacology, Inje University, College of Medicine, Busan 614-735, Korea; 3 Department of Crop Life Safety, NIAST, RDA, Suwon 441-707, Korea Exposure of operator was measured when thiophanatemethyl was applied in pepper field, using patches for dermal exposure and a personal air sampler (XAD-2 resin) for inhalation exposure. In mixing procedure, the amount of dermal and inhalation exposure was 24.7 mg and 2107.9 ng, respectively. The most of exposure was observed on hand (75.8% of total exposure).

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During the application of thiophanate-methyl using power sprayer, 53.9 mg for assistant (0.015% of total application amount), 102.6 mg for applicator (0.029% of total application amount) were observed as dermal exposure. The exposure rates were 50.1 and 94.5 mg/h, respectively. The most of exposure was observed on hand of assistant (91.7% of total exposure) and on thigh of applicator (45.0% of total exposure). The other exposed parts of body were arms and shin. The data for potential dermal exposure were highly variable. It may be due to operational condition, such as the growth stage of the crop, operator technique, and other unpredictable parameters. The inhalation exposures was observed only on applicator (472.5 ng). For risk assessment, the potential dermal exposure (PDE), the absorbable quantity of exposure (AQE) and the margin of safety (MOS) were calculated. MOS was >1, indicating of least possibility of risk. P15-11 DI-SPME/GC–MS A TOOL ANALYSIS OF CHLOROPHENOXYACETIC ACID RESIDUES WITHIN CEREAL TISSUES R. Krzyzanowski, B. Leszczynski Department of Biochemistry, University of Podlasie, 12 Prusa Str., Siedlce 08-110, Poland Accumulation of the MCPA (4-chloro-2methylphenoxy)acetic acid in leaves and roots of winter wheat Roma cultivar was investigated. Dichloromethane extracts of the wheat seedlings was performed. After evaporation the residue was redissolved in methanol, and then the solid phase microextraction was performed. Identification and quantification of the MCPA residues within the plant tissues was done using a gas chromatography–mass spectrometry (GC–MS) system with a selective ion monitoring (SIM). The highest concentration of MCPA was found within the leaf tissues on hour after treatment and was decreased during the next 72 h (0.067–0.026 ␮g/l). Much lower concentration of MCPA was found within the roots of the wheat 2 h after treatment and declined during the next 12 h. Thus, we suggest that the SPME technique is very useful analysis traces of the chlorophenoxy acid residues within the aerial and underground plant organs.


P15-12 RESIDUAL ANALYSIS OF CHLORPYRIFOS, ENDOSULFAN AND DELTAMETHRIN PESTICIDES IN PLAINS AND HILLY AREA OF KUMAON REGION OF UTTARANCHAL STATE OF INDIA AND THEIR IMMUNOTOXICOLOGICAL EVALUATION IN POULTRY S.P. Singh, L.D. Sharma, U. Kant Mishra, S.K. Pandey, G. Mehta, R. Varma, A.K. Singh Department of Pharmacology and Toxicology, College of Veterinary Science and A.H., NDUAT, Kumarganj, U.P. India A survey was undertaken to determine the residues of chlorpyrifos, endosulfan and deltamethrin pesticides in specimens of milk, poultry egg and meat, green fodder, wheat grains and water collected from different centres spread throughout the plains and hilly area of Kumaon region of Uttaranchal State of India. Procedures for extraction, clean up, detection and quantitation of residues of pesticides in above specimens were standardized. Acetone and dichloromethane for extraction and alumina and silica gel columns were used. The detection and quantitation of pesticide residues were done by HPLC. The maximum concentration of pesticides was detected as 0.9948 ␮g/g chlorpyrifos in egg, 0.9525 ␮g/g endosulfan in egg and 0.9260 ␮g/g deltamethrin in wheat grains. The results of this study revealed that the residual concentration in different above mentioned samples were found in different order for above three pesticides. However, levels of pesticide residues detected in various specimens were less than MRL as per guidelines WHO/FAO (1990). For evaluation of immunotoxic potential of these pesticides a subacute oral toxicity studies was carried out following prolonged administration of endosulfan (25 and 50 ppm), chlorpyrifos (10 and 25 ppm) and deltamethrin (10 and 25 ppm) in diet for 16 weeks in poultry. The humoral and cell mediated immune response showed a dose and time-dependent suppression in the birds in endosulfan treated groups. In chlorpyrifos fed groups, immunosupression in cellular and humoral immune response in lower dose group and immunopotentiation in higher dose group was observed. Both dietary levels of endosulphan and deltamethrin suppressed cellular immune response,


Abstracts / Toxicology Letters 158S (2005) S1–S258

however, suppression in humoral immune response was also observed in endosulphan treated birds. P15-13 EVALUATION OF PYROLISIS METHOD SAFETY UNDER ORGANOCHLORINE PESTICIDES REMOVAL L.I. Povyakel, S.V. Snoz, V. Ye Krivenchuk Medved’s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine Environmental pollution in Ukraine caused of warehousing of organochlorine-containg waste, which including useless and banned pesticides, is creating the critical ecological situation which demand of urgent solution. Thermal method of waste destruction is a perspective way of waste amount decreasing. By this way total mass of waste decreased on 80%, but it is possible the pollution of atmospheric air by toxic compounds including polychlorinated dibenzo-p-dioxins and dibenzofurans. We have analyzed fly ash samples collected during the pyrolisis of hexachlorocyclohexane and slag samples collected after the pyrolisis finishing. We have used the DF1 starter kit (“Cape Technologies”, USA) for polychlorinated dibenzo-p-dioxins and dibenzofurans enzyme immunoassay in fly ash samples and GC–MS for hexachlorocyclohexane determination in slag. According to obtained results the emission of polychlorinated dibenzo-p-dioxins and dibenzofurans in air during pyrolisis of hexachlorocyclohexane without purification of waste gases (t = 600 ◦ C) was 0.8 ng TEQ/m3 and with purification (t = 850 ◦ C) was 0.4 ng TEQ/m3 . Thus, the using of the purification system decreases the polychlorinated dibenzo-pdioxins and dibenzofurans emission in air to 50%. Hexachlorocyclohexane level in slag after 12-h pyrolisis was 250 mg/kg, but after 24-h pyrolisis this pesticide residues was not detected. Thus, the technology of multicurcuit pyrolisis is perspective for organochlorine pesticides removal. Because our investigations revealed toxic substances residues dependence from temperature and temporal conditions of pyrolisis process, for the using slag, which formed in the pyrolisis of organochlorine pesticides, it is necessary to control of pesticides residues in slag.

P15-14 KINETICS OF MANCOZEB METABOLISM AMONG OCCUPATIONALLY EXPOSED POTATO FARMERS AFTER MANCOZED APPLICATIONS IN FINLAND S. Pennanen1 , J. Liesivuori1,2 , S. Makkonen1 , O. Lankia1 , V. Turunen1 1 Finnish Institute of Occupational Health, Kuopio, Fin-

land; 2 University of Kuopio, Kuopio, Finland Ethylenethiourea (ETU) is a metabolite of ethylenebisthiocarbamate (EBDC) fungicides. ETU can be assayed in human urine samples collected after occupational exposure to EBCDs. Urinary excretion of ETU was monitored from samples given by 36 potato farmers after mancozeb applications. The urine sample was to be given 6 h after the pesticide application and the second one in the next morning. Immediately after the application period, increased ETU concentration was found in 24 exposed workers. Urinary ETU concentrations ranged from 8.7 to 429 ␮mol mol−1 creatinine. The urinary ETU level was still elevated in 12 of the subjects in the next morning. The presence of ETU in the morning urine was generally observed if the initial ETU concentration was over 80 ␮mol mol−1 creatinine. The elimination rate was calculated from the slope resulting from a linear regression analysis of the natural logarithm of two urinary ETU concentrations versus time points. A mean half-life of ETU in urine (n = 19) was about 3.6 h (±1.7 h) ranging from 1.5 to 6.9 h. This indicates that urinary ETU excretion might be dose-dependent and much slower than described in previous studies. P15-15 DRE-CALUX bioassay for the dioxin level in environmental matrices and human blood K.E. Joung, K.N. Min, J.Y. An, Y.W. Kim, K.Y. Kim, H.J. Chung, Y.Y. Sheen College of Pharmacy, Ewha Womans University, Seoul, Korea Two different methods, HRGC/MS chemical analysis and CALUX bioassay were used for testing environmental water and soil samples if CALUX bioassay is applicable for the measurement of dioxin-like compounds. Furthermore, REPs for 17 kinds of dioxin-

Abstracts / Toxicology Letters 158S (2005) S1–S258

like compounds were determined and compared with WHO-TEFs of corresponding compounds. In the water samples, HRGC/MS-TEQs ranged from 0 to 2.281 pgTEQ/L, and the highest TEQ value was 2.281 pgTEQ/L. All samples except one sample showed totalTEQ values below the 1 pg-TEQ/L. HRGC/MS-TEQ values of soil samples was in a range of 0–65.6 pgTEQ/g (dry) and the highest TEQ value was found in Maseo, where the refinery used to be near. CALUXTEQs of water samples ranged from 0.7 to 18 pgTEQ/L and CALUX-TEQs of soil samples ranged from 0.6 to 650 pg-TEQ/g (dry). The correlation between CALUX-TEQs and HRGC/MS-TEQs was very high (r2 = 0.982). However, the absolute values obtained by CALUX bioassay were always higher than that by HRGC/MS. To interpret this data, the REP values for 17 kinds of dioxin-like compounds were determined by CALUX bioassay. And REP values were compared with WHO-TEF values. The correlation between estimated REP-TEQs and HRGC/MS-TEQs turned out to be very high (r2 = 0.998) and the absolute values obtained by the analysis of HRGC/MS and estimated based on REP EC50 were appeared to be very similar. And also, there were good correlation (r2 = 0.970) between estimated REP-TEQs and CALUX-TEQs. However, the absolute TEQ values obtained by CALUX bioassay were always about ten times higher than the estimated REP-TEQ values. Thus, the CALUX bioassay can be highly recommended as an “early warning system” for routine measurement of dioxin-like compounds in environmental matrices. This research was supported by KNTP Grant from KFDA. P15-16 COMPARITION OF INHALATION OF A MIXTURE OF OXYGEN AND NITROS OXIDE WITH TRANSCUTANEOUS ELECTRICAL NERVE STIMULATION ON THE SEVERITY OF PAIN IN THE ACTIVE PHASE OF LABOR AMONG THOSE REFERING TO TEHRANS MATERNITY HOSPITAL IN 2001 F. Pazandeh, F. Firouzeh Chian, M. Sharafi, H. Alavi Shahid Behehshti University of Medical Sciences, Tehran, Iran


Pain relieving is one of the main parts of obstetrics. In maternity hospitals of Iran, pain relieving procedures during labor are not applied for different reasons. Hence, it seems necessary to evaluate the cheap, safe methods in this regard, which need no specialist. Sixty pregnant women, volunteered to take part, were randomly placed in two groups of TENS and Entonox. Mothers of both groups applied each method in the form of self administering and showed their pain severity in certain intervals (4–5 cm, 6–7 cm, 8–9 cm, 10 cm dilatations) to the researcher using visual analog scale. There was no significant difference between the two groups considering the age, education, mother’s job, spouse’s job and education, gestational age, weight to height ratio (%), number of abortions, dysmenorrheal background, decided pregnancy, weight, and apgar of the first and fifth minutes of the birth. The mean of the pain severity in the whole active phase of labor was 6.9083 (with the standard deviation of 1.03) in the TENS group and 5.18 (with the standard deviation of 1.15) in Entonox. The mean of the pain severity between the two groups was different significantly (p = 0.0001). It seems that application of Entonox relieves the pain of the active phase of labor more than TENS. P15-17 HPLC-CHIP/MS: A NOVEL MICROFLUIDIC DEVICE FOR NANOELECTROSPRAY LC-MS DETECTION OF CARBOXY-THC IN HAIR A. Fandino, M. Vollmer, G.L. Gauthier, W. Dorlijn Agilent Technologies R&D and Marketing GmbH, Hewlett-Packard Str-8, 76337 Waldbronn, Germany This presentation will review a new and highly sensitive technique in liquid chromatography/mass spectrometry (LCMS) for the detection of c-THC in hair using nanoscale chromatography, electrospray ionization and ion trap MS/MS analysis. The currently proposed SAMHSA (Substance Abuse and Mental Health Services Administration) limit of detection for screening c-THC is 0.05 pg/mg in specimens of sweat, saliva, urine, and hair, which is achievable in gas chromatography/mass spectrometry (GCMS) by derivatization (PFPA, pentafluoropropionic anhydride) with analysis run-times typically more than 10 min. To decrease sam-


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ple preparation time and increase throughput, analysis by LCMS is attractive. This work will show the comparison of present analytical methods using GCMS with ion trap mass spectrometry and nanoelectrospray LCMS using a novel microfluidic device known as HPLCChip/MS. HPLC-Chip is a new microfluidic polymer device for providing both the sample chromatography and electro-spray ionization at only few hundred nanoliters per minute flow-rates. The polymer microfluidic device (HPLC-Chip) integrates sample preparation, enrichment column, analytical column, hydraulic connections and electrospray emitter directly onto the polymer chip. A real sample from a known THC user is analyzed and quantified for the presence of c-THC with the deuterated D9 analog used as an internal standard. Fabrication, hydraulic connections and overall chromatographic performance of the HPLC-Chip will also be reviewed. P15-18 UNIFORM PROCEDURE OF 1 H NMR ANALYSIS OF RAT URINE AND TOXICOMETABONOMICS W. Schoonen1 , M. Smit1 , P. Zandberg1 , S. Horbach2 , J.P. Ploemen2 , C. Kloks3 , J.R. Mellema3 , C. Thijssen van Zuylen3 , J. Vogels4 , A. Tas4 , Joop van Nesselrooij4 1 Departments of Pharmacology, N.V. Organon, Molen-

straat 110, 5340 BH Oss, The Netherlands; 2 Toxicology and Drug Disposition, N.V. Organon, Molenstraat 110, 5340 BH Oss, The Netherlands; 3 Medical Chemistry, N.V. Organon, Molenstraat 110, 5340 BH Oss, The Netherlands; 4 Department of Food and Food supplements, TNO Food and Nutrition Research, Utrechtseweg 48, PO Box 360, 3700 AJ Zeist, The Netherlands An uniform analysis of urine using NMR pattern recognition has been performed on rats treated with 13 liver toxic and 1 non-toxic compound. Data were correlated with histopathology and clinical chemistry parameters (aspartate aminotransferase, alanine aminotransferase, bilirubine, triglycerides). For these studies animals were treated daily for at least 1 up to 4 days. On day 1 and 2 after the start of the treatment urine was collected and animals were sacrificed after 1, 2 and/or 4 days. At the high doses used, marked toxicological changes in liver were observed with carbon tetrachloride, bromobenzene, paracetamol, ANIT, and ibupro-

fen as judged from histopathology and classical clinical chemistry. Less extensive changes were observed for thioacetamide and chlorpromazine treatment. Changes with iproniazid, isoniazid, phenobarbital, ethinylestradiol and tetracycline were of minor toxicological relevance. For methyltestosterone and mianserine no relevant changes were observed. Urine NMR spectroscopy revealed significant differences in the spectra of 70 selected NMR peaks between several of the treated animals in comparison with the untreated animals. Evaluation of the pattern recognition with principal component diffraction analysis (PCDA) led to the recognition of several changes in NMR spectra as function of liver toxicity. Ibuprofen and bromobenzene treatment clustered markedly differently to all other treatments. Isoniazid, iproniazid, paracetamol and thioacetamide treated animals clustered more or less similar, while chlorpromazine, ANIT and tetracycline treated animals clustered in the proximity of the previous cluster. Mianserine treatment was hardly distinguishable from the control group. Phenobarbital, ethinylestradiol and methyltestosterone treatment, on the other hand, could be discriminated from the controls. Next, the correlation of our rat NMR PCDA data with the most characterisitic histopathological changes reported in humans and/or rats, i.e., necrosis (carbon tetrachloride, bromobenzene, paracetamol, thioacetamide, iproniazid and isoniazid), cholestasis (ANIT, chlorpromazine, ethinylestradiol, methyltestosterone and ibuprofen) and steatosis (phenobarbital and tetracycline), suggests that our rat NMR liver model may help to predict liver changes in humans and/or rats. P15-19 INTEGRATION OF BIOMONITORING EXPOSURE DATA INTO THE RISK ASSESSMENT PROCESS M.P. Holsapple, G.N. Doerrer ILSI Health and Environmental Sciences Institute, Washington, DC, USA Improved analytical methods permit the measurement of low levels of chemicals in human tissues. Despite evidence that chemicals are absorbed; it is not clear whether their presence in human tissue leads to adverse health outcomes. Furthermore, without exposure path-

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way information, it is difficult to relate biomonitoring results back to sources of exposure and to develop effective risk management strategies. On a global basis, the demand for biomonitoring data has undergone a dramatic increase, as evidenced by programs, such as the European Union’s SCALE initiative and the US government-sponsored NHANES (the CDC National Health and Nutrition Examination Survey). In September 2004, the ILSI Health and Environmental Sciences Institute (HESI) organized and co-sponsored an International Biomonitoring Workshop. Scientists from US government agencies, international groups, academia, and industry explored the processes and information needed to put biomonitoring data into perspective for the risk assessment process, with special emphasis on integrating biomarker measurements of exposure, internal dose, and potential health outcome. Criteria for applying biomonitoring data were also considered. A series of papers will be published in 2005, which will include the conclusions and recommendations from the workshop, and describe case studies which illustrate general and basis principles for the use and interpretation of biomonitoring data. This poster presentation will include information about the 2004 HESI workshop, the development of criteria for the application and interpretation of biomonitoring exposure data into the risk assessment process, and existing and future collaborative partnerships for advancing the science of biomonitoring data collection and interpretation. P15-20 DETERMINATION OF LEAD AND CADMIUM LEVELS IN IRANIAN CHAMOMILE (MATRICARIA RECUTITA L.) HERB MEDICINE M. Motlagh1 , Z. Nazari2 , A. Arzi2 1 Department of Pediatric, Ahvaz Jundishapour University of Medical Sciences, Iran; 2 Department of Toxicology and pharmacology, Pharmacy School, Ahvaz Jundishapour University of Medical Sciences, Iran

Chamomile is used for sensitive skin of infants and young children, gastrointestinal spasms and mild sleep disorders in Iran pediatric medicine. Little studies on Pb and Cd in this herb have been conducted in Iran. For this purpose, 30 samples of Iranian Chamomile were collected. An adequate amount of each milled sample was dried at 110–120 ◦ C an oven. Then vessel


was placed in a cold furnace and the temperature was set at 500 ◦ C and kept at this temperature over until white carbon free ash was obtained. Residue was dissolved in HNO3 -H2 O (1 + 9), quantitatively transferred to 50 ml volumetric flask and diluted with water. Deionized water was used for blank. Citric acid 10% and 3 drops of bromo-cresol-green 1% in ethanol were added to aliquot volumes of sample, blank and standard solution, adjusted to ca pH 5.4 by adding NH4 OH and citric acid. 5 ml APDC 2% (Ammonium pyrrolidinedithiocarbamate) and n-butyl acetate were added to them and shake vigorously 2 min. Then organic phases were separated and the amount of Cd2+ and Pb2+ in samples was measured by Flame Atomic absorption Spectrometer. The mean value of lead in investigated Iranian Chamomile was 0.49 mg/kg and the mean value of Cd in samples was 0.019 mg/kg. P15-21 BIOMONITORING OF MYOSMINE IN HUMAN MILK AND SALIVA M. Maier1 , A. Schulze2 , E. Richter1 1 Walther

Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University Munich, Germany; 2 Gynecologic Clinic, Ludwig-MaximiliansUniversity Munich, Germany The minor tobacco alkaloid myosmine is not tobaccospecific because of its occurrence in a wide variety of foodstuffs including milk and cream. Under nitrosative or peroxidative conditions myosmine gives rise to reactive intermediates binding to DNA and haemoglobin, which release 4-hydroxypyridylbutanone (HPB) after hydrolysis. These adducts are made responsible for the carcinogenicity of tobacco-specific nitrosamines (TSNA) but lack the expected specificity for smoking in human biomonitoring studies. The present study was undertaken to prove the correlation between smoking behaviour and myosmine concentrations human milk and saliva. For analysis of myosmine and cotinine by GC–MS an analytical method was developed with multiple liquid–liquid and solid-phase extraction steps and deuterated internal standards. Thirteen out of 23 milk samples had detectable levels of myosmine, 0.17 ± 0.12 ng/mL in non-smokers (n = 17), 0.30 ± 0.19 ng/mL in passive smokers (n = 5) and 1.34 ng/mL in a smoker. Cotinine was not detectable


Abstracts / Toxicology Letters 158S (2005) S1–S258

in non-smokers and reached 1.37 ± 2.09 ng/mL in passive smokers and 97.1 ng/mL in the smoker. In saliva, 16 of 22 samples had detectable myosmine concentrations, 1.58 ± 1.67 ng/mL in 14 nonsmokers, 1.89 ± 0.35 ng/mL in 2 passive smokers and 1.87 ± 1.01 ng/mL in 6 smokers. Cotinine concentrations were 0.46 ± 0.37, 2.15 ± 1.89 and 71.6 ± 41.8 ng/mL in non-smokers, passive smokers and smokers, respectively. These results indicate that myosmine exposure in humans is largely independent from smoking and may contribute to HPB-releasing adducts unrelated to TSNA uptake.

or preconcentration steps needed. For MLU, the parent and product ions were m/z 204 → 173, and the limit of detection was 5 nmol/g globin. MLU was found in most globins from the exposed subjects but not in the controls. A close correlation between the MLU and MVH levels (nmol/g) was observed: MLU = 7 + 0.48 MVH (R2 = 0.84, n = 32). In conclusion, MLU can be easily measured in globin of workers exposed to DMF. The findings also indicate a long-term persistence of MLU in human body, and, consequently, its potential as a biomarker of chronic exposure to DMF.


The study was supported by the Grant Agency of the Czech Republic (grant no. 310/00/0923).

J. Mr´az1 , J. Cimlov´a2 , V. Str´ansk´y1 , R. Kiˇcov´a1 , H. 2 ˇ Nohov´a1 , P. Simek


1 National

Institute of Public Health, Prague, Czech Republic; 2 Institute of Entomology, Academy of Sciˇ e Budˇejovice, Czech Republic ences, Cesk´

Department of Occupational Health, Faculty of Health, Hamadan University of Medical Science, Hamadan, Iran

Metabolism of the hepatotoxic solvents N,Ndimethylformamide (DMF) and N-methylformamide (MF) results in the formation of N-methylcarbamoyl adducts at the N-terminal valine and lysine in blood protein globin, of which the lysine adduct has so far only been reported in rats given high doses of both solvents (Mr´az J. et al., 2004: Chem.-Biol. Interact. 148, 1–10). Here we examined whether the lysine adduct is produced, and accessible to analysis, in humans occupationally exposed to DMF. Globin from exposed workers (n = 35) and unexposed controls (n = 5) was analyzed by two methods. Edman degradation was used as a sensitive reference method to measure the valine adduct, by converting it to 3-methyl-5-isopropylhydantoin (MVH). The MVH levels in globin of the exposed subjects were in the range of 1–441 nmol/g, in controls <1 nmol/g. The principal method of globin analysis consisted of enzymatic hydrolysis with pronase to release free N␧ -(N-methylcarbamoyl)lysine (MLU) and N-methylcarbamoylvaline (MVU), which were determined by HPLC/MS/MS, with no clean-up

Toluene is an important toxic volatile used in many industrial processes: The critical health effect of toluene is suppression of the function of the central nervous system. In order to compare the levels of hippuric acid and o-cresol from urine of taxi drivers, workers of gasoline pump stations and car painters, 127 urine samples were obtained. The results were compared with 60 non-exposed men living in rural area as control group. The urinary o-creasol and toluene in air were analysed with a gas chromatography equipped with Flame Ionization Detector. Hippuric acid was extracted from urine and analyzed with high performance liquid chromatography. The urinary o-cresol and hippuric acid were increased in urine of subjects when the concentration of toluene higher than 4 and 35 ppm, respectively. The significance differences in the levels of urinary o-cresol were found in painters and workers of gasoline stations compared to control group (p < 0.005). The significance difference in the levels of urinary hippuric acid was found between painters and control group (p < 0.005). The correlation coefficient between toluene in air and o-cresol for painters, gasoline station

Abstracts / Toxicology Letters 158S (2005) S1–S258

workers and drivers were 0.75, 0.57 and 0.08, respectively. There was not significant correlation coefficient between toluene in air and biomarkers for taxi drivers. In conclusion our results have showed that o-cresol and hippuric acid could separate the exposed to toluene from the non-exposed when toluene in ambient air was greater than 4 and 35 ppm, respectively. Hippuric acid and o-cresol are not suitable biomarkers for persons exposed to low concentration of toluene such as drivers. P15-24 EXCRETION OF UNCHANGED XYLENE AND TOLUENE IN URINE AFTER EXPERIMENTAL EXPOSURE OF HUMAN VOLUNTEERS B. Janasik, M. Jakubowski Institute of Occupational Medicine, Lodz, Poland The results reported recently in the literature show that the measurement of urinary excretion of unchanged solvents in urine provides highly sensitive and specific index of occupational exposure to volatile organic solvents (VOC’s). However, there is insufficient information at present on the kinetics of excretion of VOC’s in urine. The aim of this study was to evaluate the kinetics of urinary excretion of unchanged xylene and ethylbenzene after termination of inhalation exposure of human volunteers. The subjects of this study were male volunteers. They were exposed to xylene an ethylbenzene in concentration of 200 mg/m3 for 4 h at rest. Urine samples were collected before the onset of exposure, every 2 h during exposure and within 4 h after its termination. About 0.0005% of the absorbed dose of xylene and 0.0012% of the dose of ethylbenzene was excreted unchanged in urine during and after termination of exposure. The obtained result suggests that excretion after termination of exposure followed the first-order single compartment model with half-time values of about 1.47 h for xylene and 1.18 h for ethylbenzene. These data suggest that excretion of unchanged toluene and ethylbenzene in urine integrates the two rapid phases of elimination of these compounds from blood. It is important for the strategy of sample collection.


P15-25 LONG TERM COMPLICATION IN NEROUS SYSTEM OF MUSTARD VICTIMS Haghighi Alireza-Afshari, Poorandokht-Najin Rabiei Islamic Azad University Dezful branch The rate of disintegration of mustard gas is different in different parts of the organism. Dersch measured concentrations of mustard gas in different organs of Iranian soldiers who died following intoxication. Determinations were performed using GC-MS method of analysis. He highest concentrations were in brain (10.7 mg/l), liver (24 mg/kg), cerebrospinal fluid (109 mg/l) and in kidney (5.6 mg/l). It was a descriptive study at first we prepare a questionnaire at the next step we selected 200 chemically injured soldiers randomized and had an interview with them the time of contact with gas was 14 years ago. The average age of soldiers at the time of study were 40 and at the time of expose were 26. At the time of expose 30.7% shows cerebral symptoms and 69.3% had not any problem in his nervous system. After 14 years 50.3% of them had not any problem in nervous system and 49.7% shows some problem in their nervous system. This difference between 14 years is significant (p < 0.05). P15-26 MOLECULAR DOSIMETRY OF ALKYLATING AGENTS USING THEIR ADDUCTS AT THE NTERMINAL VALINE IN GLOBIN: AN IN VITRO STUDY J. Mr´az, R. Kiˇcov´a, V. Str´ansk´y, K. Ctibor, H. Nohov´a National Institute of Public Health, Prague, Czech Republic Several industrially important compounds or their reactive metabolites act in the organism as alkylating agents, which entail a serious health risk. Biological monitoring of internal exposure to alkylating agents is possible through determination of the respective covalent adducts at the Nterminal valine in globin. The analytical procedure (modified Edman degradation) converts the adducts to 1-alkyl-5-isopropyl-3-pentafluorophenyl-2thiohydantoins (PFPTH), which are determined by GC/MS. In this study we focused on the following agents (the resulting alkyl group is given in parenthe-


Abstracts / Toxicology Letters 158S (2005) S1–S258

ses): dimethyl sulphate (methyl-), ethylene oxide (2hydroxyethyl-), propylene oxide (2-hydroxypropyl-), acrylonitrile (2-cyanoethyl-), glycidonitrile (2-cyano2-hydroxyethyl-), acrylamide (2-carbamoylethyl-), glycidamide (2-carbamoyl-2-hydroxyethyl-), (R)- and (S)-styrene oxide (2-hydroxy-2-phenylethyl-), butadiene monoxide (2-hydroxy-3-butenyl-) and butadiene dioxide (2,3,4-trihydroxybutyl-). Several existing methods were combined to enable simultaneous determination of all the adducts studied. PFPTH compounds with hydroxy group(s) were converted to acetyl derivatives. Sensitivity and specificity of measurements employing the EI and NCI ionization techniques in both the MS and MS/MS modes were compared. Batches of globin modified with the alkyl groups were prepared by incubation of blood with the corresponding agents in vitro. Half-times of the agents in blood and the resulting adduct levels in globin were determined. We wish to prepare standards of globin with requested levels of selected alkyls for reference purposes, since such materials are not available. The study was supported by the Internal Grant Agency of the Czech Ministry of Health (grant no. NJ/7387-3). P15-27 EXPOSURE ASSESSMENT TO METHYLMERCURY THROUGH THE INGESTION OF FISH: CROSS-SECTIONAL EVALUATION AND RISK INDEXES IN YOUTHS AND ADULTS C.M.L. Carvalho, A.I.N.M. Matos, M.L. Mateus, A.P.M. Santos, M.C.C. Bator´eu CECF, Faculty of Pharmacy, University of Lisbon, Portugal Human exposure to methylmercury (MeHg) presents a high health risk due to its neurotoxicity, teratogenicity and immunotoxicity. The aim of this work was to evaluate the exposure of a potentially risky population living in Sesimbra, where fish is an important component of daily diet. In a preliminary questionnaire distributed to people professionally related to fishery, 50% of the inquired ingested an average of 6 meals of fish per week. Infants and children due to their higher food consumption rates per body weight are generally expected to have an increased relative risk due to the higher exposure level; therefore, they constitute a susceptible subset of the population. To attain youth exposure

an inquiry was distributed to 298 students of a middle + secondary school established in Sesimbra. Students’ age ranged from 10 to 20 years with 42% being 10–12-year-old. Inquiries were anonymous and were evenly distributed being 47% of the students’ female and 53% male. The average number of fish meals consumed by person was 4.15; 16% of the students stated the ingestion of 7 or more fish meals per week. Fish samples were collected in the dock of Sesimbra and total mercury was determined by FI-CV-AFS. Among the predatory fish specimens analysed two specimens, dogfish and carocho, contained 1.44 and 2.07 ppm of Hg, respectively, surpassing the legislated limit (1.0 ppm). The cross-sectional data were integrated with the fish analysis results to attain the population exposure assessment to MeHg. The indexes of risk calculated for adults and youths indicate the existence of risk in some situations. This study indicates that monitorization of mercury levels present in fish is mandatory and the diet habits of the risky populations should be investigated for risk prevention. Project POCTI/41741/ESP/2001 – Risk Assessment of Methylmercury Exposure: Integrated actions through the food chain. Eixo 2, medida 2.3 POCTI do QCA III (componentes Feder e OE). P15-28 URINARY ARSENIC SPECIATION AS A TOOL TO ASSESS THE EFFICACY OF TWO DRINKING WATER INTERVENTIONS IN BANGLADESH J. Ng1 , A.H. Milton2 , W. Smith2 , K. Dear3 , M. Sim4 , G. Ranmuthugala3 , K. Lokuge3 , A. Shraim1 , S.H. Huang1 , A. Rahman5 1 National Research Centre for Environmental Toxicology, The University of Queensland, Australia; 2 Centre for Clinical Epidemiology and Biostatistics, The University of Newcastle, Australia; 3 The National Centre for Epidemiology and Population Health, The Australian National University, Canberra, Australia; 4 Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia; 5 NGO Forum for Drinking Water Supply and Sanitation, Bangladesh

Abstracts / Toxicology Letters 158S (2005) S1–S258

Large scale arsenic contamination of tube-well groundwater in Bangladesh has attracted international attention. Through the Australian Agency for International Development special research fund, we investigated the efficacy of two commonly used water intervention options, namely dug-well water and a relatively simple and inexpensive three-pitcher filter system. In Natore district, we selected 218, 216 and 206 villagers and aligned them with 20 dug wells, 216 three-pitcher filters and control (no treatment), respectively. Arsenic speciation was measured using HPLC-ICP-MS. An initial survey of tube-wells gave an average arsenic concentration of 128 ␮g/L containing about 50% arsenite. During the trial, mean dug-well arsenic concentrations were 13.3 ␮g/L, 11.1 ␮g/L and 6.0 ␮g/L at 1, 6 and 12 months post intervention. 88 three-pitcher treated water samples were analysed for arsenic 10 months post intervention. 35% of the samples exceeded the national permissible limit of 50 ␮g/L. Urinary arsenic speciation revealed a significant decrease in both creatinine adjusted total arsenic and inorganic arsenic in the dugwell group after 1-month intervention. For the threepitcher group, no significant differences were observed after 1 month of intervention either in total arsenic or in arsenic methylation profile. The total arsenic and inorganic arsenic increased after 12 months compared to pre intervention in all groups. A survey of compliance with correct water usage was also conducted. The results on water and urinary arsenic could be partly due to poor compliance rate during the trial. This raises a question about the social acceptability of these intervention technologies and their ultimate health benefit. P15-29 DIFFERENCES IN THE URINARY EXCRETION OF PAH METABOLITES IN NONSMOKING COHORTS OF EASTERN AND WESTERN EUROPE A. Seidel, G. Dettbarn, A. John, J. Jacob Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer-Foundation, Lurup 4, D22927 Grosshansdorf, Germany Data on the excreted levels of PAH metabolites in urine of the general population in Europe are limited. In the framework of the DIEPHY project (funded by the EU) we have investigated cohorts of nonsmok-


ing women of the general population from Poland and Italy. Among the PAH metabolites naphthols and 1-hydroxypyrene have been determined that reflect the endogenous PAH body burden of each individual. Furthermore, the recently established biomarker phenanthrene-1,2,3,4-tetrol (Phen-T) has been determined that reflects the metabolic activation of carcinogenic PAH (Hecht et al., 2003). The nonsmoking status of each subject has been validated by determination of the urinary cotinine level. The medianes for the sum parameter 1–+2-naphthol and 1-hydroxypyrene of the Italian cohort were 4.76 ␮g/g Crea and 0.085 ␮g/g Crea, respectively. In contrast the corresponding medianes for the Polish cohort were 65.3 ␮g/g Crea and 0.24 ␮g/g Crea. The geometric mean for Phen-T was 1.59 ␮g/g Crea in the Polish and 0.44 ␮g/g Crea in the Italian cohort. The concentrations of PAH metabolites found in the Italian cohort compared closely to findings in Germany and United States. The higher concentrations of PAH metabolites in the Polish cohort clearly point out that the individuals are either exposed to a significant higher level of PAH from ambient air and/or contaminated food. P15-30 ENAMEL SURFACE BIOPSY TO BIOMONITORING LEAD EXPOSURE G.C.R. Almeida1,2 , J.A. Cury2 , F. Barbosa Jr.3 , M.C.P. Saraiva1 , R.F. Gerlach1 1 Dental

School of Ribeir˜ao Preto, FORP-USP, 2 FOP-Unicamp, Ribeir˜ao Preto-SP, Brazil; 3 Piracicaba-SP, Brazil; FMRP-USP, Ribeir˜ao Preto-SP, Brazil Lead (Pb) is an environmental pollutant commonly find in industrialized cities. Children living in these areas are the most affected and have systemic problems, whose origin is related to Pb exposure. Thus, there is a special interest on the evaluation of non-invasive techniques to biomonitoring Pb exposure. The present study had the following objectives: (i) Use of enamel surface deciduous teeth as a possible biomarker to diagnose lead exposure in children; (ii) Evaluation of the correlation between the amount of Pb in the enamel surface with biopsy depth. Then, we studied 250 children from pre-schools from Ribeir˜ao Preto city-Brazil (non-


Abstracts / Toxicology Letters 158S (2005) S1–S258

contaminated area), and 26 children living in Bauru city-Brazil (Pb contaminated area). Enamel Pb analysis was carried out by atomic absorption spectrometry with graphite furnace (AAS) in enamel biopsy samples. Phosphorus was also determined to calculated biopsy depth. Statistical analysis was aimed to find a significance level of 0.05. Deciduous teeth superficial enamel Pb concentration from children living in Bauru was significantly higher (median = 785.7 ␮g/g) than that found in the deciduous teeth superficial enamel of children living in Ribeir˜ao Preto (median = 204.3 ␮g/g) (p < 0.0001). Lead concentration was highly correlated to environmental exposure to lead. The more superficial the biopsy, the higher the Pb concentration found. Acknowledgment: Fapesp, CNPq. P15-31 EXPOSURE EVALUATION AT ORGANIC SOLVENTS OF WORKERS FROM ELECTRONIC FIELD Elena Teslariu1 , Veronica Mihalache1 , Oana Teslariu2

Oprea1 ,


1 Occupational Health Department, Univ. Med. Pharm.

“Gr.T. Popa” Iasi, Romania; 2 Med. Univ. Student, Iasi, Romania In this study we followed the exposure evaluation at organic solvents of a series of 20 workers from electronic industry. They use a solvent mix containing toluen, xylen, esthers and alcohols for the cleaning of some electronic plates. In order to assess the health effects, the following toxicological parameters were measured: phenols, hippuric acid, methylhippuric acid levels. Hippuric acid was determined by spectrophotometric method after the separation from urine by ionic exchange. Methylhippuric acids were determined after clean-up by C18-SPE and esterification with hexafluoroisopropanol and diisopropylcarbodiimide, by GCECD. Some lipid peroxidation degradation products (aldehydes, acetone) were also determined. The measurements of compounds of type oxime, obtained after derivatisation with pentafluorbenzyl hydroxylamine hydrochloride were made in urine samples by GCECD method. The study showed a significant difference of hippuric acid and methylhippuric acid values in the series of exposed workers compared with control group. The average values obtained for hippuric

acid were 2.63 ± 1.04 mg/l in exposed workers and 0.88 ± 0.37 mg/l in control group. The methylhippuric acid values were 11.8 ± 3.05 ␮mol/l in exposed workers and 4.3 ± 1.2 ␮mol/l in control group. Conjugated phenols values were 37 ± 7.15 mg/l in exposed workers compared with 16.7 ± 5.25 mg/l in control group. Values obtained for lipid peroxidation degration products were higher in the exposed workers group, also. P16 Toxicokinetics P16-01 THE ACTIVITY OF CYP1A1 IN DIFFERENT REGIONS OF RAT BRAIN B.C. Eke, A.C. C ¸ alıs¸kan, M. ˙Is¸can Ankara University, Faculty of Pharmacy, Department of Toxicology, Ankara, Turkey Cytochrome P-450 (CYP) and associated monooxygenases are important family of heme containing enzymes that are involved in metabolism of several endogenous and exogenous compounds. CYPs are expressed in hepatic and extrahepatic tissues including brain. However, the brain is the most complicated organ of body and its cellular composition and functions differ tremendously as compared to the liver. Recently, brain CYPs have been noted to be capable of activating/inactivating various xenobiotics. In addition, the expression of some CYPs appeared to be regionspecific indicating their distinct roles in the metabolism process. Therefore, we investigated 7-ethoxyresorufin-Odeethylase (EROD, CYP1A1) activity in different regions of rat brain. Brain CYP1A1 activity was also compared to that of liver. For comparison with rat brain regions, CYP1A1 activity of human brain in different regions was also determined. P16-02 LIGHT-DEPENDENT INFLUENCE OF NATURAL FUROCOUMARINS ON RAT CYTOCHROME P450 1A ENZYMES A. Baumgart, M. Schmidt, H.J. Schmitz, D. Schrenk Food Chemistry and Environmental Toxicology, University of Kaiserslautern, 67663 Kaiserslautern, Germany

Abstracts / Toxicology Letters 158S (2005) S1–S258

Furocoumarins are natural constituents in certain fruits and vegetables, e.g., in grapefruit, and are wellknown phototoxicants. In vitro studies have shown that individual furocoumarins are highly potent inhibitors of cytochrome P450 (CYP) enzymes, such as CYP 3A4 thus affecting drug metabolism. Little is known about the interaction of furocoumarins with CYP 1A enzymes, the expression of which is mediated via binding of the activated aryl hydrocarbon receptor (AhR) to xenobiotic responsive elements (XREs) in the promoter regions of the encoding genes. In this study, we investigated the influence of furocoumarins, together with or in the absence of daylight, on the expression and activity of CYP 1A enzymes in rat primary hepatocytes. All tested furocumarins were potent inhibitors of microsomal 7-ethyoxyresorufin O-deethylase (EROD) activity induced by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) their inhibitory potency being modulated by light. Furocoumarins except bergamottin increased XRE-driven reporter gene expression in transfected H4IIE rat hepatoma cells when light was excluded. Western blot and realtime PCR analysis showed an induction of endogenous CYP1A1 mRNA and CYP 1A proteins by furocoumarins, primarily in the dark. These findings demonstrate that natural furocoumarins can both inhibit CYP1A catalytic activity, and activate the AhR thus inducing CYP 1A gene expression in rat hepatocytes. All effects were strongly modulated by the presence or absence of light. P16-03 EXPRESSION AND VARIABILITY OF CYTOCHROME P450 (CYP) 1A ENZYMES IN 110 INDIVIDUAL HUMAN LUNG SAMPLES AND COMPARISON WITH ACTIVE SMOKING HABITS U. Bernauer1 , B. Heinrich-Hirsch1 , M. T¨onnies2 , M. Wolski1 , U. Gundert-Remy1 1 Federal Institute for Risk Assessment (BfR), Thielallee

88-92, D-14195 Berlin, Germany; 2 Zentralklinik Emil von Behring, Department Lungenklinik Heckeshorn, Zum Heckeshorn 33, D-14109 Berlin, Germany The lung represents an important target tissue for the toxic effects of chemicals. Individual susceptibility towards lung toxicants might be due to interindi-


vidual variability of local metabolism in the lung. Interindividual variability of xenobiotic-metabolizing CYP enzymes can be genetically determined or influenced by environmental factors. Besides CYP2E1, whose pulmonary varaibility has been determined for the first time at the protein level, CYP1A enzymes were determined which also play a role in the activation of environmentally relevant chemicals. In order to determine variability of CYP1A enzymes 110 different individual lung samples, we used a previously established methodology to separate CYP1A1 and CYP1A2 and compared the results with data on smoking habits. Human lung tissue was obtained after informed consent and smoking habits were recorded. Microsomes were prepared and total protein content was determined. A Western Blotting procedure using a commercially available antibody was applied to quantify CYP1A1 and CYP1A2 simultaneously and to differentiate between the two enzymes. CYP1A2 protein could be determined in all 110 individual lung samples, whereas CYP1A1 protein was detectable only in 41 of 110 samples. Comparison of these results with active smoking habits demonstrated that CYP1A1 was only expressed in lung samples obtained from smokers. This finding is remarkable, because the patients had all stopped smoking preoperatively for at least four days. There were also lung samples from smokers, in which CYP1A1 protein could not be determined. The influence of the pattern of smoking cessation on CYP1A1 expression in smokers is currently under investigation. P16-04 EXPRESSION OF CONSTITUTIVE AND INDUCIBLE CYTOCHROMES P450 IN FETAL AND NEWBORN RAT LIVER P. Czekaj, A. Wiaderkiewicz, A. Pałasz, B. Czajkowska, R. Wiaderkiewicz II Department of Histology and Embryology, Medical University of Silesia, Katowice, Poland The developing organism lacks many of cytochrome P450 isoforms detected in the adult or they are expressed at very low levels. It remains controversial whether P450 gene regulatory mechanisms are present prenatally. As a result, the catalytic function of P450s in fetal tissues has been questioned.


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The aim of the study was to evaluate CYP1A1, 1A2, 2B1/2, 2E1, 3A1 and 3A2 expression in fetal and newborn rat livers from untreated and P450-inducer-treated animals. The experiments were carried out on 16-, 18-, and 20-day-old fetuses, and newborns of SpraqueDawley rats. The following inducers were used: ␤naphthoflavone (50 mg/kg b.w./2 days), phenobarbital (80 mg/kg b.w./2 days), dexamethasone (30 mg/kg b.w./2 days) and ethanol (5% solution/3 weeks). CYP expressions were evaluated at both transcriptional (RTPCR) and protein (Western blotting) level. CYP1A2, 2B1/2, 2E1, 3A1 and 3A2 mRNA expression was detected at day 16 of gestation. CYP2B1/2 and 3A1 protein was found at day 18; CYP2E1 protein, at day 20; 1A2 and 3A2 protein, in newborns liver. CYP1A1 was detected at day 18, at both mRNA and protein level. Studied P450s demonstrated very low expression in animal tissues before and just after birth, but in most cases they were inducible. It is concluded that the inductory mechanisms of CYP1A1, 2B1/2, 3A1/2 and 2E1, but not CYP1A2 are functional in fetal liver at transcriptional or translational level. The effects of metabolic activation of CYP1A2 substrates may be reduced in fetuses. P16-05 EVALUATION OF HYDROXYIMINE AS CYTOCHROME P450-SPECIFIC PRODRUG STRUCTURE N. M¨ah¨onen1 , H. Kumpulainen2 , J. Rautio2 , M.L. Laitinen2 , H. Raunio1 , R.O. Juvonen1 , T. J¨arvinen2 1 Department

of Pharmacology and Toxicology, University of Kuopio, PO Box 1627, FI-70211, Finland; 2 Department of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FI-70211, Kuopio, Finland In order to evaluate the in vitro and in vivo usefulness of oxime structure as a prodrug structure for ketones, hydroxyimine derivatives of ketoprofen (1) and nabumetone (2) were synthetized and the release of parent drug from hydroxyimine was evaluated in vitro using isolated liver microsomes from humans, untreated rats, rats treated with CYP inducing agents and with 2 also using eight different recombinant human CYP isoforms. The formation of nitric oxide from the 2 during oxidation was also demonstrated in vitro. Finally, the bioconversion of 2 to nabumetone was determined in rats in vivo.

The oxidation of 2 occurred more than 1000-fold faster than that of 1. The main reason for the differences in the oxidation rates is likely the poor CYP substrate specificity of 1 and the effect of the neighboring aromatic rings of 1, which enhance the steric hindrance around the hydroxyimine structure. Activation of 2 to nabumetone and other metabolites occurred also in vivo in rats. Urine samples of rats treated with 2 included both nabumetone and its active metabolite, 6-MNA. The present study shows that hydroxyimine structure is a potential prodrug structure for ketones. P16-06 THE ACTIVITIES OF CYP1A1 AND CYP2A5 IN MOUSE BRAIN ¨ E. D.Ozdamar, M. ˙Is¸can, B.C. Eke Ankara University, Faculty of Pharmacy, Department of Toxicology, Ankara, Turkey Cytochrome P450 (CYP), a super family of heme containing enzymes, involves in the metabolism of several endogeneous and exogeneous compounds, such as drugs, carcinogens and environmental pollutants. Recently, several CYPs has been found to be expressed in extrahepatic tissues, such as brain in addition to liver. However, the brain has limited capacity for xenobiotic metabolism when compared to liver. We investigated coumarin 7-hydroxylase (Coh) and 7-ethoxyresorufin-O-deethylase (EROD), indicated as Cyp2a5 and Cyp1a1 respectively, activities in different regions of mouse brain. Also, Cyp1a1 and Cyp2a5 activities in mouse liver were compared with those in mouse brain. In addition to these CYP1A1 and CYP2A6 activities in human liver and different regions of human brain are compared. P16-07 IN VITRO METABOLISM OF TESTOSTERONE IN RATS AND TROUT AND ITS MODULATION BY DIFFERENT ENZYME INDUCTORS E. Fabian BASF Aktiengesellschaft, Experimental Toxicology and Ecology, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany Metabolically competent in vitro systems were prepared from Rainbow Trout and Wistar Rats. These

Abstracts / Toxicology Letters 158S (2005) S1–S258

systems were characterised for their metabolism of the model substrate Testosterone. The modulation of the metabolism by pre-treatment with the enzyme inductors Aroclor 1254, ␤-Naphtoflavone and Phenobarbital was of special interest within these studies. The conversion rates of Testosterone were generally lower in the fish systems than in the rat models. Metabolites of Testosterone in all applied trout systems were Androstane-3-17-dione, 5␣-Dihydrotestosterone, and Androstane-3␣-17␤-diol. The situation in liver systems of rats was different and the results were sex and system dependent: Testosterone was metabolically oxidised in liver microsomes and S9 fractions. Additionally to the oxidative metabolism, Androstane-3-17-dione, 5␣-Dihydrotestosterone, and Androstane-3␣-17␤-diol were formed in S9 fractions. The conversion rates in these systems were higher in males than in females. In liver slices, oxidative metabolism of Testosterone was dominant in males, whereas non-polar metabolites of Testosterone were characteristic for females. The pre-treatment of rats with the applied enzyme inductors changed the metabolisation of Testosterone in the in vitro-systems significantly. Although the induction of the total CYP 450 content and the ERODand PROD-activities was demonstrated for the corresponding trout systems, the oxidative metabolism of Testosterone remained unchanged. In conclusion, the results of these experiments indicate differences in the metabolism of Testosterone with respect to investigated species, sex, metabolic in vitrosystem and induction behaviour. P16-08 BIOTRANSFORMATION OF PARA-AMINOPHENOL AND PARA-PHENYLENEDIAMINE IN RECONSTRUCTED HUMAN EPIDERMIS AND HUMAN HEPATOCYTES G.J. Nohynek1 , D. Duche2 , A. Garrigues2 , P.A. Meunier2 , H. Toutain1 , J. Leclaire2 1 L’Or´ eal

Research and Development, Worldwide Safety Department, 92600 Asni`eres, France; 2 L’Or´eal Research and Development, Life Science Research, 92583 Clichy, France We investigated the biotransformation of the oxidative arylamine (AA) hair dye ingredients [14 C]-paraaminophenol (PAP) and [14 C]-para-phenylenediamine (PPD) in reconstructed human epidermis and human


hepatocytes. Human epidermis quantitatively transformed PAP to its N-acetylated derivative (APAP), whereas hepatocytes transformed PAP to sulfate or glucuronic acid conjugates of APAP or PAP and free APAP. Epidermis and hepatocytes converted PPD to N-mono-(MAPPD) and N,N -diacetylated (DAPPD) derivatives. At higher concentrations of PPD (250–1000 ␮M), epidermis or hepatocytes produced more of the MAPPD, whereas concentrations below 250 ␮M and lower favoured formation of the DAPPD metabolite. When compared with epidermis, human hepatocytes had a 3- or 8-fold greater capacity for generation of MAPPD or DAPPD, respectively. No evidence of transformation of PAP or PPD to Nhydroxylated derivatives was found in epidermis or hepatocytes. Our results suggest that (i) after dermal absorption of PAP or PPD, humans are systemically exposed to acetylated derivatives, (ii) current in vitro skin absorption studies may be inadapated for determination of human systemic exposure to AAs due to reduced or absent metabolic capacity of non-viable skin, (iii) due to qualitative differences between dermal and hepatic metabolism, oral toxicity studies are unsuited for the hazard assessment of dermal exposure to AAs and (iv) use of induced rodent liver S9 metabolic activation systems for in vitro genotoxicity studies may produce misleading results on the hazard of human dermal exposure to AAs. In conclusion, our data support the growing evidence that AAs are transformed in human skin and suggest that current practices of safety assessment of AAs should take these new findings into account. P16-09 PHARMACOLOGICAL BLOCKING OF ABC-TRANSPORTERS AND TOXICITY OF BENZO(A)PYRENE IN MCF-7 CELLS P. Myllynen1 , V¨ah¨akangas2


Kurttila1 ,


Vaskivuo1 ,


1 Department

of Pharmacology and Toxicology, University of Oulu, Oulu, Finland; 2 Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland Benzo(a)pyrene, a polycyclic aromatic hydrocarbon (PAH), present also in cigarette smoke, has a complex metabolism in humans. The ultimate car-


Abstracts / Toxicology Letters 158S (2005) S1–S258

cinogenic metabolite, benzo(a)pyrene-7,8-diol-9,10epoxide (BPDE), can bind to DNA. ATP-binding cassette (ABC) transporters protect cells from many foreign chemicals. In addition to specific inhibitors many widely used drugs inhibit function of these transporters. The aim of this study is to clarify whether pharmacological blocking of ABC-transporters affects benzo(a)pyrene adduct formation. MCF-7 breast adenocarcinoma cells were cultured with benzo(a)pyrene (1 ␮M) and the ABCtransporter inhibitors verapamil (0.125–100 ␮M), PSC833 (0.05–5 ␮M) or probenecid (0.05–2 mM) for 24 and 48 h. DNA was isolated from samples using a phenol extraction/ethanol precipitation method and BPDE-DNA adducts were analysed by synchronous fluorescence spectrophotometry. MTT assay was used for cytotoxicity analysis. According to preliminary results PSC 833 increases BPDE-DNA adduct formation both at 24 and 48 h (about 1.5- and 2-fold, respectively). Verapamil increases adduct formation statistically significantly at 48 h (about 2-fold). BPDE-DNA adduct formation was not affected by probenecid treatment. Additional benzo(a) pyrene slightly increased cytotoxicity but studied inhibitors were cytotoxic even without benzo(a)pyrene. According to these preliminary results it seems that certain ABC-transporters may protect cells from toxicity of benzo(a)pyrene. P16-10 EFFECT OF TOPICALLY APPLIED RETINOL, RETINYL PALMITATE OR ORALLY ADMINISTERED RETINYL PALMITATE ON ENDOGENOUS RETINOID PLASMA LEVELS IN HEALTHY WOMEN W.J.A. Meuling1 , G.J. Nohynek2 , W.H.J. Vaes1 , R.S. Lawrence3 , S.S. Shapiro4 , S. Schulte5 , W. Steiling6 , J. Bausch7 E. Gerber8 , H. Sasa9 1 TNO

Quality of Life, Zeist, The Netherlands;

2 L’Oreal, R&D, Asni` eres, France; 3 Unilever Research,

Sharnbrook, UK; 4 Johnson and Johnson, New Brusnwick, USA; 5 BASF AG, Ludwigshafen, Germany; 6 Henkel KGaA, D¨ usseldorf, Germany; 7 DSM Nutritional Products, Basel, Switzerland; 8 Beiersdorf AG, Hamburg, Germany; 9 Sisheido Co. Ltd., Yokohama, Japan

Two groups of 14 healthy female volunteers (age range: 20–36 years) received for 21 days daily topical applications of creams containing 0.30% retinol or 0.55% retinyl palmitate, respectively, corresponding to the maximal concentrations of these substances in cosmetic preparations, on approximately 3000 cm2 of their body surface area (back and thighs). After a wash-out period of 12 days, the groups were given single oral doses of 10000 or 30000 IU retinyl palmitate, respectively. Blood samples were collected over 24 h on study days, 3 (pre-study), 1, 21 (first and last days of topical treatment) or 34 (after oral administration) at 1, 2, 4, 6, 8, 12, 14, 16 or 24 h after treatment for determination of plasma concentrations of retinol, retinyl palmitate, oleate or stearate, 9-cis-, 13-cis-, all-trans-, 13-cis-4-oxo- or all-trans-4-oxo-retinoic acids (RAs). With the exception of mild and transient local irritation, no adverse local or systemic effects were observed and all subjects continued the study up to termination. Plasma retinoid levels on days 1 or 21 of topical treatment were similar to respective pre-treatment values, and no increases in CMAX or AUC0–24h values of individual endogenous retinoids were observed after topical treatment. In contrast, oral administration of 10000 or 30000 IU of retinyl palmitate produced dose-related and significant increases in CMAX and AUC0–24h values for plasma retinyl palmitate, oleate and stearate, 13-cis- and 13-cis-4-oxo RAs as well as increases in plasma levels of all-trans and 4-oxoall-trans RAs. Overall, our data demonstrate that topical administration of retinol up to 0.30% or retinyl palmitate up to 0.55% does not affect human endogenous retinoid levels, whereas oral doses at the maximum daily allowance during pregnancy (10,000 IU) or three-times higher (30,000 IU) produced significant and dose-related changes in endogenous levels. In conclusion, our data confirm that repeated topical exposure to retinol- or retinyl ester-containing cosmetic products up to the concentrations tested produces no increases in retinoid plasma levels that may produce adverse systemic or reproductive effects in humans.

Abstracts / Toxicology Letters 158S (2005) S1–S258

P16-11 INTESTINAL ABSORPTION OF XENOBIOTICS AFTER INTRADUODENAL AND RECTAL ADMINISTRATION IN RABBITS ˇ etinov´a, A. Pol´asˇkov´a, M. Nobilis, J. Kvˇetina V. Stˇ Institute of Experimental Biopharmaceutics, Joint Research Centre of the Academy of Sciences of the Czech Republic and PRO.MED.CS Praha a.s., Hradec Kr´alov´e, Czech Republic The goal of this work was to evaluate the possibility of utilizing the domestic rabbit for the purpose of studying the intestinal absorption of new generic drug formulations being developed in comparison with reference preparations. Two dosage forms of 5-aminosalicylic acid (5-ASA) were used; suppositories for rectal application, and coated tablets for application per os, administered intraduodenally to the anesthetized animals. Molsidomin in the form of tablets with controlled release, administered intraduodenally, was also studied. Blood samples for HPLC analysis of a drug level in the plasma were withdrawn from incannulated a. auricularis. While absorption and elimination were fast after the rectal application of 5-ASA supp. (tmax ca 1 h and low plasmatic levels after 4 h), cmax was reached in a time as long as 5 h, with the plasmatic level in some animals not decreasing after as many as 6 h, following the intraduodenal administration of the coated tablets. In case of molsidomin (tbl. retard), the value of tmax was about 0.7 h, while, similar to 5-ASA, we have not found a significant difference in the values of cmax and AUC as compared to those of the reference preparation. In conclusion, the rabbit appears to be a suitable species for pilot kinetic comparative studies. P16-12 THE PHARMACOKINETICS OF TMC125 IN DIFFERENT MICE STRAINS: IMPACT ON CARCINOGENICITY TESTING STRATEGY J. Verbeeck2 , A. Raoof1 , S. Lachau-Durand1 , B. Willems1 , L. De Zwart1 , M.P. Bouche1 , K. Steemans1 , H. Van Cauteren1 1 Global

Preclinical Development, Johnson and Johnson Pharmaceutical Research and Development Turnhoutseweg 30, B-2340 Beerse, Belgium; 2 Tibotec BVBA, Generaal de Wittelaan L11B3, 2800 Mechelen, Belgium


Carcinogenic potential of pharmaceuticals can be evaluated using one long-term study in a rodent species plus another short-term study using transgenic animal models. The present study aimed to investigate the pharmacokinetics of TMC125, an NNRTI in development for treatment of HIV, in Swiss CD1 mice and non-transgene rasH2 littermates produced by crossing C57BL/6J transgenic males with BALB/cByJ nontransgenic females. Animals were dosed daily with TMC125 HBr at 10, 50, 200, and 800 mg/kg to CD1 mice for 3 months and at 50, 200, 400, and 800 mg/kg to non-transgenic littermates for 1 month. Vehicle groups were included. Toxicokinetic evaluations were performed on satellite animals. TMC125 systemic exposure (Cmax and AUC) was significantly lower in the rasH2 littermates relative to CD1 mice at comparative tested doses. No change in toxicity profile was seen between the different mice strains. The study demonstrates differences in pharmacokinetics between various mouse strains. This may lead to discrepancies in the selection of the highest tolerated doses that can be employed in carcinogenicity studies. The guidelines are not clear about the suitability of a particular strain for dose range finding studies when using transgenic animals for carcinogenicity testing. Based on our data, the same strain of mouse should be used in dose range finding studies that precede carcinogenicity testing. P16-13 THE ABSORPTION AND TISSUE DISTRIBUTION OF THE PHYTOSTEROL, BRASSICASTEROL IN THE RAT L.J. Lea, H.J. Minter, D.J. Sanders Unilever Safety and Environmental Assurance Centre, Colworth House, Sharnbrook, Bedford, UK Phytosterol-esters are used as a cholesterol-lowering ingredient in yellow fat spreads, milk and yoghurts in the EU. As part of the safety evaluation of phytosterolesters the comparative absorption and tissue distribution of the main phytosterol components were evaluated (Sanders et al., 2000). Brassicasterol is a phytosterol found in rapeseed oil and therefore often present as a minor component of commercial phytosterols derived from edible vegetable oil distillates. In this study, the relative extent of intestinal absorption and tissue distribution of 14C-brassicasterol and


Abstracts / Toxicology Letters 158S (2005) S1–S258

14C-brassicasterol linoleate in the rat was investigated using radio-analytical techniques and whole body autoradiography (WBA). Total radioactivity was excreted predominantly in the faeces with <1% of the dose excreted via the urine. Excretion was rapid with the majority being excreted by 24 h post dose with ≤2% retained in the carcass after 96 h. WBA showed some absorption from the GI tract with distribution in the adrenal, ovary, spleen; lower levels were seen in the liver, lung and bone marrow. Levels were diminished after 48 h but low levels persisted in the adrenal and the ovary after 96 h. Findings were similar for brassicasterol and brassicasterol-linoleate and were consistent with those previously reported for other phytosterols. Results from mutagenicity and oestrogenicity screens and a repeat dose toxicity study on phytosterol-esters containing 8% brassicasterol were also consistent with previous studies on phytosterol-esters. P16-14 NEW HPLC METHOD TO DETERMINE EPIRUBICIN AND ITS METABOLITE EPIRUBICINOL IN DOG PLASMA M. Giorgi, V. Meucci, A. Cantini, G. Soldani Pharmacology and Toxicology Section, Department of Veterinary Clinics, University of Pisa, Vle Piagge 2, Pisa Italy Epirubicin (EPI) is a semisynthetic derivative of doxorubicin, an anthracycline widely used in human medicine and recently in veterinary medicine for treatment of cats and dogs affected by mammary cancers. Since EPI is highly toxic, monitoring drug concentrations during therapy is recommended to avoid severe adverse effects. The present study describes a rapid and simple HPLC method to detect EPI and epirubicinol (EPIol) in dog plasma. Four mongrel female dogs, 3–4 years hold, 20–25 kg, were treated intravenously with 2 mg/kg of EPI. Blood was collected at different times and centrifugated to separate plasma fraction. Samples were extracted after addiction of internal standard (daunorubicin). The mobile phase consisted in NaH2 PO4 50 mM/CH3 CN 65:35 (v/v) pH 4.00; the detector was set at 480 and 560 nm for excitation and emission, respectively. The LOQ and LOD for EPI and EPIol were 10 and 5, 1 and 0.5 ng/ml, respectively. Recovery was 85.5 ± 2.5% (SE) for both substances.

Precision and accuracy were within 10%. EPI concentration reached a plateau after 30 min, while EPIol concentration was under LOQ for the first 30 min and rose to Cmax (47 ng/ml) after 4 h. Eight hour after the administration, EPI was under LOQ, while EPIol was detected also after 56 h. As EPIol seems to be the main responsible of cardiotoxicity, this method could be used to assess the EPI and EPIol plasma concentrations in dog. P16-15 UPTAKE OF METHYLMERCURY, ETHYLMERCURY AND THIMEROSAL IN CULTURED BRAIN ENDOTHELIAL CELLS S. Larson1 , M. Aschner2 , T. Syversen1 1 Department

of Neuroscience, Norwegian University of Science and Technology, N-7489 Trondheim, Norway; 2 Departments of Pediatrics and Pharmacology, Vanderbilt University Medical Center, B-3307 Medical Center North, 1162 21st Avenue, Nashville, TN 372322495, USA Thimersoal (TM) has been used as a preservative in, e.g. vaccines. Concern has been raised as to its possible accumulation and toxic effects especially in babies and young children. As a result, its use as a preservative in vaccines has been discontinued. The active ingredient of TM is ethylmercury (EtHg). Among the alkyl mercurials (AM) it is only methylmercury (MeHg) for each comprehensive toxicity data have been generated. For other AMs we have to rely partly on limited experimental data and extrapolations to data collected for MeHg. We have used a rat brain endothelial cell line (RBE4) to compare in vitro mercury uptake after exposure to 5 ␮M of MeHg, EtHg and TM. Cells were incubated in triplicate for 30 min in Hg-containing HEPES-buffer without serum present. The uptake was 30 ± 3, 26 ± 2 and 10 ± 7 ng Hg/mg protein for MeHg, EtHg and TM respectively. Cysteine and cysteine/methionine was added to test for the relative importance of the neutral amino acid transporter (NAT) as a mechanism for cellular uptake. A significant reduction in uptake was noted for all three compounds when incubating the compounds as cysteine complexes (p < 0.05). The addition of methionine caused a further reduction in uptake for MeHg only (p < 0.05).

Abstracts / Toxicology Letters 158S (2005) S1–S258

P16-16 EFFECTS OF 2-BUTOXYETHANOL ON THE SULFHYDRYL COMPOUNDS LEVELS IN RAT ERYTHROCYTES A. Starek Department of Biochemical Toxicology, Faculty of Pharmacy Collegium Medicum, Jagiellonian University, Krak´ow, Poland 2-Butoxyethanol (BE) is well known as an industrial solvent and a causal factor of acute hemolytic anemia (AHA) in laboratory animals and in humans. AHA is a result of swelling, fragility, and intravascular lysis of erythrocytes (RBCs). Metabolic activation of BE to butoxyacetic acid is required for the development of hemolytic effect. There is a suggestion that susceptibility of RBC to hemolysis is age related. Intracellular sulfhydryl compounds may be the index of erythrocyte age. In this study, changes in erythrocytic protein and nonprotein sulfhydryl (NPSH) compounds from Wistar rats treated with single dose of BE (0.625–2.5 mmol/kg) were evaluated. The protein sulfhydryl compounds and NPSH levels in the RBCs of rats intoxicated with BE were elevated. Also, the activities of glutathione peroxidase, glutathione reductase and lactate dehydrogenase (enzymes containing –SH groups) were increased. These changes demonstrated a time- and dose-dependent relationship, suggesting recruitment of new, younger RBCs populations in hemolytic anemia induced by BE. It seems that the changes observed in this study are a result of regenerative processes. P16-17 INFLUENCE OF SODIUM DEPLETION ON THE KINETICS OF PARAQUAT IN THE ISOLATED RAT LUNG R.J. Dinis Oliveira1,2 , M. de Jes´us Valle1 , M. de Lourdes Bastos2 , F. Carvalho2 , A. S´anchez Navarro1 1 Faculty of Pharmacy, University of Salamanca, Spain; 2 REQUIMTE,

Department of Toxicology, Faculty of Pharmacy, University of Porto, Portugal Paraquat accumulates in the lung through a characteristic polyamine uptake system. It has been previously shown that paraquat uptake can be significantly prevented if extracellular sodium is reduced, although, the


available data correspond to experiments performed using tissue slices or incubated cells. This type of in vitro studies fails to give information on the actual behaviour occurring in vivo since the anatomy and physiology of the studied tissue is disrupted. Accordingly, the aim of the present study was to explore the usefulness of the isolated rat lung model when applied to characterize the kinetic behaviour of paraquat in this tissue after bolus injection under standard experimental conditions as well as to evaluate the influence of isoosmotic replacement of Na+ by lithium in the perfusion medium. The obtained results showed that the present isolated rat lung model system is useful for the stochastic analysis of paraquat toxicokinetics. It was observed that Na-depletion in the perfusion medium leads to a decreased uptake of paraquat in the isolated rat lung although it seems that this condition does not contribute to the elimination of paraquat since the paraquat tissue wash-out content is similar under both experimental conditions assayed. Ricardo Dinis, acknowledges FCT for his Ph.D. grant. (SFRH/BD/13707/2003). P16-18 THE TOXICITY PHARMACOLDRXTICS OF BUTYLBENZYL PHTHALATE BY INJECTION OF VEIN IN NICE J. Tian1,2 , F. Fu1 , M. Geng2 1 Marine

Drug and Food institute, Ocean University of China, Qingdao, Shandong, PR China; 2 School of Pharmacy, Yantai University, Yantai, Shangdong, PR China Aim: To study on the toxicity pharmacokinetics of butylbenzyl phthalate radiolabelled with 3 H in mice after a single injection of vein. Methods: After injection of 10 mg/kg and 100 mg/kg 3 H-butylbenzyl phthalate for a certain interval, activity of tracer in blood serum and organs were detected by multi-purpose scintillation counter. Pharmacokinetic compartment model was evaluated by NDST21 software, and parameters were calculated. Results: First-order linear equation and two-compartment open model were gained. 3 Hbutylbenzyl phthalate was largely absorbed by male mice especially by liver and gonad and the cumulative amounts of male mice were larger than female. Gener-


Abstracts / Toxicology Letters 158S (2005) S1–S258

ally 3 H-butylbenzyl phthalate was mainly excreted by urine within 8 h, and 80% of 3 H-butylbenzyl phthalate was excreted within 4 days. Conclusion: The pharmacokinetic processes of 3 H-butylbenzyl phthalate by injection of vein were different between male and female in vivo.


Keywords: Environmental estrogens; Butylbenzyl phthalate; Distributing; Kinetics; Eliminating

1 Department of Orthopaedic Surgery, Medical Univer-

P17 Alternative Methods P17-01 3 R COMPLIENT BIOMATERIAL TESTING: HET-CAM EVALUATION OF BIOMATERIALS GIVES COMPARABLE RESULTS TO IN VIVO MODELS REGARDING BIOCOMPATIBILITY PATTERNS C. Eder1,2 , E. Falkner2,3 , C. Chiari-Grisar1 , H. Sch¨offl2 , U.M. Losert3 , S. Nehrer1 1 Department of Orthopaedic Surgery, Medical Univer-

sity Vienna; 2 zet—Centre for Alternative and Complementary Methods to Animal Testing; 3 Core Unit for Biomedical Research, Medical University Vienna To investigate whether data determined via HET-CAM testing are comparable with data acquired in animal models, an in vivo biocompatibility experiment performed in sheep was repeated using the HET-CAM assay. Analysis of the sheep knee joint showed a good integration and a vascularisation of the implant after 1 month. HET-CAM analysis revealed a firm attachment of the samples after 3 days and a high vascularization. A synovial hypertrophia observed in the sheep was visible as hypertrophia of the reticular connective tissue in the CAM model. Both models showed an inflammatory response and a foreign body tissue reaction. Analysis of synovial smears indicated the presence of lymphocytes in the synovia. Lymphocytosis was also observed in the CAM vessels surrounding the implant. Fertilized, 10 days incubated chicken eggs can show a tissue response similar to an animal model. Biocompatibility and -incompatibility can be tested prior to in vivo implantation. The routine application of the HET-CAM test would allow the exclusion of unsuitable prototypes and facilitate the selection of the best biomaterial for animal experimentation.

C. Eder1,2 , E. Falkner2,3 , H. Sch¨offl2 , U.M. Losert3 , S. Nehrer1 sity Vienna; 2 zet—Centre for Alternative and Complementary Methods to Animal Testing; 3 Core Unit for Biomedical Research, Medical University Vienna Various biodegradable scaffolds were applied onto the CAM and maintained in ovo for 3 days prior to digital documentation, macroscopical biocompatibility evaluation and subsequent histological analysis. Collagen sponge: macroscopic analyzis demonstrated extreme rapid degradation, spontanous bleedings in the sourrounding of the implant and a vessel retraction from the implantation site. Histological analysis, in contrast, demonstrated an increase in blood vessel content. A foreign body tissue reaction was observed only histologically. Chondro-Gide® : Macroscopic evaluation showed excellent integration and biocompatibility patterns which were confirmed by histology. Bio-Gide® : Macroscopic observation showed excellent integration and significant induction of angiogenesis, which was confirmed by histology. An inflammatory infiltrate was observed in histological sections. Chondrocell® : Spontanous bleedings at the implantation site as well as vessel retraction and altered vessel courses were observed macroscopically. Histological evaluation in contrast demonstrated good angiogenetic properties. Macroscopical scoring only partially correlated to histological evaluation: The impact of spontaneous bleedings and vessel path alteration was often overestimated and a foreign body tissue reaction could not be detected after macroscopical evaluation. HETCAM biomaterial testing should therefore be combined with histological evaluation to receive the full force of expression of the CAM model.

Abstracts / Toxicology Letters 158S (2005) S1–S258


C. Nehrer1

1 Department of Orthopaedic Surgery, Medical Univer-

sity Vienna; 2 zet—Centre for Alternative and Complementary Methods to Animal Testing; 3 Core Unit for Biomedical Research, Medical University Vienna Biodegradable scaffolds are crucial for transplantation of tissue engineered cells. Animal models for scaffold evaluation usually cover the mid-term and late tissue reactions; the early phase tissue response is not documented. The HET-CAM test was used to compare the early phase tissue reaction of a synthetic (Hyalograft C® ) and a natural scaffold (Chondrocell® ) material. Samples were completely integrated into the connective tissue of the CAM and showed different degradation stages: The Hyalograft C® scaffolds were disintegrated into single fibres while the Chondrocell® scaffolds maintained their original shape. The synthetic material induced an inflammatory tissue response and thrombus formation in single vessels was observed. Fibrous tissue in the surrounding of the biomaterial showed a beginning incapsulation of the implant. The normal CAM structure was altered and atypical cells were observed. The collagen implant only provoked a mild tissue response indicated by the presence of single lymphocytes within the scaffold. CAM structure was not altered and atypical cells or vessel thrombosis were not observed. Although the long-term impact of these observations remain to be evaluated, CAM testing is a valuable tool for short term analysis and intermediate in vitro/in vivo biomaterial characterization.


P17-04. MANDATORY HET-CAM TESTING OF CELLS/CONSTRUCTS FOR TISSUE ENGINEERING E. Falkner1,2 , C. Eder2,3 , W. Fr¨oschl4 , C. Schmatz4 , H. Sch¨offl2 , U.M. Losert1 1 Core

Unit for Biomedical Research, Medical University Vienna; 2 zet—Centre for Alternative and Complementary Methods to Animal Testing; 3 Department of Orthopaedic Surgery, Medical University Vienna; 4 BAXTER Vaccine AG, Vienna The chorioallantois membrane (CAM) angiogenesis test system, originally developed for toxicity and irritation studies, was used for tissue engineering, the evaluation of biomaterials and cell transplantation. In cooperation with the BAXTER Vaccine AG, supplying the fertilized, special pathogen free (SPF) chicken eggs we established a CAM-test unit, where the angiognensis testing, cell culture and digital and histological analysis are performed: Introduction to the test system and hands-on teaching for experimentators in the field of regenerative medicine, tissue engineering, biomaterial testing a.s.o. is offered. The local animal testing advisory comittee evaluates current projekt proposals and selects those that should be subjects of HET-CAM testing prior to in vivo experiments. Standard Operating Procedure: Introduction to CAM protocols, modification to suit the particular scientific problem, setting up of a time-table, realisation of test series, discussion of the in vitro and in vivo data and the implications for originally proposed in vivo onsets that were delayed until evaluation of HET-CAM tests. Prior to tissue engineering experiments involving in vivo implantation into mammals, cells and constructs have to be evaluated upon/within the chorioallantoismembrane in vivo. Two years of experiences resulted in extensive coaching of working groups, optimized in vitro/in vivo protocols, altered time schedules, different choices of cells/media/scaffolds and in some cases, abdication of proposed animal testing systems.


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P17-05 PRIMARY CULTURE OF CORNEAL KERATOCYTES: MODEL FOR OCULAR TOXICITY ASSESSMENT IN VITRO G. Paladino, C. Marino R&D – SIFI S.p.A., Italy To develop an in vitro model, based on primary cell culture of corneal keratocytes (BCKe), to screen the proapoptotic property of molecules used in ophthalmic practice. BCKe were obtained from bovine eyes. Epithelial side was separated from the stroma, containing keratocytes, through enzymatic digestion with collagenase. When BCKes reached the confluency, plates were treated with pranoprofen, ketorolac, diclofenac, flurbiprofen, dexametasone and desonide (5, 1, 10−1 , 10−3 , 10−5 mM). Test item effects were evaluated considering the percentage of cell viability using MTT test. Nucleus morphology was evaluated using a cytological method to detect the presence of apoptotic figures using the optical microscope. The expression of p53 anti-oncogenic factor, over-expressed during apoptotic toxicity, was evaluated by RT-PCR. After 24 h of incubation with the different chemicals, all NSAIDs and desonide, but not dexametasone showed a reduction of cell viability at the highest concentration. Flurbiprofen exerted the toxicity till 10−1 mM. Using the nucleus staining diclofenac and flurbiprofen gave apoptotic signs already at 10−5 mM, while ketoralac showed less apoptotic figures at 10−3 mM. Pranoprofen, dexametasone and desonide did not present similar toxicity. The RT-PCR for p53 showed that all molecules exterted over-expression of this gene, desonide and pranoprofen only at the highest concentration. On the basis of above data, the use of these cells could represents an alternative during toxicological screening, providing information about the apoptotic potential in preclinical phases.

P17-06 DESIGN OF A CHROMATOGRAPHIC METHOD FOR STUDYING THE EFFECT OF XENOBIOTICS ON THE SECRETION OF NEUROTRANSMITTERS E. Sabater, E. Quesada, M.A. Sogorb, E. Vilanova, V. Carrera Divisi´on de Toxicolog´ıa. Instituto de Bioingenier´ıa. Universidad Miguel Hern´andez. Avda. de la Universidad s/n 03202 Elche (Alicante) Spain This work is focused in the simultaneous determination of neurotransmitters secreted by chromaffin cells in culture using liquid cromatographic coupled to mass spectrometry. The separation was performed with a C8 column (4.6 mm × 150 mm, 5 ␮m). The chromatographic separation was carried out using a gradient (methanol-3 g/l acetic, pH 2.8) that increased methanol concentration of the mixture from 0 to 60% in 6 min with a flow of 0.75 ml/min. The nebulizer worked with 12 l N2 /min at 300 ◦ C. The pressure was 50 psi and the voltage of the capillar was set at 3000 V. The detection mode was SIM positive. The secretions were quantitatively determined recording the abundance of the m/z fragments 152, 166, 137 and 160 that correspond respectively to norepinephrine (NE), epinephrine (E), dopamine (D) and serotonine (5-HT). The minimal detected concentration for NE was 2 ng ml−1 and 0.5 ng ml−1 for E, D and 5-HT. Cells were cultured in 96-well plates at 200,000 cells/well. Basal secretion was collected with Krebs bicarbonate, induced secretion was obtained with 75 mM potassium, while total intracellular contents were obtained after collecting the lysate of cells treated with 0.4 N perchloric acid. For testing the suitability of the method, chromaffin cells in culture were incubated with 75 ␮M mipafox, an organophosphorus compound, during 60 min. After this treatment, basal secretion, induced secretion and total intracellular content of 5-HT were, respectively 50, 50 and 6 times higher than controls. This method can be used to study the effect of other xenobiotics on neurotransmitters secretion. E. Quesada is granted by Spanish Ministry of Culture. E. Sabater is enjoying a fellowship from Ramon Areces Foundation.

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Researches, Prevor, Talence, France;

2 Scientific communications, TCMTS Inc, Elk Moutain,

WY, USA The aim of this study is to show by in vitro experiments the penetration of a corrosive, such as sodium hydroxide (NaOH) and to evaluate the interest of a rinsing with an active solution. In Vitro studies were performed: (1) Penetration of NaOH through the tissues was simulated through a semi-permeable membrane depending on the concentration. A volume of NaOH is put at the surface of the membrane and the evolution of the pH is measured in a compartment containing sodium chloride (420 mosmoles/Kg). (2) A model of semi-permeable membrane was used to simulate a complete rinsing of 2 N NaOH exposure. Comparison was made between a water rinsing and an active rinsing solution, such as Diphot´erine® . Two times of exposure was tested, 20 s and 1 min. The penetration of sodium hydroxide depends on its osmotic pressure. For 0.1 N and 0.2 N NaOH concentrations, the penetration was delayed and a final pH value after 900 s was, respectively about 9 and 10. When the NaOH concentration is 1 N, the penetration is faster due to a higher osmotic pressure than cornea; the final pH value after penetration is 11.5 at 900 s. For 2 N and 5 N concentrations, the penetration is really higher than for 1 N concentration and a final pH value is, respectively 12 at 900 s and 12.3 at about 600 s. The complete rinsing showed better efficacy with Diphot´erine® rinsing versus water rinsing at all times of exposure. For a 20 s time of contact and after 3 min of rinsing, the external pH value was, respectively about 9 for Diphot´erine® and about 12.8 for water. After 45 min, the internal pH value is about 9 with Diphot´erine® rinsing and 11.5 with water rinsing. For 1 min of contact, the curves of pH are similar with a delayed decrease of pH. This in vitro model has shown to be depending on the concentration of the corrosive and the balance of


osmotic pressure. The simulation of a complete rinsing showed better efficacy with an active rinsing solution versus water rinsing after NaOH exposure. P17-08 APPLICATION OF WAVE BIOREACTOR FOR THE PRODUCTION OF BHK 21 AND CHO CELLS BIOMASS I. Slivac, V. Gaurina Srcek, K. Radosevic, Z. Kniewald Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia Stable bioprocess systems are a demand for cultivation and maintaining of various cell lines used for pharmaceutical, toxicological or biomedical applications. The Wave bioreactor 20 SPS as a novel bioprocess system was used for biomass production of CHO-K1 and BHK 21 C13 cells. During the period of 62 days a repeated fed-batch cultivation of suspended BHK 21 C13 cells was performed. Maximum cell concentration of 5.2 × 106 was achieved with viability of more than 90%. The viability of the cells was determined using trypan blue exclusion method. The scale-up of 1:10× of that system provides a significant amount of cells. The CHO-K1 cells were attached on Cytoline 1 microcarriers, which have macroporous structure suitable for cultivation of adherent cells. We monitored their growth on the microcarriers in order to determine time needed for cells to overgrow them. The number of cells for total overgrowing is approx. 8.000/microcarrier. Still, limited data on viability, and quantification of cells attached to the Cytoline 1 are available Cell harvesting with 0.25% trypsin shows just an outer layer of cells due to macroporous structure of the microcarriers. In order to avoid this limitation we performed standard MTT viability assay to determine number of viable cells grown on the microcarriers. The obtained BHK21 C13 cells and CHO-K1 cells were later used for toxicological investigations and were proved as a standard quality. This system provides possibility for the production of other cells faster and cheaper than with previous systems.


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P17-09 TESTING OF ATRAZINE CYTOTOXICITY ON CHINESE HAMSTER OVARY (CHO-K1) CELL LINE I. Kmetiˇc, T. Bituh, J. Kniewald Faculty of Food Technology and Biotechnology, Laboratory of Toxicology, Zagreb, Croatia Atrazine, as s-triazine herbicide, selectively controls broadleaf weeds and certain grass weeds in corn and sorghum fields. An estimated 34.5 million kg of atrazine are applied to cropland each year in the United States, and it is also one of the most used herbicides in Europe. The US EPA does not consider likely the atrazine to be a human carcinogen. However, its effects on other physiological processes point to atrazine as an endocrine disruptor. In order to determine possible toxic effect at ovarian cellular level, in this study Chinese Hamster Ovary (CHO-K1; CCL-61) cell line was used. Cells grew in monolayer at 37 ◦ C in the atmosphere of 95% air and 5% CO2 , in the medium Dulbecco’s MEM with 5% newborn calf serum. The biomass was produced in T-bottles. Cells were separated in the early logarithmic phase of growth and seeded on multiwell plates in concentration of 1.5 × 104 cells/mL of cultivating medium. Cytotoxic effect of atrazine at the concentration range of 10–160 ␮g/mL was determined by colorimetric methods after 24, 48 and 72 h. IC50 value was determined from the slope of % inhibition versus log dose values. Cell viability and the number of cells were measured by: Trypan Blue exclusion method (IC50 95.3 ± 9.25 ␮g/mL); Kenacid Blue R binding method change in total cell protein (IC50 173.85 ± 13.35 ␮g/mL); lyzosomal activity by Neutral Red method (IC50 196.24 ± 9.8 ␮g/mL); MTT assay, the ability of viable cells to convert a soluble tetrazolium salt into insoluble formazan precipitate (IC50 157.17 ± 14.14 ␮g/mL). Application of in vitro assays in evaluation of the toxicity should be an important contribution in elucidating cellular and molecular mechanisms of atrazine.

P17-10 PREDICTION OF THE HUMAN ACUTE TOXICITY BY THE Hep G2/24 h TOTAL PROTEIN ASSAY, MEASURING PROTEINS WITH 3-(4-CARBOXYBENZOYL)-QUINOLINE-2CARBOXALDEHYDE P.J. Dierickx Institute of Public Health, Wytsmanstraat 14, 1050 Brussel, Belgium In our previously described Hep G2/24 h/total protein assay the proteins were measured with the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) study. In order to increase the MEIC database with a wider range of chemicals, we were interested in introducing the more practical CBQCA [3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde]-method for the quantitation of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity are obtained with the CBQCA-method. The cells were treated for 24 h. The cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method with 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large, unexpected, differences when using both protein assays. However, when comparing the logPI50c with the human toxicity, a correlation coefficient r2 = 0.761 (n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than for the Lowry method (r2 = 0.695). The Hep G2/24 h/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large scale analyses.

Abstracts / Toxicology Letters 158S (2005) S1–S258




E. Petrikou1 , A. Tzimogianni2 , M. Tzatzarakis3 , E. Karageorgou1 , E. Charvalos2 , A.M. Tsatsakis3 , G. Petrikkos1

N.V. Kokshareva, P.G. Zhminko, N.P. Dmitrenko, M.L. Zinovieva, O.P. Zhminko, T.O. Kishko, E.N. Strumenska

1 Research Laboratory of Antimicrobial Chemotherapy

Medved’s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine

and Infectious Diseases, University of Athens, Greece; 2 School of Health and Caring Professions, Technological Educational Institution of Athens, Greece; 3 Center of Toxicological Sciences and Research, Department of Medicine, University of Crete, Greece Four types of water-soluble polyvinylpyrrolidone (PVP) complexes of nystatin were prepared and their biological activity was compared with that of free nystatin against 16 strains of Candida (C. albicans, C. glabrada, C. krusei, C. lusitania and C. parapsilosis). The prepared nystatin complexes were pure substances, free from contaminants; with relatively simple, fast and cheap production process and they were soluble in aqueous solutions. The molecular weight of nystatin formulations NC1, NC2, NC3 and NC4 were 10 kDa, 24 kDa, 40 kDa and 360 kDa, respectively. Susceptibility testing for yeasts was performed according to the National Committee for Clinical Laboratory Standards guidelines (NCCLS MA 7A 1997). MICs of nystatin complexes were equal or slightly higher compared to those of nystatin, (with NC2 showing the lowest MICs and MFCs) against Candida spp. Cytotoxicity was evaluated by measuring the release of LDH from J774 macrophages after 4 h incubation with 40 ␮g/ml of each compound. J744 cells were cultured at 37 ◦ C in 95% air/5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% foetal calf serum. LDH release was considerably lower (up to 60% lower compared to nystatin) for the complexes than for nystatin in J774 macrophages. The activity of nystatin polymeric complexes in our in vitro study depended on the molecular weight of the polyvinylpyrrolidone. The present project is 75% financed by the European Union Education Program “Archimedes” and 25% by the Greek Government.

Two models as a suspension of thymocites of rat and a culture of infusoria Tetrhymena pyriformis were tested for evaluation of acute toxicity of a number of drugs substances in comparison with acute toxic effect on rats in vivo. Study in vivo showed low toxicity (LD50 > 1400 mg/kg b.w.) of metamizole sodium, acetylsalicylic acid, nitrate sodium, paracetamol, papaverine hydrochloride, benzylpenicillin; moderate toxicity of diclofenac, nitrite sodium, neostigmine bromide (LD50 is 57 mg/kg b.w.; 150 mg/kg b.w.; 160 mg/kg b.w., respectively). Study of drugs acute toxicity in vitro on rat thymocite suspension showed a low toxicity for metamizole sodium, acetylsalicylic acid, nitrate sodium, a high toxicity was shown for diclofenac, nitrite sodium, neostigmine bromide. Study on another alternative in vitro test-system as infusoria Tetrahymena pyriformis culture revealed a low toxicity of metamizole sodium, acetylsalicylic acid, nitrate sodium, benzylpenicillin, neostigmine bromide, a moderate toxicity of paracetamol, papaverine hydrochloride, nitrite sodium, high toxicity of diclofenac. Correlation coefficient (r) between means of LD50 in study on rats in vivo and LC50 in study on thymocites suspension as well as on infusoria culture is 0.85–0.95. Thus, the appliance of alternative in vitro models is prospective for the forecast of acute toxicity of drugs. P17-13 EFFECT OF HEAVY METALL COMPOUNDS ON THE MEMBRANE PERMEABILITY AND BARRIER FUNCTION OF EPIDERMIS IN VITRO O. Roik, M. Prodanchuk, O. Gudz 6th Heroyv Oborony St., L.I. Medved’s Institute of Ecogygiene and Toxicology, Kyiv, Ukraine


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Alternative to animal models were used to investigate the effect of low dose exposure (maximum authorized concentrations in finished cosmetic product and lowest concentrations) of such cosmetic ingredients, as Pyrithione zinc (authorized in cosmetic products rinsed off), Lead acetate (field of application, for hair dyeing) and Strontium chloride hexahydrate (field of application, toothpaste, shampoo and face care product) on the membrane permeability and barrier function of epidermis: modification BRC-test and biphasic water-lipid model of the epidermis. Depending of chemical structure, these cosmetic ingredients have been shown to increase or decrease membrane permeability in BRCtest. Lead acetate at maximum authorized concentrations (0.6% calculated in lead) led to strong damage of plasmatic membrane of erythrocytes. Exposure erythrocytes to low concentration of this compound (0.0125%) caused 1.5-fold higher levels of erythrolysis than control solvent. At minimum concentration (0.00625%) it did not effect on membrane permeability. Erythrocytes treated with Strontium chloride, did not differ significantly from control group in their membrane permeability. Pyrithione zinc has the ability to increase membrane permeability with high efficiency. Topical application of Lead acetate at concentration 0.1% on lipid layer of the biphasic water-lipid model of the epidermis did not effect on the level TEWL. At the similar conditions Strontium chloride demonstrated a tendency to protect water losing, respectively by 19%. Our in vitro results suggest those both inorganic and organic heavy metals’ cosmetic ingredients, such as Lead acetate and Pyrithione zinc can induce injury changes in barrier function of biological membrane. P17-14 THE USE OF TETRAHYMENA PYRIFORMIS W. AS ALTERNATIVE TEST-OBJECT FOR DETERMINE TOXICITY OF CHEMICAL SUBSTANCES O. Zhminko L.I. Medved’s Institute of Ecohygiene and Toxicology, 6, Heroiv Oborony Str., 03680 Kyiv, Ukraine The use of the Tetrahymena pyriformis W. infusoria, as alternative test-object, enables in short time to deter-

mine toxic properties of chemical substances and predict their danger for a human. The researches on determination of acute toxicity of some derivative of pyridine was carried out and also comparative estimation of findings for Tetrahymena pyriformis W. and for animals was conducted. Death of cells, change of infusoria form and movement testify to toxic of substance. Objects of researches, infusorian Tetrahymena pyriformis W., formalin, trichlorfon and some pyridine derivative: pyridine, 2,6-dimethylpyridine, N-oxide-2-methylpyridine, 2,6-dimethyl-N-oxide pyridine, aqua-N-oxide-2-methylpyridine-manganese-2-chloride, bis(N-oxide-2-methylpyridine), Di(N-oxide-2methylpyridine) zinc (II) iodide. It is shown that formalin, trichlorfon, pyridine and 2,6-dimethylpyridine are the a middle-toxic substances for Tetrahymena pyriformis W., N-oxide-2-methylpyridine, 2,6-dimethyl-N-oxide pyridine, aqua-N-oxide-2-methylpyridine-manganese-2-chloride, bis(N-oxide-2-methylpyridine), Di(N-oxide2-methylpyridine) zinc (II) iodide, small-toxic substances. Comparison of toxicity values of the investigated substances for Tetrahymena pyriformis W. and animals showed that those chemicals which behave to the middle-toxic substances for animals, they are the same and for the Tetrahymena pyriformis W. Thus, findings and also these literatures allow to use the Tetrahymena pyriformis W. infusoria as testobject for determination of acute toxicity of chemical substances. P17-15 EFFECT OF LINEZOLID ON HEMATOPOIETIC PROGENITORS


S.G. G´omez1 , J.A. Bueren1 , C. Lagunas2 , B. Albella1 1 Hematopoiesis Division/Fundaci´ on Marcelino Bot´ın.

CIEMAT. Avda Complutense, 22, 28040 Madrid, Spain; 2 Laboratorios SALVAT S.A. Barcelona, Spain Linezolid is a synthetic antimicrobial of the oxazolidinone class that has rapidly become an important and widely used drug in the United States since its approval in 2000. Diarrhea, nausea, and headache are the most common adverse reaction associated with the

Abstracts / Toxicology Letters 158S (2005) S1–S258

use of Linezolid. Moreover, with increased use of this antibiotic, reports of dose-limiting anaemia and thrombocytopenia have been published. The aim of the present study was to evaluate the effect of Linezolid on human hematopoietic committed progenitors. Since the haematological effects on patients were detected after prolonged periods of treatment (>2 weeks), human hematopoietic cells from umbilical cord blood were exposed to different concentrations of linezolid for two weeks and CFUGM (myeloid progenitors), BFU-E (erythroid progenitors) and CFU-Meg (megakaryocytic progenitors) were scored. Survival curves were obtained and IC50, IC70 and IC90 values were calculated. Our results show similar survival curves for myeloid and megakaryocytic progenitors with IC90 values of 0.60 ± 0.08 and 0.51 ± 0.01 mM for CFU-GM and CFU-Meg respectively. However, erythroid progenitors showed a higher sensitivity with IC90 of 0.24 ± 0.01 mM. The higher sensitivity of erythroid progenitors to Linezolid may account for the appearance of anaemia in treated patients. Further research needs to be done on the megakaryocytic progenitors in order to clarify whether or not the thrombocytopenia in patients is a consequence of a damage on megakaryocytic compartment. P17-16 EFFECTS OF CHEMOPREVENTIVE AGENTS ON IN VITRO N7 -METHYLGUANOSINE ADDUCTS FORMATION INDUCED IN RAT HEPATOCYTES BY N-NITROSODIMETHYLAMINE


against NDMA deleterious effects. Since candidates for chemoprevention are numerous, we have developed an in vitro pre-screening test capable of rapidly identifying and selecting promising agents for further in vivo studies, reducing the number of animal experiments. It was based on the compound’s ability to inhibit CYP2E1 and therefore to inhibit DNA adducts formation in rat hepatocytes incubated with NDMA. Quantification of N7 -methylguanosine adducts was carried out by an ELISA developed by our laboratory. This screening allowed the detection of potential candidates for chemoprevention: ellagic acid, resveratrol, green tea catechins, ginkgolides, theaflavins. In conclusion, this method constitutes a suitable in vitro pre-screening test for identifying chemopreventive agents capable of exerting protective effects against N7 -methylguanosine adducts formation induced by NDMA. Moreover, this type of assay could be applied to other compounds exerting their carcinogenic effects by forming DNA adducts. P17-17 IN VITRO MAN MADE MINERAL FIBERS (MMMF) DEGRADATION FOLLOWING MONOCYTE ACTIVATION BY AN ESCHERICHIA COLI EXTRACTS H. Dika Nguea1 , A. De Reydellet2 , P. Lehuede3 , A. De Meringo3 , A. Robe1 , A. Le Faou1 , B. Rihn4 1 Laboratoire

1 Laboratoire

de Bact´eriologie Virologie, Facult´e de M´edecine, Universit´e Henri Poincar´e -Nancy 1, 54501 Vandoeuvre-l`es-Nancy, France; 2 Saint Gobain Insulation, Les Miroirs, 92400 Courbevoie, France; 3 Saint Gobain Recherches, 93300 Aubervilliers, France; 4 Facult´e de Pharmacie, Universit´e Henri Poincar´e—Nancy 1, 54000 Nancy, France

N-nitrosodimethylamine (NDMA), a probable human carcinogen, is widely present in food items and tobacco smoke. It requires a metabolic activation by the cytochrome P450 2E1 (CYP2E1) to produce DNA adducts, N7 -methylguanosine being the major one, considered as an early event in carcinogenesis. Since NDMA cannot be completely eliminated from our environment, it would be of a great interest to identify chemopreventive agents effective for protecting

Toxicity of mineral fibers, whether they are natural or man made (MMMF), is usually evaluated by tests of biopersistence using rodents. A cellular model would be convenient to reduce, refine and replace animal models. As a matter of fact, macrophages are the cells of choice as MMMF toxicity is the consequence of interactions of fibers with alveolar macrophages. Indeed, we developed an in vitro assay of MMMF biodegradation. We activated U-937 monocytes with several factors, such as Escherichia coli extracts, interleukine 6 (IL-6), lipopolysaccharide (LPS), and tumor necro-

V. Niot-Mansart1 , V. Niot2 , J.P. Arnould1 de Toxicologie, Facult´e de Pharmacie, Amiens, France; 2 Laboratoire de Biologie des Plantes, Facult´e des Sciences, Amiens, France


Abstracts / Toxicology Letters 158S (2005) S1–S258

sis factor ␣ (TNF-␣). We assessed MMMF chemical biodegradation by performing induction coupled plasma atomic emission spectrometry to measure silicon release in extracellular medium and scanning electron microscopy of fibers. RNA extraction from E. coli-activated U-937 monocytes and native U-937 monocytes in presence of MMMF allowed us to measure gene expression with 20 K-microarrays. E. coli supernatant induced U-937 monocytes differentiation into active macrophages as shown by light microscopy. Tested MMMF displayed surface degradation manifested as holes when incubated with E. coli- or with E. coli supernatant-activated U-937 monocytes, whereas natural mineral fibers, namely crocidolite, do not show any surface erosion. In our assay, silicon release from tested MMMF was volume and time-dependent. The comparison of gene expression of E. coli activated macrophages and quiescent cells was obtained using 20 K-microarrays. Of the 1037 genes with expression differences, 56 were considered pertinent as related to inflammation, oxidative stress, tissue remodeling, and an indicator of presence of the bacteria. Then the susceptibility of MMMF to degradation evaluated in vitro providing activation of macrophages can be obtained and the results were consistent with what has been observed in vivo. E. coli contact procured macrophage activation and further characterization of the active compound in the culture supernatant is in progress. P17-18 U937 CELL LINE: A TOOL FOR THE IDENTIFICATION OF CONTACT ALLERGENS F. Python1,3 , C. Goebel2 , P. Aeby1 1 Cosmital

SA (Wella AG), Marly, Switzerland; of product safety, Toxicology, Wella AG, Darmstadt, Germany; 3 Supported by The European Cosmetic Toiletry and Perfumery Association (COLIPA) 2 Department

The role of dendritic cells in the initiation of allergic contact hypersensitivity is well documented. Our group and others reported that sensitization tests using human peripheral blood monocytes derived dendritic-like cells (PBMDCs) may represent an alternative to replace animal tests. To solve sourcing issues and donor-to-donor variability, the U937 cell line was evaluated as an alternative to PBMDCs. U937 cells were cultured with

low concentration of interleukin-4 (IL-4) in 12-well plate and exposed to test items. After exposure, cells were harvested for flow cytometric measurements of CD86 and quantitative real time Reverse transcriptasePolymerase Chain Reaction analysis of IL-1␤ and IL-8 gene expressions. In a dose effect study, cells were exposed to standard sensitizers/irritants and five oxidative hair dye precursors at non-toxic to sub-toxic concentrations for 48 h. Thus, an optimal non-toxic dose was determined for each test item and then evaluated in a kinetic experiment for 24, 48 and 72 h exposure. Based on the test items specific modulation of the chosen markers, a classification scheme could be proposed. The described in vitro test was able to discriminate weak/strong contact allergens from irritants. Moreover, the five oxidative hair dye precursors were identified as potential sensitizers, confirming the results obtained with the murine local lymph node assay. P17-19 DEVELOPMENT OF IN VITRO HAPTENATION ASSAYS FOR SKIN SENSITIZATION THROUGH CHEMICAL AND METABOLIC UNDERSTANDING C.K. Smith Pease1 , M. Divkovic1 , O. Payne1 , J.D. Vassallo2 , E. Coulter3 , D.J. Naisbitt3 , B.K. Park3 , M. Panico5 , A. Dell5 , H. Morris5 , R. Alvarez Sanchez4 , E. Gim´enez-Arnau4 , J.P. Lepoittevin4 , G. Frank Gerberick2 , D.A. Basketter1 1 Unilever,

Colworth House, Sharnbrook, Beds, UK; and Gamble, Cincinnati, Ohio, USA; 3 University of Liverpool, UK; 4 Universit´ e Louis Pasteur, Strasbourg, France; 5 Imperial College London, UK 2 Procter

With publication of the 7th Amendment to the Cosmetics Directive invoking an EU ban on in vivo testing of cosmetic ingredients, the search for practical alternative approaches for safety evaluation of chemicals has high priority within industry. For skin sensitization, the development of novel in vitro assays may be achieved through better understanding of cellular and molecular processes. A key molecular event initiating skin sensitization is protein haptenation, i.e., the chemical modification of skin protein(s). Two hypotheses remain to be proven: (1) that all skin sensitizers (with or without metabolic activation) haptenate pro-

Abstracts / Toxicology Letters 158S (2005) S1–S258

teins and that non-sensitizers do not; (2) that the extent or type of protein haptenation can be related to in vivo skin sensitization hazard and potency. We have investigated the binding mechanisms of a variety of different sensitizers (e.g. DNCB, cinnamaldehyde, isothiazolinones, PPD, phenylsalicylate) to intact proteins (such as human serum albumin and collagen) or peptides and analysed adducts by modern proteomics and analytical techniques. We have shown that protein/peptide haptenation can follow a variety of adduction mechanisms at preferred amino acid residues. Using our collective knowledge of haptenation, we have begun to profile the type and extent of protein modification (including glutathione, model nucleophiles, carefully designed peptides and intact proteins) by a broad range of different sensitizers and non-sensitizers, using standard analytical read-outs, such as HPLC or ELISA techniques. So far, both hypotheses above have been supported by protein binding data on >90 chemicals. If the hypothesis of protein haptenation can be supported robustly, then this principle could be used to develop novel, cheap and efficient in vitro assays to predict the sensitization potential of chemicals. P17-20 THE MUTZ-3 CELL LINE AS AN IN VITRO MODEL FOR THE SCREENING OF CONTACT SENSITIZERS J.L. Peiffer1 , P. Azam1 , D. Chamousset1 , M.H. Tissier1 , P.A. Bonnet1 , L. Vian2 , I. Fabre1 , J.C. Ourlin1 1 AFSSAPS, Unit´ e BCM, 635 Rue de la Garenne 34740

Vendargues, France; 2 Laboratoire de Toxicologie, Facult´e de Pharmacie (UM1), 15 av Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France In vitro methods for the prediction of the skin sensitization potential of chemicals are still lacking a valuable model capable of mimicking the activation process of Langerhans cells (LC). In this study, we have tested the MUTZ-3, a cytokine-dependent human monocytic cell line, as a potential in vitro model through various approaches. Undifferentiated MUTZ-3 cells were tested against a panel of sensitizers and irritants on a broad spectrum of cell surface markers including HLA-DR, CD54, CD86, CD40, B7-H1, B7-H2, B7-DC. With CD86, all sensitizers from weak to strong were positively


detected, including benzocaine, a classical false negative in animal sensitization tests. Irritants were not detected. The other markers reacted in a more variable manner. The CD86 overexpression in response to DNCB was confirmed at the mRNA level by real-time PCR analysis. Moreover, MUTZ-3 cells were tested for their potential to differentiate into dendritic-like cells. Up to 80% CD1a+ cells were detected in a protocol using GM-CSF, IL-4 and low TNF-␣. Taken together, our data suggest that MUTZ-3 cells may represent a valuable in vitro model for the screening of sensitizers. However, our most recent data suggest also that the cell line is subject to variations over passages, and optimization of the cell culture conditions is required. P17-21 VITAMINS AS AN ALTERNATIVE TO REDUCE GENOTOXIC RISK OF FLUOROQUINOLONES TREATMENT M. Arriaga-Alba1 , J. Flores-Lozada1 , R. Flores-Paz1 , R. Rivera-S´anchez1 , N. Ruiz-Perez1 , M. Gonz´alez2 , R. Cari˜ ´ Avila no-Cort´es3 1 Laboratorio

de Investigaci´on Microbiol´ogica, Hospital Ju´arez de M´exico; 2 Universidad Polit´ecnica de Pachuca; 3 Escuela Nacional de Ciencias Biol´ogicas (IPN) The Norfloxacin (NOR), as other flour-quinolones antibiotics, has been reported to induce mutagenesis on the Salmonella typhimurium TA102 strain. Further studies have shown that the mutagenic effect was inhibited by antioxidants, such as ␤-carotene and the natural antioxidants of Roheo discolor. The combined therapy of norfloxacin and vitamins is studied to evaluate, vitamins C and E, employed as an alternative to reduce genotoxic risk due to fluoroquinolones treatment. The antimutagenicity of piridoxal (vitamine B6), ␣-tocoferol (vitamin E) and ascorbic acid (vitamin C), was evaluated on the S. typhimurium TA102 with S9 against mutations induced by the fluoro-quinolone norfloxacin (NOR). In vitro, MIC values of (NOR), was obtained employing the plate dilution method. Vitamin B and Vitamin E 1.0 mM induced an statistical reduction of that mutagenicity value induced by NOR on S. typhimurium TA102. While Vitamin C,


Abstracts / Toxicology Letters 158S (2005) S1–S258

reduced 15.86% under the standard Ames test condition. The antioxidant properties of these vitamins were demonstrated by the antioxidant reduction of the DPPH. Any of these vitamins reduced the obtained MIC value neither for NOR against 25 uropathogenic Escherichia coli strains. These results suggest that Vitamines E and B6 might be a good alternative to reduce genotoxic risk in patients with treatment of fluoroquinolones. The marginal antimutagenic effect of Vitamin C on the Ames test might induce a Fenton reaction, if divalent cations on distillated water are present, it could also be a pro-oxidant rather than an antioxidant. Ascorbic acid should be further evaluated under different divalent cations concentrations. P17-22 ASSESSMENT OF THE INVOLVEMENT OF THE P38 MITOGEN ACTIVATED PROTEIN KINASE (MAPK) PATHWAY IN THE INDUCTION OF IL-1␤ EXPRESSION IN MURINE J774A.1 MACROPHAGES EXPOSED TO TWO DIFFERENT PLATINUM COMPOUNDS J. Arkusz, D. Lewi´nska, M. Sta´nczyk, M. St˛epnik Department of Tocixology and Carcinogenesis, Nofer Institute of Occupational Medicine, Lodz, Poland P38 MAPK is one of the most important pathway involved in the activation of interleukin-1␤ (IL1␤) transcription. Induction of IL-1␤ expression is considered to be useful marker for predictive skin sensitization test. The involvement of p38 MAPK in the induction of il-1␤ expression in murine J774A.1 macrophages exposed in vitro to two different platinum compounds: potassium tetrachloroplatinate (TCPP) and cis-platin was investigated. Cytotoxicity of cisplatin for the macrophages was much higher than of TCPP (the IC50 value: 23 ± 4 ␮M and 238 ± 30 ␮M, respectively) as was assessed in the 24-h MTT assay. It has been demonstrated that the contact of the macrophages with each of tested platinum compounds caused the induction of il-1␤ mRNA expression but only TCPP induced the synthesis of IL-1␤ protein. The production of IL-1␤ was detected in the lysates of the cells treated with TCPP (150–350 ␮M) for 24 h (22–106 pg/ml). Results of the study revealed that the treatment of the macrophages with each of tested

platinum salts resulted in the phosphorylation of p38 MAPK and blocking of p38 MAPK activity with specific inhibitor (SB203580) down-regulated the il-1␤ expression induced by these compounds. In contrast, the iritant sodium dodecyl sulfate that did not induce the il-1␤ expression, also did not trigger p38 MAPK. These data suggest that, p38 MAPK is essential for the induction of il-1␤ mRNA expression in J774A.1 macrophages exposed to each of tested platinum compounds. Although both tested platinum compounds induced il-1␤ mRNA expression, only TCPP activated translation into the IL-1␤ protein. P17-23 THE INFLUENCE OF SELENIUM ON CADMIUM TOXICITY IN CULTURED WEHI 164 CELLS M. Sta´nczyk, J. Gromadzi´nska, W. W˛asowicz Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, Lodz, Poland Selenium is an essential trace element that occurs in active site of antioxidative enzymes directly involved in redox reactions. Numerous literature data indicate also that selenium plays an important role in protection against toxic effects of cadmium, which is one of the most toxic substances in the environment. The aim of our study was to evaluate the effect of 48-h preincubation of WEHI 164 mouse fibrosarcoma cells in the presence of sodium selenite (0.3 ␮M) and medium containing 2% foetal bovine serum (FBS) (with decreased selenium concentration) on their viability after exposure for 24 h to cadmium chloride (7 ␮M). In order to intensify the effect of cadmium, hydrogen peroxide (H2 O2 ) at the concentration of 0.1 mM was added to culture medium. Cytotoxicity of the chemicals was determined using MTT reduction assay. We observed that the viability of WEHI 164 cells pre-treated with selenite and exposed to cadmium was increased in comparision to the cells exposed to cadmium alone. We also showed that the viability of the cells growing in medium containing 2% of FBS was significantly decreased in comparision to control cells cultured in the presence of 10% FBS. In our study H2 O2 had no significant effect on cadmium cytotoxicity. These results suggest that the concentration of selenium in culture medium can have a significant influence on WEHI

Abstracts / Toxicology Letters 158S (2005) S1–S258

164 cells viability and decrease cadmium cytotoxicity. This work was supported by grant from the Polish Scientific Research Committee (PB 0548/P05/2003/25). P17-24 MITOCHONDRIAL MEDIATED EFFECTS OF SURFACTANTS ON MA-10 CELL STEROIDOGENESIS D.R. Farmer1 , S.L. Levine1 , W.F. Heydens1 , R. Garnett1 , Z. Han2 , V. Papadopoulos2 1 Monsanto

Company, St. Louis, MO, USA; University Medical Center, Department of Biochemistry and Molecular Biology, Washington D.C., USA 2 Georgetown

A decrease in progesterone synthesis in MA-10 Leydig cells treated with cytotoxic concentrations of a concentrated Roundup herbicide formulation, led Walsh et al. 2000 to suggest that the formulation of Roundup they tested had endocrine disrupting potential by effecting expression of the steroidogenic acute regulatory (StaR) protein. The authors demonstrated that the formulation (Roundup Concentrate Herbicide 180 g a.s./L, 6.5 g/L POEA surfactant), not glyphosate, was responsible for the concentration-dependent but reversible decline in progesterone synthesis. We repeated this experiment in MA-10 cells, and included several household surfactants and a formulation blank not containing glyphosate. A sensitive cytotoxicity assay that indexed mitochondrial membrane damage was also included in our experiments. The results of our study demonstrate that decreased progesterone synthesis in MA10 cells results from surfactant-induced mitochondrial membrane damage, not a direct effect on the StAR expression. Assays conducted with household surfactants and the formulation blank produced a comparable effect on progesterone synthesis, which was coincident with mitochondrial membrane damage. The results of this study underscore the non-specific action of a variety of surfactants on cellular function in an in vitro test system and how this secondary activity can confound the interpretation of effects on biochemical processes when a suitable cytotoxicity assay is not used.


P17-25 CARBON MONOXIDE IN ANGIOGENESIS RESEARCH AND THE HET-CAM BIOASSAY E. Falkner1,2 , G. Holzer3 , C. Eder2,4 , O. Wagner3 , H. Schoeffl2 , U.M. Losert1 1 Core

Unit for Biomedical Research, Medical University Vienna; 2 zet—Centre for Alternative and Complementary Methods to Animal Testing; 3 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Vienna; 4 Department of Orthopaedic Surgery, Medical University Vienna The Hen’s Egg Test Chorioallantois Membrane (HETCAM) angiogenesis system, originally developed for irritation studies, was used for evaluation of effects of exponation of early breeding stage chicken egg CAMs (up to incubtion day 10) to carbon monoxide (CO): quantitative/qualitative effects of low doses (50–500 ppm) upon development of vessels were evaluated. CO application of doses at this concentration are reported to effect cell cultures in vitro, modulating/triggering expression of heme oxygenase enzymes and therby effecting angiogenesis in vivo. The suitability of the in ovo HET-CAM system for, e.g. angiogenesis/hypoxia but also CO-induced embryo toxicity testing is discussed. P17-26 TERBIUM CHELATES AS MARKERS FOR IRREVERSIBLE DNA CONFORMATIONAL CHANGES I. Cristescu, M. Ilie, D. Balalau, D. Baconi, A. Popa “Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Toxicology Department, Bucharest, Romania Lanthanide chelates are among the promising substances to be used in photodynamic therapy. Even if intensively studied, their interaction at the molecular level with proteins and/or DNA has still unclear aspects. The purpose of the study was to emphasise the DNA denaturation by using UV absorption spectrophotometry and fluorescence spectroscopy. As a fluorescence marker, the lanthanide chelate of terbium–diethylentriaminopentaacetic acid (TbDTPA) has been used. Experiments have been per-


Abstracts / Toxicology Letters 158S (2005) S1–S258

formed on aqueous double stranded, single stranded and denatured calf thymus DNA solutions, having in view the UV absorption, the fluorescence emission of Terbium and the scattering spectra. The experiments showed a DNA concentration-dependent fluorescence resonance energy transfer process in which the macromolecule is the donor and the metal is the acceptor. Results suggest that terbium binds both to the double stranded and the single stranded DNA molecule, the denatured DNA being more efficient in forming association complexes with the lanthanide chelate than the double stranded DNA. P18 Apopthosis and Cell Death P18-01 CELLULAR APOPTOSIS IN MICE BY INHALATION OF GLUES A. M´endez-Perez, M. G´onzalez, A. Casillas, G.P. Carreira S. Huerta QC Universidad Veracruzana, M´exico According to UNICEF in Latin America there are 30 million children addicted to toxic inhalations, made from toluene-benzene base glues. The constant inhalation of these lead to a cerebral organic syndrome characterized by the loss of intellectual capacity, motor disfunction, muscular spasms and ataxia. The inhalant lesions in 8-week-old Balb C mice was caused with industrial glues using the intoxicating technique of street children. The changes at the central nervous system level were characterized by mononuclear inflammatory cells, desmielinization, degeneration and axonal loss. The stress state and hypoxia generated the synthesis of HSP70. This in turn activated the microglia mediated by the presence of TLR 4 with induction of innate immunity. This indicates a polarization toward a cerebral immuno-competency substituting the normal state of immuno-suppression. In the tenth week of the mice samples a depopulation of 35% of oligodendrocyte cells and neurons was found. The neurons revealed the presence of liberated cytochrome C, apaf-

1, and important concentrations of caspasa 9. At the same time the neuronal growth factor diminished and Bim was activated leading to neuronal and oligodendrocytes apoptosis. P18-02 APOPTOSIS VERSUS CELLULAR PROLIFERATION IN GALACTOSAMINE INDUCED HEPATOTOXICITY B. S˜ao Braz1 , A. Alves2 , A.J. Silva1 1 Nucleus

of Pharmacology and Toxicology, CIISA/FMV, Lisboa, Portugal; 2 Dpt. Patologias e Cl´ınicas Veterin´arias, UTAD, Vila Real, Portugal Hepatotoxicity by galactosamine has been used as a model of acute hepatitis on a multiple kind of pharmaco-toxicological studies. However, is important to know how cellular hepatic population deals with this toxic insult. In this study we intended to evaluate both cellular death through apoptosis (TUNEL, Caspase-3) and cellular proliferation (PCNA). Animals (Wistar, adult male rats) were exposed (IP) to galactosamine (400 mg kg−1 b.w.), test group; control group received only saline (0.9%). At intervals of 24, 48, 72 and 96 h groups of animals are sacrificed and livers removed and fixed in 10% of neutral buffered formalin. Paraffin sections were made and stained for immunochemical markers of PCNA, Caspase-3 and DNA fragmentation (TUNEL). The intensity for Caspase-3 was scored as undetectable (−), weak (+) and moderate/strong (++). For TUNEL and PCNA 1000 (minimum) nucleus was counted and percentage of positive nucleus was established. As expected in control group both cellular death and proliferation are almost absent in hepatic tissue. At 24–48 h apoptosis achieved maximum expression (TUNEL and caspase-3), and at 96 h these markers expression was identical to control group. For cellular proliferation maximum expression was registered at 72 h but at 96 h remains elevated. So it can be concluded that in galactosamine hepatotoxicity sequential cellular death (through apoptosis) and proliferation occurs.

Abstracts / Toxicology Letters 158S (2005) S1–S258




1 “Carol

Davila” University of Medicine and Pharmacy, Toxicology Department, Bucharest, ROMANIA; 2 “Victor Babes” National Institute, Bucharest, Romania

1 Institute

Opioid receptors are expressed on a large panel of cells, beside those of the central nervous system. Recent evidence exists regarding an opioid mechanism of controlling inflammation and tumor progression. We investigated the effects exerted by agonists and antagonists of opioid receptors (methadone and naloxone) on the growth of the human lymphoblastic cell line K562. Cell proliferation and viability were measured by the tritium-labeled uridine incorporation method and the MTS reduction test. Apoptosis was evaluated by flow cytometry using annexin V and propidium iodide. Treatment of K562 cells with methadone (20–2000 ng/mL) inhibits uridine incorporation, without affecting cell viability. On the other hand, targeting of tumor cells with the opioid receptor antagonist naloxone (2–2000 ng/mL) also hinders cell proliferation, but another mechanism seems to be active, namely naloxone alters cell viability and favors apoptosis. Stimulatory effects of naloxone on uridine incorporation were also registered in some experiments, associated with an increase of apoptosis, suggesting a reparatory response of the cells to apoptotic signaling. Correlation analysis indicates that both methadone and naloxone preferentially inhibit highly proliferating cells. Our experimental results indicate that targeting opioid receptors expressed on neoplastic immune lymphocytes with agonists (methadone) and antagonists (naloxone) might control the tumor growth tumors (at least the lymphoblastic cell line K562).

Dendritic cells (DCs) are used in immunotherapy for the treatment of different tumors, of infectious diseases caused by viral, bacterial, parasitic and fungal pathogens, of autoimmune diseases or of transplant rejection. DCs are antigen-presenting cells, which initiate and regulate the immune response via T cell activation. The maturation of monocyte-derived DCs can be stimulated by cytokines. To study the impact of DNA repair capacity and apoptosis in cytokine-induced differentiation of monocytes into DCs, we compared monocytes, immature DCs and mature DCs as to the expression and activity of DNA repair enzymes and their sensitivity to alkylating drugs. Here we show that cytokine-induced differentiation of monocytes affects the activity and expression of O6 -methylguanine-DNA methyltransferase (MGMT). Further, we observed a change in the expression of DNA repair enzymes involved in base excision repair, such as FEN1. Different expression levels of DNA repair enzymes might influence DNA repair capacity and sensitivity of cytokine-stimulated cells to alkylating agents. Thus, we conducted cell sensitivity assays in order to determine cell survival and apoptosis upon treatment of monocytes and immature DC with alkylating agents and performed comet assays for the investigation of DNA repair capacity. Overall, the available data shows that during maturation of monocytes into dendritic cells a change of expression of DNA repair enzymes occurs having impact on the sensitivity of monocytes and differentiated DCs to genotoxic agents.

of Toxicology, Johannes Gutenberg University, D-55131 Mainz; 2 Department of Dermatology, Johannes Gutenberg University, D-55131 Mainz, Germany


Abstracts / Toxicology Letters 158S (2005) S1–S258



R. Zdanowski, M. Krzy˙zowska1 , J. Bany, M. Niemiałtowski2 , M. Kowalczyk

ˇ R. Fuchs, A.-M. Domijan, A. Luci´c VrdolV. Zlender, jak, M. Peraica, B. Radi´c

1 Department

Institute for Medical Research and Occupational Health, Ksaverska c. 2, Zagreb, Croatia

of Pharmacology and Toxicology, Military Institute of Hygiene and Epidemiology, Kozielska 4, 01-163 Warsaw, Poland; 2 Division of Immunology, Department of Pre-Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Ciszewskiego 8, 02-786 Warsaw, Poland For a past few years several papers have reported that nicotine possesses angiogenic properties, which appear to be mediated through non-neuronal nicotinic acetylcholine receptors. There are also several investigations showing involvement of cholinergic system in regulation of immune activity, angiogenesis and subsequent development of cancer. Cholinergic receptors are present in various effector cells of the immune system, such as lymphocytes, macrophages, mast cells, etc. Among others, acetylcholine modulates release of cytokines, proliferation or activation and inhibition of several immune cell types. In this study we evaluated the influence of irreversible acetylcholinesterase inhibition by acute isopropyl-methylphosphonofluoridate (IMPF) intoxication on the angiogenesis in mice with tumour. Sarcoma L1 cells (106 per animal) were given 14 days before starting each experiment. IMPF was administered subcutaneously into the dorso-lateral trunk of tumourus mice at the dose of 100 ␮g/kg. Liver, spleen and tumour cells were harvested at 4, 12, 24 h after injection with IMPF and subjected to measurement of mitochondrial potential with JC-1 stain and annexinV/propidium iodide staining. Results showed that IMPF intoxication led to induction of apoptosis in tumour cells, whereas liver and spleen cells showed decrease in apoptotic cells counts.

Ochratoxin A (OTA) is a mycotoxin produced by several Aspergillus and Penicillium species. It is known as a worldwide common food contaminant. This toxin has received considerable attention because of its deleterious effects on human and animal health. It has immunotoxic, hepatotoxic, teratogenic, neurotoxic and carcinogenic properties in different experimental animals, with the kidney being its main target organ. The role of OTA as a possible causative agent involved in the etiology of human endemic nephropathy has still not been ruled out. To study adverse effects of OTA on cellular level, three renal cell lines (CV-1, Hek293, LLC-PK1) were cultivated and exposed to the different OTA concentrations, ranging from 0.1 to 50 ␮M, respectively. Metabolic activity was measured by using thiazolyl-tetrazolium bromide mitochondrial dehydrogenase activity test (MTT) over 72 h. Remaining cell viability after treatment with 20 ␮M OTA amounted to 18.2% for CV-1 and 64.9% for LLC-PK1. The same OTA concentration induced cell death of almost 43% Hek293 cell population. Induction of apoptotic and necrotic cell death was measured by anexin V cell labeling and flow cytometry analysis after being exposed to treatment in the same OTA concentration range over 24 h. Results suggest to apoptosis and necrosis induction in OTA dose-dependent manner. P18-07 FLY ASH LEACHATE INDUCES CYTOTOXIC EFFECT IN CULTURED HEPATOCYTES OF FRESH WATER FISH CHANNA PUNCTATA (BLOCH.) AN IN VITRO ASSESSMENT M. Ali, A. Sageer, Khan Hasib-ur-Rehman, S. Raisuddin Ecotoxicology and Immunotoxicology Lab., Faculty of science, Hamdard University, New Delhi-110062, India

Abstracts / Toxicology Letters 158S (2005) S1–S258

Fly ash is a complex mixture of heavy metals, dioxins, difurans, PCBs, PCDs, PAHs and PHAH produced by combustion of coal. Recently, we reported its effect on antioxidant level of fresh water fish Channa punctata (Bloch.) (Ali et al., 2004). Cultured hepatocytes of Channa punctata (Bloch.) was used to assess cytotoxicity of fly ash leachate (FAL). Cells were treated with different concentration of FAL (10, 20, 50 and 100%) for 24 and 48 h. The LDH, H2 O2 superoxide ions generation and apoptosis were estimated by standard protocols. We were also interested to measure the lipid peroxidation. The production of H2 O2 superoxide and level of Lipid Peroxidation (LPO) increased significantly (p < 0.01, 0.001) with concentration. Lactate dehydrogenase (LDH) release and Percentage of apoptotic cells were also increased significantly (p < 0.01, 0.001) with concentration. It may be concluded that FAL causes LPO by producing ROS, such as H2 O2 superoxide ions. Moreover, LDH release and induction of apoptosis in the cells may also be due to ROS. This is first kind of study to assess the cell death by complex mixture of toxicant like fly ash. Further investigations are under process. P18-08 THE EFFECT OF NCX 4016 AND NCX 4040, TWO NITRIC OXIDE-DONATING ASPIRIN DERIVATIVES, ON APOPTOSIS OF NEUTROPHIL GRANULOCYTES IN VITRO S. Vasilev1 , I. Majstorovic1 , D. Vucevic1 , S. Gasic1 , S. Vasilijic1 , B. Bufan1 , V. Cupic2 , M. Colic1 1 Institute

for Medical Research, MMA, Belgrade, Serbia and Montenegro; 2 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Belgrade, Serbia and Montenegro Apoptosis of neutrophil granulocytes is a critical event in the resolution of inflammation. In this work we studied the effect of two nitric oxide(NO)-donating aspirin derivatives (NCX 4016 and NCX 4040) on apoptosis of inflammatory granulocytes. These aspirin analogues have been primarily synthesized to reduce the side effects of aspirins on the gastrointestinal tract. Inflammatory granulocytes have been isolated from implanted polyvinyl sponges under skin of Albino Oxford (AO) rats. Apoptosis was measured after incubation of the cells with different concentrations of NO-


aspirins. Apoptosis was detected by staining cells with propidium iodide and analysis by flow cytometry, as well as, by morphological criteria. We found that NCX 4016 and NCX 4040 at concentrations 50–500 ␮M and 0.25–10 ␮M respectively, induced apoptosis of rat inflammatory granulocytes in a dose-dependent manner. Aspirin induced apoptosis of these cells only at high pharmacological concentration (10 mM). NCX 4016 at lower concentrations (0.5 ␮M–5 ␮M) inhibited apoptosis. NO-aspirins differently modulated apoptosis of inflammatory granulocytes, as well as production of TNF␣, IL-1␤ and NO, depending on applied dose, suggesting that their mode of action on these cells are not the same. In conclusion, our results suggest that anti-inflammatory properties of NO-aspirins are additionally potentiated by their proapoptotic effect on granulocytes. Thiscould possibly represent a novel mechanism of their action. P18-09 ASSESSMENT OF NUCLEAR FACTOR KAPPA B (NF-␬B) INVOLVEMENT IN APOPTOSIS INDUCED BY TRIBUTYLTIN M. St˛epnik, M. Sta´nczyk, J. Arkusz, D. Lewi´nska Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, Łodz, Poland The results of numerous studies provide evidence that after inhibition of NF-␬B activity the level of apoptosis in some cancer cells increases. However, the role of NF␬B in the induction of apoptosis is still unclear and it likely depends on the cell type and the type of inducer. The aim of our study was to investigate the role of NF-␬B in apoptosis induced by tributylin chloride (TBT) the known proapoptotic agent. Cytotoxicity of the compound on Jurkat cells was assessed with MTT reduction assay. Sulindac and pyrrolidinedithiocarbamate (PDTC) were used as inhibitors and phorbolmyristate acetate (PMA) was used as an activator of NF-␬B. We observed that cytotoxicity of TBT (1.3 ␮M) after 24 h of incubation in the presence of sulindac (0.5 mM) was significantly increased. In TUNEL staining significant increase in the number of apoptotic cells was shown (control cells 2 ± 0.9%, sulindac 2 ± 0.5%, TBT 28 ± 13%, TBT+sulindac 43 ± 8%). The changes were associated with increased activity of caspase-3 in the


Abstracts / Toxicology Letters 158S (2005) S1–S258

exposed cells. PDTC in non-cytotoxic concentration of 10 ␮M had no effect. PMA (100 ng/ml) showed clear protection against cytotoxicity of the chemical on Jurkat cells. It significantly decreased the number of apoptotic cells (control cell 2 ± 0.4%, PMA 1 ± 0.8%, TBT 25 ± 11%, TBT + PMA 19 ± 5%) and caspase-3 activity. The results suggest that activation of NF-␬B may protect Jurkat cells against proapoptotic effects of TBT while its inhibition may enhance the cytotoxicity.


P19 Clinical Toxicology

The purpose of this study is to evaluate the acute adult poisoning data and identify the risk factors of mortality during 6 years at a university emergency department in Turkey. In this survey, data collected from 2229 adult patients admitted to the Emergency Department within January 1st 1997 and December 31st 2002. Of all emergency admissions during 6 years percent of poisoning was 1.6%. Of the total poisoning cases, 725 (32.5%) were males and 1504 were females. Mean age was 29.3 ± 13.2 for males and 23.8 ± 9.6 for females (p = 0.001). Majority of the cases (76.4%) were suicidal poisoning. Attempted suicide ratio is higher in women (gender ratio; 2 .4) and youth. Drugs were the most frequent cause of poisoning (59.0%), pesticides was the second (26.4%). Psychoactive drugs were the most common agent (33.5%) among drugs with its rate increasing by years. Eighty-seven patients died (3.9%) and alcohol (methanol) was the most frequent (20.6%) cause of death followed by mushroom (11.5%), carbon monoxide (10.3%) and pesticides (8.3%). Mortality rate decrease by years was significant (p = 0.001). More men than women died by poisoning (OR: 1.8 CI: 1.1–3.1). Gender, age, season, clinical status, initial emergency care and type of substance were significant risk factors for mortality. This study points out that organic phosphorus is a serious risk for poisoning and mortality in this area. Providing satisfactory public and emergency staff assistance and education on the subject may reduce the mortality rate.

P19-01 LIBIDO CHANGES IN MUSTARD VICTIMS IN KHOZESTAN PROVICE, IRAN P. Afshari Islamic Azad University Dezful Branch Chemical gases are defined as materials that are used in military operation and cause: lethal effects, damaging effects and incapacitating effects. Dr. Balali and Dr. Moorie in a research called “Third research on long term effects of poisoning with MS” mentioned their action. Decrease in libido sequence registered in %52 of soldiers. Interesting point about 4 patients (9.7%) was increasing of libido that it reported in no publication. To determine the effect of mustard on libido we did this research. It was a historical cohort study. At first we prepared a questionnaire in the next step we selected 200 chemically injured soldiers randomized and 200 soldiers who were at war but were not exposed to mustard. We had an interview with them. The time after exposure to gas was 14 years ago. Data analyses by SPSS statistical softwar. The mean age of soldiers at the time of study in case group was 40 and in control group were 38.8. Their wives age in case group were 33.6 and in control group it was 31.9. In case group 71.6% had decrease in libido, 9.7% increse in libido and 19.6% not had any changes in libido and in control group it was 16.2%, 1.2%, 77%. Statistical tests showed significant differences between the two groups and in case group between before and after exposure. This research showed that exposure to mustard can reduce the libido in mustard victims.

G. Seydaoglu1 , S. Satar2 , N. Alparslan1 1 Cukurova University, School of Medicine Department

of Biostatistics, Balcali/Adana, Turkey; 2 Cukurova University, School of Medicine, Emergency Department, Balcali/Adana, Turkey

Abstracts / Toxicology Letters 158S (2005) S1–S258

P19-03 ACUTE INTOXICATION WITH DOXAZOSIN: A CASE REPORT S. Satar, A. Sebe, A. Avci, H. Yesilagac, Y. Gokel Cukurova University, School of Medicine, Department of Emergency Medicine, Balcali/Adana, Turkey Experience with overdosage and toxicity with the alpha adrenoreceptor antagonists remains very limited in the literature. In this paper, the second case in the literature with doxazosin overdosage is reported. Supportive treatment was given to the patient and the patient was discharged after 48 h of admission to the emergency department. P19-04 INTRACRANIAL HEMORRHAGE ASSOCIATED WITH METHANOL INTOXICATION S. Satar, A. Sebe, B. Uzun, M. Topal, H. Yesilagac Cukurova University, School of Medicine, Department of Emergency Medicine, Balcali/Adana, Turkey Methanol is a common component of gasoline, antifreeze, wash fluid, perfume, household cleaners and various other industrial products. Acute methanol poisoning produces severe metabolic acidosis, serious neurologic sequelae and rarely imaging findings. In this paper, we described a 35-year-old man with methanol intoxication who was in vegetative stage and computer tomography showed brain edema and hemorrhage in the temporal lobe. P19-05 PROGNOSTIC FACTORS IN UNINTENTIONAL CARBON MONOXIDE POISONING D. Chaparoska Department of Urgent Internal Medicine and Toxicology, University Clinical Centre, Skopje, R. Macedonia The aim of the study is to identify early predictors of recovery from mild carbon monoxide poisoning and to search for qualitative interactions between subsets of patients and treatment effects. Eleven victims of unintentional, fire-related, in house, smoke inhalation poisoning, were evaluated if the time elapsed from end of exposure to hospital


admission was less than 1 h, the carboxyhemoglobin level was greater than or equal to 20% for smoker or non-smoker, respectively, and if they did not suffer a loss of consciousness. 100% oxygen therapy was given. Diagnosis of inhalation injury from smoke inhalation is generally clinical. Acidosis, hypoxia and hypercarbia in arterial blood gas measurements. In this study that obtain blood samples of smoke inhalation victims at the scene found that a plasma lactate concentration above 10 mmol/l, was a sensitive indicator of CN intoxication independent of hypoxemia. The direct measure of carboxyhemoglobin was >20%. Patients with inhalation injury had negative initial chest radiograph. Apathy and headaches were the main moderate sequel. The multivariate analysis selected dizziness before admission and headaches upon hospital admission as jointly associated with persistent neurological symptoms. One month after mild carbon monoxide intoxication, victims who presented dizziness before hospital admission or headaches upon admission have only an increased risk of minor persistent neurological symptoms, but almost all patients could resume their former occupation. P19-06 SEVERE HEPATITIS CAUSED BY AMANITA PHALLOIDES POISONING D. Chaparoska, V. Jordanov Department of Urgent Internal Medicine and Toxicology, University Clinical Centre, Skopje, R. Macedonia Toxic mushroom poisoning leeds to a variety of clinical outcomes ranging from self-limited gastrointestinal symptoms to fulminant hepatic failure requiring orthotopic liver transplantation. We reviewed the outcomes of patients with severe acute hepatitis secondary to amanita phalloides poisoning, treated with contemporary modalities. We retrospectively reviewed patients admitted to our institution over a 5-year period (2000–2005) with elevated transaminase levels (>1000 IU/L) attributed to recent mushroom ingestion. The patient’s clinical course, laboratory data, and treatment regimen were recorded and analyzed. The mean peak serum levels were: aspirate transaminase 6520 IU/L, alanine transaminaze 8860 IU/L, total bilirubin 11.5 mg/dL, creatinine 535 Mmol/L, and pro-


Abstracts / Toxicology Letters 158S (2005) S1–S258

thrombin time-international normalized ratio (PT-INR) >1.0. Three patients developed acute renal failure requiring hemodialysis and hemoperfusion. The other 17 patients survived without significant morbidity. Patients with severe hepatitis from amanita phalloides poisoning are thought to have a poor prognosis and frequently need liver transplantation for survival. We suggest that with early and aggresive multidisciplinary care, such patients recovered from severe hepatitis caused by amanita phalloides poisoning, without liver transplantation.

SSRIs antidepressant paroxetine and venlafaxine were similar in effectiveness for depressive minor syndrome to chronic addicted patients. We have established the improvement of parameters related to quality of life over the entire period of study.


1 Department

D. Ionescu1 , C. Cristescu2 1 Department of Toxicology, Faculty of Pharmacy, Uni-

versity of Medecine and Pharmacy “Victor Babe¸s” Timi¸soara, Roumanie; 2 Department of Pharmacology, Faculty of Pharmacy, University of Medecine and Pharmacy “Victor Babe¸s” Timi¸soara Selective Serotonin Reuptake Inhibitors (SSRIs) represent the most commonly prescribed class of antidepressants. This study compares the effectiveness of paroxetine (Seroxat) and venlafaxine (Efectin) in the case of chronic ethylic patients diagnosed with minor depressive syndrome. We have achieved a prospective clinical study during 9 months in Department of Psychiatry focused in chronic ethylic pathology and minor depressive comorbidity. A number of 129 patients were comprised in this programme: 64 (49.62%) were treated with paroxetine and 65 (50.38%) with venlafaxine. All patients were identified and validated according to DSM-IV and ICD-10 criteria. At the beginning and to 1, 3, 6 and 9 months of therapy we performed a separate assessment by means of Hamilton, Beck scales and statistics instrument EPI-Info6. It comes out that both groups have comparable scores on all measures and at all time points of our study. The incidence of adverse effects and abandon rates was higher in the case of patients with venlafaxine therapy.

P19-08 AN UNUSUAL INTOXICATION BY Hyoscyamus albus: ABOUT TWO CASES C. Hamouda1 , H. Ghorbal2 , K. Cherni2 , M. Allouche1 , A. Moklin1 , A. H´edhili2 , M. Amamou1 of Intensive care, CAMU, Tunis, 1008 Tunisia; 2 Laboratory of toxicology, CAMU, Tunis, 1008 Tunisia In Tunisia plants were always used as drugs in traditional medicine and aliments. Many cases of accidental intoxications are reported every year; Most of them are caused by confusion between comestible plants and toxic plants. They also may be caused by an overdose in plant’s use. In this work, we report two poisoning cases by Hyoscyamus albus “Jusquiame”. The two patients were hospitalized in unit of intensive care of the antipoisons center of Tunis, because of collective ingestion of a vegetable stock containing Chinese leaves and jusquiame leaves. The patients are two males aged, respectively 26 and 61 years. The intoxication diagnosis was based on anamnesis, physical examination and toxicological analysis. The medical investigation revealed a confusion between chinese leaves and jusquiame leaves. The principal clinical symptom was an anticholinergic syndrome (mydriasis, mouth dryness, retention of urines). For these patients no neurological symptoms were observed by factory medical staff. Toxicological analysis based on the alkaloids detection in urines by thin layer chromatography showed the presence of atropine. After symptomatic treatment, the anticholinergic symptoms and digestive disorders disappeared into 24 h, therefore the prognosis of these two cases was favourable.

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P19-09 SNAKEBITE DURING PREGNANCY A. Sebe, S. Satar, A. Acikalin, F. Icme Cukurova University, School of Medicine, Department of Emergency Medicine, Balcali/Adana, Turkey Little is known about snakebites during pregnancy. The fact that venomous snakebites during pregnancy result in a high fetal wastage and that venomous snakebites may cause maternal mortality, makes this an important, albeit, uncommonly encountered entity in emergency. Only few cases of snakebite in pregnancy have been reported in the literature and in this paper we reported 3 cases with snakebite in the pregnancy, treated successfully. P19-10 DETERMINATION OF OXIDATIVE STRESS, SOME CLINICAL BIOMARKERS AND BLOOD ALCOHOL LEVEL IN CHRONIC ALCOHOLIC PATIENTS ADMITTED TO A PSYCHIATRY CLINIC 2 , S. Yalc ˙ Ilhan ˙ S¸.S¸. C ¸ ec¸en1 , A. Karakus¸1 , I. ¸ ın1 , E. Dural1 , Y. Do˘gan2 , T. S¨oylemezo˘glu1 1 Ankara

University, Institute of Forensic Medicine, Dikimevi, Ankara, Turkey; 2 Ankara University, Faculty of Medicine, Department of Psychiatry, Alcoholism Department, Dikimevi, Ankara, Turkey The objective of this study was to evaluate oxidative damage in 20 male patients who were admitted to a psychiatry clinic with alcoholism diagnosis and compared with 20 healthy male who drink occasionally. Blood specimens of both groups were tested for alcohol by headspace gas chromatography upon first admission to the clinic. Malondialdehyde (MDA) and total sulphydryl levels (TSH) were determined as biomarkers of oxidative stress. Patients were also subjected to detailed laboratory investigation. Transaminases (AST, ALT), gama glutamyl transferase (GGT), mean corpuscular volume (MCV) were measured for this aim. The results showed chronic alcohol consumption enchanced MDA level (p = 0.021) and reduced TSH levels significantly


(p = 0.047) in plasma when compared to control. A significant increasein alcoholic patient was observed when AST (p = 0.035) and GGT (p = 0.000) values were compared with the control group. However, no significant difference was noted in ALT levels (p = 0.226). A notable value was observed in MCV (92.53 ± 4.69) in alcoholic patients because it was found to be very close to the upper reference value (81–101 fL). P19-11 FLUORESCENCE STUDY OF METHOTREXATE LEVELS IN CHILDREN TREATED FOR DIFFERENT NEOPLASTIC DISEASES M. Barca1 , D. Balalau1 , M. Ilie1 , D.L. Baconi1 , N. Mitrea1 , C. Dancus2 , E. Gutescu1 1 Carol

Davila, University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest, Romania; 2 Al. Trestioreanu, Oncologic Institute, Bucharest, Romania Methotrexate (amethopterine, 4-amino-N-10methylpteroyl glutamic acid) is a well known antineoplastic agent. The administration of methotrexate in high dosage has the potential of serious intoxications, mainly in children, and the therapist needs to correlate the methotrexate plasmatic concentration to the treatment protocol. The paper presents such a study aimed at the quantification of the methotrexate level in peripheral blood plasma of children treated with the agent in view of determining the patients having high methotrexate blood levels and/or a too slow elimination of the drug. The method implies the methotrexate oxidation with potassium permanganate in acetate buffer (pH 5) in presence of ascorbic acid, followed by fluorescence quantification (excitation 380 nm, emission 457 nm). The method is fast, accurate, requires rather small quantities of sample, inexpensive reagents and devices. By using the method concentrations of 0.01 micrograms/mL could be accurately determined. The study showed there is a great inter-individual variability in methotrexate metabolisation, stressing once again the necessity of monitoring methotrexate therapy.


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P19-12 APPLICATION OF COMMERCIAL HPLC METHOD FOR ANALYSIS OF ANTIEPILEPTIC DRUGS S. Yakis¸tiran1 , M. Kurt1 , O. G¨uven1 , S¸.S¸. C ¸ ec¸en2 , 2 T. S¨oylemezo˘glu 1 Ministry of Health, Refik Saydam Hygiene Center, Poi-

son Research Department, Ankara, Turkey; 2 Ankara University, Institute of Forensic Medicine, Dikimevi, Ankara, Turkey To determine antiepileptic drugs, such as carbamazepine, a carbamazepine metabolite carbamazepine 10,11-epoxide, which is believed to have a contribution to its therapeutic action and side effects, phenytoin, phenobarbital, ethosuximide, primidone and lamotrigine in serum, an HPLC method was established by using a commercial procedure, Chromsystems. It is important to monitor antiepileptic drug levels routinely for the correct medication or to avoid serious side effects or toxicity. For all indicated drugs limit of detection ranged between 0.3–0.5 mg/L except for ethosuximide (2.5 mg/L). Calibration graphs were linear for all the drugs with R2 values greater than 0.99 and recovery rates were found to be over 95%. In the serum of 29 epilepsy patients admitted to Ministry of Health, Refik Saydam Hygiene Center, Poison Research Department were screened for antiepileptic drug levels by a commercial extraction technique with HPLC at 204 nm. In some patients more than one of the drugs were detected according to the medication given. Mean concentration range of the drugs in serum of the epilepsy patients carbamazepine (7.81 ± 3.82), carbamazepine 10, 11 epoxide (2.50 ± 0.27), phenytoin (17.24 ± 18.50), phenobarbital (24.06 ± 11.72), ethosuximide (11.20 ± 1.85) and primidone (8.71 ± 4.63). P19-13 THE EFFECT OF DRESSING WITH HONEY ON HEALING OF ORTHOPAEDIC WOUNDS M. Fakoor Department of Orthopaedy, Ahwaz Jondishapour University of Medical Sciences, Ahwaz, Iran Using antimicrobial effect of plants and natural material was recommended from ancient years. Now, it is used by many people from all over the word. New

researches in this grade show such property of applications because herb and medicinal plants have not side effect. One of the oldest material in traditional medicine has been for treatment of infections is honey. Honey is a natural valuable gift that has been diagnosed as the best food production. It is clear that effects of honey on all human physiological systems are important. In old times medicine put more emphasis on medical effects of honey. The aim of this study was to show the effect of dressing with honey on healing of wounds. The type of study was clinical trial. One hundred and thirty seven patients were studied. All of the patients had an orthopaedic surgery and had discharge from incision after surgery. They took antibiotic, but this therapy could not help them alone. We added dressing with honey to their treatment. All of the were patients visited every day and had their dressing changed. After two weeks we analyzed the data. 137 patients (M = 81%, F = 19%) were studied. The mean age was 47. 96.3% and 3.17% of patients had fracture and surgery on lower extremity and upper extremity respectively. 31.3% of patients had diabetic gangrene in the foot. 69.4% had open fracture (grade III). After 2 weeks discharge deceased and the patients became well. 100% of patients were satisfied with dressing with honey. Honey has several antiseptic and antibiotic properties, which are not harmful to maul beside it has a bacteriostatic (inhibitive) effect. There is very little water in honey therefore it’s osmotic pressure is very high and dehydrates bacteria which leads them to their death or becoming inactive and it seems that the cause of well being of our patients were this effect of honey. P19-14 DYNAMICS OF PSYCHOACTIVE DRUGS ABUSED IN LITHUANIA WITHIN 1992–2004 Z. Minkuvien˙e, R. Damijonaitien˙e, A. Benoˇsis University of Lithuania, Institute of Forensic Medicine, Vilnius, Lithuania Within the period 1990–2003 the number of drug abusers in Lithuania has increased by over ten times, i.e., from 13.2 cases in 1990 up to 135.2 cases per 100 000 inhabitants in 2003.

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The analysis of data on 8203 persons’ biological mediums investigations performed at the Toxicology laboratory of the Institute of Forensic Medicine during the decade (1992–2001) has revealed the following data: most frequently the following narcoticpsychotropic materials were identified in the investigated samples: opiates, benzodiazepines and psychostimulators, respectively 79.6%, 31.9% and 6.6%; in the majority of cases narcotic-psychotropic materials in biological samples were identified alone, in 60.3% of cases, in combinations of two materials, in 26.8% of cases and in combinations of three and more materials in one sample, in 12.9% of cases; the majority of narcotic-psychotropic material abusers were 15–34year-old male (72.1%) and female (70.9%). The analysis of data on 5543 persons’ biological mediums investigation performed at the Toxicology laboratory of the Institute of Forensic Medicine during the recent 3 years (2002–2004) has revealed changes in the spectrum of materials identified in biological mediums: opiate abuse remains stably high, 78.07%, but benzodiazepines group material abuse has a tendency to decrease to 16.06%. Whereas, the psycho-stimulator group materials abuse has significantly increased up to 14.93%. The cannabis products abuse is also increasing: given that within the period 1992–2001 among the spectrum of the identified psychoactive materials they made only 1.1%, in 2002–2004 the number has increased up to 5.17%.

In proportion to Greek legislation for drugs of abuse drug abusers are ranged into three groups: drug addicts, simple users and non-users. The Court should discriminate between these attributes. The law demands an expert report (i.e medical, psychiatric, clinical, toxicological or other, e.g segmental hair testing) to be conducted. Ninety-three cases related to the judicial verification of addiction in Crete were studied. Information from each file about the process, the decision, the police record, the indictment, the expert reports, the defendant’s individuality, the crimes, the penal confrontal, etc. were classified into 11 groups. Conclusions on addiction and particularly about the conduction of the expert reports and theirs evident value is presented. 6 cases are methodically examined and analytically presented. The Court is aloud to justify this decision in other conclusive evidence besides an expert report. The conclusion of the report occurs in the disengaged and incontrollable judgment of the Court. In case of opponent expert reports the Court is allowed to reject one based on special justification and under specific preconditions. Evaluation of the 9 common scientific criteria of addiction. The usefulness and credibility of the dependence criteria are trusted on the reliability and trueness of the defendants answers. The medical procedure for the diagnosis of addiction that the law enforces in none case was abided by. The chronic frames and the examination by several experts are impossible to be kept to. Contrarily hair testing is accepted in Court, although is not specially refereed in any Law.



M.A. Savvopoulos1 , M.N. Tzatzarakis2 , E. Pallis2 , K. Giannakoudakis2 , N. Paritsis3 , A.M. Tsatsakis2 1 Center

of Toxicological Sciences and Research, Department of Medicine, University of Crete, Heraklion, Greece, PhD Candidate, Member of the Heraklion of Greece Bar Association; 2 Center of Toxicological Sciences and Research, Department of Medicine, University of Crete, Heraklion, Greece; 3 Department of Psychiatry and Behavioral Sciences, Faculty of Medicine, University Hospital of Heraklion, Crete, Greece

C. Cristescu1 , M. Voicu1 , D. Ionescu2 , L. Dr˘agan3 1 Department

of Clinical Pharmacy, Faculty of Pharmacy, Timi¸soara, Romania; 2 Department of Toxicology, Faculty of Pharmacy, Timi¸soara, Romania; 3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Timi¸soara, Romania Clinically manifested cardiac failure can be observed at a significant percentage (30%) of patients who underwent an acute myocardial infarction (AMI), half of


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them passing away in the first year after the debut of coronarian accident. Administrated a few days after the AMI, angiotensin-converting enzyme (ACE) inhibitors have favorable effects concerning the quality of patient’s life and surviving hope. The goal of this research was to study the adverse effects of ACE inhibitors administration in the acute stage of MI. The group investigated comprised 31 patients, hospitalized for AMI. The treatment with ACE inhibitors was initialized between days 1 and 11 from the debut of AMI. The patients were hospitalized for a mean interval of 8 days. After externalization they were followed up for 10 months. During the hospitalization, the treatment with ACE inhibitors was interrupted in four patients (14%) due to the apparition of some adverse effects (symptomatic hypotension, pectoral angina, irritative cough). For the rest of patients who continued the treatment with ACE inhibitors (86%) was observed a significant improvement of left ventricle ejection fraction, from 48 ± 11% at the debut of study at 52 ± 7% after 1 month of treatment. The small incidence of adverse effects in investigated patients might be explained by their careful selection at the choice of optimum treatment moment, after their hemodynamical stabilization.

The most results among the men have occured in the age groups of 20–29, 30–39 and 40–49. It should be mentioned that the number of cases has been growing especially rapidly among the age groups of 20–29, as well as 40–49 and 60–69. The most results among the women have occured in the age groups of 50–59 and the number has continue growing in the age groups of 20–29 and 40–49, while, the number in the age groups of 0–19 has been stable growing down. During the analysed period in the men groups the number has been growing in cases of founded alcohol, drugs and psychotropical materials, however, the number has been growing down in cases of founded medicine. During the analysed period in the women groups the number has been growing in cases of founded medicine, drugs and psychotropic materials, however, the number has been growing down in cases of founded alcohol. In conclusion, the results of the analysis have highlighted that there is a tendency appearing of the growing number among young people (20–29) as well as middle-age people (40–49).


2 Pfizer

P. Vainauskas, A. Jasulaitis, V. Stonkus The goal of the research, to analyse the results carried out by the laboratory of toxicology of the Forensic Medicine Institut of the University of Justice of Lithuania in 2001–2003. The aim of the results, to find out the relationship between men and women involved in the reserach during the analysed period, their position and the change of dynamics among the age groups as well as the most popular materials groups discovered among them. In 2001–2003 have been carried out 498 positive results of the toxicological analysis by the postmortem material. Of the analysis by the men the number of positive results has been growing during the whole analysed period, however, the number of the results by the women has remained stable.

P19-18 ESTABLISHMENT OF ECHOCARDIOGRAPHY IN THE MARMOSET G. Rochefort1 , V. Eder1 , M. Gautier1 , P. Bonnet1 , G. Hanton2 1 LABPART,

Facult´e de M´edecine, Tours, France; Global Research and Development, Amboise,

France The aim of the study was to set up the methodology for echocardiographic recording in marmosets and to establish a preliminary database for major echocardiographic parameters. Echocardiographic examinations were conducted on males and females marmosets using a 13 MHz sector scan transducer. The animals were anesthetized and the heart was visualized in two-dimensional (2-D) echocardiography in right parasternal incidence allowing long and short axis section and in apical (left parasternal) incidence for 4-cavity sections. The long axis section was used for time-movement (TM) echocardiography across either right and left ventricles or aorta and left atrium. Systolic and diastolic dimensions of these cavities and indicators of contractility (fractional shortening) were measured. The short axis section done in

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the upper part of the heart for visualizing the arterial trunk vessels was used for Doppler investigation of pulmonary flow. The apical incidence was used for measurement of the area of the four cavities (and subsequent calculation of left ventricle volume) and for Doppler investigation of aortic, mitral and tricuspid flows. For the atrioventricular flows, the maximum velocity (Vmax) of both E and A wave, the deceleration time of E wave and the velocity time integral (VTI) of the flow were measured. For the arterial flows, Vmax, VTI as well as pre-ejection, acceleration and ejection times were measured. Using 11 marmosets, the method for 2-D, TM and Doppler recording was set up in this species. In particular, the optimal position and incidence of the transducer for visualizing the heart were established. The 2-D views were then easily obtained and allowed the assessment of a number of parameters characterizing the function and morphology of the marmoset heart. A preliminary database for these parameters has been established. Standardization of the methodology was found critical for Doppler recording. In conclusion, we have established the method for echocardiographic recording in marmosets, together with a preliminary database for major parameters of cardiac function and morphology in this species. P19-19 SONOGRAPHIC EXPLORATION OF THE SUPERIOR MESENTERIC ARTERIAL BLOOD FLOW IN ADULT RATS S. Serriere1 , F. Tranquart1 , G. Hanton2 1 Facult´ e

de M´edecine, University of Tours, France; 2 Pfizer Global Research and Development, Amboise, France The mesenteric arteries of the rat are the toxicological target of a number of drugs, in particular some subtypes of phosphodiesterase inhibitors and these adverse effects are possibly related to local haemodynamic changes. The aim of this study was therefore firstly, to establish the method for echographic examination of mesenteric arteries in rats, using color coded Doppler and secondly to measure changes in mesenteric flow following treatment with reference vasoactive drugs. Doppler sonography was done in adults SpragueDawley rats under gaseous anesthesia (Aerrane® , 1.5%


in oxygene 2 L/min) using Technos scanner (ESAOTE, Firenze, Italy) equipped with an 8–14 MHz linear probe. Doppler examination was performed on longitudinal view of abdominal aorta with angle correction and a sample gate of 2 mm positioned at the origin of the superior mesenteric artery. All animals were in dietary status (T1). PSV (peak systolic velocity), EDV (peak diastolic velocity) and RI (resistance index) were determined as standard parameters of the mesenteric flow. A good reproducibility of results was obtained from different groups of rats. In follow up experiment, the 3 rats received intravenous injections of noradrenalin (NOR, vasoconstrictor) and/or risordan (RIS, vasodilator). These treatments produced marked changes in mesenteric velocities 1 min after drug injection. Mean PSV was 63 cm/s before treatment and, respectively 52 cm/s and 108 cm/s after injection of NOR and RIS respectively. Mean EDV was 14 cm/s before treatment and 12.63 and 31.33 cm/s after treatment with NOR and RIS respectively. In conclusion we have established the ultrasonographic method for recording and characterizing mesenteric flow in untreated rats. Application to this methodology to rats treated with vasodilators or vasoconstrictors showed that it could evaluate drug-induced changes. P19-20 INFLAMMATION REACTIONS MEDIATE INDUCTION OF MRP EXPRESSION IN HUMAN LUNG CELLS A. R¨amisch1 , A.R. Wageeh Torky1 , S. Hofmann2 , H. Foth1 1 Institute

of Environmental Toxicology, MartinLuther University Halle/Saale, Germany; 2 Institute of Cardiothoracic Surgery, Martin-Luther University Halle/Saale, Germany The multidrug resistance related proteins are membrane-bound efflux transporters. About their transcriptional regulation only minor information is available. There is evidence that inflammation reactions play a role in the regulation of MRP. In our study, we investigated the effect of prostaglandins E2 and F2␣ on the expression of MRP1, 3, 4 and 5 in human lung cells. We used normal human


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bronchial epithelial cells (NHBEC), peripheral lung cells (PLC) and the tumour cell line A549 as test tools. We could show that MRP1 reacts with increase of expression after prostaglandin E2 treatment only in NHBEC whereas PLC and A549 did not react. This could be correlated with higher transport-activity of MRP1. Prostaglandin F2␣ did not show any effect in all used cell-types? We could also prove that MRP3 and 5 but not MRP4 react to Prostaglandin E2 treatment but in different grades. Preliminary expeiments using the inflammation mediators EGF and Interleukin 1␤ did not show any effect on MRP1- but on MRP3expression. MRP4 and 5 showed a suspicious reaction to EGF Treatment. In bronchial epithelial cells MRP4 and 5 were induced by IL-1␤. Because of the variable response of the different transporter isoforms to inflamatory mediators, we suggest the involvment of different signal transduction pathways as regulatory factors for MRP-proteins. P19-21 PERSPECTIVES OF AN AGED PRIMATE COLONY FOR THE STUDY OF METABOLIC DISORDERS J. Henken, P. Nowak, R. Korte, F. Vogel, G.F. Weinbauer Covance Laboratories GmbH, Kesselfeld 29, 48163 M¨unster, Germany Life expectency of mankind is predicted to attain about 100 years by 2050 and the proportion of people older than 60 years is estimated to exceed 20% of the population by 2050(1). Inevitably, the prevalence of type diabetes 2, metabolic syndrome and obesity will increase with great bearing on society and economy necessitating the need for development of relevant animal disease models. The aim of the present work was to screen a colony of aged nonhuman primates for the prevalence of metabolic diseases. Mauritian male and female cynomolgus monkeys (Macaca fascicularis) were retained as retired breeders (animals generally >13 years of age) and screened for fastened blood sugar levels, HbA1c, cholesterol, triglycerides, insulin, C-peptide and glucagon. Evidence for diabetes during initial screening was obtained in approx. 7% of animals. Whereas cholesterol levels remained mainly within the normal range, triglyceride levels

increased with age. Obese animals had elevated blood sugar, Hb1Ac and triglyceride levels compared to nonobese animals. In addition, oral and intravenous glucose tolerance tests were performed. The screening program is being continued but our preliminary data suggest that aging cynomolgus monkeys can be used to develop spontaneous disease models for type 2 diabetes, metabolic syndrome and obesity. Reference: Oeppen, J., Vaupel, J.W., 2002. Science 296:1029–1031.

P19-22 WOUND HEALING AND TOXICITY EVALUATION OF IRANIAN HYPERICUM PERFORATUM EXTRACT AND OLIVE OIL CREAM FORMULATION IN HUMAN A.H. Moghbel, H. Aghel, A. Ghalambor, N. Aghel, M. Latifi, Z. Nazari, F. Saberpour Pharmacy School, Ahvaz Jundishaour University of Medical Sciences, Ahvaz, Iran Heypericum Perforatum is familiar to St. John’s wort in USA. From 2000 years ago with olive oil have been used for wound burn healing. The aim of this study was formulating a topical cream with antiinflammatory and wound burn healing effects from extract of Hypericum Perforatum and olive oil. For this purpose after providing of herb, primary identical tests for flavonoids, alkaloids, anthraquinones and tannins and determination of ash were done. Then extraction by soxhlet method prepared and the extract was dried. Among 15 different water in oil bases formulation, the best product with 2% extract concentration (Hufarighon) was selected. The clinical effect investigation was done as such: Hufarighon cream along with olive oil (main drug), base cream (negative control), and A + D vitamin ointment (positive control) were used as three unknown products with code number 1, 2, 3 and in case of double blind trail, by patients. The statistical results showed that there was significant difference between effects of Hufarighon and olive oil with those of base cream (P = 0.000) and there was not any with significant difference between effects of this product those of A + D vitamin ointment (P = 0.6). Toxicity evaluation of the subject showed neither soft tissue

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injuries or skin diseases producing rashes, swelling, inflammation, redness, irritation, itching nor any other toxic symptoms. P19-23 MERCURY AND DENTAL PATIENTS: TOXICOLOGY, IMMUNOLOGY AND GENETIC CONNECTION G. Guzzi1 , C. Minoia2 , P.D. Pigatto3 , S. Lucchiari4 , G. Severi5 1 Italian

Association for Metals and Biocompatibility Research—AIRMEB, Milan, Italy; 2 Laboratory of Environmental Hygiene and Toxicology Testing IRCCS, “S.Maugeri”, Pavia, Italy; 3 Department of Dermatological Sciences, University of Milan, Maggiore Hospital IRCCS, Milan, Italy; 4 Department of Neurological Sciences, University of Milan, Maggiore Hospital IRCCS, Milan, Italy; 5 Cancer Epidemiology Centre, The Cancer Council Victoria, Melbourne, Australia Mercury(s) adverse events (MAE) are associated with amalgams. We analyzed features of the immunotoxicology of mercury and their relation to the characteristics of the patients. We studied 236 patients with MAE, 18–74 years of age, woman to man ratio 2.4:1. We assessed samples of stimulated saliva for the presence of total mercury (n = 236). At baseline, we also measured organic mercury species (n = 20). We used spectrometry methods as well as neutron activation. We did patch-testing (n = 127) to evaluate the allergy to mercurys. We examined, into a subgroup, haplotype (n = 25) and lymphocyte proliferation tests with metals (n = 111). In our survey, toxic reactions and/or immunological sensitization to mercurys occur in 83% of sample. We noted MAE in subjects with normal mercury levels in blood, serum, urine, and hair. In the 236 saliva samples analyzed, total mercury concentrations ranged from 2.5 ␮g/L to 780 ␮g/L. Of 236 patients studied 21 had oral lichen lesions (8.9%) and two also had lichen cutaneous. 46 had burning mouth syndrome (14.8%); 13 oral ulcers (5.5%); 8 oral leukoplakia (3.4%), 24 thyroid disorders (10.1%), 5 baboon syndrome, 3 angioedema. Some subjects with MAE have underlying defects in copper metabolism (6 of 22). In our 12 cases and 13 controls, HLA-DRB1*07 might


confer susceptibility to mercury. Complement C3-C4 were depleted (5 of 18), 3 patients of 4 returned to normal after amalgams removal. CD3+, CD4+ were diminished (7 of 12). Thyroid-related autoimmunity cases were 24; no evidence of renal autoimmunity. Two had reverse autoimmune reactions (anti-DNA antibodies and ?) cleared after removal. In our ongoing investigation, plasma homocysteine levels of post-amalgam removal group were comparatively higher (7 of 21) than pre-amalgam removal group (3 of 13). The standard treatment of MAE relied on safe and well performed dental amalgam removal. During mastication, the saliva has the highest level of total mercury released from amalgams with respect to other matrices. Exposure to mercurys from amalgams and, subsequently immunotoxicology reactions may determine local and systemic mercurys adverse effects in genetically susceptible individuals. P19-24 A TOXIC EVENT SURVEILLANCE SYSTEM IN THE EMERGENCY DEPARTMENT OF SPANISH HOSPITALS A. Ferrer-Dufol1 , S. Nogu´e-Xarau2 , R. RoyoHernandez1 , E. Civeira-Murillo1 , F. MarquesMarques3 , O. Castillo-Soria3 , the members of the Toxic Surveillance System Program 1 Clinic

Hospital, Zaragoza, Madrid, Spain; 2 Clinic Hospital, Barcelona, Madrid, Spain; 3 Clinic Hospital, Ministry of Health, Madrid, Spain We present the profile of the toxic incidents caused by chemicals in the Emergency Departments of Spanish Hospitals, in the frame of a program developed by the Health Ministry and the section of Clinical Toxicology of AETOX during 6 years. There are 20 participant hospitals and 3358 cases. Mean age is 37.73: males 50.20% and females 49.80%. Exposure has been a domestic accident in 2225 cases, suicidal in 426, occupational 554, other 132 and unknown in 14. The main chemical compounds are: toxic gases (1243), caustics (955), solvents (320), detergents (216) and pesticides (360). The route of exposure is oral (1343), respiratory (1425), cutaneous (156) and ocular (504). 2915 cases have had symptoms: neurologic 801, respiratory 742, digestive 1064, cutaneous 112 and ocular 504, renal 5, cardiovascular 76. Some treatment has been used


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in 2800 cases: gastric decontamination in 298, cutaneous or ocular decontamination in 344, antidotes in 827, enhanced elimination in 41 and symptomatic in 1895 cases. There are 57 deaths, caused by methanol (7), perchloroethylene (1), paraquat (16), other pesticides (6), HCl (13), CO (8), and other (6). Most of the non-lethal cases have had a good outcome with a few minor sequels. P19-25 DECOMPENSATED LIVER CIRRHOSIS LEADING TO DEATH AS THE FIRST PRESENTATION OF WILSON‘S DISEASE IN AN ADULT T. Lech1 , P. Hydzik2 1 Institute

of Forensic Research, Krak´ow, Poland; of Clinical Toxicology, Collegium Medicum of Jagiellonian University, Krak´ow, Poland 2 Department

Wilson‘s diseases is autosomal recessive disorder of copper metabolism resulting from the dysfunction of a copper transporting P-type ATPase encoded on chromosome 13. This ATPase transports copper into ceruloplasmin and helps in copper excretion into the bile (progressive copper accumulation in the liver). When capacity for hepatic storage is exceeded, copper starts manifesting a toxic action (cell death ensues from copper release into the plasma, hemolysis, and tissue deposition). If a patient is not diagnosed (decreased ceruloplasmin, increased urinary copper concentration, and elevated hepatic content), a rapidly progressive liver disease may happen. In this paper, a case of an unexpected death of a young women (22-year-old student) is presented. The women with no past medical history of underlying liver disease was hospitalized for 7 days before death because of a strong hemolytic anaemia of unknown origin. An acute poisoning from a heavy metal was suspected. In the Institute of Forensic Research in Krak´ow, Poland, on the basis of examination of blood and urine samples taken from the patients, using the AAS method, intoxication by lead, mercury, cadmium, thallium, zinc, chromium, manganium and arsenic compounds, was excluded. The high levels of copper in blood and urine, however, were found (blood: 3.90 ␮g/ml, urine: 8.10 ␮g/ml, 12140 ␮g/24 h). The symptomatic treatment connected to multi organs failure was used immediately, but, unfortunately, in spite of

intensive care, after 6 days the patient died. Postmortem findings revealed changes in liver characterized for Wilson’s disease (orange colour of internal organs, cirrhotic liver), and the content of copper (␮g/g) in the internal organs were the following: brain, 26.8; liver, 89.8; kidney, 111; spleen, 8.95; lung, 6.66; heart, 6.12; small intestine, 7.79; large intestine, 5.55). In the literature, the postmortem distribution of copper in body tissues of people who died from Wilson‘s disease, are given very rarely (here some data are reported). P19-26 ALKALI OCULAR BURNS IN MARTINIQUE (FRENCH WEST INDIES): EVALUATION OF DIPHOTERINE® AS THE DECONTAMINANT H. Merle1 , A. Donnio1 , L. Ayeboua1 , F. Michel1 , F. Thomas2 , J. Ketterle1 , Ch. Leonard1 , P. Josset3 , M. Gerard4 , A. Hall5 , F. Burgher6 Hospital Fort de France; 2 Fire Brigade Martinique; 3 Trousseau Hospital, Paris; 4 Cayenne Hospital, France; 5 TCMTS, Inc., Elk Mountain, WY, USA; 6 Prevor Laboratory France

1 University

Alkali cause serious injury to stroma, and corneal endothelium. Comparison is made of two decontamination solutions: normal saline and Diphoterine® . Cases of alkali ocular burns were collected from 01/01/98 to 12/21/01. Injuries were classified with Roper-Hall grades. The therapeutic protocol was standardized with injury grade. Sixty-six cases of ocular alkali burns (overall total of 104 eyes). 46% decontaminated with normal saline, 54% with Diphoterine® . Injuries were: grade I, 52; grade 2, 32; grade 3, 12; grade 4, 8 eyes. For grades 1 and 2, the time to reepithelialization was significantly shorter with Diphoterine® (p = 10−7 and p = 0.02). There were insufficient cases in grades 3 and 4 for statistical comparison. Complications occurred in 11 cases: corneal opacities (8); perforations (3). Diphoterine® is an amphoteric, slightly hypertonic, polyvalent, chelating agent. Animal studies with Diphoterine® found: (1) faster return to normal intraocular physiological pH; (2) faster and steeper decrease in the intraocular pH curve; (3) lesser stromal edema with more preservation of endothelial cells. The elapsed time to reepithialization was shorter in Grade 1 and 2 burns with Diphoterine® .

Abstracts / Toxicology Letters 158S (2005) S1–S258

P19-27 ACUTE PARAQUAT POISONING: REPORT OF A SURVIVAL CASE FOLLOWING INTAKE OF A POTENTIAL LETHAL DOSE R.J.D. Oliveira1 , A.M. Sarmento2 , P. Reis2 , A. Amaro2 , F. Remi˜ao1 , M. de Lourdes Bastos1 , F. Carvalho1 1 REQUIMTE,

Department of Toxicology, Faculty of Pharmacy, University of Porto, Portugal; 2 Intensive Care Unit, Pedro Hispano Hospital, Porto, Portugal Presented report describes a successful clinical case, regarding the intoxication of a 15-year-old girl by a presumed lethal dose of paraquat. The adolescent referred the ingestion of approximately 25 ml of a commercialized concentrate (20%) formulation of paraquat. High urinary levels of paraquat confirmed the bad prognosis. However, the therapeutical protocol followed in the present clinical case, after the initial basic treatment measures, lead to a positive outcome: (i) cyclophosphamide (15 mg/kg, i.v., OD after haemoperfusion during the first 2 days); (ii) methylprednisolone (15 mg/kg, i.v., OD for three consecutive days always after haemoperfusion); (iii) dexamethasone (5 mg i.v. TID, beginning at the fourth day, during the next 5 days); (iv) desferrioxamine (100 mg/kg, i.v., during 24 h in single administration started after the first haemoperfusion session); (v) vitamin-E (300 mg per os BID after haemoperfusion) (vi) N-acetylcysteine (150 mg/kg, i.v., for 3 h, after the first haemoperfusion session, and thereafter 300 mg/kg i.v., 21 ml/h during 3 weeks). In most cases of paraquat moderate to severe poisonings, death usually results from hypoxemia secondary to lung fibrosis. However, in the present clinical case, the patient survived, with no lung sequelae.


We describe five cases of biguanide poisoning; all patients presented important lactic acidosis (lactate range 18–33 mmol/l, pH 6.62–7.24). The serum quantification was performed on HPLC–UV after deproteinization. At admission, metformine concentration was 10.4 mcg/ml (therapeutic: 0.18–1 mcg/ml) in an intentional overdose case. Levels between 63 and 100 mcg/ml were detected in three intoxications secondary to accumulation during chronic therapy as consequence of renal impairment; an accumulation case regarded phenformine, whose serum level was 0.38 mcg/ml (therapeutic: 0.16–0.24 mcg/ml). In the first case, the renal function was normal, the patient was treated with bicarbonates and the drug was eliminated in 36 h. The patients with metformine accumulation were also dialyzed; the one with serum level of 100 mcg/ml died. The phenformine-intoxicated patient was treated with bicarbonates and the acidosis was resolved in 12 h. In our cases metformine levels above 10 mcg/ml were related to important toxic effects. The appearance of renal impairment during therapy was the most relevant cause of severe biguanide toxicity. Serum phenformine level of 1.5 times above the normal range is related to severe lactic acidosis. The method is suitable in emergency for the diagnosis of acute poisoning: it is simple results are obtained in 30 min and has sufficient specificity and sensibility for toxic concentrations. P19-29 ADVERSE EVENTS AFTER NALOXONE TREATMENT IN OPIOID OVERDOSE D. Chaparoska, V. Jordanov

Ricardo Dinis Oliveira, acknowledges FCT for his Ph.D. grant. (SFRH/BD/13707/2003).

Department of Urgent Internal Medicine andToxicology, University Clinical Centre, Skopje, R. Macedonia


The aim of this study was to determine the frequencies and characteristics of adverse events related to the hospital administration of naloxone by medical doctors. A five-year prospective observational study from 2000 to 2005 was performed in patients suspected to be acutely overdosed by an opioid. A total 600 episodes treated with naloxone admitted in the Emergency Department, were included. The main outcome variable was adverse events observed immedi-

A. Valli1 , M.L. Baldi1 , P. Papa1 , A.F. Castoldi2 , C. Locatelli2 1 Legal

Medicine and Toxicology Service, IRCCS Policlinico S. Matteo, Pavia (Italy); 2 Pavia Poison Center, Toxicology Unit, IRCCS Maugeri Foundation and University of Pavia, Italy


Abstracts / Toxicology Letters 158S (2005) S1–S258

ately after the administration of naloxone. The mean age of patients included was 28 years, and 77% were men. Adverse events suspected to be related to naloxone treatment were reported in 45% of episodes. The most common adverse events were related to opioid withdrawal (33%) such as gastrointestinal disorders, aggressiveness, tachycardia, shivering, sweating and tremor. Cases of confusion/restlessness (30%) might be related either to opioid withdrawal or to the effect of the heroin in combination with other drugs. Headache and seizures (27%) were probably related to hypoxia. Most events were non-serious. In five episodes (0.5%) the patients were hospitalized because of adverse events. Although adverse events were common among patients treated for opioid overdose in hospital setting, serious complications were rare. A hospital naloxone treatment by medical doctors seems to save several lives a year without a high risk of serious complications. P19-30 DOES RESVERATROL PREVENT THANE HEPATOTOXICITY?


¨ Erdem, O. ¨ N. Kul, S. Ya˘gar, S. Turan, T. Aldemir, O. Erdemli Exposure of human individuals to halothane causes, in about 20% of all cases, mild transient form of hepatotoxicity. Halothane causes impairment in the hepatic antioxidant defense system and accelerates peroxidation reactions. As a result some ultrastructural changes in hepatic tissues occur during halothane treatment. Resveratrol (trans-3,4,5 trihidroxystilbene) is a phytoalexin found in high concentration in the skin of grapes and red wines which has been shown to have antiinflammatory, anticancerogen and antioxidant properties. Therefore, it was the goal of the present study to determine whether prophylactic resveratrol treatment prevents hepatotoxicity of halothane. 30 Male Wistar rats were divided into 3 groups. In the control group rats were exposed for 1 h to 50% oxygen, halothane group were exposed for 10 min to 3% halothane in 50% oxygen and halothane concentration was decreased to 1% and exposure duration was 1 h. In the resveratrol group, 50 mg/kg oral resveratrol was adminestered 3 days before the same halothane treatment. After 1 h all group rats were sacrificied. Livers of rats were excised, stained, and evaluated for hepatotoxicity using a histopathological 0

(normal) to 3 (severe damage) point categorical scale and for as a peroxidation index the determination of malondialdehyde (MDA) levels. Histopathologically, in contrast to control group nuclear pleomorphism was observed in both halothane receiving groups. In resveratrol group nuclear pleomorphism was significantly lower than halothane only group. There were no significant difference between other histopathologic parameters (hidrophic degeneration, portal lenfocytic infiltration, portal neutrophilic infiltration). In comparison with control group MDA concentration were both increased in liver tissues halothane and resveratrol groups significantly. In resveratrol group the amount of MDA increase was significantly lower then halothane group. We concluded that prophylactic resveratrol treatments can somehow diminshes peroxidative damage in liver but it does not ameliorate ultrastructural changes caused by halothane exposure. P19-31 SUICIDE BY FORMALIN INGESTION M. Petcu1 , A. Enache2 , O.A. Baboiu3 , M. Ciocani1 , F. Chatzinikolaou4 1 Institute

of Legal Medicine Timisoara, Romania; of Medicine and Pharmacy Victor Babes Timisoara, Forensic Department Romania; 3 SUNNY University, USA; 4 Aristotle University, Department of Forensic Medicine and Toxicology, Thesalonik, Greece

2 University

Aim: is to highlight the signs of acute intoxication with formalin and to suspect this intoxication in suspicious deaths or in suicides. Materials and methods: the retrospective study was made at IML Timisoara, where between 1995 and 2004, we performed autopsy of two suspicious deaths. These were toxicologically investigated and many histopathological investigations were carried out. Results and discussions: the solution of formaldehyde 40% (formalin) is a local and systemic toxic. The acute intoxication by formalin ingestion is dominated by the direct lesional effect on the digestive mucus, where necrotico-haemoragic lesions appear and may determine even perforations. The toxic action also manifests itself through the making of the degenerative lesions on the liver, kidney, heart and brain. Though rare, because of the characteristics of formalin, this type of intoxication determined

Abstracts / Toxicology Letters 158S (2005) S1–S258


the death of two persons, which used the substance for suicide. One of the persons was brought to the hospital, but the other one was found dead at home. The histopathological examinations highlighted: cerebral edema, acute pulmonary edema, interstitial miocardic edema, gastric haemoragy, esophageal ulcerations, hepatic necrosis, hemorrhagic pancreatitis, shock kidney and visceral stasis. Conclusions: the existence of corrosive esophageal lesions associated with visceral edema, hepatic necrosis and hemorrhagic pancreatitis are important evidence for the presence of the intoxication with formalin.

to appear. Conclusions: the intoxication with lead tetraethyl is rarely met and presents uncharacteristically morphological changes, which do not suggest the diagnosis. The presence of the toxicological exam in the forensic examination is compulsory for establishing the etiological diagnosis.


National Poison Control Centre, Military Medical Academy, Belgrade, Serbia and Montengro

M. Petcu1 , A. Enache2 , O.A. Chatzinikolaou4 , M. Tatulescu1

Baboiu3 ,


1 Institute

of Legal Medicine Timisoara, Romania; of Medicine and Pharmacy Victor Babes Timisoara, Forensic Department Romania; 3 SUNNY University, USA; 4 Aristotle University, Department of Forensic Medicine and Toxicology, Thesalonik, Greece 2 University

Aim: the hightlight of the particularities of lead tetraethyl intoxication. Materials and methods: we retrospectively analyzed the forensic autopsies performed during 1995–2004, period during which we met a low number of cases of death by lead tetraethyl intoxication. The victims were men and the ingestion of the substance was either accidental or for suicidal purposes. Discussions: lead tetraethyl is an organic compound with 64% lead, which can be easily obtained. The absorbtion is done through the airways, through skin and specially by ingesting it. Lethal intoxication is produced after the absorption which is either accidental or in suicidal purposes; the substance affects the brain, liver, muscles and fat tissue. At the autopsied cases the discovered morphological modifications were cerebral, pulmonary and hepatho-renal. After the ingestion, the patients were brought in the hospital and survived for about 16–18 h. The necroptic examination showed cerebral edema, poliserosis, epichardic blood suffusions, bronchopneumonia, intestinal mucus necrosis and hemorrhagic pancreatic. The survival period was long enough for the hepatho-renal cellular changes


Acute poisonings of adolescents are serious sociological and health problem. Frequency of admission in emergency room is rising and the etiologies are diversifying due to many challenges for substance abuse. We retrospectively analysed the incidence, severity and outcome of acute poisonings of adolescents aged 14–18 years, treated in Clinic of Toxicology of National Poison Control Centre in Belgrade during 1year period. The severity of poisoning was estimated by Poisoning Severity Score (PSS). During follow up period 374 patients or 13.5% of all patients were adolescents. Except 3 cases of accidental poisoning by chlorine gas, all other cases were due to deliberate ingestions or substance abuse. The most frequent was ethanol abuse, in 172 patients (46%). Suicidal ingestion of psychotropic medications was on the second place (102 patients or 27%), and opiate and street drugs overdose on the third place (30 patients or 8%). The rest were poisonings by other medications, corrosives and pesticides. The severity of poisoning at admission to emergency room was estimated as following: mild (PSS 1), 61%; moderate (PSS 2), 21%; severe (PSS 3), 18%. Most cases were managed in emergency room (78%) as the outpatients, while 84 (22%) were admitted to hospital for further treatment. There was single case of lethal outcome and it was due to Ecstasy overdose. The ethanol abuse is the most common cause of adolescents acute poisoning, probably due to lack of legal prevention.


Abstracts / Toxicology Letters 158S (2005) S1–S258



N. Bekarovski1 , S. Jurukov, L. Meloska1

N. Bekarovski1 , S. Radulovik-Bekarovska2 , D. Chaparoska1 , L. Meloska1 , I. Jurukov1 , N. Popovski1

Radulovik-Bekarovska2 ,


1 Clinic

1 Clinic

of Toxicology, Clinic Center Skopje, Macedonia; for Neurology, Clinic Center Skopje, Macedonia

of Toxicology, Clinic Center Skopje, Macedonia; 2 Clinic for Neurology, Clinic Center Skopje, Macedonia

Introduction: Frequent and enormous prescription made the benzodizepines the main drug for suicidal attempts. Aims: The study is to establish the level of the correlation between the applied dose of the Flumazenil and the severity of acute poisoning with benzodiazepines. Materials and methods: The analyzed group consists of 135 patients. Clinical presentation was evaluated with 7-point level of consciousness scale (7LCS) and Glasgow Coma Score (GCS). Doses of 0.30 mg flumazenil, till total recovery of conscious, were applied in all patients with severity clinical expression. Results: 106 patients (78.5%) were female. The average age of the group was 19 ± 3.25 years. According to the 7LCS 37 patients of the group were with easy clinical expression, 57 with average clinical expression, 29 were with severe disorders and 12 in the group with extremely severe clinical expression. According to the GCS 37 of the group were with easy clinical expression, 58 with average and 40 in the group with severe clinical expression. 49 patients have ingested Diazepam, 41 Bromazepam, 22 Lorazepam and 23 Diazepam and Bromazepam. The specific antidote Flumazenil was applied at 42 patients. 8 patients received 0.30 mg, 11 patients 0.60 mg, 12 patients 0.90 mg, 9 patients 1.50 mg and two patients 2.00 mg. The level of the correlation between the clinical expression and applied doses of Flumazenil was R0 = 0.836; DF = 133; p < 0.01. Conclusions: There is a high level of correlation between the clinical expression of poisoning and the applied dose of the antidote.

Introduction: Abstinence syndrome after chronic use of benzodiazepines is a well-known fact, but there are not any studies of developing of this syndrome after acute poisonings with benzodiazepines. Aim: The study is to establish the frequency of after poisoning abstinence syndrome in the benzodiazepine poisonings. Materials and methods: The analyzed group is consisted of 135 patients. The interview by psychiatrist, special questionnaire and the control of all parameters was made 1 month after the intoxication to all patients. Results: 106 patients (78.5%) were female. The average age of the group was 19 ± 3.25 years. 49 patients have ingested Diazepam, 41 Bromazepam, 22 Lorazepam and 23-mixed Diazepam and Bromazepam. 13 Patients (9.63%) developed abstinence syndrome, 7 in the group with Lorazepam poisoning and two patients in all other groups. Extremely high percent of abstinence syndrome (31.81%) was noticed at the patients with lorazepam poisoning. All the patients who developed abstinence syndrome were treated with Flumazenil in the detoxification procedure. Average interval of the existing of abstinence syndrome was 12 days and developed among 4th and 14th day after intoxication. Conclusions: Abstinence syndrome is a rare phenomenon after acute poisonings with benzodiazepines treated with Flumazenil, except in the lorazepam poisonings.

2 Clinic

P19-36 ANTIDOTES AVAILABILITY AND SUPPLY IN EMERGENCY SETTINGS IN ITALY C. Locatelli1 , V. Petrolini1 , R. Butera1 , S. Arrigoni1 , J. Georgatos1 , D. Lonati1 , L. Mela1 , A.F. Castoldi, T. Coccini1 , A. Volpini2 , L. Manzo1 1 Pavia Poison Centre, IRCCS Maugeri Foundation and

University of Pavia; 2 Department of Civil Protection, Rome, Italy

Abstracts / Toxicology Letters 158S (2005) S1–S258

Essential antidotes are frequently inadequately stocked in hospital emergency setting. A survey on the availability of antidotes has been recently conducted in Italy by the Pavia Poison Center (PC). Three hundred and thirty seven emergency services participated to the survey: all investigated antidotes resulted insufficiently stocked in the majority of hospitals. Actions were addressed to improve both educational aspects (40 educational courses all-over-Italy in 2 years) and antidotes availability and supply. Three models for antidotes supply were implemented. The first is a national database accessible through website ( that includes information on antidotes stocks of all departments participating to the survey. A simple query allows the registered users to identify hospitals provided with antidotes. This database can be also used to ameliorate antidotes procurement. The second, actually operating in 44 major chemical plants all-over Italy, consist of local antidotes stockpiles according to specific risks. This allowed in some accidents a timely and proper management of cases of acute occupational poisoning. The “national antidotes stockpile for NBCR emergencies” is the third model actually operating within the context of an agreement between the Italian Department of Civil Protection and the Pavia PC. Supported by grants from Italian Ministry of Health, National Research Council and Department of Civil Protection. P19-37 THE CONTRIBUTION OF THE FORENSIC EXPERT TO THE PREVENTION OF CARBON MONOXIDE (CO) INTOXICATION A. Enache, M. Petcu, O.A. Baboiu, V. Belei, F. Chatzinikolaou 1 University

of Medicine and Pharmacy Victor Babes Timisoara, Forensic Department Romania; 2 Institute of Legal Medicine Timisoara, Romania; 3 SUNNY University, USA; 4 Aristotle University, Department of Forensic Medicine and Toxicology, Thesalonik, Greece Aim: was to observe the risk factors and those determining death by CO intoxication. Materials and methods: the study was done retrospective on the cases which had the autopsies performed in a period of 3 years (2001–2003); 2 9 violent deaths due to CO intoxication


were necroptically