Abstracts to BTS Annual Congress S.I.

Toxicology 194 (2004) 197–272

Invited speaker’s abstracts

Cloning, Stem Cells and Pharmacogenetics Ian Wilmut Roslin Institute, Roslin Midlothian, EH 25 9PS New opportunities in pharmacogenetics will be provided by the derivation of hepatocytes from human ES cell lines. This will provide for the first time the opportunity to use the same cell population over a period of many months for studies of drug metabolism. While the same cell population will be available over prolonged period, it will be impossible to pre-select the genotype of the cells as they will be derived from donated embryos. However, in the longer term it may be possible to derive cell lines from cloned human embryos. At this point it would become possible to have routine access to Hepatocytes of a variety of known genotypes. Realisation of these new opportunities depends upon three procedures, cloning of human embryos, derivation of embryos stem cell lines and the controlled differentiation of human embryo stem cells to hepatocytes. Progress in these three areas of research will be reviewed. Genomic and Functional Proteomic Analysis of Stress-Induced Gene Regulation Jonathan G. Moggs1 , Neil Macdonald1 , David Clynes1 , Catherine A. Hazzalin2 , Angela Hollis2 , Stuart Thomson2 , Louis C. Mahadevan2 and George Orphanides1 1 Syngenta Central Toxicology Laboratory, Alderley Park, Cheshire, SK10 4TJ, UK; 2 Department of Biochemistry,

University of Oxford, OX1 3QU, UK Mammalian cells respond to a wide range of external stimuli, including growth factors, peptide hormones, cytokines, osmotic stress, heat shock, pharmacological agents and toxicants, via multiple signalling pathways. Genome-wide transcript profiling simultaneously monitors the gene expression programs downstream of all signal transduction pathways and can identify novel molecular targets for stress-inducing signals. Our laboratories have combined transcript profiling of cytotoxic compounds with experimental systems in which signalling components are disrupted (e.g. small molecule protein kinase inhibitors) in order to identify genes regulated by specific signalling pathways during cellular responses to toxicant-induced stress. The stress-like pharmacological agent anisomycin, a potent activator of stress-activated and mitogen-activated protein (MAP) kinase cascades in mammalian cells, induced the up-regulation of more than 15 genes, including several immediate early genes (C-FOS; CYR61; MPK-1) and an EST encoding a novel protein. The anisomycin-induced expression of this EST was insensitive to the p38 MAP kinase inhibitor SB203580, implying that it may be regulated by a distinct molecular mechanism to the majority of anisomycin-induced genes we observed. The final step in stress-activated protein kinase cascades is the post-translational modification of transcription factors and chromatin components that occupy gene regulatory regions (e.g. promoters) of stress-response genes. Chromatin consists of repeating units called nucleosomes (each consisting of a core histone octamer containing 0300-483X/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2003.09.003

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histones H2A, H2B, H3 and H4) and plays a key role in regulating gene expression (Orphanides and Reinberg, 2002). Indeed, chromatin-modifying proteins including histone acetyltransferases and histone kinases are known to be key components of stress-activated signalling pathways (Thomson et al., 2001). Together, these enzymes “write” a specific mark, or code, onto the N-terminal tails of histone H3 that determines the transcriptional status of stress-response genes. We have developed functional proteomic approaches that have allowed us to identify novel proteins that can “read” and “write” histone H3 tail modifications on the promoters of stress-responsive genes. Together, these genomic and biochemical approaches reveal the molecular mechanisms used to finely tune alterations in gene expression, enabling cells to react in an appropriate manner to external stress-stimuli. A more detailed understanding of molecular mechanisms underlying transcriptional regulation of stress response genes will enhance our understanding of xenobiotic responses in mammals and should facilitate the development of compounds with favourable toxicity profiles. References Orphanides, G., Reinberg, D., 2002. Cell 108, 439–451. Thomson, S., Clayton, A.L., Mahadevan, L.C., 2001. Molecular Cell 8, 1231–1241. Role of Nrf2 in Mediating Regulation of Cytoprotective Genes by Chemopreventive Agents J.D. Hayes1 , M. McMahon1 , I.R. Jowsey1 , Q. Jiang1 , P. Nioi1 , L.G. Higgins1 , L.I. McLellan1 , C.J. Henderson1 , K. Itoh2 and M. Yamamoto2 1 Biomedical

Research Centre, Ninewells Hospital & Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK; 2 Center for TARA and Institute of Basic Medical Sciences, University of Tsukuba, Tennoudai, Tsukuba 305, Japan The administration of a chemical, or dietary components, to prevent the onset or to inhibit the progression of neoplastic disease is referred to as cancer chemoprevention (Wattenberg, 1985). Chemopreventive agents can inhibit cancer at several stages of the carcinogenic process. Compounds which prevent the production of mutations are referred to as blocking agents, and include coumarins, curcumin, dithiolethiones, flavones, indoles, isothiocyanates, organosulphides, phenols, tannins and terpenes. These chemicals block carcinogenesis either by inducing proteins involved in detoxifying carcinogens, by inhibiting the activation of carcinogens, by preventing uptake of carcinogens, or by enhancing DNA repair. Compounds which inhibit post-mutational events in the neoplastic process are referred to as suppressing agents, and include difluoromethylornithine, isothiocyanates, carotenoids, curcumin, dehydroepiandrosterone, indoles, inositol, NSAIDs, phytate, polyphenolics, retinoids, selenium, terpenes and vitamin A. These compounds have diverse actions and can act by producing differentiation, by inhibiting oncogene activation, by inhibiting the proliferation of initiated cells and by enhancing apoptosis. A large body of literature has revealed that blocking agents prevent carcinogenesis by increasing the expression of a range of detoxication enzymes and antioxidant proteins. Typically, glutathione S-transferase (GST) isoenzymes, aldo-keto reductase (AKR) and NAD(P)H:quinone oxidoreductase (NQO) have been found to be highly inducible by many chemopreventative blocking agents. Similarly, glutamate cysteine ligase (GCL) and heme oxygenase (HO) are also inducible. The change in gene expression elicited by chemopreventive blocking agents appears to confer resistance to chemical and oxidative stress. Evidence suggests transcriptional activation of GST, NQO, GCL and HO genes occurs through the antioxidant responsive element (ARE; 5
-A/GGTGACnnnGCA/G-3
) (Rushmore et al., 1991). Work from our laboratories, using a knockout mouse, has demonstrated that the Nrf2 bZIP protein mediates induction of many GST, NQO and GCL genes by the phenolic antioxidant butylated hydroxyanisole (BHA) (Itoh et al. 1997; McMahon et al. 2001; Chanas et al., 2002). These studies have also indicated that Nrf2 regulates induction of cytoprotective genes by other chemopreventive agents that are structurally distinct from the phenolic antioxidant BHA, including angelicalactone, ethoxyquin, coumarin, limettin, oltipraz, indole-3-carbinol, sulforaphane and kahweol/cafestol (McMahon et al. 2001; Chanas et al., 2002). Thus, a wide range of chemicals can activate Nrf2. The molecular basis for the diverse

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effects of Nrf2 on expression of detoxication/antioxidant genes is poorly understood. This is because relatively few of the genes that appear to be regulated by Nrf2 have been demonstrated to contain functional AREs. In conclusion, it is clear Nrf2 makes a major contribution to detoxication and antioxidant defences, but for many genes it is unclear whether the effects of Nrf2 are direct or indirect. References Chanas, S.A., Jiang, Q., McMahon, M., McWalter, G.K., McLellan, L.I., Elcombe, C.R., Henderson, C.J., Wolf, C.R., Moffat, G.J., Itoh, K., Yamamoto, M., Hayes, J.D., 2002. Biochem. J. 365, 405–416. Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., Nabeshima, Y.-i., 1997. Biochem. Biophys. Res. Commun. 236, 313–322. McMahon, M., Itoh, K., Yamamoto, M., Chanas, S.A., Henderson, C.J., McLellan, L.I., Wolf, C.R., Cavin, C., Hayes, J.D., 2001. Cancer Res. 61, 3299–3307. Rushmore, T.H., Morton, M.R. & Pickett, C.B., 1991. J. Biol. Chem. 266, 11632–11639. Wattenberg, L.W., 1985. Chemoprevention of cancer. Cancer Res. 45, 1–8. Molecular Chaperones and the Prevention of Proteotoxic Damage J. Paul Chapple Institute of Ophthalmology, Division of Pathology, University College London, Bath Street, London EC1V 9EL When organisms are exposed to environmental stress, such as heat shock or toxins, they respond by synthesising proteins which function to protect against proteotoxic damage. These stress or heat shock proteins (HSPs) prevent the accumulation of abnormal proteins and increase stress-tolerance. They are essential for survival and environmental adaptation. For example, changes in the levels of chaperones such as Hsp70 closely correlate with seasonal changes in temperature and thermotolerance in eurythermal ectotherms such as the marine bivalve Mytilus edulis, Chapple et al. (1998). HSPs function as molecular chaperones which are facilitators of protein conformational change. Molecular chaperones have a principal role in protein folding, where they are involved in the de novo synthesis of polypeptides, transport across membranes and the refolding of proteins denatured by unphysiological conditions. The best studied and mechanistically understood chaperones are the chaperonins and Hsp70s. These chaperones recognise and bind to unfolded or partially folded polypeptides by binding to exposed hydrophobic regions preventing them from aggregating and maintaining them in a folding competent state, Bukau and Horwich (1998). For many chaperones, cycles of client protein binding are dependent on the hydrolysis and exchange of ATP that is regulated by cochaperones. Cochaperones function synergistically with the major chaperones in protein folding and often have independent chaperone activity, but their major role might be to provide these folding machines with specificity. For example, the Hsp70 protein machinery achieves multiple cellular functions because of various cochaperones such as the DnaJ/Hsp40 family which stimulate Hsp70 ATP hydrolysis. The misfolding of proteins due to disease, caused by ageing or inherited genetic mutation, is another form of proteotoxic damage, for example, the misfolding of mutant rhodopsin is believed to lead to photoreceptor cell death by apoptosis. Thus, the manipulation of molecular chaperones may represent a potential therapy for diseases of protein misfolding, such as rhodopsin retinitis pigmentosa, Chapple et al. (2001). References Bukau, B., Horwich, A.L., 1998. Cell 92, 351–366. Chapple, J.P., Smerdon, G.R., Berry, R.J., Hawkins, A.J.S., 1998. J. Exp. Mar. Biol. Ecol. 229, 53–68. Chapple, J.P., Grayson, C., Hardcastle, A.J., Saliba, R.S., van der Spuy, J., Cheetham, M.E., 2001. Trends Mol. Med. 7, 414–421.

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Ionizing Radiation as a Model DNA-Damaging Agent: Global Gene Expression Responses S.A. Amundson,1 M. Bittner,2 A.J. Fornace Jr.1 1 Gene

Response Section, National Cancer Institute; 2 National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA Ionizing radiation provides a useful tool for the study of DNA damage responses, as it causes a variety of lesions in DNA, while minimizing problems of dosimetry, metabolic processing, and damage to other cellular compartments that may be issues with many other genotoxic agents. Many of the physiological responses to ionizing radiation exposure, including apoptosis, cell cycle arrest, and DNA-damage repair, are mediated in part by changes in gene transcription. Gene transcription patterns, in turn, reflect the complex interplay of multiple signal transduction pathways, and vary with factors including genetic background and cell or tissue type. For example, in the transcriptional response associated with radiation-induced apoptosis, induction of some genes is limited to p53 wild-type cells from tissue types with the ability to undergo rapid apoptosis after irradiation. In contrast, other genes are triggered after irradiation in cell lines undergoing rapid apoptosis regardless of p53 status. With the advent of the genomic era, modern techniques, including microarray analysis, can monitor changes in expression in large sets of genes in a single experiment. We have previously used microarray analysis to identify a set of genes regulated by exposure to high doses of ionizing radiation Amundson et al. (1999a), as well as to explore the differential effects of various DNA-damaging stresses and treatment conditions. With increasing interest in the direct measurement of effects of low doses of ionizing radiation, we have demonstrated reproducible changes in gene expression at doses as low as 2 cGy Amundson et al. (1999b), and have demonstrated a linear response for induction of multiple stress genes in the human p53-wt myeloid ML-1 line, and in human peripheral blood lymphocytes (PBL) irradiated ex vivo. We have also used cDNA microarray hybridization analysis to identify radiation-regulated genes that could potentially serve as informative biomarkers of radiation exposure Amundson et al. (2000). Three of these genes were shown to be induced in a linear fashion between 0.2 and 2 Gy at 24 and 48 h after treatment. These results support the use of peripheral blood cells as an accessible and sensitive indicator of radiation exposure, and begin laying the foundation for expression profiles that may someday provide signatures for past radiation exposure. The ability to measure changes in expression of large numbers of genes following small exposures holds great promise for future studies of diverse molecular signaling pathways, inter-individual variations in response, and other important aspects of DNA damage-induced stress responses. References Amundson, S.A., Bittner, M., Chen, Y., Trent, J.M., Meltzer, P., Fornace Jr., A.J., 1999a. Oncogene 18, 3666–3672. Amundson, S.A., Do, K.T., Fornace Jr., A.J., 1999b. Radiat. Res. 152, 225–231. Amundson, S.A., Do, K.T., Shahab, S., Bittner, M., Meltzer, P., Trent, J.M., Fornace Jr., A.J., 2000. Radiat. Res. 154, 342–346. Survival Signaling by Cell Adhesion Receptors Bob van de Water Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands Cell–cell and cell-extra-cellular matrix (ECM) interactions are critical in the maintenance of cellular integrity and are involved in activation of signaling networks that are important in cell migration, proliferation and survival. Loss of cell survival, i.e. induction of apoptosis, is generally preceded by loss of cellular interactions. The cytoskeletal network plays an important role in the regulation of these interactions, and loss of cytoskeletal organization provides a basis for disruption of these cellular interactions and subsequently loss of survival signals with apoptosis as a final end-point. We are interested in the role and mechanisms of loss of both cell-cell and cell-ECM interactions

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in (xenobiotic-induced) apoptosis of epithelial cells with either a normal (renal proximal tubular epithelial cells) or malignant (mammary adenocarcinoma cells) phenotype, both in vitro and in vivo. Cell survival signaling through integrin receptors is mediated through focal adhesion kinase (FAK), a non-receptor tyrosine kinase that is central in cell migration, proliferation and survival. We have investigated the role of FAK in the control of cytotoxicity. FAK is dephosphosphorylated in cytotoxicity in renal cells in association with loss of focal adhesion organization prior to the onset of the apoptotic program (van de Water et al., 1999). Inhibition of FAK function using dominant negative acting FAK deletion mutants, sensitizes renal cells towards the onset of apoptosis (van de Water et al., 2001). We have further analyzed the signal transduction pathways that are controlled by FAK and involved in the regulation of toxicant-induced apoptosis of renal epithelial cells. Moreover, we are analyzing the role of FAK in controlling acute renal failure caused by nephrotoxicants. These investigations will be important to better understand the role of cell adhesion-mediated survival signaling in cytotoxicity and provide a basis for the identification of ‘drugable’ targets to prevent unwanted side-effects of xenobiotics and/or develop strategies to circumvent the drug-resistant phenotype of tumor cells. References van de Water, B., Mulder, G.J., Stevens, J.L., 1999. J. Biol. Chem. 274, 13328–13337. van de Water, B., Houtepen, F., Huigsloot, M., Tijdens, I.B., 2001. J. Biol. Chem. 276, 36183–36193. Acknowledgement Supported by grant 902-21-208 from the Dutch Organization for Scientific Research (NWO) and a fellowship from the Royal Netherlands Academy for Arts and Sciences (KNAW). Recognition of Adverse and Non-Adverse Effects in Toxicity Studies: A Problem of Consistency Richard W. Lewis Syngenta Health Assessment and Environmental Safety, Alderley Park, Cheshire, UK One of the most important quantitative outputs from toxicity studies is identification of the highest exposure level (dose or concentration) that does not cause treatment related effects relevant to human health risk assessment. A review by European Centre for the Toxicology and Ecotoxicology of Chemicals (ECETOC) (Lewis et al. 2002), of regulatory and other scientific literature and of current practices has revealed a lack of consistency in definition and application of frequently used terms such as ‘No Observed Effect Level’ (NOEL), ‘No Observed Adverse Effect Level’ (NOAEL), ‘adverse effect’, ‘biologically significant effect’ or ‘toxicologically significant effect’. Moreover, no coherent criteria were found that could be used to guide consistent interpretation of toxicity studies, including the recognition and differentiation between adverse and non-adverse effects. In order to achieve consistency in interpretation, a standard set of definitions are proposed for key terms such as NOEL and NOAEL that are frequently used to describe the overall outcome of a toxicity study. Secondly, a coherent framework is outlined that can assist the toxicologist in arriving at consistent study interpretation. This structured process involves two main steps. In the first, differences from control values are evaluated in order to decide if they are treatment related effects or chance deviations. In the second step, only those differences judged to be effects are further evaluated in order to discriminate between those that are adverse and those that are not. For each step criteria are defined that can are used to make consistent judgements. In differentiating an effect from a chance finding, consideration is given inter alia to dose response, spurious measurements in individual parameters, the precision of the measurement under evaluation, ranges of natural variation and the overall biological plausibility of the observation. In discriminating between the adverse and the non-adverse effect consideration is given to: whether the effect is an adaptive response, whether is it transient, the magnitude of the effect, its association with effects in other related endpoints, whether is it a precursor to a more significant effect, whether it has an effect on the overall function of the organism, whether it is a specific effect on

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an organ or organ system or secondary to general toxicity or whether the effect is a predictable consequence of the experimental model. In interpreting complex studies, it is recognised that a weight of the evidence approach, combining the criteria outlined above to reach an overall judgement, is the optimal way of using the process. It is believed that the use of such a scheme will help to improve the consistency of study interpretation that is the foundation of hazard and risk assessment. Reference Lewis, R.W., Billington, R., Debryune, E., Gamer, A., Lang, B., Carpanini, F., 2002. Toxicol. Pathol. 30 (1), 66–74. Using Toxicogenomics to Distinguish Adverse from Adaptive Effects in the Liver Kevin T. Morgan1,2 , Nanxiang Ge1 , Katja Kotlenga3 , and Zaid Jayyosi1 1 Aventis

27612;

Pharmaceuticals, Bridgewater, NJ, USA; 2 Mailing Address, 5020 F. Edwards Mill Road, Raleigh, NC Pharmaceuticals, Frankfurt, Germany

3 Aventis

E-mail address: [email protected] The distinction of adaptive from adverse responses can be challenging in any organ system. The key to this diagnostic dilemma lies in comprehension of the underlying biological mechanisms. Toxicogenomics (Toxicology via the transcriptome) can reveal clues as to the nature of both normal and abnormal physiology. Meaningful interpretation of transcriptional responses induced in the liver by environmental, including chemical, stresses begins with thorough knowledge of this complex organ. For a brief introduction to the anatomy, physiology, phylogeny, embryology, and function of the liver, see Arias et al. (2001). The liver performs many critical functions, including secretion of bile, synthesis of clotting factors, compliment components and many other blood proteins, storage of iron and glycogen, cholesterol, vitamin and amino acid biosynthesis, detoxification and metabolism of xenobiotics, whilst maintaining blood glucose levels and the Cori cycle, and being a key component of the immune system. All of these functions and many others, being reflected in the liver transcriptome, are available for the trained observer to interpret. A process is needed, however, for the intelligent development of this powerful tool. Such a process should step through (1) careful experimental design, (2) visual triage of the large-scale differential gene expression data sets generated, using such tools as clustered heat maps (Eisen et al., 1998) and principal component analysis, (3) statistical triage (Box et al., 1978), employing both liberal and conservative approaches, (4), functional triage, (5) RT-PCR confirmation, prior to (6) detailed interpretation, (7) correlation with individual animal clinical data, (8) hypothesis generation, (9) follow-up functional studies, and (10) comparison with the output of mathematical models, with due regard to transcriptome dynamics, finally leading to (11) improved experimental design as the process is repeated. One important objective of all this work is to provide a comprehensible risk assessment to an appropriately educated risk manager. With respect to Hepato-Toxicogenomics, this process will need education on all fronts, along with effective collaboration between scientists with divergent training. The ‘omics’ revolution is real; you can put it off, but you cannot escape! References Arias, I.M., Boyer, J.L., Chisari, F.V., Fausto, N., Schachter, D., Shafritz, D.A., 2001. The Liver, Biology and Pathobiology. Lippincott Williams & Wilkins, Philadelphia, p. 1064. Box, G.E.P., Hunter, W.G., Hunter, J.S., 1978. Statistics for Experimenters. Wiley, New York. Eisen, M.B., Spellman, P.T., Brown, P.O., Botstein, D., 1998. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. U.S.A. 95, 14863–14868.

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The Mechanistic Basis of Adverse Effects John R. Foster AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK The discipline of Toxicology normally involves the use of surrogates for human exposure, such as cell cultures or laboratory animals, and the skill in the discipline comes with the interpretation of adverse results in the context, initially of the laboratory studies themselves, and finally of their relevance to man. Much of this extrapolation will involve subsequent experimentation to test the relevant hypothesis obtained from previous experiments. Some of the strategy for preventing human toxicity involves avoidance of human exposure but in many instances this is not practical, and management of the hazard is the alternative strategy. An essential part of this management strategy will be a more in depth understanding of the reasons for the observed toxicity. Determinants of targeted, adverse, effects include some, or many, of the following features: exposure of the particular chemical or drug to the target; an appropriate means by which the chemical enters the target cells—a receptor or similar uptake system; a metabolic system that preferentially activates, rather than detoxifies the chemical; and a molecular target with which chemical interaction results in toxicity to the cell. An in-depth understanding of the mechanism by which administration of a chemical induces toxicity, specific to a particular target tissue or cell, is the critical first step in determining whether or not the observed toxicity will translate into a similar adverse effect on exposure in man. The identification of a qualitative species-specific mechanism, such as metabolism, will allow a particular toxicity, observed in laboratory species, to be put into context for human exposure. A quantitative species difference in toxicity will enable an informed risk assessment to be made. In the field of drug development, a mechanistic understanding of why a particular therapy induces toxicity in non-disease tissue can aid in developing a therapeutic strategy that provides adequate margins of safety. Similarly an understanding of the reason for unexpected animal, or human, toxicity can provide a strategy for avoidance, e.g. by changing the chemistry involved or by altering the therapeutic schedule. The application of toxicology has altered from being an empirical discipline, where conservative approaches deemed that all animal toxicity necessarily translated into a similar hazard to man, to a sophisticated science whereby the identification of a toxicity in the laboratory is only the first step in an iterative process of hypothesis testing to assess the full meaning to man. The vast majority of toxicities remain unexplained but progress thus far should give us optimism that future toxicology will, through improved technology and more relevant testing systems, become less empirical and more mechanistically based, as our understanding of basic biological processes, and the ways in which they can be perturbed, improves. References Goddard, M.J., Krewski, D., 1995. The future of mechanistic research in risk assessment: where are we going and can we get there from here? Toxicology 102, 53–70. Kelloff, G.J. et al., 1995. Approaches to the development and marketing approval of drugs that prevent cancer. Cancer Epidemiol. Biomarkers Prev. 4, 1–10. Reproductive Toxicology in the 21st Century: Being Pecked to Death by Chickens Robert E. Chapin Investigative Developmental Toxicology Laboratory, Pfizer Global R&D, Eastern Point Road, MS 8274-1336, Groton, CT 06340, USA E-mail address: robert e [email protected] Adaptive changes are always a puzzle. They indicate an effect, but is this necessarily a bad change? Adaptation in reproduction or development may be similar to some adaptive changes in other organs in that what we see is

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“better biology”. Examples might be elevated LH in the face of normal levels of testosterone and normal numbers of Leydig cells, or perhaps greater post-natal weight gain in pups from treated dams. The only logical way to view such changes is that they indicate that the organism can compensate for an exposure. This in itself is not adverse, though it may make the animal more susceptible to additional insults in the future. It’s this shadow of possibly-poor future response which bedevils adaptive changes. The other issue in reproductive toxicology, composed of small measures which have big impacts, is endocrine disruption and its correct detection. A severe perturbation of endocrine signaling during fetal development has lifetime consequences which we all agree are incontrovertibly awful. Simultaneously, reproductive toxicity studies are among the more expensive to conduct, and when faced with limited financial resources, we need “the most data out for the money we put in”. This often leads to shortened assays relying on measuring sentinel or intermediate endpoints which are mechanistically linked to the final apical outcome. Unfortunately (though perhaps not surprisingly, given a capricious and malicious fate), there is always some degree of disconnect between these early, easy-to-measure endpoints, and the final, wholistic, apical function with which we are concerned. Examples might be (respectively) age at preputial separation and normal male reproductive function, or vaginal opening and normal female reproductive function. So what to do when the early measure responds differently than the wholistic functional assay? First, do not think of small, simple endpoints as true indicators of large complex ones. Second, the wrong response is “ Any change in a reproductive endpoint is adverse”. As scientists, the right response is: fully evaluate the system; use the redundancy to your advantage (i.e., generate more data). As regulators, the right response is to ask for more data (fertility, sperm measures, adult histopathology). At this point, we know of several occasions when these disconnect, but we lack a clear understanding of all the conditions leading to mis-predictions. For a limited time, we have a golden opportunity to arrange a multi-lab effort to conduct a number of truly comprehensive studies, intended to define the circumstances in which the intermediate endpoints are correctly predictive, vs. those in which they err. A prospective, inclusive effort to produce a comprehensive dataset from which a consensus could emerge is the only way to remove emotion and innuendo from this issue, and re-insert science and data as the prime drivers. And as Albert Einstein observed: “One thing I have learned in a long life: that all our science, measured against reality, is primitive and childlike—and yet it is the most precious thing we have”. The Mechanism of Carcinogenicity of Tamoxifen and its Implications David H. Phillips Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG, UK The widely used antioestrogen tamoxifen is a potent carcinogen in rat liver. It also induces tumours in the uterus of the rat. Women who take the drug are at increased risk of endometrial cancer. The key question is whether the mechanism of these events is the same, and understanding the answer has implications for assessing the safety for other selective oestrogen receptor modulators (SERMs). Tamoxifen is activated to DNA binding products via a reactive ester of ␣-hydroxytamoxifen in rat liver. Although ␣-hydroxytamoxifen has intrinsic reactivity and can form DNA adducts without further metabolic activation, it is unlikely that at physiological concentrations and under physiological conditions this will occur to a significant extent. In our hands 32 P-postlabelling analysis has revealed the formation of tamoxifen-derived adducts in rat liver in vivo and in rat hepatocytes in vitro treated with tamoxifen or ␣-hydroxytamoxifen. Many of the adducts formed have undergone N-demethylation. The R-isomer of ␣-hydroxytamoxifen is significantly more active in forming DNA adducts in rat hepatocytes than the S-isomer. Similar results have been obtained with the enantiomers of N-desmethyltamoxifen. Where we have compared our adduct determinations with other methods of analysis, good quantitative agreement has been obtained both with immunological methods and with electrospray ionisation tandem mass spectrometry. We have not found consistent evidence for DNA adduct formation by tamoxifen or ␣-hydroxytamoxifen in any other rat tissues. This tissue specificity in DNA adduct formation is unusual for a

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genotoxic carcinogen. It is normal to detect adducts distributed in many tissues. Significantly, DNA adducts have not been detected in rat uterus, nor have mutations been detected by others in this tissue, yet this is a site of tumour induction by tamoxifen. The suspicion is that tamoxifen might be a non-genotoxic carcinogen at this site, due to its uterotrophic and oestrogenic activity on the developing organ. In rat liver ␣-hydroxytamoxifen is activated by an isoform of SULT2A1. This enzyme is induced in male rats by tamoxifen, leading to its activation in both sexes. There is little or no expression of SULT2A1 in other rat tissues. No human SULT, including SULT2A1, showed any activity in causing DNA binding by ␣-hydroxytamoxifenin either V79 cells or S. typhimurium strains expressing the enzymes, nor was any mutagenicity observed. On the other hand, expression of rat SULT2A1 in the same systems led to both adduct formation and mutagenicity. We have not detected tamoxifen-DNA adducts in human endometrium or peripheral blood lymphocytes. Human endometrium does not express SULT2A1. Although synthetic ␣-acetoxytamoxifen is a highly electrophilic compound, we have found no evidence that ␣-hydroxytamoxifen is activated by acetyltransferases. If tamoxifen does form DNA adducts in human tissues, the mechanism of activation is unlikely to be the same as in rat liver. Our own results do not provide evidence for DNA adduct formation by either tamoxifen or ␣-hydroxytamoxifen in any tissues other than rat and mouse liver. Therefore we favour the hypothesis that tamoxifen is, uniquely, a genotoxic carcinogen in rat liver, but primarily a non-genotoxic, hormonal carcinogen in rat uterus and human endometrium. Reference Phillips, D.H., 2001. Understanding the genotoxicity of tamoxifen? Carcinogenesis 22, 839–849. Types Of Biomarker and Challenges for New Biomarkers John Timbrell Pharmacy Department, King’s College London E-mail address: [email protected] There are three types of biomarker (as defined by the US National Academy of Sciences Committee on Biological Markers (National Research Council, 1989) in relation to chemical exposure: biomarkers of exposure, of response and of susceptibility. For the evaluation of the toxicity of a chemical and for risk assessment, all three biomarkers can be used because the study of toxicity requires: (a) a knowledge of the dose or level of the substance to which the animals or patient is exposed; (b) a means of detecting and quantifying the pathological response; (c) an understanding of the factors which affect the occurrence of the pathological response. Biomarkers of exposure can be divided into markers of internal dose such as metabolites, and markers of effective dose such as adducts. For example S-phenyl N-acetylcysteine, a urinary metabolite of benzene, is used as a biomarker of internal dose of the solvent whereas a N7 guanine adduct is a marker of the effective dose of aflatoxin B1 to which humans have been exposed which can be detected in urine. Biomarkers of response cover a much greater variety of types of parameter. Thus such biomarkers range from simple non-invasive markers such as body weight, invasive markers such as detectable pathological changes in a tissue, changes in enzymes or in specific endogenous metabolites. Some of these are well known, for example, serum enzymes such as lactate dehydrogenase and the transaminases for detection of tissue damage or serum levels of metabolites such as urea and creatinine for evaluation of kidney function. However, there are many organs and tissues for which there are few if any biomarkers. For example, testicular damage is difficult to detect apart from using invasive techniques such as histopathology. A potential biomarker, urinary creatine has been investigated. This seems to be more sensitive than other markers and is reasonably specific for the testis, being elevated after acute doses of a variety of different testicular toxicants and after repeated exposure to 2-methoxyethanol and cadmium chloride.

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The detection and utilization of tissue/organ specific metabolites or enzymes is therefore one important avenue for the development of new biomarkers. Similarly for particular types of toxicity specific biomarkers need to be discovered, developed and validated. For example, there are no established, validated biomarkers for the non-invasive detection of fatty liver or phospholipidosis, both common toxic responses,. However, in both cases, there are biochemical markers associated with these phenomena. Thus for example urinary taurine is increased by chemicals causing fatty liver. However, it is also elevated by other types of liver dysfunction and is not therefore specific for this lesion. Phospholipidosis will be discussed later in this session. Biomarkers of susceptibility are inherent in the organism, typically enzymes involved in the metabolic activation of a chemical showing genetic polymorphisms. Other types are DNA repair enzymes which can show genetic variability and hence alter susceptibility to cancer, oncogenes and gene products, tumour suppressor genes, receptors or specific tissue types. More than one susceptibility factor and hence biomarker may be involved in the toxicity of a chemical. For example the lupus erythematosus syndrome induced by the drug hydralazine is associated with three susceptibility markers, the slow acetylator phenotype, the HLA type DR4 and female gender. Waterfield, C.J. Timbrell, J.A., 1999. Biomarkers: an overview. In: Ballantyne, B., Marrs, T., Turner, P. (Eds.), Textbook of Applied and General Toxicology, second ed. Macmillan, New York. Novel Biomarkers of Phospholipidosis and Peroxisome Proliferation Catherine J. Waterfield Safety Assessment, GlaxoSmithKline, Park Road, Ware, Herts SG12 ODP, UK We have been investigating the use of 1 H NMR spectroscopy to help classify subtle histological lesions that are not associated with any gross or overt toxicity, where a definitive diagnosis may be dependent on a biopsy and electron microscopy or direct biochemical analysis of the tissues. Changes in endogenous metabolite profiles have been identified using 1 H NMR spectroscopic analysis of urine from rats treated with compounds that cause either abnormal accumulation of phospholipids (PLs) or peroxisome proliferation (PP).Phenylacetylglycine (PAG) was identified as a potential urinary marker for phospholipidosis following the routine analysis of rat urine by 1 H NMR spectroscopy of rats treated with a series of new chemical entities (Nicholls et al., 2000) and subsequently chloroquine and amiodarone which caused PL accumulation. Routine screening of urine for PAG from treated rats correctly identified 14 compounds that subsequently caused PL accumulation, 127 that were identified as being unlikely to cause PL accumulation and incorrectly identified 4 as false positives. Those incorrectly identified caused other forms of lipidosis. As yet we have been unable to identify the mechanism by which the PAG is produced. A “targeted approach” to identify biomarkers in pathways that are perturbed has also been used to identify biomarkers of PL accumulation. The analysis by LC/MS of PL profiles in tissues from animals which have PL accumulation showed an increase in the total amount of PLs, as would be expected. However, a catabolically resistant species of PL, lysobisphosphatidic acid (LBPA), which is normally present in very low amounts, increased dissproportionately when phospholipids accumulate (Reaves et al., 2000). It is therefore suggested that LBPAis a potential biomarker of phospholipidosis (Mortuza et al., 2003) and investigations are ongoing as to the feasibility of measuring this in the plasma. 1 H NMR spectroscopy has also highlighted metabolites in the tryptophan/quinolate pathway as possible biomarkers of PP. Metabolites in the pathway, including the end product, N-methylnicotinamide (NMN), were found to correlate very strongly with PP and better than any other parameters of other pharmacological change (R2 = P > 0.97). NMN was subsequently measured in plasma samples taken at autopsy using LC–MS methods and correlated with urinary NMN excretion and PP. The mechanism responsible for the increased flux of tryptophan to NMN appears to be the down-regulation by PPARs, of the enzyme of aminocarboxymuconate-semialdehyde decarboxylase (ACMSD, EC 4.1.1.45) (Ringeissen et al., 2003).

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The true value of these molecules as biomarkers is currently being investigated in a series of validation studies. References Mortuza, G.B., Neville, W.A., Delaney, J., Waterfield, C.J., Camilleri, P., 2003. Molecular and cell biology of lipids BBA. Mol. Cell Biol. Lipids 1631, 136–146. Nicholls, A.W., Nicholson, J.K., Haselden, J.N., Waterfield, C.J., 2000. Biomarkers 5, 410–423. Reaves, B.J., Row, P.E., Bright, N.A., Luzio, J.P., Davidson, H.W., 2000. J. Cell Sci. 113, 4099–4108. Ringeissen, R., Connor, S.C., Brown, H.R., Sweatman, B.C., Hodson, M.P., Kenny, S.P., Haworth, R., McGill, P., Price, M.A., Aylott, M.M.C., Nunez, D.J., Haselden, J.N. Waterfield, C.J., 2003. Biomarkers 8 (3–4), 240– 271. Serum Pneumoproteins as Non-Invasive Markers of Lung Epithelium Damage Alfred Bernard Unit of Toxicology, Catholic University of Louvain, 30.54 Clos Chapelle-aux-Champs, B-1200 Brussels, Belzium E-mail address: [email protected] The assessment of lung toxicity in humans largely relies on indicators which although sometimes sensitive do not accurately reflect lung epithelial damage. Recent research in the field of biomarkers has led to the development of a new non invasive approach allowing the detection of subclinical changes of the respiratory epithelium caused by air pollutants and other types of injury. This approach, referred to as pneumoproteinemia, is based on the determination in serum of lung-specific secretory proteins reflecting the permeability or the cellular integrity of the lung epithelial barrier (Hermans and Bernard, 1999). Several pneumoproteins have already been validated as blood markers of increased permeability of the airways epithelium (Clara cell protein or CC16) or of the alveolar epithelium (surfactant-associated proteins A and B or SP-A and SP-B). A transient or chronic elevation of serum pneumoproteins has been reported following exposure to various lung toxicants such as tobacco smoke, firesmoke, ozone, LPS and more recently nitrogen trichloride, an irritant gas released in indoor chlorinated pools. In rats or mice acutely exposed to ozone or systemic lung toxicants (ANTU, MMT, 4-ipomeanol), the intravascular leakage of CC16 correlates with the elevation of albumin in lung lavage fluid. In humans, these permeability changes have been observed in asymptomatic subjects with normal or only slightly impaired lung function. For instance, in chlorinated pool attendees, the assay of serum SP-A or SP-B is presently the only test enabling the detection of an increased alveolar epithelium permeability caused by nitrogen trichloride (Carbonnelle et al., 2002). Alterations of airways epithelium resulting in a transient elevation of serum CC16 have been observed in humans and mice exposed to ozone levels below current standards. Serum CC16 also appears as the first non-invasive test to have evidenced a disruption of airways epithelium barrier during heavy exercise (Carbonnelle et al., 2002). Serum pneumoproteins can also serve as specific markers of damage or dysfunction of epithelial cells. A decrease of serum CC16, reflecting most likely a progressive destruction of Clara cells, has been found in smokers as well as in subjects chronically exposed to asbestos, crystalline silica, foundry dust and other lung irritants. Because of their sensitivity, non invasiveness and repeatability, serum pneumoproteins thus represent new tools which should improve the assessment of toxic lung injury and therefore contribute to a better protection of populations at risk. References Hernans, C., Bernard, A. 1999. Am. J. Resp. Crit. Care Med. 159, 646–678. Carbonnelle et al., 2002. Biomarkers 7, 1–15. Acknowledgement Research supported by the European Union (ENV-CT96-171 and QLK4-99-1308).

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Novel Biomarkers of Nephrotoxicity Detected Using Proteomics Mike D. Kelly1 Lasantha R. Bandara1 , Edward A. Lock2 , Sandy Kennedy1 1 Oxford GlycoSciences (UK) Ltd., The Forum, 86 Milton Park, Abingdon, Oxon OX14 4RY, UK; 2 Syngenta CTL,

Alderley Park, Cheshire, SK10 4TJ, UK 4-Aminophenol (4-AP) and d-serine are established rodent nephrotoxins that selectively damage renal proximal tubules (Gartland et al., 1989). As part of a mechanistic investigation into a range of organ toxicities, we took a high throughput proteomics approach to profile protein changes in the plasma of animals treated with each compound. Male F344 and Alpk rats were treated with increasing doses of 4-AP or d-serine and plasma samples were collected over time. Control groups received either saline or the non-toxic enantiomer, l-serine. Using high throughput 2D gel analysis (Kennedy, 2001), we identified a number of plasma proteins that displayed dose and time dependent regulation. One toxicity associated plasma protein was identified as the cellular enzyme fumarylacetoacetate hydrolase (FAH), which is known to be required for tyrosine metabolism. The FAH gene is mutated in the human genetic disorder type I tyrosinaemia resulting in liver and kidney abnormalities and neurological disorders (Kvittingen et al., 1991; Sun et al., 2000). FAH was elevated in the plasma of 4-AP and d-serine treated animals at early time points and returned to base line levels after 3 weeks. The protein was not elevated in the plasma of control animals or those treated with l-serine. The presence of FAH in plasma is intriguing, as it is normally a cellular enzyme with no known function in plasma. It is possible that 4-AP and d-serine may work through a previously unknown mechanism in the kidney through regulation of tyrosine metabolism or regulation of FAH activity. The link between kidney toxicants and inherited tyrosinaemia also raises the possibility that FAH may be a marker of kidney toxicity in man. These observations highlight the value of proteomics in identifying new biomarkers and providing new unprecedented insights into complex biological mechanisms. References Gartland, K.P., Bonner, F.W., Timbrell, J.A., Nicholson, J.K., 1989. Arch. Toxicol. 63 (2), 97–106. Kennedy, S., 2001. Toxicol. Lett. 120 (1–3), 379–384. Kvittingen, E.A., Talseth, T., Halvorsen, S., Jakobs, C., Hovig, T., Flatmark, A., 1991. J. Inherit. Metab. Dis. 14 (1), 53–62. Sun, M.S., Hattori, S., Kubo, S., Awata, H., Matsuda, I., Endo, F., 2002. J. Am. Soc. Nephrol. 11 (2), 291–300. Paediatric Studies—Are They needed? A Clinical Perspective Klaus Rose Novartis Pharma AG, 4002 Basel, Switzerland E-mail address: [email protected] Traditionally, most drugs are developed in adults first. Development in children is usually considered once the drug proves to be safe in adults and a reasonable paediatric market exists. Exceptions were specific pediatric diseases. This has led to today’s high incidence of unlicensed and off-label drug use in intensive-care units for neonates as well as in general paediatric practice. The multiple metabolic pathways involved in absorption, distribution and eleminination of drugs do not mature synchronously. Sulphatisation is increased in neonates and infants whereas glucuronidation is reduced. Children may use another main pathway for metabolisation of a specific drug than an adult. Especially in newborns absorption and elimination differ considerably from adults. Dose extrapolation from adults is of limited value, and specific testing in children is needed where a drug has therapeutic potential. PK/PD studies may suffice where the mechanism of disease is the same, clinical efficacy studies are needed in case of differences regarding pathophysiology or clinical presentation of a disease.

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Paediatric studies need to follow the same rules and guidelines as studies in adults. Among the many additional aspects are: children cannot give informed consent, parents must understand and support the study, personnel needs specific experience, children need time, blood should be drawn as rarely as possible in small volumes. The US pediatric exclusivity act 1997 offers extended market exclusivity for drugs for which additional paediatric clinical research is performed. This incentive beyond the mere size of the pediatric market resulted in a sharp increase in paediatric studies. The paediatric rule, introduced little later, required assessment of new compounds’ therapeutic potential in children. Although this rule was struck down October 2002 by a federal judge, ruling that FDA overstepped its statutory authority, pharmaceutical industry will nevertheless have to assess early potential use of new drugs in children. Industry must plan years in advance, and it is likely that paediatric development will be again be required by US legislation in not too far future. This is even more critical as similar efforts exist in Europe. The EU Commission’ (EUC) consultation paper ‘Better Medicines for Children’ published February 2002 will be followed by a formal EUC proposal and then a complicated decision making process. The barriers to paediatric clinical research include scepticism, uncertainty, overprotection, lack of experience, lack of political will, and many more. All layers of society including parents, clinicians, ethical committees, health authorities, legislators and pharmaceutical industry have contributed to the insufficient availability of drugs properly tested for use in children. Development of better medicines for children will need a strong public opinion in favour of more clinical and basic research. References Conroy, S., McIntyre, J., Chonara, I., 1998. Arch. Dis. Childhood Fetal Neonatal Ed. 80, F142–F145. http://www.ich.org. http://www.fda.gov/cder/pediatric/. Turner, S., Longworth, A., Nunn, A., Choonara, I., 1998. BMJ 316, 343–345. Paediatric Drug Development—A Regulatory View Henry E. Stemplewski Medicines Control Agency, London, UK E-mail address: [email protected] Most of the medicinal products used in paediatric patients have not been developed or licensed for use in this age group. In most cases, an extrapolation from the adult data is used to support the use in children. However, insufficient information on which to base an appropriate dosage regimen may lead to underdosing with a resulting lack of efficacy or to overdosing with a resulting increase in toxicity. There may also be unexpected toxicity unique to the paediatric population. It is difficult to quantify the extent of the unlicensed prescribing, as there appear to be no national surveys published. Results of hospital based surveys indicate that “off label” prescribing is extensive, e.g. about 90% of neonates in a neonatal intensive care unit and about 70% of children in an intensive care unit. There is little evidence from the MCA spontaneous reporting system of harmful effects arising from “off-label” use. However, this may be due to prescribing clinicians being unwilling to report adverse reactions resulting from such use. The Committee for Proprietary Medicinal Products (CPMP) have issued guidelines which define paediatric age bands, encourages the pharmaceutical industry to conduct trials in the paediatric population where appropriate and states that the regulatory authorities expect paediatric data where paediatric use is likely. A recently published analysis of 45 substance licensed by the EMEA between January 1995 and April 1998 showed that although 29 (64%) were of possible use in children, only 10 were licensed for paediatric use. There is currently no legal obligation for a company to apply for a license in the paediatric population or to provide information on paediatric use, nor an obligation to develop appropriate formulations/ dosage strengths. The current national legislation allows a physician

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to prescribe unlicensed medicines or to use licensed medicines “off-label”. Many medicines are prescribed to the paediatric population under the exemption. There may be many reasons to explain the reluctance of pharmaceutical companies to develop medicines for the paediatric population. The most important may be the lack of financial incentive: the market for children is smaller than the adult market. The European Commission is interested in encouraging paediatric medicine development by adopting a modified US model of incentives (additional market exclusively) but also of obligations (inclusion of paediatric studies for every new active substance and line extension). Recently (November 2001), the CPMP Safety Working Party (SWP) considered the non-clinical safety testing requirements of medicines intended for use in paediatric patients. The SWP issued a concept paper which recommended that the circumstances in which studies in juvenile animals are likely to be valid should be investigated. To date, studies in juvenile animals appear not to have been well validated for their ability to predict human toxic responses. This may not be surprising since the inherent species differences are augmented by differences in growth and development. Therefore, since adult animals predict only 50–70% of human toxic responses, the utility of juvenile animals for human responses appears to be uncertain. The question still remains under which circumstances the use of juvenile animals may be of value. Arguably, at present the only circumstances would be in the evaluation of animal/human concordances, for example, assessing juvenile animal models for well documented effects in children of individual medicines e.g. Reye’s Syndrome and aspirin. References Note for Guidance on Clinical Investigation of Medicinal Products in the Paediatric Population, 1999. CPMP/ ICH/2711/99. Concept Paper on the Development of a CPMP Points to Consider on the Evaluation of the Pharmacokinetics of Medicinal Products in the Padiatric Population, 2002. CPMP/EWP/968/02. Acknowledgement Concept Paper on the Development of a CPMP Note for Guidance on the Need for Preclinical Testing of Human Pharmaceuticals in Juvenile Animals, 2001. EMEA/CPMP/SWP/3404/01. Keywords: Paediatric; Juvenile Animals in Paediatric Drug Development, Predictive Models Or Confounding Factors? Jim Ridings Covance Laboratories, Otley Road, Harrogate, North Yorkshire, HG3 1PY, UK The use of juvenile animals in drug development is not a new discipline but in recent years has become the focus of increased attention. High profile issues related to off-label paediatric therapy and, more specifically, the FDA’s final Paediatric Rule have led to an ever-greater debate about obtaining toxicology data from juvenile animals. Using juvenile animals in toxicology studies presents the challenge of obtaining data from an immature model without any regulatory guidance and creates a unique set of ethical and emotional issues. The selection of the correct species represents a balance between allowing comparison with adult toxicology data, the ability to dose the required age by the chosen route and formulation, and the practicalities of maintaining the mother and offspring. It could be argued that intra-species comparisons of adult-juvenile responses are more relevant than juvenile animal-human comparisons. The age of the offspring at the start of treatment and the duration of treatment have been the subjects of greatest debate. Treatment must not only cover the equivalent developmental period to that targeted in Man but must also try and predict any subsequent impact (e.g. behaviour, reproduction) in adulthood. Large knowledge gaps in comparative stages of development are being addressed by organisations such as the International Life Sciences Institute (ILSI) but the picture remains incomplete. Providing the correct study design is used, toxicology studies in juvenile animals are equally predictive and no more confounding for paediatric therapy than are studies in adult animals for adult therapy. Studies in adult animals, including those associated with pre- and postnatal

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development, are less predictive for paediatric therapy yet there remains a reluctance to conduct studies involving direct administration to juvenile animals. Juvenile Animal Toxicity Studies For Strattera® : A Non-Stimulant Drug for Attention-Deficit/Hyperactivity Disorder (ADHD) David O. Clarke, John-Michael Sauer, Joseph P. Tizzano Lilly Research Laboratories, Eli Lilly and Company, Indiana, USA Strattera® (atomoxetine HCl) is a selective norepinephrine reuptake inhibitor, recently approved in the United States for the treatment of Attention-Deficit/Hyperactivity Disorder (ADHD) in children, adolescents, and adults. To support the pediatric indication, a programmatic and comprehensive toxicologic evaluation of atomoxetine was conducted in young rats and dogs of analogous ages to the intended patient population. Definitive studies conducted in young Fischer 344 rats (10–20/sex/group) assessed general toxicity, reproduction and fertility, and neurobehaviour. Oral atomoxetine HCl doses of 0, 1, 10 or 50 mg/kg per day were administered from postnatal day 10 for approximately 11 weeks throughout sexual maturation to early adulthood, including mating through implantation in the fertility assessment. Plasma concentrations of atomoxetine increased with dose; exposure was initially high compared to adult rats. The highest dose produced generalized toxicity, indicated by a low incidence of clinical signs and decreases in body weight gain and food consumption. Growth, indicated by femur length, was not affected. Learning, memory, and sensory motor function were normal throughout the study; a slight, non-persistent increase in motor activity occurred at the higher doses. Evaluation of multiple brain sections, with emphasis on the major norepinephrine-containing regions, demonstrated no toxicity. Other major organs were also unaffected. Onset of vaginal patency and preputial separation were slightly delayed; however, all rats matured and there were no malformations of the vagina or prepuce/glans penis. Vaginal cytology and estrus cycling assessed during the final 2 weeks were normal. Spermatogenesis was normal, while cauda epididymal sperm count was slightly lower at the higher doses. Reproductive performance and fertility were unaffected. The toxicity of atomoxetine HCl was also assessed in 8-week old beagle dogs (4/sex/group) that received oral doses of 0, 4, 8, or 16 mg/kg per day for the next 4 weeks, a significant growth period. Identical doses were previously administered to adult dogs for up to 1 year, enabling comparison of findings in young versus adult dogs. Plasma concentrations of atomoxetine increased with dose; initial exposures were slightly lower than in adult dogs and approached adult levels with repeated administration. Atomoxetine was well tolerated at all doses, effects being limited to dose-related clinical signs, e.g. mydriasis, tremors, and emesis, as seen in adult dogs and attributed to the pharmacology of atomoxetine. No persistent abnormalities were noted during physical, neurological, or ophthalmic examinations. There was no major organ toxicity (again, multiple brain sections were evaluated), no effect on body weight gain, and no electrocardiographic abnormalities attributed to atomoxetine. The data generated from dosing young rats and dogs with atomoxetine HCl suggest that clinically important effects on reproductive and neurobehavioral development would not be expected. We conclude that these studies have provided valued information for the development, registration, and approval of Strattera® for the treatment of pediatric ADHD. Drug Hypersensitivity Reactions in the Skin: Understanding Mechanisms and the Development of Diagnostic and Predictive Tests Dean J. Naisbitt Department of Pharmacology, Ashton Street, The University of Liverpool, Liverpool, L69 3GE The skin is a common target for drug hypersensitivity reactions. Skin damage is often accompanied by eosinophilia, fever and derangement of liver function tests. Identification of drug-specific T-cells in peripheral blood and skin of hypersensitive patients provides evidence for the role of T-cells in the pathology of the reaction. The nature of

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the drug signal presented to T-cells, the mechanism of drug presentation and why drug-specific T-cells frequently target skin is not fully understood. Immunochemical theory suggests that drug metabolism and the formation of a protein reactive intermediate is a prerequisite to T-cell activation; the haptenated protein is processed and presented on MHC molecules to T-cells. Recently, an alternative mechanism has been proposed, which states that the drug can bind directly, in a non-covalent manner to MHC. We have developed an animal model to investigate the mechanism of drug presentation to the immune system. T-cells proliferated in the presence of MHC-restricted antigen derived from both viable and dead cells haptenated with low and high levels of drug metabolite (Naisbitt et al., 2002). Direct modification of MHC was not the mechanism of antigen presentation. This contrasts with human studies where an in vitro cellular response to the parent drug predominates. Proliferation of human lymphocytes is seen at therapeutic concentrations in the absence of metabolism and covalent binding. Diagnosis of drug hypersensitivity is difficult since available tests have a low specificity and sensitivity. Recently, we have used in vitro cell culture methods to diagnose drug hypersensitivity. Lymphocytes from 20/22 hypersensitive patients and 0/12 controls proliferated when rechallenged with drug. Identification of the culprit drug prevented unnecessary re-exposure and the clinician was able to continue administration of non-allergenic drugs in patients on multiple dosing regimens. By cloning drug-specific T-cells, we have characterized different forms of cutaneous reactions in terms of their cellular pathophysiology. This presentation will focus on anticonvulsant hypersensitivity. Over eighty drug specific T-cell clones have been generated; most of which were CD4+ cells (Naisbitt et al., 2003a,b). Usage of V␤ 5.1 seems to be of particular importance since a number of T-cell clones were T-cell receptor V␤ 5.1 positive. T-cells recognized drugs in an MHC-restricted fashion; activated T-cells killed target cells and secreted perforin, IFN-␥, IL-5 and MIP-1␣, MIP-1␤ and I-309. The T-cell receptor was able to accommodate small changes in the structure of the drug antigen. Drug-specific T-cells also expressed increased levels of the skin homing molecule cutaneous lymphocyte antigen and a range of chemokine receptors that may explain the organ specificity of anticonvulsant hypersensitivity. Presently no tests are available to predict the ability of a drug to stimulate an immune response. The focus of our current research is to use sulfamethoxazole (SMX) and it’s nitroso metabolite as a paradigm, to study the ability of a drug (metabolite) to stimulate T-cells: (1) in vivo, by measuring mouse lymph node cell proliferation and cytokine polarisation; and (2) in vitro, using human lymphocytes from drug-na¨ıve individuals. Administration of nitroso SMX, but not the parent drug stimulated lymph node cell proliferation and proliferating cells secreted high levels of IL-5 and IFN-␥. These data are consistent with a hapten mechanism. Similarly, lymphocytes from 10/10 drug na¨ıve individuals responded to nitroso SMX. Cells from 3 patients also responded with SMX. These cells were CD4+ and secreted IFN␥, IL-4 and IL-5. These data confirm the ability of chemically inert drugs and drugs that gain their immunogenicity by the formation of a hapten carrier complex to stimulate T-cells. The long-term aim of these studies is to provide test systems for the evaluation of drug safety and patient susceptibility to drug hypersensitivity. References Naisbitt, D.J., Farrell, J., Gordon, S.F., Maggs, J.L., Burkhart, C., Pichler, W.J., Pirmohamed, M., Park, B.K., 2002. Mol. Pharmacol. 62 (3), 628–637. Naisbitt, D.J., Britschgi, M., Wong, G., Farrell, J., Depta, J.P.H., Chadwick, D.W., Pichler, W.J., Pirmohamed, M., Park, B.K., 2003. Mol. Pharmacol., in press. Naisbitt, D.J., Farrell, J., Wong, G., Depta, J.P.H., Dodd, C.C., Hopkins, J.E., Gibney, C.A., Chadwick, D.W., Pichler, W.J., Pirmohamed, M., Park, B.K., 2003. J. Allergy Clin. Immunol., in press.

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Polymer-coated Adenovirus Can Be Programmed to Infect Cells Using Selected Ligands and Shows Extended Plasma Circulation Following Intravenous Injection Leonard W. Seymour Department of Clinical Pharmacology, Oxford Unversity, Oxford OX2 6HE, UK Human type 5 adenovirus can be surface-modified with multivalent reactive polymers based on poly[N-(2-hydroxypropyl)methacrylamide] designed to form covalent bonds to amino groups exposed on the virus surface. The resulting dense polymer coating can lead to effective masking of surface epitopes, protecting the virus from antibody-binding and neutralisation of infection. The coating also masks receptor-binding sites in fibre and penton, preventing normal receptor-mediated cell entry and ablating virus infection (1). Linkage of new ligands to the outside of the polymer coating has the potential to mediate cellular entry via novel receptors, and in many cases this can lead to efficient restoration of infection through target-associated receptors. Several growth factors have been shown to act as successful retargeting ligands, certain antibodies (notably against Her2) and laminin fragments, although optimal activity often depends on careful removal of residual free polymer and non-incorporated retargeting ligand. Receptor-mediated retargeting can be performed successfully in the presence of significant levels of anti-adenovirus neutralising antibodies. Polymer-coated adenovirus shows decreased association with human peripheral blood mononuclear cells, notably CD14-positive cells, and no transgene expression is detected. Following intravenous injection to mice there is a complete loss of hepatic infection, reflected in an extension of its circulation in the blood plasma. This combination of antibody-resistance, tropism ablation and reprogramming together with systemic bioavailability makes this an enticing technology platform enabling application of therapeutic adenovirus for treatment of widespread and disseminated disease. References Fisher, K.D., Stallwood, Y., Mautner, V., Ulbrich, K., Etrych, T., Strohalm, J., Seymour, L.W., 2001. Polymer-coated adenovirus permits efficient retargeting and evades neutralising antibody. Gene Therapy 8, 341–348. Applications of Cytochrome P450 Gene Therapy in Cancer Therapeutics David J. Waxman, Youssef Jounaidi, Chong-Sheng Chen, Hong Lu, Christopher H. Hurst, Pamela S. Schwartz Department of Biology, Boston University, Boston, MA 02215, USA Several commonly used cancer chemotherapeutic prodrugs, including cyclophosphamide and ifosfamide, are activated via a liver cytochrome P450 (CYP)-catalyzed prodrug activation reaction. Rodent and human hepatic P450s display clear similarities in their catalytic specificities towards these anti-cancer prodrug substrates. Preclinical studies have shown that the chemosensitivity of tumors to P450 prodrugs can be dramatically increased by transfer of a rodent or human CYP gene, which confers on the target tumor tissue the capacity for localized prodrug activation and, in the case of cyclophosphamide, induces a mitochondrial/caspase 9-dependent pathway of apoptotic cell death. This P450 gene-directed enzyme prodrug therapy (P450 GDEPT) greatly enhances the therapeutic effect of P450-activated anti-cancer prodrugs without increasing host toxicity associated with systemic distribution of active drug metabolites formed by the liver. The efficacy of P450 GDEPT can be augmented in several ways: (1) by further increasing the partition ratio for tumor:liver prodrug activation in favor of increased intratumoral metabolism, e.g., by co-expression of the flavoenzyme NADPH-P450 reductase, by localized prodrug delivery or by the selective pharmacologic inhibition of liver prodrug activation; (2) by anti-angiogenic scheduling of prodrug administration, which substantially magnifies the anti-tumor effect and leads to major regression of established tumors; and (3) by co-expression of an anti-apoptotic factor, which inhibits tumor cell apoptosis in a manner that prolongs the generation of soluble, bystander cytotoxic metabolites but does not ultimately block tumor cell death. P450 GDEPT prodrug substrates are diverse in their structure, mechanism of action and choice of optimal prodrug-activating P450

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gene; they include both established and investigational anti-cancer prodrugs, as well as bioreductive drugs that can be activated by P450/P450 reductase in a hypoxic tumor environment. Several strategies have now been employed to achieve the tumor-selective gene delivery that is required for the success of P450 GDEPT; these include the use of tumor-targeted cellular vectors and tumor-selective oncolytic viruses. Overall, P450-based GDEPT presents several important, practical advantages over other GDEPT strategies that may facilitate the incorporation of P450 GDEPT into existing cancer treatment regimens. Recent reports of clinical efficacy in P450-based phase I/II gene therapy trials for breast and pancreatic cancer patients support this conclusion. Supported in part by NIH grant CA49248 (to D.J.W.). References Chen, L., Waxman, D.J., 2002. Curr. Pharm. Des. 8, 1405–1416. Jounaidi, Y., Waxman, D.J., 2001. Cancer Res. 61, 4437–4444. Schwartz, P.S., Chen, C.S., Waxman, D.J., 2002. Cancer Res. 62, 6928–6937. Virus-directed Enzyme Prodrug Therapy for Cancer: Clinical Trials with an Adenovirus Encoding Bacterial Nitroreductase (Ad-ntr) Vivien Mautner Cancer Research UK Institute for Cancer Studies, University of Birmingham, UK The bacterial enzyme nitroreductase (ntr) converts the prodrug CB1954 into a highly toxic short-lived bifunctional alkylating agent, which kills both dividing and non-dividing cells and has a strong bystander effect. An E1, E3 deleted adenovirus vector encoding ntr under control of the CMV promoter (CTL102) has demonstrated in vivo efficacy in xenograft mouse models, after single intratumoural injection of virus and systemic delivery of CB1954 (Weedon et al., 2000; Djeha et al., 2001). A phase I trial of CTL102 to determine the maximum tolerated dose has been undertaken in patients with hepatic metastatic colorectal cancer awaiting surgical resection. Escalating single doses of virus (range 108 to 5×1011 virus particles, three patients per dose) have been administered percutaneously into the tumour under ultrasound guidance. Liver resection is performed at 48-96hr after virus injection, and the tumour examined for ntr expression. Patients are monitored post-injection for overt toxicity (which has been minimal; 1 patient with systemic flu-like symptoms); for virus shedding (no virus detected in urine, stool, throat swab or blood at 24 h post-injection); for viral DNA dissemination by quantitative PCR of venous blood (detectable but cleared by 8hr in most patients); and for antibody responses to adenovirus and ntr (detectable and rising in most patients). Distribution and extent of ntr expression has been demonstrated and quantitated by immunohistochemistry of fixed tumour sections; it is seen in tumour and non-tumour cells within the tumour margin, but not in adjacent normal liver cells. Significant expression has been achieved at a dose of 1011 virus particles, and the trial is now extended to patients with non-resectable tumours, who will receive virus and a single dose of CB1954 (24 mg/m2 i.v.). A phase I trial has also been initiated in prostate carcinoma patients, where similar observations have been made; results from the liver and prostate trials will be presented. References Weedon, S.J., et al., 2000. Int. J. Cancer 86, 848–854. Djeha, A.H., et al., 2001. Mol. Ther. 3, 233–240. 1 The

following people contributed to the preclinical and clinical studies: Nicholas James, Sarah White, Ratna Rajaratnam, Daniel Palmer, Graham Young, Stefan Hubscher, Lawrence Young, Peter Searle, David Kerr∗ (Cancer Research UK Institute for Cancer Studies, University of Birmingham, UK); Simon Olliff, Darius Mirza, Mike Wallace (Queen Elizabeth Hospital, Birmingham, UK); John Ellis, Andrew Mountain, Peter Harris, Hakim Dejha, Mark Horne, Simon Hill, Christopher Wrighton (ML Laboratories, Keele, UK). ∗ Present address: Department of Clinical Pharmacology, University of Oxford, UK. 2 Supported by the Medical Research Council.

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Prospects of Immune Gene Therapy for Cancer Nicola Hardwick, Lucas Chan, David Darling, Joop Gäken, Joanna Galea-Lauri, Farzin Farzaneh Department of Molecular Medicine, GKT School of Medicine, King’s College London, UK Tumour cells express a range of potential targets for immune mediated recognition and rejection of the malignantly transformed cells. Such targets include mutated gene products, viral antigens, abnormally glycosylated proteins, etc. Professional antigen presenting cells (APC) such as dendritic cells (DC), play crucially important roles in the immune recognition of foreign antigens and rejection of cells expressing such antigens (e.g. rejection of mismatched transplanted organs). Therefore, enhanced expression and/or display of tumour antigens by the APC, or expression of appropriate APC specific gene products by tumour cells, may provide effective means of generating tumour cell vaccines. Such vaccines are now beginning to demonstrate evidence of efficacy in the induction of immune mediated rejection of already established tumours (therapy) and/or prevention of tumour development (prophylaxis). The basic principles and some of the strategies being developed for the induction of immune mediated tumour rejection will be discussed. Gene Therapy—Is It Safe? Lincoln Tsang Arnold & Porter, Tower 42, 25 Old Broad Street, London EC2N 1HQ, UK E-mail address: lincoln [email protected] Whilst gene transfer technology offers potentials for treating certain life-threatening and debilitating diseases, it should be recognised that we are still at the stage of validating its full potentials for use in a clinical setting. Patient safety is of paramount importance in any clinical studies. Therefore, due consideration should be given to ensuring appropriate quality and non-clinical pharmaco-toxicological testing is carried out. Studies should be aimed at characterising the product in order to address specific questions concerning the pharmaco-toxicology of the product. Gene transfer is defined in the European regulatory guidance as the deliberate introduction of genetic material into cells for diagnostic, prophylactics such as DNA vaccines and therapeutic products such as cancer vaccines. Gene transfer products belong to a sub-set of the recombinant DNA derived products and biologicals. Genetic materials can be transferred using physico-chemical means or a micro-organism such as virus or a cell-based substrate for in situ gene transfer. The underpinning regulatory science with respect to the safety, quality and efficacy is not substantially different whilst recognising that the active ingredient in the finished product consists of genetic material. That said, there are, however, specific quality and safety issues surrounding the clinical use of gene transfer medicinal products. In this presentation, I will attempt to provide an overview of the European regulatory and legal framework and to highlight the salient points arising from the established regulatory guidance governing gene transfer medicinal products. These include: Product characterisation and control. Pre-clinical pharmaco-toxicology such as the relevance of bio-distribution in the assessment of safety of gene transfer medicinal products. Clinical safety. Keywords: Regulatory; Gene transfer; Safety

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Cytochrome P450 reductase (CPR) knockouts from yeast to C. elegans Steven Kelly, Diane Kelly, Andrew Warrilow, David Lamb Wolfson Laboratory of P450 Biodiversity, Institute of Biological Sciences, University of Wales at Aberystwyth, Aberystwyth, Wales SY23 3DA, UK The cytochrome P450 superfamily is central to the metabolic fate of organic chemicals and biochemical warfare between organisms as well as synthesising cholesterol, hormones and signalling molecules. Yeast has served as a useful model of eukaryotic biology and has an elegant genetic system. In eukaryotes, CPR catalyses electron-transfer from NADPH to CYP during the monooxygenation catalytic cycle as well as being involved in electron transfer to other enzymes such as squalene epoxidase in sterol biosynthesis. In gene knockout studies, it was apparent that yeast was not dependent on CPR for essential CYP function. Indeed, it was observed that growth and sterol synthesis occurred and that cytochrome b5 and NADH cytochrome b5 reductase could support CYP51 activity (Venkateswarlu et al., 1998, Lamb et al., 1999). This was contrary to dogma suggesting that CPR was needed at least to donate the first electron of the catalytic cycle. Data suggesting this might not be true for all mammalian CYPs exists, but a mouse CPR knockout was lethal (Shen et al., 2002). The completed Caenorhabditis elegans genome revealed the presence of 80 putative cytochrome P450 (CYP) genes, but as usual only one NADPH cytochrome P450 reductase (CPR) gene. We investigated further the biodiversity of essentiality of CPR using C. elegans and RNAi gene inactivation technology. As with yeast where gene deletion on a genomic scale has been achieved, RNAi is a rapid method to investigate gene function. Two such studies have found RNAi strains for CPR had an embryonic lethal phenotype. In contrast our independent work using a different methodology showed viable worms exhibiting defects in reproductive development and the formation of inclusion bodies along the gastrointestinal tract, the initial interface for xenobiotic metabolism of ingested material. This suggested a knock-down phenotype had been achieved and interference with CYP activity might be responsible for the phenotype. By varying the nucleic acid used in the RNAi approach it may therefore be possible to obtain clues to function of essential genes in the nematode and the studies on CPR from C. elegans and mouse suggest that this gene may be essential across the animal kingdom. References Lamb, D.C., Kelly, D.E., Manning, N.J., Kaderbhai, M.A., Kelly, S.L., 1999. FEBS Lett. 462, 283–288. Shen, A.L., O’Leary, K.A., Kasper, C.B., 2002. J. Biol. Chem. 277, 6536–6541. Venkateswarlu, K., Lasmb, D.C., Kelly, D.E., Manning, N.J., Kelly, S.L., 1998. J. Biol. Chem. 273, 4492–4496. Mouse Hepatic P450 Reductase Knockout Colin J. Henderson, Diana M.E. Otto, Dianne Carrie, Aileen W. McLaren, Jane Ross, Ian Rosewell1 , C. Roland Wolf. Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Level 5, Ninewells Hospital & Medical School, Dundee, UK; 1 Cancer Research UK Transgenic Services, Clare Hall Labs, Potters Bar, Herts, UK Cytochrome P450s represent a major defence against chemical challenges from the environment; these haemoproteins constitute a supergene family, and are classified as Phase I drug metabolising enzymes, catalysing the insertion of an atom of molecular oxygen into a huge range of diverse substrates (Omura, 1999). In addition, P450s carry out a wide variety of ‘housekeeping’ functions, including hormone metabolism and bile acid and cholesterol biosynthesis (Pikuleva and Waterman, 1999). Our laboratory has been working on the expression and regulation of P450 enzymes for many years, in particular, the relationship between P450 expression and fundamental cellular processes such as carcinogenesis and drug resistance (Wolf et al., 1994; Henderson et al., 2000). Although several P450 genes have been ‘knocked out’, these studies have been relatively uninformative: deletion of P450s involved in essential endogenous functions tends to result in lethality, whereas inactivation of drug

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metabolising P450s leads to little or no obvious phenotypic changes unless challenged chemically (McKinnon and Nebert, 1998). This is in the main due to the overlapping substrate specificity demonstrated by P450s, and the ability of other isoenzymes to at least partially compensate for the absence of individual enzymes. In order to advance our knowledge of P450 function, particularly in relation to carcinogenesis, we have deleted the sole electron donor to cytochrome P450s, cytochrome P450 reductase (CPR) (Smith et al., 1994) specifically in the liver. These hepatic CPR null mice grow and develop normally; however, they have enlarged, fatty livers, an almost complete lack of bile production, and a significantly lowered serum lipid profile. Hepatic CPR activity is reduced by >95%, and the ability of liver microsomes from mice lacking hepatic CPR to hydroxylate testosterone is essentially abolished. When challenged with a dose of pentobarbital non-narcotic to wild-type mice, hepatic CPR null mice sleep for a period in excess of 2 h, and furthermore fail to activate paracetamol to a hepatotoxic intermediate. These data demonstrate not only that the hepatic P450 system is not essential for survival in the postnatal period, but that hepatic CPR null mice will be a good model to study the role of hepatic and extrahepatic P450s in drug disposition. References Henderson, C.J., Sahrouei, A., Wolf, C.R., 2000. Biochem. Soc. Symp. 28, 42–46. McKinnon, R.A., Nebert, D.W., 1998. Clin. Exp. Pharmacol. Physiol. 25, 783–787. Omura, T., 1999. Biochem. Biophys. Res. Commun. 266, 690–698. Pikuleva, I., Waterman, M., 1999. Mol. Aspects Med. 20, 33–42, 43–37. Smith, G.C., Tew, D.G., Wolf, C.R., 1994. Proc. Natl. Acad. Sci. U.S.A. 91, 8710–8714. Wolf, C.R., Smith, C.A., Forman, D., 1994. Br. Med. Bull. 50, 718–731. Transient Humanization of Nuclear Receptor CAR in Mouse Liver M. Negishi, R. Moore, T. Sueyoshi Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA The nuclear receptor CAR is a coordinate regulator of hepatic gene expression in defense against chemical toxicity. While CAR activity is tightly regulated in liver in vivo, the receptor displays high constitutive activity in a cell-based in vitro assay. Thus, development of an in vivo system is essential for investigating the cellular and molecular mechanisms that regulate CAR function. For this purpose, we have been using tail vein injection of plasmids (Sueyoshi et al., 2002). Mouse CAR is sequestered in the liver cytoplasm and translocates into the liver nucleus in response to drug treatments. Tail vein injection of GFP-tagged human CAR expression plasmid resulted in the cytoplasmic expression of GFP-CAR in the mouse liver. Moreover, the GFP-CAR was accumulated in the liver nucleus of the drug-treated mice, allowing us to identify the C-terminal Leu-rich peptide as a xenochemical response signal of the nuclear translocation (Zelko et al., 2001; Sueyoshi et al., 2002). Co-injection into CAR-null mice of the CAR-expression and PBREM-Luc reporter plasmids provided us with an in vivo CAR-mediated trans-activation assay system, with which an in vivo role of a given amino acid residue in the receptor activation can be investigated. The in vivo expression of human CAR turned the CAR-null mice into a transient humanization of the receptor. Both PB and TCPOBOP treatments accumulated human CAR in the nucleus of the CAR-null mice, while the only PB treatment activated the human receptor-mediated trans-activation of the reporter gene. These indicate that the nuclear translocation and trans-activation activities are distinctly regulated in mouse liver in vivo. Using tail vein injection of expression plasmids into mice, various recombinant receptors can directly be expressed in the livers, offering us an in vivo transient transfection assay system. References Sueyoshi, T., Moore, R., Pascussi, J.M., Negishi, M., 2002. Methods Enzymol. 356, 205–213. Zelko, I., Sueyoshi, M., Kawamoto, T., Moore, R., Negishi, M., 2001. Mol. Cell. Biol. 21, 2838–2846.

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Seeing is Believing—Transgenic Promoter/Reporter Mice and Bioluminescent Imaging Anthony M. Lynch Safety Assessment, GlaxoSmithKine, Park Road, Ware, Herts SG12 0DP, UK The metabolism of luciferin in cells or animals transgenic for luciferase produces light that may be detected in vitro (cell culture) or trans vivo in living animals by an intensified CCD camera (biophotonics) and captured by image analysis (Zhang et al., 2001). Thus, the generation of transgenic promoter-luciferase animals for genes regulated by specific toxic processes, coupled with real-time evaluation of site-specific gene expression may provide novel, non-invasive in vivo biomarkers which may be predictive of developing toxicity or provide information regarding drug metabolism. For example, studies in HO-1.luc mice, transgenic for the murine haem oxygenase-1 promoter show that the transgene is upregulated by treatment with cadmium chloride, chloroform, allyl alcohol, bleomycin, and benzofuran. Comparison of the HO-1.luciferase response with markers of toxicity measured ex vivo (RT-PCR differential gene expression, clinical chemistry and pathology) confirm that the expression of the transgene is correlated with the expression of the endogenous HO-1 gene and with more traditional endpoints of toxicology. In the CYP3A4-luc rat, hepatic expression of the transgene was upregulated by various xenobiotics (rifampicin, nifedipine, phenobarbital, clotrimazole, dexamethasome-t-butylacetate) and the ability to evaluate extra-hepatic enzyme induction in the whole animal, in real time, may provide a significant advantage for understanding developing toxicity and evaluating drug-drug interactions. The development of luciferase tagged cells has been used to screen for drug efficacy in Oncology (Voojis et al., 2002) and Infectious Disease models (Bhaumik and Gambhir, 2002), but also, these may be used to evaluate host defence responses in immunotoxicology. The use of promoter/reporter cell lines may also permit in vitro and in vivo correlates (Esposito et al., 2002). Such studies demonstrate the potential for biophotonic image analysis to capture real-time, site-specific, gene expression in living animals, which may be predictive of developing toxicity and/or drug metabolism. References Bhaumik, S., Gambhir, S.S., 2002. PNAS 99, 377–382. Voojis, M., Jonkers, J., Lyons, S., Berns, A., 2002. Cancer Res. 62, 1862–1867. Zhang, W., Feng, J.Q., Harris, S.E., Contag, P.R., Stevenson, D.K., Contag, C.H., 2001. Transg. Res. 10, 423–434. Esposito, L., Kwon, P., Yu, C., McMullen, D.J., Sambucetti, L.C., 2002. Abstract at SOT 41, Nashville, TN. Cancer Genetics and Acceptable Exposure Limits Julian Peto Section of Epidemiology, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK Acceptable exposure limits for carcinogens are based on average cancer rates in exposed populations. A few individuals are at very high risk for certain cancers due to known susceptibility genes, and a considerably larger proportion may be at quite high risk due to combinations of less penetrant genes. Mechanistic considerations and epidemiological evidence suggest that hereditary cancer syndromes may confer greatly increased susceptibility to certain carcinogens. The ability to identify susceptible individuals by genetic testing will raise difficult ethical and legal issues. Those who suffer substantially higher risks than the general population can be protected either by identifying and protecting them, or by reducing exposure limits for occupational and environmental exposures for the whole population.

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Visualizing the regulatory network of Ah receptor signaling using a combined experimental and bioinformatics approach Guang Yao, Christopher A. Bradfield McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Avenue, Madison, WI, 53711, USA A high throughput approach was developed that allowed the use of a genetically-convenient yeast deletion array to identify 66 novel modifiers of the mammalian AHR pathway. To characterize these modifiers of AHR signaling, an integrated approach was established that combines multiple bioinformatic and experimental techniques. By overlapping the data from these methods, we were able to classify AHR modifiers into several functional groups. These groups can be described as those that regulate receptor folding, nuclear translocation, transcriptional activation, receptor level, and one unknown function related to the PAS domain. Based on these results, an expanded model of AHR signaling has been proposed. It is suggested that receptor folding involves Hsp90 cochaperones Sti1p, Cpr7p, and that transactivation requires the SAGA/PCAF complex subunits Gcn5p, Spt3p, and Spt8p, as well as the SWI/SNF complex subunits Snf12p and Swi3p. The approaches that we have developed in this study can be applied to other mammalian proteins to obtain a global view of their signaling mechanisms in a timely manner. Aryl Hydrocarbon Receptor (AhR) Biology David R. Bell School of Life and Environmental Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK E-mail address: [email protected] Polycyclic aromatic compounds and dioxins are present as ubiquitous contaminants; of these, 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is a highly potent congener, and is the most extensively studied. TCDD can cause toxicity in animals, including tumour promotion, embryotoxicity/teratogenesis and a severe wasting syndrome followed ultimately by death (Schmidt and Bradfield, 1996; Gu, Hogenesch et al., 2000). The toxic effects of TCDD are mediated by binding to a cytosolic protein, aryl hydrocarbon receptor (AhR). When AhR binds the ligand, it initiates the dioxin-signalling pathway through transcriptional activation. Cytochromes P450 1A1, 1A2 and 1B1, and many phase II detoxification genes are induced through this pathway (Gu, Hogenesch et al., 2000). TCDD’s toxic effects are highly conserved in evolutionary terms. TCDD is acutely lethal to the invertebrate, the crayfish (Pacifastacus leniusculus), with an LD50 <30 mg/kg body weight; this lethality is characterised by a chronic wasting syndrome, reminiscent of mammalian toxicity (Ashley, Simpson et al., 1996). However, the C. elegans AhR is reported to be unable to bind dioxins (Powell-Coffman; Bradfield et al., 1998). We show that P450s are induced by aryl hydrocarbons in C elegans, but not by dioxins; thus the effects of dioxin appear to have evolved with the invertebrates. The dioxin receptor’s (AhR) biochemistry shows distinct differences between the invertebrates and vertebrate. The C. elegans AhR is unable to bind the co-chaperone protein, AIP (AhR-Interacting Protein—also know as ara9 or XAP2), but mammalian and fish AhR proteins bind AIP. The significance of this discovery lies in the fact that the AhR requires an extensive chaperone pathway, involving hsp90, AIP and p23, for folding of the AhR into a ligand-binding form. The AIP protein makes contact with hsp90 through its tetratricopeptide domain, and makes contact with the AhR through a C-terminal ␣-helical domain, making a multi-protein complex (Bell and Poland, 2000). The ligand-binding domain (LBD) of the AhR has a key regulatory role, as the LBD maintains the AhR in the “off” conformation. After activation of the LBD by ligand, the AhR undergoes nuclear translocation and undertake signal transduction. We are therefore attempting to understand the molecular mechanisms of ligand binding to the

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AhR. Minimal domains of the AhR LBD were expressed in the yeast, Pichia pastoris, or in baculovirus. Both systems produced high level of recombinant protein, and in the yeast, this was essentially all insoluble. There was little or no detectable AhR LBD in cytosol extracts. However, in Sf9 cells, soluble AhR LBD was expressed in cytosol at high levels (>0.1% of total cytosolic protein), as determined by western blotting. These proteins were capable of binding ligand, and their application for structural biology studies will be discussed. References Ashley, C.M., Simpson, M.G., et al., 1996. Aquat. Toxicol. 35 (3–4), 157–169. Bell, D.R., Poland, A., 2000. J. Biol. Chem. 275 (46), 36407–36414. Gu, Y.Z., J.B. Hogenesch, et al., 2000. Annu. Rev. Pharmacol. Toxicol. 40, 519–561. Powell-Coffman, J.A., Bradfield, C.A., et al., 1998. Proc. Natl. Acad. Sci. U.S.A. 95 (6), 2844–2849. Schmidt, J.V., Bradfield, C.A., 1996. Annu. Rev. Cell. Dev. Biol. 12, 55–89. Keywords: Ah Receptor; TCDD; Ligand binding domain; Expression Multigene Interaction Leading to Porphyria and Liver Injury Caused by Dioxin A.G. Smith MRC Toxicology Unit, Leicester University, Leicester LE1 9HN, UK The risk assessment for dioxin and its analogues usually focuses on the affinity for the aryl hydrocarbon receptor (AHR) that controls expression of many genes including ones for drug metabolism, development and cell growth. In addition, polymorphism of the gene for AHR with different resulting affinities is often proposed as a dominant factor in determining susceptibilities of individuals. However, molecular mechanisms of toxic agents in vivo are rarely likely to be the result of changes in expression governed by a single polymorphic controlling gene such as a transcription factor; interaction of the AHR with other genes has been known for a long time (Knutson and Poland, 1982). Even apparently simple actions of chemicals may be the consequence of genetically variable multigene expression. Genetic variation in mice and toxicogenomics were used to explore mechanisms of gene interaction leading to cell malfunction and injury in the liver caused by dioxin interacting with iron metabolism. C57BL/6J mice null (−/−) for the Ahr were virtually refractory to the hepatic effects of dioxin even with additional iron treatment, but the low response of +/− mice was markedly influenced by iron status despite no changes in expression of the cytochrome P4501A isoforms. Using polymorphic DNA microsatellites, genetic analysis of F2 crosses between C57BL/6J and SWR mice with the resistant DBA/2 strain demonstrated susceptibility loci pertinent to the development of porphyria (a disruption of haem synthesis) and liver injury on chromosomes 1,9,11 and 14, besides that of the Ahr gene on chromosome 12 (Robinson et al., 2002). Despite experiments using mice with the knockout of the gene for CYP1A2 showing protection from porphyria and to some extent liver damage (Smith et al., 2001), the Cyp1a2 gene was not a susceptibility locus in these crosses and there was no correlation with expression. cDNA arrays of candidate genes in haem, iron and drug metabolism, oxidative stress, cell cycle, DNA repair, apoptosis etc. were used to compare multiple gene expression in the parent strains relative to their initial hepatic response, e.g. of cytochrome P450 isoforms and to the subsequent development of porphyria and liver injury. Some genes regulated by Ahr and by the antioxidant transcription factor Nrf2 were induced by dioxin at an early time point in both susceptible and resistant strains of mice whereas expression of some others such as haem oxygenase, were associated with porphyria development. In summary, although the AHR clearly plays an important part in the porphyria and liver injury induced by dioxin in mice, polymorphisms in other gene products can play significant roles in modulating the response. References Knutson, J. C., Poland, A., 1982. Cell 30, 225–234. Robinson, S.W., Clothier, B., Akhtar, R.A., Yang, A.L., Latour, I., Van Ijperen, C., Festing, M.F., Smith, A.G., 2002. Mol. Pharmacol. 61, 674–681.

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Smith, A.G., Clothier, B., Carthew, P., Childs, N.L., Sinclair, P.R., Nebert, D.W., Dalton, T.P., 2001. Toxicol. Appl. Pharmacol. 173, 89–98. Rat Strain Difference-Based Animal Model for Mechanistic Studies of Dioxin Toxicity Raimo Pohjanvirta1,2,3 , Mikko Unkila4 , Jouni T. Tuomisto3 , Matti Viluksela3 , Allan B. Okey5 , Jouko Tuomisto3 1 National

Veterinary and Food Research Institute, Kuopio Department, P.O. Box 92, FIN-70701 Kuopio, Finland;

2 Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Helsinki,

Finland; 3 Laboratory of Toxicology, National Public Health Institute, Kuopio, Finland; 4 Hormos Medical Ltd., Turku, Finland; 5 Department of Pharmacology, University of Toronto, Toronto, Canada Our understanding of the molecular mechanisms of dioxin toxicity has been furthered remarkably by studies in two inbred mouse strains, C57BL/6 and DBA/2. These strains exhibit an approximately 10-fold difference in their sensitivity to a number of toxic and adaptive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) including acute lethality, thymic atrophy, teratogenicity and CYP1A1 induction. The difference is quantitative but not qualitative, and in genetic crosses of these strains, susceptibility to TCDD is inherited as an autosomal dominant trait. The strain divergence has been shown to arise from structural differences in the mediator of most biological effects of dioxins, the AH receptor (AHR). A single point mutation in the ligand-binding domain of the AHR results in a diminished binding affinity of the receptor to TCDD in DBA/2 mice largely accounting for the reduced sensitivity of this strain to the biochemical and toxic impacts of TCDD. In the late 1980s, another intraspecies sensitivity difference to TCDD was found. An inbred strain of rat, Long-Evans (Turku/AB) (L-E) was shown to be highly sensitive to the acute lethality of TCDD (LD50 10–20 ␮g/kg), whereas an outbred strain, Han/Wistar (Kuopio) (H/W) was extremely resistant (LD50 > 9600 ␮g/kg). This 1000-fold difference did not appear to be due to kinetic factors, and it was specific to dioxins. Among dioxins, however, there was a good correlation with AHR binding affinity of a congener and the degree of susceptibility difference between the strains to it. Several salient features distinguished the rat model from its mouse counterpart: (1) in hybrid rats, resistance to TCDD was inherited as an autosomal dominant trait; (2) the resistant H/W rats showed normal responsiveness to, e.g., CYP1A1 induction and thymic atrophy; (3) in some cases, a clearly qualitative divergence was seen; e.g., the teratogenic manifestation was cleft palate in L–E rats but hydronephrosis and intestinal bleeding in H/W rats. Subsequent studies showed that although the binding affinities to TCDD of the AHRs from the two strains were similar, the H/W AHR was smaller (ca. 98 kDa versus 106 kDa in L–E rats) and displayed peculiar sensitivity to metal oxyanions and high salt concentrations. Molecular cloning disclosed a point mutation in the first invariant nucleotide of intron 10 of H/W rat AHR resulting in usage of cryptic splice sites and production of two distinct AHR proteins of similar mass both harbouring a restructured transactivation domain. The deviant AHR proved to be the major reason for H/W rat resistance, although there is another, at present unknown, contributing gene “B”. The rat model has implied that transactivation domain structure in the AHR may have a prominent role in dioxin resistance and may further divide responses into those (type II effects; e.g. acute lethality) critically dependent on it and into those (type I effects; e.g. CYP1A1 induction) refractory to it. The rat model will next be exploited in an attempt to pinpoint the changes in gene activities decisive to acute lethality of TCDD. Recent Risk Assessments Of Dioxins A.G. Renwick Clinical Pharmacology Group, Allergy and Inflammatory Sciences Division, School of Medicine, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, UK Polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated planar biphenyls are potent toxins that share a common mechanism of toxicity, and are known as “dioxins”. They are degraded very slowly, and their wide distribution in the food supply results in daily intake by humans. Risk assessments are based on the

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toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most extensively studied congener, which is one of the most potent and has one of the longest half-lives. Extensive bioaccumulation occurs in humans, and a steady-state body burden is not achieved until about 30–40 years of age. Dioxins produce a number of effects in experimental animals including developmental and reproductive effects, immunotoxicity and cancer, which are believed to be mediated via binding to a ligand-activated transcription factor, known as the Ah-receptor or AHR. The toxic equivalency factors (TEQs) of different congeners are usually based on their interactions with the AHR. Until recently, the tolerable intake for humans was based on the dose–response data for liver cancer in rats dosed with TCDD. In 1992, adverse effects were reported on spermatogenesis in young rats following a single oral dose of TCDD given during gestation, but this observation could not be interpreted in relation to a steady-state body burden that would arise during chronic daily oral administration. Recent in utero toxicokinetic studies have allowed estimation of the fetal burden arising from a single dose given during gestation and from chronic oral administration. In consequence the single dose in utero effects on the male reproductive system have become the focus of recent evaluations by the FAO/WHO JECFA (Canady et al., 2002), the European SCF (SCF, 2001) and the UK COT (COT, 2001). Recent evaluations have been based on calculations of the body burden in pregnant rats that would be associated with adverse effects on the male reproductive system in utero following repeated daily doses to steady-state. This body burden, expressed per kg body weight, was calculated using the in utero single dose–response data, with correction for the difference in fetal body burdens following repeated daily doses to steady-state compared with a single dose. The corrected maternal body burden in rats was divided by an uncertainty factor of 10 to allow for the fact that the risk characterisation was based on an effect level not a no-effect level, and for possible human variability in toxicokinetics. The daily intake by humans that would result in this adjusted body burden (per kg body weight) was estimated taking into account the very long half-life (about 7.5 years) and extensive accumulation in humans. The resulting tolerable intake was expressed as TEQ/kg body weight on a daily basis (2pg-COT), weekly basis (14 pg SCF) or monthly basis (70 pg JECFA). References Canady, R., Crump, K., Feeley, M., Freijer, J., Kogevinas, M., Malisch, R., Verger, P., Wilson, J., Zeilmaker, M., 2002. WHO Food Additives Series, No. 48, pp. 451–664. COT, 2001. Annual Report of the Committees on Toxicity, Mutagenicity and Carcinogenicity of Chemicals in Food, Consumer Products and the Environment, 2001. Food Standards Agency/Department of Health, pp. 61–83. SCF, 2001. Opinion on the Risk Assessment of Dioxins and Dioxin-like PCBs in Food—Update. Adopted 30 May 2001. CS/CNTM/DIOXIN/20 final. ORAL COMMUNICATIONS An Analysis of the Need for an Additional Toxicokinetic Safety Factor for Neonates Namali V. Corea, Andrew G. Renwick Clinical Pharmacology Group, Allergy and Inflammatory Sciences Research Division, School of Medicine, University of Southampton, Bassett Crescent East, Southampton, S016 7PX, UK E-mail address: [email protected] The current risk assessment process for chemicals in food involves the use of default uncertainty factors to allow for inter- and intra-species variability when converting data from an animal study into a health-based guidance value for human exposure. The proposal that an additional factor of 10 should be applied for determining acceptable exposures for infants and children under the Food Quality Protection Act (1996) in the USA, implies that the early stages of human development may not be adequately protected by the default inter-species toxicokinetic factor of 4 (WHO, 1999).

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There are limited data on the kinetics of food additives, but extensive published data for prescribed drugs in human neonates and adults. There are few corresponding data for the equivalent life-stages in the rat. The adequacy of the default uncertainty factor could be assessed by comparison of the in vivo pharmacokinetics of selected probe drugs in young (10-day-old) and adult rats with the equivalent published pharmacokinetic parameters for the same compounds in human neonates and adults. Toxicokinetic profiles were determined in 10-day-old (n = 3 per sex, per timepoint) and adult rats (n = 6 per sex, consecutive blood samples taken). Animals received a single intraperitoneal dose of 50 mg/kg theophylline (to represent metabolism by CYP1A2) or 200 mg/kg chloramphenicol (to represent glucuronidation). Plasma was analysed using validated HPLC methods, and pharmacokinetic parameters calculated using WinNonlin, version 1.5. Clearance values in young and adult rats (mean ± S.D.) were compared to equivalent published data in human neonates and adults (Dorne et al., 2001a,b). The ratio represents the difference between rats and humans. Comparison

Clearance (ml/min kg)

Ratio

Theophylline: adult rat cf. adult human Theophylline: young rat cf. human neonate Chloramphenicol: adult rat cf. adult human Chloramphenicol: young rat cf. human neonate

0.75 (0.11) cf. 1.0 (0.29) 0.44 cf. 0.35 (0.11) 13 (3.5) cf. 3.2 (1.3) 6.9 cf. 1.1 (0.60)

0.8 1.3 4.0 6.3

The interspecies comparison for theophylline indicated that the ratios for adult and young clearance values were less than the default interspecies toxicokinetic factor of 4. For chloramphenicol, the adult clearance ratio was equivalent to the default factor. Clearance was more efficient in young rats compared to human neonates and consequently the default safety factor was exceeded slightly. The data to date do not support the need for an extra 10-fold factor for human neonates (or infants and children—data not shown) in relation to toxicokinetic differences. References Dorne, J.L.C.M., Walton, K., Renwick, A.G., 2001a. Food Chem. Toxicol. 39, 68–96. Dorne, J.L.C.M., Walton, K., Renwick, A.G., 2001b. Food Chem. Toxicol. 39, 1153–1173. WHO, 1999. Environmental Health Criteria 210. World Health Organisation, Geneva. Keywords: Risk assessment; Food additives; Safety factors; Pharmacokinetics; Neonates; Metabolism Synergistic Activation of an Endogenous Estrogen Receptor Target Gene by Hormonal Signals and Chemical Stress Damian G. Deavall, David N. Hickinson, Jonathan G. Moggs, John W. Edmunds, Ian Kimber, George Orphanides Syngenta CTL, Alderley Park, Cheshire SK10 4TJ, UK E-mail address: [email protected] Mammalian cells integrate responses to many stimuli including cellular stress, hormonal signals, growth factors and toxicants. In the physiological milieu, such stimulation simultaneously activates a range of signaling mechanisms. Thus, the effects of estrogen receptor ligands could be modulated by other signaling pathways, perhaps amplifying the tissue- and developmental stage-specific effects of these compounds. We are using genomic and biochemical techniques to investigate cellular responses to combinations of such signals, acting through nuclear hormone receptors. We have focused on transcriptional induction by 17␤-estradiol (E2) and 12-O-tetradecanoylphorbol-13-acetate (TPA) of a classical estrogen-responsive gene, pS2 (TFF1), in a model system, the ER␣-positive MCF-7 breast cancer cell line. TPA is a well-characterised cellular stressor and protein kinase C agonist capable of activating mitogen-activated protein kinase cascades (Parker et al., 1989; Liu et al., 2002). Northern blot analysis revealed

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Table 1 Increase in pS2 mRNA abundance in response to E2 and TPA Treatment (10−10

E2 M) TPA (10−7 M) E2/TPA (10−10 M/10−7 M)

Increase in pS2 mRNA above vehicle control 73 ± 13.98 135 ± 2.61 343 ± 8.74

Values are mean ± S.E.M.; n = 3.

that E2 and TPA are both capable of increasing pS2 mRNA abundance independently, and that a combination of the two stimuli induce transcription in a synergistic manner across a range of E2 doses from 10−7 to 10−11 M, being maximal at 10−10 M E2 (Table 1). Thus, TPA is capable of potentiating E2 responsiveness to a level above that achievable with E2 alone. Epidermal Growth Factor (100 ng/ml) similarly elevated pS2 mRNA abundance both alone and synergistically with E2. Chromatin immunoprecipitation (X-ChIP) assay revealed differences in pS2 promoter occupancy by ER␣ in response to E2/TPA compared to E2 or TPA alone, suggesting that the combined stimuli elicit more rapid recruitment of ER␣ to the pS2 promoter. Western blot analysis of ER␣ indicated that TPA (10−7 M) resulted in a rapid increase in phosphorylation at Ser118, a MAPK p42/p44 target (Kato et al., 1995) within the ligand-independent transactivation domain (AF1) of ER␣. Phosphorylation at ER␣ Ser118 was greatest in the presence of the combined E2/TPA stimulus. We conclude that synergistic activation by estrogen and stress stimuli is observable with an endogenously expressed gene. We are now investigating the significance of phosphorylation within the AF1 domain of ER␣ to the transcriptional synergy response, and are using the X-ChIP to investigate differences in promoter occupancy by other transcription factors and components of the transcriptional machinery in response to E2 and TPA alone and in concert. Further insights into the molecular mechanisms underlying transcriptional synergy observed with estrogen receptor ligands and stress signals may help to explain differences in tissue- and developmental stage-specific potency of estrogens. References Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawadhima, H., Metzger, D., Chambon, P., 1995. Science 270, 1491–1494. Lui, J., Crepin, M., Liu, J., Barritult, D., Ledoux, D., 2002. Biochem. Biophys. Res. Commun. 293 (4), 1174–1182. Parker, M., Kour, G., Marais, R., Mitchell, F., Pears, C., Schaap, D., Stabel, S., Webster, C., 1989. Curr. Opin. Cell. Biol. 5, 499–504. Keywords: Estrogen; Synergy; Phosphorylation Ochratoxin A—Induced Tumour Formation: DNA Adducts or Oxidative Stress? Angela Mally, Herbert Zepnik, Paul Wanek, Wolfgang Voelkel, Erwin Eder and Wolfgang Dekant Department of Toxicology, University of Wuerzburg, Versbacher Strasse 9, 97078 Wuerzburg, Germany E-mail address: [email protected] Ochratoxin A (OTA) is a mycotoxin produced by several species of Penicillium and Aspergillus fungi which contaminates a variety of food items, resulting in chronic human exposure. In rodents, OTA is a potent nephrotoxin and renal carcinogen, but the mechanism of OTA tumourigenicity is not known. OTA is not mutagenic in the Ames test and negative in several other genotoxicity tests. However, conflicting results have been obtained regarding the potential of OTA to bind to DNA. In addition, oxidative stress and lipid peroxidation have been implicated in OTA carcinogenicity. The aim of this study was to characterise the role of direct binding and oxidative DNA damage in OTA induced adduct formation.

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Table 1 DNA-adducts formed by oxidative stress in rats exposed to ochratoxin A for 2 weeks 8-oxodG/106 dG

Control 250 ␮g/kg bw 500 ␮g/kg bw

HNE-dG/109 nucleotides (kidney)

Kidney

Liver

5.8 ± 1.7 6.0 ± 0.5 4.8 ± 0.6

5.6 ± 0.5 5.6 ± 0.5 5.0 ± 0.4

11.8 ± 5.6 7.3 ± 1.2 14.3 ± 1.2

Values are means ± S.D. 14 C-Accelerator Mass Spectrometry (AMS) is a highly sensitive method for quantifying extremely low concentra-

tions of radiocarbon (Turteltaub et al., 1998) and was used to investigate the potential of OTA to bind to DNA. Male and female F344 rats (n = 5 per group) were treated with a single dose of 14 C-OTA (500 g/kg bw; 0.25 mCi/mmol) in corn oil by gavage and sacrificed 72 h post-dosing. DNA was isolated from livers and kidneys and the 14 C content in graphitized DNA samples, was measured by AMS. No difference was observed in the levels of 14 C between OTA treated animals and respective vehicle controls, with a calculated limit of detection of 4 adducts/109 nucleotides for kidney and 8 adducts/109 nucleotides for liver. To determine the extent of oxidative DNA damage induced by OTA, male F344 rats (n = 3 per group) were administered 0, 250 and 500 mg/kg bw OTA in corn oil by gavage for 2 weeks (5 days per week). Animals were sacrificed 72 h after the final dose and DNA was isolated from livers and kidneys. 8-Oxo-7,8-dihydro-2
deoxyguanosine (8-oxodG), a commonly used biomarker of oxidative stress and DNA damage, was measured by LC/MS–MS. In addition, 1,N2 -propanodeoxyguanosine (HNE-dG), which results from binding of the lipid peroxidation product trans-4-hydroxy-2-nonenal to DNA, was analysed in kidney DNA using 32 P-postlabelling (Wacker et al., 2000). Treatment with OTA did not result in increased levels of 8-oxodG and HNE-dG. In contrast, i.p. administration of ferric nitrilotriacetic acid (15 mg Fe/kg body weight; sacrifice 5 h post-dosing) which is known to induce 8-oxodG formation in the kidney, led to a 1.7-fold increase in 8-oxodG in kidney (6.1 ± 1.5 dG versus 3.7 ± 1.0/106 dG). Although oxidative stress has been associated with OTA-mediated toxicity and carcinogenicity, the lack of oxidative DNA damage in this study could indicate that chronic effects due to accumulation of OTA in the kindeys play a crucial part and that prolonged treatment or higher doses may be required. However, our results do not support the role of direct DNA binding in OTA induced tumour formation. References Turteltaub, K.W., Dingley, K.H., 1998. Toxicol. Lett. 102–103, 435–439. Wacker, W., Schuler, D., Wanek, P., Eder, E., 2000. Chem. Res. Toxicol. 13, 1165–1173. Keywords: Ochratoxin A; DNA adducts; Oxidative stress; AMS; 8-oxodG; HNE-dG Acknowledgement This work was supported by the European Union 5th RTD Framework Programme and ISIC/PEC.

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Early Markers of Nephrotoxicity Following Exposure of Human and Rat Proximal Tubular Cell Cultures to Platinum Analogues Richard D. Wainford1 , Gerrard Descotes2 , Richard Weaver3 , Henri Merdjan3 , Gabrielle M Hawksworth1 1 Deptartments of Medicine & Therapeutics and Biomedical Sciences, University of Aberdeen, Aberdeen AB25 2ZD,

UK;2 Drug Safety Assessment Institute, Servier, 92415 Courbevoie, France;3 Drug Metabolism and Development, Servier SL3 6HH, UK E-mail address: [email protected] Cis-platin is used to treat 70–90% of testicular cancers and brain, ovarian, bladder and breast cancer in combination with other pharmaceutical agents. Cis-platin exhibits dose limiting nephrotoxicity (Murzano et al., 2002). Within the kidney the proximal tubular cells are a frequent target for drug-induced toxicity due to their metabolic and transport characteristics. In response to a toxic insult cells synthesise specific proteins which are detectable before toxicity is apparent. These include the products of the immediate early genes (IEGs), c-fos and c-jun. IEG transcriptional activation is one of the earliest nuclear responses to a toxic insult (Joannidis et al., 1997), but under normal conditions the IEGs play a role in cellular function, influencing the cell cycle. Toxicity of cis-platin, trans-platin and carboplatin to human (HPT) and rat proximal tubular (RPT) cell cultures was assessed by the MTT assay. IEG levels were quantified by RT-PCR, during a 3 h exposure to 500 ␮M cis-platin, and by immunoblotting using nuclear extracts taken over a 12 h time-course following 2 h exposure to 500 ␮M cis-platin. Cis-platin concentration corresponds to the maximum level detected in plasma post-injection. RPT cells exhibited greater sensitivity to platinum analogues than HPT cells (Wainford et al., 2002). c-jun mRNA was maximal at 2 h and remained elevated at 3 h in RPT cells, but c-jun protein was not detected at any time-point with any of the tested platinum analogues in RPT cells. In HPT cells c-jun protein levels correlated with toxicity, cis-platin exposure resulted in the greatest c-jun protein response. Maximal c-jun response in HPT cells was at 4 h (ODU per mm2 0.18 ± 0.04 cis-platin, 0.08 ± 0.01 cis-platin and 0.07 ± 0.01 carboplatin (n = 4 ± S.D.)). Increases in c-fos mRNA were maximal at 1h and declined by 3 h during a 3 h cis-platin (500 ␮M) exposure in RPT cells. c-fos protein levels were maximal at 8h in RPT cells, (ODU per mm2 ) 0.37 ± 0.05 cis-platin, 0.24 ± 0.04 cis-platin. No signal was detected with carboplatin treatment (n = 3). In HPT cells c-fos protein levels correlated with toxicity. Maximal response was at 4 h (ODU per mm2 0.41 ± 0.04 cis-platin, 0.13 ± 0.03 cis-platin and 0.12 ± 0.02 carboplatin (n = 4 ± S.D.)). In HPT cultures c-fos and c-jun protein correlated with, and were detectable before, platinum analogue toxicity. In RPT cultures c-fos protein was detectable within 2 h but c-jun protein was not detected at any time-point. AP-1, the product of the c-fos and c-jun proteins, which initiates a protective response within the cells may not be formed in RPT cells due to a lack of c-jun protein. RPT cells exhibit greater sensitivity to platinum analogues than HPT cells, this may be due to an absence of the AP-1 protective response. References Joannidis, M., Cantley, L.G., Spokes, K., Stuart-Tilley, A.K., Alper, S.L., Epstein, F.H., 1997. Kidney Int. 52, 130–139. Murzano, C., Trevisan, A., Giovagnini, L., Fregona, D., 2002. Toxicol. In Vitro 16, 413–419. Wainford, R., Desoctes, G., Merdjan, H., Weaver, R., Hawksworth, G., 2002. Drug Met. Rev. 34, 99. Keywords: Cis-platin; Nephrotoxicity; IEG; Biomarkers; Proximal tubule

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Gene Expression Changes in the Mouse in Response to Toxic and Non-Toxic Doses of Paracetamol Dominic P. Williams, Claudia Garcia-Allan1,2 , Geoff Johnston1 , Dennis A. Smith2 , B. Kevin Park Department of Pharmacology and Therapeutics, The University of Liverpool, Liverpool, UK; 1 Genetic Technologies; 2 PDM Pfizer, Sandwich, Kent, UK E-mail address: [email protected] Hepatotoxicity is a major cause of patient morbidity and mortality, and can preclude effective drug treatment (Lazarou, et al., 1998; Pirmohamed, et al., 1998). Such reactions are a major impediment to drug development and may lead to drug withdrawal even if they occur only in a minority of patients (Jefferys et al., 1998). Microarray analysis is becoming increasingly employed for mechanistic studies in toxicology (Bulera et al., 2001). Genetic knowledge of hepatocellular responses to toxicants is required to create effective drug development screening assays and ultimately the “designing-out” of toxicophores in potential candidate drugs. Mice (male; CD-1; 25–30 g; n = 3 or 6) were administered either vehicle or paracetamol (1 mmol/kg and 3.5 mmol/kg; i.p.). Animals were killed after 1, 4 and 24 h, livers were removed, and ALT activity (n = 6) was measured using the Sigma GP transaminase kit. Global gene expression patterns were assessed according to the manufacturer’s protocol using the Affymetrix murine gene chip (n = 3; U74Av2: 6000 genes and 6000 ESTs). One chip per mouse was used, thereby preventing RNA masking. Mean plasma ALT values (Table 1) indicated that there was no toxicity at any of the time points after 1 mmol/kg and 3.5 mmol/kg after 1 h. However, 4 h after 3.5 mmol/kg paracetamol, significant toxicity is seen. Table 1 Mean plasma ALT values Time point (h)

Dose (mmol/kg)

Mean ALT (IU/ml)

1 4 24 1 4 24

1 1 1 3.5 3.5 3.5

51.9 ± 46.2 40.5 ± 28.5 41.3 ± 21.8 26.1 ± 7.9 219 ± 139.4∗ 954 ± 627.8∗

∗P

< 0.05, Mann–Whitney U-test.

Stringent data reduction was achieved by using Bonferroni’s probabilities (with 12,499 genes, significance is achieved when P < 4.1 × 10-6 ). The number of significantly regulated genes (across each dose and time point) was reduced to 789 when a P-value of 4.1 × 10-6 was used as a cut-off. With the exclusion of ‘absent to absent’ genes and genes which were not present in at least 2/3 calls, the number of significantly altered genes was further reduced to 626. The genes were then categorized into seven categories (Table 2).

Table 2 Categorization of significantly (P < 4.1 × 10−6 ) regulated genes Functional categories

Number of genes

Metabolism-related Transcription factor-related Apoptosis-related Antioxidant/defence-related Immune-related Glutathione-related Miscellaneous/EST

104 85 47 39 18 6 327

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In conclusion, paracetamol regulates numerous genetic changes within the murine liver. Whether these gene changes represent an adaptive cellular response to oxidative stress, mediated by glutathione depletion and/or covalent binding, remains to be investigated. References Bulera, S.J., Eddy, S.M., Ferguson, E.T.A., Jatkoe et al., 2001. Hepatology 33 (5), 1239–1258. Jefferys, D.B., Leakey, D., Lewis, J.A., Payne, S., Rawlins, M.D., 1998. Br. J. Clin. Pharmacol. 45, 151–156. Lazarou, J., Pomeranz, B.H., Corey, P.N., 1998. JAMA 279 (15), 1200–1205. Pirmohamed, M., Breckenridge, A.M., Kitteringham, N.R., Park, B.K., 1998. Br. Med. J. 316, 1295–1298. Non-Steroidal Anti-Inflammatory Drugs Cause Polyamine Depletion and Apoptosis in Human Bladder Cancer Cells Robert A. Lockhart1 , Alun Hughes1 , Anna Milsom1 , Lucy Crow1 , A. Alhasso2 , James N’Dow3 , Heather M. Wallace1 1 Departments

of Medicine & Therapeutics and Biomedical Sciences, 2 Urology and 3 Surgery, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK E-mail address: [email protected]

Ninety thousand new cases of bladder cancer were predicted in the USA for 2002 (Jemal et al., 2002). As recurrence rates remain high at 50–75% (Heney et al., 1983) chemoprevention could be an invaluable new strategy in the treatment of this disease. The non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to be effective chemopreventative agents against colorectal cancer and their frequent use may also prevent bladder cancer (Grubbs et al., 2000). It is thought that the NSAIDs mediate their effect through inhibition of cyclo-oxygenase (COX-2), which supports aberrant cell growth through production of the prostaglandins. However, previous work has shown that the NSAIDs are cytotoxic to colon cancer cells that lacked both COX enzymes in vitro (Hanif et al., 1996). We have previously demonstrated that the polyamine metabolic pathway is modulated by the NSAIDs and may provide an alternative pathway for their action in colon cancer (Smith et al., 1999). The aim of this research was to determine if the NSAIDs were cytotoxic to bladder cancer cells in vitro and if polyamine content was modulated in response. The cytotoxicity of three NSAIDs (sulindac, naproxen and MF-Tricylic) were determined by cell counts, protein content and MTT assay in three bladder cancer (RT112, RT4, 304) and one colon cancer (HCT-15) cell lines. Cells were seeded, allowed to attach for 4 h and grown for 24 h prior to addition of drug. MTT assays were carried out after 48 h exposure and the IC50 values calculated. DAPI staining was used to observe the morphological signs of apoptosis. Polyamine content was quantified by HPLC analysis. All three NSAIDs tested caused comparable toxicity in bladder and colon cancer cells after 48 h exposure with the order of potency being MF-tricyclic > sulindac > naproxen (Table 1). Cytotoxicity was evident at the Table 1 Toxicity and polyamine depletion caused by 48 h NSAID treatment in bladder and colon cancer cells Cell line and cancer type

RT112 (bladder) RT4 (bladder) 304 (bladder) HCT-15 (colon)

MTT activity IC50 (␮M)

Total polyamine content (nmol/mg)

Sulindac

Naproxen

Control

Sulindac (IC50 )

750 650 75 >1000

3000 4500 n.d. 4000

43.0 ± 1.8 n.d. 41.8 ± 3.6 23.2 ± 3.1

23.6 ± 2.4 n.d. 24.2 ± 2.1 7.9 ± 1.8

Results are n = 3; n.d.: not determined. IC50 values calculated by GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego California, USA). Variance was less than 5%.

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229

IC50 dose with sulindac causing approximately 15% apoptosis at an IC50 dose and over 30% at an IC90 . NSAIDs decreased intracellular polyamine content to between 35 and 58% of control in both bladder cancer and colon cancer cells (Table 1). Our results indicate that the NSAIDs are cytotoxic in vitro and modulate polyamine metabolism in bladder cancer cells as they do in colon cancer cells. This suggests that the polyamine metabolic pathway may also provide an alternative mechanism through which the NSAIDs cause chemoprevention in bladder cancer cells. References Grubbs, C.J., Lubet, R.A., Koki, A.T., et al., 2000. Cancer Res. 60 (20), 5599–5602. Hanif, R., Pittas, A., Feng, Y., Koutsos, M.I., et al., 1996. Biochem. Pharmacol. 52 (2), 237–245. Heney, N.M., Ahmed, S., Flanagan, M.J., Frable, W., et al., 1983. J. Urol. 130 (6), 1083–1086. Smith, N.I., Hughes, A., Wallace, H.M., 1999. Scottish Med. J. 44 (4), 124. Jemal, A., Thomas, A., Murray, T., Thun, M., 2002. CA Cancer J. Clin. 52 (1), 23–47. Posters Cryptolepine Induces G1 Arrest and Cytotoxicity in Human Hepatoma HepG2 Cells Charles Ansah, Nigel J. Gooderham Molecular Toxicology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, South Kensington, London SW7 2AZ, UK E-mail address: [email protected]

Viabillity as % of Untreated Control

Cryptolepine (CLP), the anti-malarial alkaloid from the West African plant Cryptolepis sanguinolenta may be a candidate for cancer chemotherapy (Ansah and Gooderham, 2002). We have been studying the toxicity of the aqueous root extract of the plant (CSE) which is the traditional clinical formulation (Boye and Ampofo, 1983) and CLP on a variety of human cancer cell lines. Here, we report the toxicity and molecular effects of CSE and CLP on human hepatoma HepG2 cells. Cell viability was assessed by the Resazurin reduction method, cell cycle changes were monitored by flow cytometry and protein expression profiles by Western blotting.

120

—♦—24h — • —48h

100

∗∗ 80

∗∗ ∗∗

60

∗∗ 40

∗ significant (student-t test) 20

∗∗

compared to control

0 0

0.5

1

2.5

Fig. 1. Effect of CLP on HepG2 viability.

5

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CLP (0.5–5 ␮M) and CSE (5–100 ␮g/ml) caused a dose and time-dependent loss of viability in HepG2 cells (Fig. 1). In flow cytometry studies using propidium iodide staining, incubation of HepG2 cells with CLP (0.5–5 ␮M) and CSE (5–100 ␮g/ml) for 24 h led to accumulation of cells in the G1 phase of the cell cycle. P53 protein levels increased as early as 4 h and peaked at 12 h, and an associated accumulation of the cyclin dependent kinase (CDK) inhibitor, p21 was consistent with the G1 arrest. At higher doses, the G1 peak diminished in favour of a profound increase in the sub-G1 peak, suggesting cell death. Microscopic examination of treated cells showed membrane blebbing but, Western blot analysis of treated cells failed to show caspase-3 activation or PARP cleavage, typical hallmarks of apoptosis. These results show that CSE/CLP are toxic to HepG2 human hepatoma cells and suggest that mechanisms other than apoptosis contributed to the cell death. Since our present findings are inconsistent to a previous report of CLP induced apoptosis in HL-60 leukaemia cells (Dassonneville et al., 2000), we suggest that the mechanisms of CSE/CLP induced cell death may be cell-type specific. References Ansah, C., Gooderham, N.J., 2002. Toxicol. Sci. 70 (2), 245–251. Boye, G.L., Ampofo, O., 1983. Proceedings of the First International Symposium on Cyptolepine, Abstract No. 4, Kwame Nkrumah University of Science & Technology, Kumasi, Ghana. Dassonneville, L., Lansiaux, A., Wattelet, A., Wattez, N., Mahieu, C., Van Miert, S., Pieters, L., Bailly, C., 2000. Eur. J. Pharmacol. 409, 9–18. Keywords: Cryptolepine; Cryptolepis sanguinolenta; Cytotoxicity; G1 arrest; Hepatoma cells Acknowledgement We wish to thank the Commonwealth Scholarships Commisssion for financial support. Glutathione-dependent Cytotoxicity of Sodium fluoroacetate in Rat and Human Hepatoma-derived Cell Cultures Paul J. Dierickx Laboratory of Biochemical Toxicology, Institute of Public Health, Wytsmanstraat 14, B-1050 Brussel, Belgium E-mail address: [email protected] Sodium fluoroacetate (SFA) is a toxic chemical primarily used as a rodenticide. It is converted in vivo to fluorocitrate, which inhibits mitochondrial aconitase, and so blocks the Krebs cycle. The only report on an interaction between glutathione (GSH) and SFA concerns the higher citrate production in GSH-depleted rats (Mead et al., 1985). The GSH-dependent cytotoxicity of SFA in rat (Fa32) and human (HepG2) hepatoma-derived cultured cells is reported here, together with the influence of SFA on the cytochrome P450-dependent ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depentylase (PROD) and GSH transferase (GST) activity, respectively phases I and II drug metabolising enzymes. Cells were seeded in microtiter plate wells at a density of 60,000/well and toxicity was measured as the inhibition of neutral red uptake. After 24 h, cells were treated for 24 h. The results were quantified as the SFA concentration reducing the neutral red uptake by 50% (NI50). Further experiments were performed using approximately 2/3 of the NI50 values as the maximal concentrations: 70 mM for Fa32 and 55 mM for HepG2 cells. GSH content was measured with monochlorobimane (Dierickx, 1998a). Near confluent monolayers were treated for 24 h to quantify the GST activity, as measured with 1-chloro-2,4-dinitrobenzene as second substrate. EROD and PROD activity were measured fluorimetrically as decribed (Dierickx, 1998b). The cytotoxicity of SFA is given in Table 1 for normally cultured cells and in the presence of 50 ␮M l-buthionineS,R-sulfoximine (BSO). SFA reduced the GSH content by 27% after 1 h and 19% after 24 h in Fa32. In contrast, the GSH content was increased by 23% after 1h in HepG2 and was re-established to the normal level after 24 h. The GST activity was unchanged after 24 h in both cell lines. The EROD activity was unaffected by SFA in Fa32,

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Table 1 NI50 values (mM) after 24 h SFA

Control With 50 ␮M BSO

Fa32

HepG2

104 ± 10 39 ± 3

84 ± 4 50 ± 2

and slightly increased (43%) for PROD. However, both EROD and PROD activity were dose-dependently activated until respectively 5.7× and 3.3× the control value in HepG2 cells. The cytotoxicity of SFA strongly increased in GSH-depleted Fa32 and HepG2 cells. This increase was proportional to the differing GSH depleting capacity of BSO in both cell lines. A direct, dose-dependent GSH-depletion by SFA was only observed in Fa32 cells. Altogether, GSH-dependent cytotoxicity was demonstrated in both rat and human hepatoma-derived cell lines. A direct interaction with GSH was evident in Fa32 cells; a more indirect interaction was found in HepG2 cells, maybe by influencing other mechanisms such as reactive oxygen species formation, since SFA increased the endogenous GSH content. Although both cell lines interacted with GSH, several differences were observed (e.g. for the EROD and PROD activation). These differences probably reflect the species differences observed for the toxicity of SFA in vivo. References Dierickx, P.J., 1998a. Chemosphere 36, 1263–1274. Dierickx, P.J., 1998b. FEBS Lett. 422, 185–188. Mead, R.J., Moulden, D.L., Twigg, L.E., 1985. Aust. J. Biol. Sci. 38, 139–149. Keywords: Sodium fluoroacetate; Cytotoxicity; Fa32; HepG2; Glutathione; Depletion; EROD; PROD Gene Expression Screening in Primary Hepatocytes Sharon L. Doughty, Victor I.C. Oreffo, Jane A. Wrigley, Allison Stewart, Clive N. Kind Molecular Toxicology, SAUK, AstraZeneca R&D Charnwood, Loughborough LE11 5RH, UK E-mail address: [email protected] Many new potential drugs are produced each year, which go through a rigorous discovery and development process. The progression of any compound is based on various criteria—including its efficacy and toxicity. Xenobiotics are often metabolised in the liver by cytochrome p450 enzymes, and occasionally cause hepatotoxicity. The propensity for compounds to induce p450 enzymes may therefore be a significant potential liability, and an indication for compound de-selection. The aim of this work was to provide an in vitro screen for key genes to monitor chemical induction of Cyp p450 expression. Taqman® 5
Nuclease and Quantigene® Assays were set up to detect induction of rat P450 genes in Percoll purified primary rat hepatocytes, cultured on collagen coated 96-well plates. Results were normalised against the housekeeping gene GAPDH. Known inducers of the genes of interest were used as positive controls, pregnenealone-16-alpha-carbonitrile (PCN) and phenobarbital (PB) for Cyp 3A1 and Cyp 2B1, respectively. As expected PCN and PB were shown to be selective inducers of Cyp 3A1 and Cyp 2B1. Fig. 1 shows fold induction over DMSO control in rat hepatocytes treated with 10 ␮M PCN, or 200 ␮M PB, measured using the Quantigene system. PCN was then included in each subsequent experiment as a positive control and used to calculate a ‘relative induction’ value for test compounds. Taqman and Quantigene systems were run in parallel initially. An example of a test compound dose response is shown in Fig. 2, expressed as percentage of PCN Induction. Results were then confirmed by Taqman 5
nuclease assay. Whereas the sensitivity and dynamic range of the Taqman system were greater than those of the Quantigene system, the higher throughput (2 days compared with 5 days) of the latter enables six test compounds to be screened

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Fold Induction of Cyp 2B1 and 3A1 in response to PCN and PB 35.00

N=3, values +/- sd Fold change

30.00 25.00 20.00 15.00 10.00 5.00 0.00

2b PCN

3a1

2b PB

3a1

Fig. 1. Quantigene system—positive control compounds.

Fig. 2. Quantigene & Taqman systems—test compound profile at 0–100 ␮M concentrations.

routinely per week at a range of concentrations using hepatocytes from a single liver and ranked according to their induction profiles. The data generated has utility in determining potential liability of compounds at early stages in the drug discovery and development pipeline. Keywords: Primary hepatocytes; p450; Cyp induction; Taqman; Quantigene assay Cadmium Chloride-Induced DNA And Lysosomal Damage In HTC Cells George Fotakis1 , Eduardo Cemeli2 , Diana Anderson2 , John A. Timbrell1 1 150

Stamford street, Franklin Wilkins Building, Pharmacy Department, King’s College London, London SE1 8WA, UK; 2 Biomedical Sciences, University of Bradford, Richmond Building, BD7 1DP, UK E-mail address: [email protected] The aim of this study was to compare the effects of cadmium chloride on DNA and lysosomes in rat hepatoma cells (HTC cells). HTC cells were cultured as monolayers in 12 well plates (105 cells/ml) and exposed to different concentrations of cadmium chloride (40, 80, 200, 500 ␮M) for 5 and 8 h. DNA damage was assessed with the single cell gel/comet assay. Cells were scored (50) per dose of cadmium chloride, for each experiment and the DNA tail extent moment

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was calculated. Cytotoxicity was assessed with the LDH leakage assay. The formation of reactive oxygen species (ROS) was also determined under the same experimental conditions in order to assess its involvement in DNA damage following exposure to CdCl2 . For this purpose, the 5- (and 6)-carboxy-2
,7
-dichlorodihydrofluorescein diacetate was used as an indicator for reactive oxygen species. Additionally, lysosomal damage was quantitated with the neutral red assay (NR) and qualitative assessment was made with fluorescent microscopy using acridine orange as lysosomal stain. Table 1 Exposure of HTC cells to 40, 80, 200, 500 ␮M CdCl2 for 8 h: LDH leakage assay–Neutral Red assay (NR)–Comet assay–reactive oxygen species assay (ROS) CdCl2 (␮M)

Comet assay ROS (% control) NR (% control) LDH (% control)

40

80

200

500

1.86 ± 0.18 115 ± 7.63 53.4 ± 6.53∗∗ 100.01 ± 3.71

3.48 ± 1.58 127 ± 11.06 36.06 ± 1.07∗∗∗ 99.31 ± 3.84

2.69 ± 0.79 126 ± 7.23 20.05 ± 4.57∗∗∗ 99.08 ± 3.82

9.08 ± 1.93∗∗∗ 164 ± 23.77∗ 41.15 ± 3.60∗∗∗ 86.29 ± 3.59

HTC cells (n = 3); data presented as mean value ± standard error mean (SEM); ∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ P < 0.001.

Our results indicate that lysosomal damage occurs at a lower concentration than DNA damage in HTC cells. Exposure of HTC cells to 40 ␮M CdCl2 for 8 h resulted in 46.53% loss of lysosomal integrity (NR assay; P = 0.006) whereas the same concentration had no effect on DNA as indicated by the comet assay (tail DNA extent moment: 1.86 ± 0.18; P = 0.49). DNA damage was only observed when cells were exposed to 500 ␮M CdCl2 for 8 h (tail DNA extent moment: 9.08 ± 1.93; P < 0.001). This effect was accompanied by an increase of reactive oxygen species as shown in Table 1 without any significant LDH leakage whereas lysosomal damage was significant. Fluorescent microscopy of cells following staining with acridine orange revealed that exposure of HTC cells to 40, 80 and 200 ␮M CdCl2 for 5 h resulted in lysosomal damage whilst there was no significant LDH leakage or formation of reactive oxygen species. The data suggest that DNA damage may be due to the formation of reactive oxygen species. However lysosomal damage is probably an earlier event in the toxicity than DNA damage. Keywords: Cadmium; Reactive oxygen species; Lysosomes Association of DNA damage to human peripheral lymphocytes with age and MnSOD and OGG1 genotype Vanessa R. Gage1 , Peter G. Blain, Julian B.S. Leathart, Ann K. Daly, Faith M Williams1 School of Clinical and Laboratory Sciences1 , University of Newcastle, Medical school, Framlington Place, Newcastle Upon Tyne NE2 4HH, UK E-mail address: [email protected] There have been conflicting reports on the influence of increasing age on DNA damage. Factors such as increased reactive oxygen species and reduced DNA repair may contribute to increased damage. 8-Oxoguanosine glycosylase 1 (OGG1) which is responsible for the repair of the 7-hydroxy-8-deoxyguanosine lesion exhibits a polymorphism leading to a ser326 cys change. Manganese superoxide dismutase (MnSOD) which is responsible for de-activating the superoxide anion in the mitochondria exhibits an ala/val change. Thirty elderly (>65-year-old) and 30 young (20–40-year-old), healthy, urban dwelling volunteers were recruited for the study. DNA damage was measured by the COMET assay (Singh et al., 1988), in freshly isolated lymphocytes. Images of 50 cells per individual were captured by confocal microscopy and analysed using the Kinetic Komet software. DNA damage was expressed as the tail distributed moment (TDM). Genotyping for MnSOD and OGG1 was performed on lymphocyte DNA by PCR followed by RFLP using the restriction enzyme BsaW1 for MnSOD and

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Table 1 Genotype distribution and DNA damage MnSOD Ala/Ala Genotype Old Young DNA damage Old Young

OGG1 Ala/Val

Val/Val

Ser/Ser

Ser/Cys

Cys/Cys

5 (17%) 8 (27%)

21 (70%) 10 (33%)

4 (13%) 12 (40%)

19 (63%) 14 (46%)

9 (30%) 14 (46%)

2 (7%) 2 (7%)

30.6 ± 0.9 23.5 ± 2.2

30.2 ± 1.3 30.9 ± 3.1

35.7 ± 2.2 24.9 ± 1.5

30.4 ± 1.2 25.7 ± 1.7

32.3 ± 2.2 27.1 ± 2.5

30.0 ± 5 6 28.2 ± 3.8

TDM mean ± S.E.M. for old and young subjects.

Fnu4H1 for OGG1.DNA damage in lymphocytes from the elderly volunteers was 31.0 ± 1.0 (TDM mean ± S.D.) and greater than for the young group 26.5 ± 1.4 (TDM mean ± S.E.M.), (unpaired t-test, P < 0.05). The genotype frequencies were in accordance with published data both for MnSOD (Degoul et al., 2001) and OGG1 (Le Marchand et al., 2002). There were no differences in the distribution of the MnSOD genotype between the old and young volunteers (OR = 0.55, P = 0.53), or the OGG1 genotype (OR = 2.26, P = 0.20). There were no differences in the distribution of DNA damage levels between genotype for either young or elderly. These results confirmed increased lymphocyte DNA damage with increased age that was not associated with genotype for MnSOD or OGG1. References Sutton, D.F., Mansouri, A. Cepanec, A., Degott, C., Fromenty, C., Beaugrand, B., Valla, M., Pessayre, D., 2001. Gastroenterology 120, 1468–1474. Le Marchand, L., Donion, T., Lum-Jones, A., Seifried, A., Wilkens, L., 2002. Cancer Epidemiol. Biomarkers Prev. 11, 409–412. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. Exp. Cell Res. 175, 184–191. Keywords: DNA damage; Age; OGG1; MnSOD; Polymorphism The Effects of Inducing Agents on the Metabolism of Pentamidine by Isolated Rat Hepatocytes C. Atsriku1 , M.H.Grant 2 , G.G. Skellern1 , D.G. Watson1 1 Department

of Pharmaceutical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR UK; Unit, Wolfson Centre, University of Strathclyde, Glasgow G4 0NW, UK

2 Bioengineering

E-mail address: [email protected] The aromatic diamidine, pentamidine isethionate, has been used for over four decades for the chemotherapy of African human trypanosomiasis (sleeping sickness) in affected areas world-wide (Goa and Campoli-Richards, 1987). Pentamidine (P) is also used for the treatment of Pneumocyctis carinii pneumonia commonly associated with AIDS victims and over 45% of patients who receive the drug are known to suffer severe adverse reactions (Sands et al., 1985). However, emergence of drug resistant strains of trypanosomes has become a major barrier to the efficacy of trypanocidal diamidines (Peregrine, 1994). In view of poor prospects for new drug development, attempts to sustain efficacy of the drug have focussed on research into the causes of parasite drug resistance as well as drug toxicity. The inhibition or induction of hepatic drug metabolising enzymes following multiple drug therapy or exposure to environmental chemicals can alter the metabolism profile and pharmacokinetics of a given drug, leading to either a toxicological response or therapeutic failure. The aim of this study was to investigate the effects of two classical cytochrome P450 inducers, phenobarbitone (PB) and 3-methylcholanthrene

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Table 1 Metabolism of pentamidine (P) in hepatocytes from inducer-treated rats Treatments

P remaining (%)

M1

M2

M3

Control 3-MC PB DM

3.2 ± 2.3 25.4 ± 1.6∗ 15.5 ± 3.1∗ 7.1 ± 1.2∗

11.9 ± 2.2 38.9 ± 2.0∗ 12.4 ± 1.2 9.9 ± 0.9

14.8 ± 2.7 51.5 ± 2.1∗ 16.9 ± 1.6 18.5 ± 3.2

22.0 ± 2.9 4.4 ± 0.2∗ 26.1 ± 1.5 32.5 ± 2.1∗

Results are means ± S.D. of three experiments. Total metabolism is indicated by the percentage of the initial amount of P remaining in the incubations after 3 h, and the formation of the three metabolites is expressed as a percentage of the initial concentration of P. ∗ P < 0.05, compared with control incubations by one-way ANOVA.

(3MC), as well as a pyrethroid insecticide, deltamethrin (DM), on the metabolism of P by isolated rat hepatocytes. Hepatocytes were isolated from male Sprague Dawley rats by collagenase perfusion, and cells from control, PB treated (1 mg/ml in drinking water for 3 days), 3MC treated (40 mg/kg i.p. in olive oil) or DM treated (50 mg/kg i.p. in olive oil) rats were exposed to 100 ␮M P for 3 h in suspension at 37 ◦ C. Six metabolites of P were identified by LC–MS/MS. The three major metabolites were 1,5-bis(4
-amidinophenoxy)-2-pentanol (M1), 1,5-bis(4
-amidinophenoxy)-3-pentanol (M2) and 5-(4
-amidinophenoxy)pentanoic acid (M3). Although there was significant induction of cytochrome P450 levels in hepatocytes from PB and 3-MC pre-treated rats, pre-treatments with PB, 3-MC and DM led to inhibition of the total metabolism of P in isolated hepatocytes to different extents, and significant differences between the profiles of the three major metabolites of P formed (Table 1). Significantly higher levels of a nephrotoxic metabolite of P, M3, were formed in hepatocytes from DM pre-treated rats. Differences in the metabolic profiles of P as a result of concomitant exposure to environmental chemicals and pesticides could have toxicological and pharmacological implications. References Goa, K.L., Campoli-Rchards, D.M., 1987. Drugs 33, 242–258. Peregrine, A.S., 1994. Vet. Parasitol. 54, 223–248. Sands, M., Kron, M.A., Brown, R.B., 1985. Rev. Infect. Dis. 7, 625–634. Keywords: Pentamidine metabolism; Inducing agents; Hepatocytes Heat Shock Protein Induction by Sodium Arsenite and Cadmium Chloride in Rat Hepatocytes and Rat FGC4 Hepatoma Cells Steven Pratt1 , Elke Gottschalg, Mukadder Atmaca, Clive N. Kind2 , Jeffrey R. Fry 1 School

of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, UK; R&D Charnwood, Safety Assessment, Loughborough, LE11 5RH, UK

2 AstraZeneca,

E-mail address: [email protected] The expression of heat shock proteins in response to chemical and environmental stressors represents a highly conserved cellular protective mechanism in all organisms which has been used as a marker of xenobiotic exposure in a variety of studies (Feder and Hofmann, 1999). In this study, FGC4 rat hepatoma cells and primary rat hepatocytes were exposed to toxic and non-toxic concentrations of cadmium chloride (CdCl2 ) and sodium (meta) arsenite (AsNaO2 ). The aim of this study was to determine whether any differences occurred in expression of a range of Heat shock proteins (Hsps) in hepatocytes and hepatoma cells following toxicant exposure. Levels of metallothionein, Hsp25, Hsp32 (heme-oxygenase-1), Hsp40, Hsp60, Hsp70, Hsp90, and Hsp110 were all observed in response to toxicant exposure.

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Table 1 Hsp fold increase relative to control ± standard deviation in treated hepatocytes Sodium arsenite

0 h-control

6 h-control

6 h-30 mM

6 h-100 mM

24 h-control

24 h-30 mM

24 h-100 mM

Ratio for Hsp70 Ratio for Hsp60 Ratio for Hsp25

1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0

1.0 ± 0.1 0.9 ± 0.2 1.0 ± 0.1

1.4 ± 0.2 0.9 ± 0.2 1.1 ± 0.1

1.0 ± 0.1 0.9 ± 0.0 1.0 ± 0.1

1.0 ± 0.1 0.9 ± 0.2 1.0 ± 0.1

11.5 ± 3.4 1.2 ± 0.2 5.3 ± 2.4

1.0 ± 0.1 2.2 ± 0.2 1.3 ± 0.1

Cadmium chloride

0 h-control

6 h-control

6 h-3 mM

6 h-10 mM

24 h-control

24 h-3 mM

24 h-10 mM

Ratio for Hsp70 Ratio for Hsp60 Ratio for Hsp25

1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0

0.9 ± 0.1 1.0 ± 0.0 1.0 ± 0.1

1.0 ± 0.2 1.0 ± 0.1 1.0 ± 0.1

1.1 ± 0.1 1.2 ± 0.1 1.0 ± 0.1

1.0 ± 0.0 1.1 ± 0.4 1.0 ± 0.0

1.6 ± 0.5 1.3 ± 0.4 1.6 ± 0.5

1.0 ± 0.1 2.2 ± 0.2 1.0 ± 0.1

Table 2 Hsp fold increase relative to control ± standard deviation in treated FGC4 cells Sodium arsenite

0 h-control

6 h-control

6 h-10 mM

6 h-30 mM

24 h-control

24 h-10 mM

24 h-30 mM

Ratio for Hsp70

1.0 ± 0.0

1.0 ± 0.2

1.5 ± 0.1

8.0 ± 1.4

1.0 ± 0.1

3.9 ± 1.2

21.4 ± 1.8

Cadmium chloride

0 h-control

6 h-control

6 h-10 mM

6 h-15 mM

24 h-control

24 h-10 mM

24 h-15 mM

Ratio for Hsp70

1.0 ± 0.0

1.0 ± 0.2

1.0 ± 0.1

1.0 ± 0.1

1.0 ± 0.1

2.5 ± 0.6

5.6 ± 1.2

Effects on cell survival and induction of Hsps were assessed at 6 and 24 h by the lactate dehydrogenase (LDH) cell viability assay and by enzyme linked immunosorbant assay (ELISA), respectively. Increases in Hsps are reported as mean fold changes (of three separate experiments) relative to control ± standard deviation. Cells were exposed to the highest sub-lethal concentration of both compounds, at 6 and 24 h, and to a concentration which produced a 30–50% decrease in cell viability at 24 h, but had no effect at 6 h. Rat hepatocytes were treated using 3 and 10 ␮M CdCl2 , and 30 and 100 ␮M AsNaO2 . Concentrations used for treatment of FGC4 cells were 10 and 15 ␮M CdCl2 and 10 and 30 ␮M AsNaO2 . Arsenite and cadmium are believed to exert their toxicity through similar mechanisms, namely oxidative stress through cellular depletion of thiols and/or glutathione. Using an experimental design that allowed comparison of both compounds on two cell types at similar levels of cytotoxicity, it was apparent that clear compound- and cell type-related differences occur in Hsp response. In the hepatoma cells, Hsp70 induction was most apparent at concentrations of cadmium and arsenic that caused demonstrable cytotoxicity at 24 h, whereas only sub-lethal concentrations of cadmium and arsenic elicited Hsp70 induction in hepatocytes. No other Hsp was affected in the hepatoma cell line, however Hsp60 was induced by toxic doses of both compounds in hepatocytes, and Hsp25 was induced by sub-lethal concentrations of both compounds. Stressor specific patterns of induced Hsps have been proposed as possible biomarkers of exposure (Gibney et al., 2001). However, the data suggest that caution should be exercised in the use of Hsps as a ‘universal’ indicator of toxicant exposure. References Feder, M.E., Hofmann, G.E., 1999. Annu. Rev. Physiol. 61, 243–282. Gibney, E., Gault, J., Williams, J., 2001. Biomarkers 6 (3), 204–217. Keywords: Heat shock protein; Arsenite; Cadmium chloride; FGC4 cells; Hepatocytes; Cytotoxicity Acknowledgements Alison Hammond and Magda Zeraia are thanked for their assistance with hepatocyte isolation. The financial assistance of AstraZeneca and FRAME is gratefully acknowledged.

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Differential Effects of Cannabidiol (CBD) and
9 -Tetrahydrocannabinol (THC) on Induction of Rat Cytochrome P450s (CYPs) Following In Vivo Administration 1 K.E.

McArdle, 1 R.P. Pertwee, 2 G.W. Guy, 2 B.A. Whittle, 1 G.M. Hawksworth

1 Departments

of Medicine and Therapeutics and Biomedical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK; 2 GW Pharmaceuticals, Science Park, Porton Down, Salisbury, Wiltshire SP4 0JO, UK E-mail address: [email protected] The co-administration of two or more cannabinoids (e.g. CBD and THC) presents the potential for drug-drug interactions, which could lead to altered pharmacological and therapeutics effect. CBD has been shown to differentially inhibit the oxidative metabolism of THC in human hepatic microsomes (McArdle et al., 2001; Jaeger et al., 1996). The aim of this study was to evaluate the ability of CBD and THC to induce CYPs in vivo in rat. Male Sprague–Dawley rats (n = 4) were administered intraperitoneally (i.p.) either CBD or THC (15, 50, 150 mg/kg in corn oil (CN)) once daily for 3 days. Dexamethasone (DEX 150 mg/kg in corn oil), sodium phenobarbitone (PB 80 mg/kg in saline (SAL)) or ␤-naphthoflavone (BN 40 mg/kg in corn oil) were also administered i.p. once daily for 3 days, as positive controls. The livers were removed on day 4 and hepatic microsomes were prepared. The extent of CYP induction was measured by Western immunoblotting using 1 ␮g protein.

Fig. 1. Induction of rat cytochrome P450 proteins by CBD.

There was no induction of CYP1A1 with CBD treatment. However, CYP1A2 was induced in a dose-dependent manner (maximum at 150 mg/kg) (Fig. 1). This suggests that CBD induction of CYP1A2 may be regulated via an AhR-independent pathway. CBD also induced rat CYP3A23 and CYP2B1/2 with the greatest increase at 150 mg/kg (Fig. 1). In contrast, THC failed to induce any of the CYPs assessed, despite being structurally related to CBD. Although the inducing effects of CBD on human CYPs have not yet been determined, these results highlight the potential for CBD to interact with other co-administered drugs. References McArdle, K., Mackie, P., Pertwee, R., Guy, G., Whittle, B., Hawksworth, G., 2001. Toxicology 168, 133–134. Jaeger, W., Benet, L., Bornheim, L., 1996. Xenobiotica 26, 275–284. Keywords: THC; CBD; CYPs; Induction, Rat

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Paracetamol-Induced Hepatotoxicity: Protection By ␣1 -Antagonists Laura E. Randle, Carrie L. Greenough, Dominic P. Williams, Yuri N. Clement, Neil R. Kitteringham, Robert Elsby, B. Kevin Park Department of Pharmacology and Therapeutics, University of Liverpool, L69 3GE, UK E-mail address: [email protected] Phenylephrine and phenylpropranolamine, alpha (␣) adrenergic agonists have been shown to potentiate paracetamolinduced hepatotoxicity. The mechanism of potentiation is not fully understood but believed to be a result of altered glutathione (GSH) homeostasis (James et al., 1993). Previous work within this department has demonstrated that prazosin (PZ), a selective ␣1 -antagonist can abrogate paracetamol (APAP)-induced hepatotoxicity (Clement et al., 2001). This study investigated whether this heptoprotection was afforded by an alteration in APAP metabolism as a result of PZ administration. We also examined whether this was a class specific effect. Male CD-1 mice received a 1hr pre-treatment with a pharmacologically relevant dose of the ␣1 antagonists, PZ or doxazosin, (PZ & DOX; 15 mg/kg) prior to the administration of a toxic dose of APAP (>530 mg/kg, 5 h). Serum ALT levels were measured as an indicator of hepatic injury. Hepatic glutathione (1 and 5 h; Vandeputte et al., 1994) and APAP–GSH conjugate levels were measured (150 and 530 mg/kg, 1 h; hepatic and urine) by HPLC. Elimination of APAP by conjugation to glucuronic acid was also determined by HPLC (150 and 530 mg/kg, 1 h; hepatic and urine; Elsby et al., 2001). Additionally, UGT enzyme kinetics were assessed in microsomes prepared from controland PZ-treated liver. CYP2E1 enzyme activity was assessed in rat hepatocytes incubated with PZ (Koop, 1986). Table 1 Serum ALT values (U/L ± S.E.M.) after treating mice with either APAP and/or ␣1 -antagonists

Control PZ APAP APAP + PZ

1h

5h

69 ± 25 48 ± 13 149 ± 52 49 ± 4

94 ± 42 26 ± 5§ 1276 ± 556∗ 69 ± 34§

Significant different from controls: ∗ P < 0.05, U-test.

∗∗∗ P

5h Control DOX APAP APAP + DOX

17 ± 5 82 ± 44§ 752 ± 185∗∗∗ 66 ± 17§§§

< 0.001; significant different from APAP: § P < 0.05, §§§ P < 0.001, Mann–Whitney

Co-treatment of mice with PZ resulted in a significant decrease in the hepatotoxicity of APAP as indicated by serum ALT levels at 5hrs. PZ alone was not toxic (Table 1). PZ pre-treatment did not prevent the depletion of hepatic GSH attributable to APAP (5 h, percentage of control, 66.19 ± 14.44; 51.27 ± 9.97%, respectively). PZ prevented the increase in the GSSG:GSH ratio. DOX was administered as a pre-treatment prior to toxic APAP to determine whether this was a class specific effect. Serum ALT values remained similar to controls and hepatic GSH levels were depleted to a similar level as APAP alone (5 h APAP, 64 ± 17.8%; APAP + DOX 61 ± 3.62%). No difference was observed in APAP glucuronidation in microsomes prepared from control- and PZ-treated livers, with the glucuronidation kinetics, Vmax and Km , remaining the same (Fig. 1). PZ appeared to increase the biliary formation of APAP-glucuronide (1 h, 530 mg/kg, liver). This was actually a decrease in glucuronidation in the mice dosed APAP alone. This decrease was thought to be a result of the toxicity, as there was no difference at a non-toxic dose of APAP. There was also no effect of PZ upon GSH-conjugate formation in both urine and bile. PZ did not provide protection by altering CPY2E1 activity, as no effect was seen in the hydroxylation of p-nitrophenol between PZ-treated and control groups. These results show that the ␣1 -antagonists do not protect against hepatotoxicity by increasing GSH-conjugation or by increasing glucuronidation. There were also no indications of oxidative stress when mice were pre-treated with PZ alone. The data suggests that the protection by PZ and DOX is an ␣1 -antagonist, class-specific effect. It is proposed that ␣1 -antagonists may protect against APAP-induced toxicity by preventing vasoconstriction caused by stress-induced

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factors released during a toxic insult. This would lead to decreased delivery of blood-borne protective factors, thereby hindering cellular recovery. However, this hypothesis requires further investigation. References Clement, Y.C., Kitteringham, N.R., Park, B.K., 2001. Toxicology 168, 111–112. Elsby, R., Maggs, J.L., Ashby, J., Park, B.K., 2001. J. Pharmacol. Exp. Ther. 297, 103–113. James, R.C., Harbison, R.D., Roberts, S.M., 1993. Toxicol. Appl. Pharmacol. 118, 159–168. Koop, D.R., 1986. Mol. Pharmacol. 29, 399–404. Vandeputte, C., Guizon, I., Genestie-Denis, I., et al., 1994. Cell Biol. Toxicol. 10, 415–421. Keywords: Paracetamol; Prazosin; Doxazosin; Metabolism; Toxicity; Hepatoprotection C-S Lyase is Not Involved in Cis-platin Nephrotoxicity in the Rat Richard D. Wainford1 , Keith N. Stewart1 , Gerrard Descotes2 , Richard Weaver3 , Henri Merdjan3 , Gabrielle M. Hawksworth1 1 Departments

of Medicine & Therapeutics and Biomedical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK; 2 Servier International Research Institute, 92415 Courbevoie Cedex, France; 3 Drug Metabolism and Development, Servier SL3 6HH, UK E-mail address: [email protected] Cis-platin exhibits dose limiting nephrotoxicity. This may be through formation of a GSH/cis-platin conjugate. Hanigan et al. (1994, 2002) investigated the biotransformation of this conjugate and concluded that inhibition of C–S lyase protects against cis-platin toxicity in mice. The aim of this study was to examine protection against cis-platin toxicity in male Sprague–Dawley rats in vivo and in rat proximal tubular (RPT) cells. Cis-platin toxicity (6 mg/kg, i.p.) in male Sprague–Dawley rats (n = 4 ± S.D.) and the effect of acivicin (AC) (10 mg/kg i.p.), which inhibits ␥GT, aminooxyacetic acid (AOAA) (50 nmol/kg i.p.), which inhibits ␤-lyase, and bestatin (2.5 mg/kg i.p.), which inhibits aminopeptidase M, co-administration was assessed over a 5-day-period. BUN and serum creatinine levels were measured and kidneys taken for histopathological examination were stained with H&E. AOAA and bestatin did not protect against cis-platin toxicity, whereas AC and did protect against toxicity (Table 1). Cis-platin toxicity in RPT cells (50 and 100 ␮M), concentrations corresponding to cis-platin plasma levels in experimental animals, and the effect of AC (250 ␮M) or AOAA (250 ␮M) pre-treatment was assessed by the MTT assay at 24, 48 and 72 h (n = 5 ± S.D.). AOAA and AC increased cis-platin toxicity at all time-points (Wainford et al., 2002). Table 1 Serum biochemical markers of proximal tubular damage BUN (␮g/ml)

Control (saline) Cis-platin AC + saline AC + cis-platin Bestatin + saline Bestatin + cis-platin AOAA + saline AOAA + cis-platin

Serum creatinine (␮g/ml)

Day 1

Day 3

Day 5

Day 1

Day 3

Day 5

7±1 8±1 13 ± 1 9±1 8±1 7±1 7±0 8±2

7±2 17 ± 3∗ 11 ± 1 10 ± 2 5±1 19 ± 5∗ 8±1 22 ± 3∗

8±1 36 ± 4∗ 12 ± 1 12 ± 3 5±1 33 ± 6∗ 9±1 35 ± 3∗

41 ± 1 50 ± 2 50 ± 1 51 ± 2 19 ± 4 22 ± 6 20 ± 2 32 ± 4

35 ± 3 119 ± 3∗ 48 ± 3 50 ± 2 11 ± 1 98 ± 19∗ 25 ± 2 106 ± 2∗

34 ± 1 158 ± 4∗ 45 ± 1 45 ± 1 21 ± 1 207 ± 35∗ 30 ± 4 143 ± 8∗

N = 4 ± S.D.: ∗ P < 0.05 ANOVA, Scheffe.

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When ␥-GT was inhibited in vivo cis-platin toxicity was prevented, suggesting that ␥-GT activity is required for cis-platin toxicity to occur. AOAA in vivo data contradict existing data in which AOAA protected mice from cis-platin toxicity (Hanigan and Townsend 2002). The difference in the role of AOAA between rats and mice may be explained by differences in C–S lyase levels between species. The conflicting in vitro and in vivo data presented here require further investigation. References Hanigan, M., Gallagher, B.P., Large, M., 1994. Cancer Res. 54, 5925–5929. Hanigan, M., Townsend, D., 2002. J. Pharmacol. Exp. Ther. 300, 142–148. Wainford, R., Descotes, G., Merdjan, H., Weaver, R., Hawksworth, G., 2002. Toxicology 178, 59–60. Keywords: Cis-platin; in-vivo; Nephrotoxicity; Protective mechanism; Biotransformation Sodium Benzoate Attenuates D-Serine-Induced Nephrotoxicity Rebecca E. Williams1 , Hugh Eyton-Jones1 , Edward A. Lock1 1 Syngenta

Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK

E-mail address: [email protected] d-Serine causes selective necrosis to the straight portion of the renal proximal tubules in the rat (Ganote et al., 1974). The reason for the selective vulnerability of this region of the kidney to d-serine is of interest as the mechanism of action is not currently fully understood. The onset of renal tubular necrosis is rapid, occurring within 3–4 h post-dosing (400 mg/kg i.p.; male Wistar rats) (Wachstein and Besen, 1964). The damage is accompanied by proteinuria, glucosuria and aminoaciduria, the latter preceding the onset of necrosis (Carone and Ganote, 1975). It has been suggested that the metabolism of d-serine by d-amino acid oxidase (d-AAO) may be important (Silbernagl et al., 1999). d-AAO is localized within the peroxisomes of renal tubular epithelial cells and it has been shown that D-serine reabsorption is colocalised to the site of metabolism (Silbernagl et al., 1999). Sodium benzoate (SB) has been shown to be a potent, competitive inhibitor of kidney d-AAO in vitro (Arnold et al., 1979) and liver and brain d-AAO in vivo (London and Gabel, 1988; Moses et al., 1996). To determine whether the metabolism of d-serine by d-AAO is important in the mechanism of nephrotoxicity, we have examined the effect of SB on the development of renal injury. Male, Alderley Park rats (n = 3) were exposed to a single dose of D-serine (500 mg/kg i.p.) 1 h after exposure to either SB (1000 mg/kg i.p.) or saline. Urine samples, collected 0–8 h after dosing with d-serine, were examined using 1 H magnetic resonance spectroscopy (MRS). In addition, standard clinical chemistry measurements were carried out on urine and terminal plasma samples and the kidneys were examined for pathological changes. The animals were killed 8 h after receiving d-serine. Clinical chemistry analysis showed that SB was able to prevent d-serine-induced elevations in plasma creatinine and urinary glucose and protein (see Table 1). Furthermore, 1 H MRS analysis of urine, combined with principal component analysis, revealed that urine collected from animals treated with d-serine and SB clustered with those treated with SB alone, separate from the d-serine treated urines. Gross examination of the kidneys indicated that SB prevented the tubular damage observed in animals treated with d-serine alone. Table 1 Clinical chemistry parameters

Plasma creatinine (␮mol/l) Urine glucose (mmol/l):creatinine (mmol/l) Urine protein (g/l):creatinine (mmol/l) Data expressed as mean ± S.D.

Control

SB

d-serine + SB

d-serine

33.2 ± 3.0 0.91 ± 0.08 0.83 ± 0.16

49.3 ± 1.53 1.69 ± 0.34 2.25 ± 0.16

40.0 ± 2.0 1.43 ± 0.30 2.05 ± 0.41

102.3 ± 16.2 37.11 ± 10.9 4.28 ± 0.41

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These results demonstrate that prior exposure to SB prevents d-serine induced nephrotoxicity over the first 8 h post-dosing. One hour after dosing with SB, d-AAO activity in the kidney was decreased by approximately 50% suggesting that inhibition of d-AAO may be responsible for the observed protection. However, SB alone produced diuresis and elevated urinary N-acetyl glucosamidase, urea, glucose and protein and increased plasma urea. Thus SB alters renal function in its own right, which could modulate the handling of and hence exposure of the kidney to d-serine. This should be considered in the interpretation of these results. References Arnold, G., Liscum, L., Holtzman, E., 1979. J. Hisotchem Cytochem. 27 (3), 735–745. Carone, F.A., Ganote, C.E., 1975, Arch. Pathol. 99, 658–662. Ganote, C.E., Peterson, D.R., Carone, F.A., 1974. Am. J. Pathol. 77, 269–282. London, R.E., Gabel, S.A., 1988. Biochemistry 27, 7864–7869. Moses, J., Siddiqui, A., Silverman, P.B., 1996. Neurosci. Lett. 218 (3), 145–158. Silbernagl, S., Volker, K., Dantzler, W.H., 1999. AJP—Ren. Physiol. 276 (6), F857–F867. Wachstein, M., Besen, M., 1964. Am. J. Pathol. 44, 383–393. Keywords: Tubular necrosis; d-Serine; d-Amino acid oxidase; Magnetic resonance spectroscopy; Sodium benzoate The Effects of Glyceryl Trinitrate, Lovastatin and Aspirin on the Proliferation of Human Aortic Smooth Muscle Cells Poh Lin Winn1 , Karen Kelso2 , M. Helen Grant1 1 Bioengineering

Unit, University of Strathclyde, Glasgow G4 ONW, UK; 2 Vascutek, Inchinnan Industrial Estate, Paisley PA4 9PR, UK E-mail address: [email protected]

Glyceryl trinitrate (GTN) and lovastatin are known to inhibit proliferation of smooth muscle cells (SMCs) (Gonzalez et al., 1996). The mechanisms responsible for this inhibitory effect involve an increase in cyclic guanidine monophosphate formation by guanylate cyclase in the case of GTN, and inhibition of mevalonate synthesis, a precursor of isoprenoids vital for DNA synthesis, by blocking 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the case of lovastatin. Interaction of graft materials causing uncontrolled proliferation of SMC in vivo contributes to failure of cardiovascular prostheses. To improve biocompatibility, grafts may be coated with aspirin to minimise platelet aggregation and to inhibit cell proliferation (Landy et al., 1991). The aim of our study was to determine whether a combination of aspirin and either GTN or lovastatin would act in synergy to inhibit proliferation of SMC. Human aortic smooth muscle cells obtained from the American Tissue Culture Collection were seeded on 96-well plates at 104 cells/cm2 in Ham’s F12-K medium with 10% (v/v) foetal calf serum. After allowing cells to adhere for 24 h, aspirin (1–3000 ␮M), GTN (3–200 ␮M), lovastatin (1–10 ␮M) or a combination of the drugs with aspirin was added. Medium and drug solutions were replenished every 2 days, and after 7 days, cell proliferation was measured by the Neutral Red (NR) assay and the (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

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Fig. 1. MTT Assay in HA-VSMC after 1-week drug treatment with GTN ± aspirin (A) and with lovastatin ± aspirin (B) ((+) P < 0.05, comparing the combination treatment and either or both of the single drug treatment groups. (*) P < 0.05, compared with the control values. ANOVA followed by Dunnett’s test. Results are mean ± S.E.M. of 10 measurements).

All three drugs inhibited cell proliferation. Figs. 1A and 1B show cell proliferation expressed as a percentage of control cell growth measured by the MTT test for cells exposed to GTN or lovastatin, alone, or in combination with aspirin. At low concentrations, aspirin consistently caused an increase in proliferation measured by both NR and MTT assays. The concentration of GTN causing 50% inhibition of cell growth (MTT) was 80 ␮M alone, and 150 ␮M in presence of aspirin. Corresponding concentrations of lovastatin were 1 and 2 ␮M. Our data indicate that there is no synergy between either drug and aspirin with regards to SMC proliferation, and that lovastatin is a potent inhibitor by itself. At present, the mechanism(s) responsible for these effects is not clear. References Gonzalez, J.M., Badimon, L., 1996. Eur. Clin. Invest. 26, 1023–1032. Landymore, R.W., MacAulay, M.A., Fris, J., 1991. Can. J. Cardiol. 7, 87–90. Keywords: Glyceryl trinitrate; Lovastatin; Aspirin; Smooth muscle cell proliferation Vaccines and Pyridostigmine Interactions in a Marmoset Model: Preliminary Results Andrew P. Bowditch, Susan Chamberlain, Gareth D. Griffiths, Rebecca J. Hornby, Thomas M. Mann, Peter C. Pearce, Daniel J. Stevens, Leah Scott, Kate E. Williams Dstl Biomedical Sciences, Porton Down, Salisbury, Wilts SP4 0JQ, UK E-mail address: [email protected] British troops deployed during the 1990/1991 Gulf Conflict were administered a range of vaccines. A number also received pyridostigmine bromide (PB) as a pretreatment against possible organophosphonate nerve agent poisoning. Following this active service, a number of UK and US veterans presented with a diverse range of symptoms which have become collectively known as Gulf Veterans’ Illnesses. The administration of vaccines and/or PB has been implicated as a possible cause of these illnesses. The aim of this study is to determine, in a marmoset model, whether combinations of relevant vaccines, with and without PB, give rise to long term adverse health effects; it is not the intention to create a model of Gulf Veterans’ Illnesses. Male (24) and female (24) common marmosets (Callithrix jacchus) were used. The experimental period was divided into seven 3-month periods. Before the first (baseline) period, animals trained to perform a complex be-

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243

havioural task were implanted with a radiotelemetry transmitter (DSI, USA), and assigned to one of 4 treatment groups (12 animals per group; group A: vaccines (V) + PB; group B: V + saline (S); group C: S + PB; group D: S + S). Behavioural, physiological and immunological parameters were monitored in each animal throughout the seven experimental periods. Over the first 51 days of the second period, animals were vaccinated (at 1/5th human dose, not corrected for body weight) according to the schedule shown below. On day 15, animals were implanted with mini-osmotic pumps (Alzet Corp., USA) containing PB (500 ␮g/kg per day; A and C) or sterile saline (B and D), which were removed on day 44. Day

Vaccine

0 3 6 23 51

Anthrax 1, pertussis 1, yellow fever, polio Typhoid, tetanus, hepatitis B Meningococcal meningitis, cholera Plague 1, anthrax 2, pertussis 2 Plague 2, anthrax 3

This poster will present the behavioural and physiological results for periods 1 and 2 (the immunology results will be reported elsewhere). Body weight: The mean body weight of each treatment group did not fluctuate by more than 30 g (<7% of initial mean weight) throughout the two periods and no differences between treatment groups were apparent. Gross muscle function: Once per week, animals performed a simple weight-pulling task for a food reward. Performance of this task was stable for all treatment groups throughout the two periods. The mean weight pulled at the outset of the baseline period (A: 490 ± 25 g (S.E.M.); B: 432 ± 35 g; C: 560 ± 38 g; D: 475 ± 60 g) had improved for all groups by the end of the second period (A: 578 ± 20 g (S.E.M.); B: 640 ± 48 g; C: 660 ± 53 g; D: 638 ± 52 g). Behavioural task: Animals were presented with an attentional set-shifting task from the Cambridge Neurological Test Automated Battery (CANTAB) on a daily (Monday–Friday) basis. There were no apparent differences between treatment groups in performance of the task. EEG: One EEG per week per animal was recorded whilst the animal was performing the behavioural task. Comparison following fast Fourier transform analysis (FFT) revealed no apparent difference in EEG frequency bands between groups during and following vaccine/PB administration. Sleep: One night-time EEG recording per animal per month was analysed to enable production of a hypnogram. Sleep parameters (sleep efficiency, light sleep, slow wave sleep, REM sleep, no. of REM cycles) were remarkably stable throughout the two periods, and no differences between treatment groups were apparent. Acetylcholinesterase (AChE) activity: The inhibition of red blood cell AChE activity on day 29 of the vaccines/PB administration schedule was—A: 30.5 ± 4.0% (S.E.M.); B: 8.0 ± 2.4%; C: 30.6 ± 3.7%; D: 2.5 ± 1.3%. Overall, the preliminary results of this study indicate there were no acute adverse health consequences 0–3 months following vaccine administration and/or PB that were detectable using a sophisticated marmoset model. Investigations are ongoing. Keywords: Vaccines; Pyridostigmine; Marmoset; Long-term effects Cloning and Characterisation of Class Kappa Mitochondrial GlutathioneS-Transferases Rachel E. Thomson, Ian R. Jowsey, Terry C. Orton1 , John D. Hayes, Cliff R. Elcombe Biomedical Research Centre, Ninewells Hospital & Medical School, University of Dundee, Dundee DD1 9SY, UK; Alderley Park, Macclesfield, Cheshire

1 AstraZeneca,

E-mail address: [email protected] A novel glutathione S-transferase (GST) was previously isolated from rat liver mitochondria (Harris et al., 1991) and designated class Kappa (rGSTK1) (Pemble et al., 1996). We have identified a possible mouse orthologue of this

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Table 1 Activity of rat and mouse class kappa mitochondrial glutathione S-transferases Substrate

1-Chloro-2,4-dinitrobenzene Km (mM) Vmax Cumene hydroperoxide Ethacrynic acid

Specific activity (␮mol/min mg protein) Rat

Mouse

74 ± 1 3.4 171 0.11 ± 0.02 0.44 ± 0.12

182 ± 4 3.0 201 1.03 ± 0.02 0.29 ± 0.08

gene. The aim of this work was to clone and characterise the novel mouse GST Kappa and further characterise the rat GSTK1. Regulation of these genes and their response to both hepatotoxins and oxidative stress inducers will be investigated. A mouse Expressed Sequence Tag (EST) clone has been identified exhibiting high nucleotide sequence similarity to rGSTK1. The EST has an open reading frame of 678 bp encoding a peptide of 226 amino acids with a calculated molecular mass of 25.7 kDa. Analysis of rat and mouse genomic DNA has revealed that the rGSTK1 gene and the gene encoding the mouse protein each consist of eight exons and span approximately 4.5 kb of genomic DNA. Comparisons between the peptide sequences of the rat and probable mouse GST Kappa reveal 86% sequence identity. This mouse protein and the rGSTK1 protein were heterologously expressed in Escherichia coli and purified by affinity chromatography. Antibodies have been generated against the mouse recombinant protein. Enzyme assays and kinetics revealed that both the mouse and rat Kappa proteins are highly active using typical GST substrates (Table 1). The enzymes exhibited high activity with other aryl halides, such as 1-iodo-2,4-dinitrobenzene and 1-bromo-2,4-dinitrobenzene. No appreciable activity was obtained with either enzyme using the substrates t-butylhydroperoxide or 4-hydroxynonenal. In both species, mRNA encoding the Kappa proteins is highly expressed in liver, kidney, heart and skeletal muscle. Mouse kappa protein is highly expressed in liver, kidney, heart and lung. Comparisons between the rat and mouse proteins with respect to cDNA sequences, genomic organisation, peptide sequences, enzyme activities and tissue specific expression reveal a high degree of similarity between these species. We therefore conclude that this mouse protein represents a novel class kappa GST (designated mGSTK1). References Harris, J.M., Meyer, D.J., Coles, B., Ketterer, B., 1991. Biochem. J. 278 (1), 137–141. Pemble, S.E., Wardle, A.F., Taylor, J.B., 1996. Biochem. J. 319 (3), 749–754. Keywords: Glutathione S-transferase; Oxidative stress; Mitochondria Activation of c-Jun N-Terminal Kinase in A549 Lung Cells by Sodium Dichromate is Associated with Dissociation of Ask-1 from Thioredoxin Daniel J. Smart, Nikolas J. Hodges, James K. Chipman School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK E-mail address: [email protected] Hexavalent compounds of chromium (CrVI ) including sodium dichromate are human genotoxic pulmonary carcinogens (IARC, 1990) capable of exerting an oxidative stress on cells. Dissociation of the MAP kinase kinase kinase (MAPKKK) Apoptosis signal-regulating kinase-1 (Ask-1) from its physiological inhibitor thioredoxin (Trx) is believed to be involved in cellular response to oxidative stress, including H2 O2 (Ichijo et al., 1997). We wished

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245

Fig. 1. Dichromate-mediated dissociation of Ask-1 from Trx as assessed by immunoprecipitation.

to investigate sodium dichromate-mediated activation of c-Jun N-terminal kinase (JNK), c-Jun, p38 MAP kinase and the possible intermediary role of Ask-1. Confluent A549 human lung carcinoma cells were treated with sodium dichromate in culture (DMEM supplemented with foetal bovine serum, penicillin (100 ␮l/ml) and streptomycin (0.1 mg/ml)). Viability of cells based on ATP levels (measured by an ATP-Bioluminescence kit, Sigma) and on the level of apoptosis (TiterTACS assay, Trevigen) remained above 87% of controls and was not significantly reduced up to 100 ␮M for 16 h. Significant cytotoxicity was observed at 500 ␮M (P < 0.01). After treatments, whole cell protein extracts were prepared and analysed by Western blotting. Immunoprecipitation of thioredoxin was achieved using a mouse anti-thioredoxin antibody and the precipitated protein was also used for analysis by western blotting. There was a marked increase in the immunodetectable levels of both phospho-JNK isoforms (46 and 55 kDa) in a time- and dose-dependent manner. Activation was seen within 1 h and the lowest concentration of sodium dichromate that provided activation was 25 ␮M. No such increase in phosphorylated p38 was seen. There was also an induction of the c-Jun protein (2 h at 100 ␮M) and it’s phosphorylated form (4 h at 25 ␮M). This provides evidence that the activation of the JNK pathway involved the dissociation of the physiological inhibitor thioredoxin (Ichijo et al., 1997) from Ask-1 such that the homodimer is able to activate the downstream target JNK without an apparent activation of the alternative p38 pathway. The activation of the MAP kinase pathway may contribute to the carcinogenicity of sodium dichromate. Associated with these changes, we found that immunoprecipitated thioredoxin was no longer bound to Ask-1 following treatment with sodium dichromate (100 ␮M) in a time-dependent manner. Within 4 h, associated Ask-1 protein was no longer detectable (Fig. 1). References Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., Gotoh, Y., 1997. Science 275, 90–94. International Agency for Research on Cancer, 1990. vol. 49. Keywords: Chromium; Ask-1; c-Jun; Thioredoxin; MAP kinase Validation of the WPI REsistance Measuring System for Measuring Transepithelial Resistance in Human Bronchial Epithelial Cells (16HBe140−) Azmina Mather, Chloe Day, Simon Carter, Brian Sweatman, Pauline Lee GlaxoSmithKline, In Vitro Models, Ware, UK E-mail address: azmina.j.mather @gsk.com Transepithelial resistance (TER) is commonly measured using a Millicell–ERS resistance meter. This method limits the number of samples that can be tested. Electrode positioning can also vary. To increase sample throughput the semi automated WPI REsistance Measuring System (REMS) has been evaluated. To validate the use of the REMS compounds from previous work (Westmoreland et al., 1999) were used: sodium chloride and titanium dioxide (non-irritants), sodium carbonate (respiratory irritant), compounds A and B shown to induce respiratory toxicity

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Table 1 Concentrations causing a decrease in TER and IC50 values determined by MTT Test material

Concentration (␮g/ml)

IC50 values (␮g/ml)

Sodium carbonate Sodium chloride Titanium dioxide Compound A Compound B

5000 5000 10 500 All concentrations

>5000∗∗∗ >5000∗∗∗ >50∗ 444∗∗∗ 12.4∗∗∗

∗P

< 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 (ANOVA + Dunnett’s test).

in the rat during pre-clinical safety assessment. 16HBe140 (HBe)-cells a transformed cell line was maintained in MEM/EBSS modified medium (supplied by Hyclone). For TER assessment 250 ␮l of cell suspension at a density of 1 × 106 cells/ml was seeded into the inserts of HTS Transwell—24-well plates. For cytoxicity assessment, 200 ␮l of the cell suspension at a density of 1 × 105 cells/ml was seeded into 96-well plates. All plates were incubated at 37 ◦ C for 5 days to reach confluency. Evaluations were also made microscopically. TER was measured prior to treatment (time 0), and 2, 4, 6, and 24 h following treatment. Cytotoxicity was assessed 20 h following treatment using MTT reduction. Three repeat experiments were carried out. Each experiment had three replicates per treatment for TER and six for cytotoxicity assessment. Osmolality and pH were measured for all test and control solutions. ANOVA followed by a one sided lower Dunnett’s t-test was carried out on the TER and MTT data. Results indicate good correlation with published data (Westmoreland et al., 1999). Table 1 shows concentrations where a significant decrease in TER was observed compared to control 2 h following treatment. Compound A (500 ␮g/ml) (P < 0.001) and compound B at all concentrations (P < 0.001) produced a significant decrease in MTT reduction. From Table 1, it can be seen that a significant decrease in TER was observed for sodium chloride this was associated with an osmolality value higher than control. This was comparable to published studies but no recovery was observed after 24 h (Westmoreland et al., 1999). In the present experiment titanium dioxide produced a decrease in TER but no decrease in MTT reduction was observed. Replicate values were more consistent with the REMS than with the Millicell –ERS resistance meter (as evidenced by small standard errors). Reference Westmoreland, C., Walker, T., Matthews, J., Murdock, J., 1999. Toxicol. In Vitro 13, 761–764. Keywords: 16HBe14o−; In vitro; Transmembrane epithelial resistance; Respiratory irritants Identification of Potential Non-Invasive Biomarkers of Peroxisome Proliferation in the Rat Stephanie Ringeissen, Susan C. Connor, Hansa Thakkar, Brian C. Sweatman, Mark P. Hodson, Kathryn A. Hutton, Steve P. Kenny, Paul McGill, Derek J. Nunez, John N. Haselden, Catherine J. Waterfield Safety Assessment, GlaxoSmithKline, Park Road, Ware, Herts SG12 ODP, UK E-mail address: [email protected] Evaluation of hepatocellular peroxisome proliferation (PP) currently depends on peroxisome counts derived from sections of liver tissue, and transmission electron microscopy (TEM) is currently the “gold standard” tool for estimating PP. There is as yet no non-invasive biomarker/pattern of biomarkers of PP available that would permit monitoring for this response in rodents or in other species. We are currently looking for individual biomarkers of PP. Compounds known to cause PP or other pharmacological effects were given to rats at effective doses in two studies, A and B (five per group, males, 250–350 g) for 7 days. In study A, compounds GW␣, GW␦ and GW␥ were used. In study B, other compounds, known to cause PP, to affect fatty acid metabolism or mitochondrial function were dosed to rats to investigate changes which may compromise the utility of a biomarker for PP. These were diethylhexylphthalate

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Table 1 Urinary excretion of NMN and 4PY, peroxisome counts and ACMSD mRNA Compounds

Mean NMN excretiona

Peroxisome % of control∗

1

100

1

1

Plasma NMN at autopsyb

Study A

Control

Dosing twice daily

GW␣ GW␦

16.9 9.47

3.43 3.48

234 287

−9.10 −3.69

41.3 14.3

Han Wistar

GW␥ Fenofibrate

1.09 23.5

1.11 3.19

114 321

1.05 −10.67

1.7 61.0

Study B

Control

Dosing once daily

Fenofibrate DEHP

Sprague–Dawley

Simvastatin Hydrazine Chlorpromazine LCFA

1

ACMSDb mRNA

Mean 4PY excretiona

1

100

1

ND

11.1 5.95

1

3.94 4.21

560 243

−10.39 −3.09

ND ND

2.46 1.19 1.77 1.13

2.03 0.86 1.62 1.22

273 120 103 155

−1.83 −1.51 2.79 −3.44

ND ND ND ND

N = 5 except ∗ N = 2; a ratio day 6/day 1 normalised to control and expressed as ␮mol per day per kg body weight, b fold-change over control; ND: not determined, NA: not applicable.

(DEHP), simvastatin, hydrazine, chlorpromazine and long-chain fatty acids (LCFA). Fenofibrate (mostly PPAR␣ activity) was used as a positive control in both studies. Urine was collected continuously over the 7 days. In study A, urine was analysed by 1 H NMR spectroscopy and multivariate statistical data analysis was used to identify potential biomarkers of PP. Following the identification of urinary metabolites from the tryptophan pathway, levels of N-methylnicotinamide (NMN) were measured by HPLC coupled with spectrofluorimetry, in selected plasma samples taken at necropsy. In study B, urinary levels of NMN and N-methyl-4-pyridone-3-carboxamide (4PY) were measured by LC–MS/MS. The mRNA expression of aminocarboxymuconate-semialdehyde decarboxylase (ACMSD, EC 4.1.1.45), one of the key enzymes in the tryptophan pathway, was assessed in liver taken at necropsy using RT-PCR (TaqMan® ). Peroxisome counts were made using transmission electron microscopy. Two end products of the tryptophan-NAD+ pathway, NMN and 4PY were identified in urine as potential biomarkers of PP amongst a number of possible markers. The correlation coefficient between total NMN excretion over 7 days and the peroxisome count was r = 0.87 in study A. There was a good correlation between the levels of urinary and plasma NMN, suggesting that changes in urinary NMN were not due to altered renal handling. There was a better correlation between PP and NMN excretion than any of the pharmacological effects (e.g. lipid lowering). The down-regulation of ACMSD by compounds that caused PP provided a mechanism for the increased urinary NMN and 4PY. Keywords: 1 H NMR; Biomarkers; Peroxisome proliferation; N-methylnicotinamide Development of a Multivariate Model to Predict Peroxisome Proliferation in the Rat Using1 H NMR-Based Metabonomic Analysis of Urine Mark P. Hodson, Susan C. Connor, Brian C. Sweatman, Stephanie A. Ringeissen, Derek J. Nunez, Catherine J. Waterfield, John N. Haselden Department of Safety Assessment, GlaxoSmithKline Research and Development, Park Road, Ware, Herts, UK Evaluation of hepatocellular peroxisome proliferation (PP) currently depends on peroxisome counts derived from sections of liver tissue, and transmission electron microscopy (TEM) is currently the “gold standard” tool for estimating PP. There is as yet no non-invasive biomarker of PP available that would permit monitoring for this

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Model-Predicted PxC

140 120 100 80 R2 = 0.9833 60 40 20 20

40

60

80 Observed PxC

100

120

140

Fig. 1. Observed versus model-predicted peroxisome counts.

response in rodents or in other species. This work aims to evaluate the ability to improve the monitoring of PP using multiple related markers. An investigative study was carried out. Male Wistar Han rats were dosed orally, twice daily for 7 days with one of four known Peroxisome Proliferator-Activated Receptor (PPAR) agonists, namely GW␣ GW␦, GW␥ and fenofibrate (predominantly a PPAR␣ agonist). Urine was collected continuously in separate 8 h collections during dosing. Previous work on this study had highlighted two endogenous biochemicals present in the urine, which were then investigated further as potential biomarkers of PP in the Wistar Han rat (Ringeissen et al., in Press). The aim of further analysis of these data was to identify common 1 H NMR spectral regions affected by PPAR␣ compounds, which may be related to PP and therefore be used to form a pattern of markers of PP in rodents, rather than a single marker. A predictive model of PP was generated based on the regions that showed significant changes and would predict PP and/or its associated liver weight increase in the rat for use in subsequent studies. A combination of statistical analyses, incorporating both multivariate (Principal Components Analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA)) and univariate (Student’s t-test) methods were used to “pare down” the data, removing unwanted/unimportant variation to highlight the most important discriminating regions. This approach resulted in a total of 39/250 spectral regions being selected as important discriminators of PPAR␣ activity and possibly peroxisome proliferation in the rat. These regions included several Tryptophan-NAD+ pathway metabolites and dicarboxylic acids from the associated glutarate pathway. The PLS modelling of NMR data versus peroxisome counts (two components; R2Y = 0.983; Q2(cum) = 0.854) gave predictions for peroxisome count which, when plotted against observed peroxisome counts, produced a plot with an R2 value of >0.98 (Fig. 1), and this remained above 0.97 during cross-validation. This compares favourably with the correlation of total N-methylnicotinamide excretion over 7 days with peroxisome count, which gave an R2 of 0.757 and has been proposed as an individual biomarker of PP. The use of multivariate data analysis (MVDA) and subsequent modelling improves the predictivity that can be extracted from NMR data. A simple correlation of a single endogenous excreted metabolite versus an endpoint can be vastly improved by building a pattern of a number of potential markers and performing multivariate PLS modelling to predict the endpoint. Such an approach, once validated using independent test sets of data, may provide a model and/or a set of markers that could be used as a predictive screen for the endpoint, in this case peroxisome counts and thus, peroxisome proliferation. Keywords: Peroxisome proliferation; PPAR; NMR; Biomarker; Predictive screen

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249

Table 1 Effect of PPAR agonists on hepatocellular DNA synthesis (S-phase) Hepatocytes

Labelling index (% of cells undergoing S-phase) Controla

Rat Guinea pig Human 1 Human 2 Human 3

5.8 ± 3.4 14 ± 0.20 0.86 ± 0.40 0.93 ± 0.55 1.1 ± 0.37

WYb

ROSIb 1.8∗∗

12 ± 18 ± 1.5∗∗ 0.43 ± 0.34 0.35 ± 0.05∗∗ 0.56 ± 0.29∗

2.5∗

10 ± 13 ± 5.3 0.0 ± 0.0∗∗∗ 0.0 ± 0.0∗∗∗ 0.47 ± 0.21∗∗

TROGb

EGF

3.1 ± 13 ± 3.2 0.0 ± 0.0∗∗∗ 0.0 ± 0.0∗∗∗ 0.11 ± 0.19∗∗∗

20 ± 5.4∗∗∗ 27 ± 3.6∗∗∗ 3.0 ± 0.92∗∗∗ 5.8 ± 0.50∗∗∗ 4.6 ± 0.99∗∗∗

0.80c

Values are mean ± S.D. (n = 4). a DMSO vehicle control; b 100 ␮M; c 12%; ∗ 12% and ∗∗ 20% at 1 and 5 ␮M, respectively. Significantly different to respective vehicle control, ∗ P < 0.05; ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Different Effects of PPAR␣ and PPAR␥ Agonists on Hepatocyte S-phase and Apoptosis Barbara M. Elcombe1 , Adam Hey2 , Clifford R. Elcombe1 1 CXR

Biosciences Ltd., James Lindsay Place, Dundee, DD1 5JJ; 2 Novo Nordisk A/S, 2760 Maaloev, Denmark

E-mail address: [email protected] These studies were designed to investigate species differences in the “liver growth” effects of peroxisome proliferator activated receptor (PPAR) agonists. Male rat, guinea pig and human hepatocytes (UK Human Tissue Bank) were isolated and cultured in Leibowitz CL15 medium for 4 days. Wy14643 (WY) was utilised as a prototypical PPAR␣ “selective” agonist and rosiglitazone and troglitazone were used as PPAR␥ agonists. EGF (25ng/ml) and TGF␤ (5 ng/ml) were used as positive controls. BrdU was included in the medium for the last 3 days of culture. S-phase was determined by BrdU incorporation and light microscopy. Apoptosis was determined by fluorescence microscopy following fixation and staining with Hoechst 33258. No overt cytotoxicity was observed by microscopy. All compounds stimulated S-phase in the rat hepatocytes at the lowest concentrations (0.1–100 ␮M) tested. Little effect was seen in guinea pig hepatocytes, while S-phase was strongly inhibited in the human hepatocytes, particularly by ROSI and TROG (Table 1). In rat and guinea pig hepatocytes, WY suppressed apoptosis, while ROSI and TROG stimulated apoptosis. WY had little effect on apoptosis in human hepatocytes, but ROSI and TROG markedly stimulated apoptosis in two out of the three human hepatocyte preparations (Table 2). Therefore, it is apparent that distinct species differences in response to PPAR␣ and PPAR␥ agonists exist. Keywords: Hepatocytes; S-phase; Apoptosis; PPAR

Table 2 Effect of PPAR agonists on apoptotic index measured using Hoechst 33258 Hepatocytes

Rat Guinea pig Human 1 Human 2 Human 3

Apoptotic index (% of cells) Controla

WYb

ROSIb

TROGb

TGF␤

0.53 ± 0.22 1.7 ± 0.66 3.6 ± 0.96 10 ± 3.3 2.5 ± 0.34

0.17 ± 0.09∗∗∗ 0.77 ± 0.24∗∗∗ 3.8 ± 0.44 5.4 ± 0.35∗ 2.9 ± 0.90

2.4 ± 0.43∗∗∗ 2.9 ± 0.26∗∗∗ 9.1 ± 1.1∗∗∗ 13 ± 0.87 7.4 ± 0.50∗∗∗

2.3 ± 0.73∗∗∗ 3.7 ± 0.28∗∗∗ 26 ± 2.7∗∗∗ 9.7 ± 4.7 10 ± 1.8∗∗∗

3.1 ± 0.40∗∗∗ 3.2 ± 0.49∗∗∗ 7.1 ± 1.5∗∗∗ 21 ± 0.65∗∗∗ 6.5 ± 1.1∗∗∗

Values are mean ± S.D. (n = 4). a DMSO vehicle control; b 100 ␮M. Significantly different to respective vehicle control, ∗ P < 0.05; ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

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LDH Leakage

40

* *

30 20 10

*

*

*

100

150

200

0 0

50

75

Drug Concentration (uM)

Fig. 1. Fold increase in LDH leakage compared with control cultures. TGZ (䊏), RGZ (䉬), Vit E (䉱). Mean ± S.E.M., n = 3. ∗ P < 0.05, one way ANOVA with Dunnett’s post-hoc test (compared to control samples).

The Toxicity of Troglitazone, Rosiglitazone and Vitamin E in HepG2 Human Hepatoma Cells Alison J. Ball1 , Justice N.A. Tettey1 , M. Helen Grant2 1 Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow G4 0NR, UK; 2 Bioengineering Unit,

University of Strathclyde, Glasgow G4 0NW, UK Troglitazone (TGZ), a member of the thiazolidinedione class of drugs used to treat type-II diabetes, has been associated with hepatotoxicity (Biswas et al., 2001). TGZ differs structurally from the newer non-hepatotoxic glitazone, rosiglitazone (RGZ), in that it contains the chromanol moiety of Vitamin E (Vit E). This study compares the toxicity of TGZ, RGZ and Vit E in human HepG2 cells. Cells were seeded at 2.5 × 105 cells/cm2 in Dulbecco’s medium and after 24 h drugs (0–200 ␮M, in dimethylsulphoxide (0.1%, v/v)) were added for 4 h in serum-free media. Viability was measured by leakage of lactate dehydrogenase (LDH) into the medium and intracellular reduced glutathione (GSH) by fluorimetry after derivatization with o-phthaldehyde. Fig. 1 shows the effect of the three compounds on viability in terms of LDH leakage. The effect was apparent at 100 ␮M with TGZ and 150 ␮M with RGZ. Leakage was greatest with TGZ (28.5-fold increase compared with controls) followed by RGZ (7.9-fold increase) at 200 ␮M. Vit E also increased LDH leakage but only caused a 1.9-fold increase compared with controls. Both TGZ and RGZ depleted intracellular GSH (Fig. 2) after exposure to concentrations at and above 75 ␮M. The effect of TGZ was greater. After exposure to 200 ␮M TGZ, cells contained 0.54 ± 0.19 nmol per well GSH compared with 3.60 ± 0.27 nmol per well after exposure to 200 ␮M RGZ and 6.10 ± 0.90 nmol per well in control cells. In contrast, Vit E did not deplete GSH. These data suggest that the chromanol moiety associated with Vit E in TGZ is not involved in glitazone toxicity. The depletion of GSH by the glitazones may reflect conjugation of the thiazolidinedione ring (Tettey et al., 2001). However, the possible oxidation to GSSG cannot be ruled out. References Biswas, P., Wilton, L.V., Shakir, S.A., 2001. Drug Safety 24, 149–154. Tettey, J.N., Maggs, J.L., Rapeport, W.G., Pirmohamed, M., Park, B.K., 2001. Chem. Res. Toxicol. 14, 965–974. Keywords: Troglitazone; Rosiglitazone; Vitamin E; Cytotoxicity

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251

GSH (nmol/well)

10

*

5

* *

*

* *

*

150

200

0 0

50

75

100

Drug Concentration (uM)

Fig. 2. GSH content. TGZ (䊏), RGZ (䉬), Vit E (䉱). Mean ± S.E.M., n = 3. ∗ P < 0.05, one way ANOVA with Dunnett’s post-hoc test (compared to control samples).

Activation of DNA Damage Response Proteins in Human Breast Epithelial Cells Treated with the Cooked Meat Mutagen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) Stuart Creton, Nigel J. Gooderham Molecular Toxicology, Division of Biomedical Sciences, Imperial College of Science Technology and Medicine, London SW7 2AZ, UK The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of meat, poultry and fish, induces prostate, colon and mammary gland tumours in rats (Ito et al., 1991; Shirai et al., 1997). Whilst the metabolism and mutational characteristics of PhIP have been well defined, the cellular and genomic responses prior to neoplastic transformation are not yet fully characterised. MCF10A cells, an adherent human breast epithelial line which displays the characteristics of normal breast cells, were treated with PhIP and the effects upon cell growth, cell cyle and the expression of key proteins were examined. Because these cells are unable to metabolise PhIP, they were cocultured in the presence of a human lymphoblastoid suspension cell line MCL-5, which constitutively express CYP1A1, and have been transfected to express human CYPs1A2, 2A6, 3A4 and 2E6. The MCL-5 cells were irradiated (2000 rad) prior to coculture, in order to sterilise the cell, rendering it unable to replicate yet still metabolically competent. MCF10A cells were treated (in the presence of MCL-5 cells) with PhIP (5–100 ␮M) and harvested at various timepoints. Cells were then assessed for viability before Western blot or cell cycle analysis. Treatment with PhIP resulted in a significant dose-dependent fall in cell viability following 24 and 48 h treatments compared with DMSO control. Cells treated for 48 h then cultured in the absence of PhIP (or MCL-5 cells) for a further 6 days continued to show a dose-dependent reduction in viability. Flow cytometric analysis of propidium iodide staining indicated that PhIP treatment (48 h) resulted in an accumulation of cells in the G1 population of the cell cycle (100 ␮M; 22.4 ± 7.8% increase) again in a dose-dependent manner. Western blotting revealed the expression of both p53 and the cyclin-dependent kinase inhibitor p21 was elevated following 24 and 48 h treatment. Levels of MDM2, and the hypophosphorylated form of Rb were also seen to be elevated. This finding that PhIP triggers G1 cell cycle checkpoint, via the expression of DNA damage response proteins, in a target cell line is in line with the anticipated response to PhIP mediated DNA damage (adduct formation). These cell cycle effects enable the cells to effect genome repair, acceptance of mutation or elimination of excessively damaged cells by necrotic or apoptotic cell death. References Ito, N., Hasegawa, T., Sano, M., Tamano, S., Esumi, H., Takayama, S., Sugimura, T., 1991. Carcinogenesis 12, 1503–1506. Shirai, T., Sano, M., Takahashi, S., Hirose, M., Futakuchi, M., Hasegawa, R., Imaida, K., Matsumoto, K., Wakabayashi, K., Sugimura, T., Ito, N., 1997. Cancer Res. 15, 195–198.

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Fig. 1. Effect of PhIP upon cell viability.

Keywords: PhIP; G1 Checkpoint; DNA damage Measurement of Allergen-Induced Lymphocyte Proliferation Using Flow Cytometry N. Humphreys, R.J. Dearman, I. Kimber Syngenta Central Toxicology Laboratory, Macclesfield, UK E-mail address: [email protected] The murine local lymph node assay (LLNA) is a method for the prospective identification of chemical contact allergens (Basketter et al., 2002). The current validated protocol assesses lymphocyte proliferation induced in the draining lymph node by in situ incorporation of radiolabelled thymidine. We have explored the possibility of using an alternative non-radioisotopic marker of cell division, the membrane-permeable cytoplasmic dye carboxyfluoresein succinimidyl ester (CSFE). When the cells divide, the CSFE-labelled cytoplasmic proteins are distributed equally between the daughter cells, thus the number of divisions each cell has undergone can be tracked. BALB/c strain mice (n = 3–5) were exposed topically to various concentrations of the contact allergen 2,4-dinitrochlorobenzene (DNCB) or to the non-sensitizing skin irritant methyl salicylate (MS) or to vehicle (acetone:olive oil; AOO) alone. Five days later, lymph nodes were pooled on an experimental group basis, a single cell suspension of lymph node cells (LNC) prepared and labelled with CSFE. After 96 h of culture, LNC were incubated with fluorescent labelled anti-CD4 (T helper) and -CD8 (T cytotoxic) cell antibodies and the percentage of proliferating CD4+ and CD8+ cells within the total viable LNC population analyzed by flow cytometry. In LNC populations derived from vehicle-treated animals (n = 4 experiments), less than 1% of either cell type had undergone one cell division or more. Topical exposure to MS did not increase the frequency of proliferating cells. Exposure to DNCB, however, resulted in marked increases in the percentage of CD4+ and CD8+ proliferating cells recorded in cell divisions 1 to 4. Additional experiments are required to examine further the selectivity and sensitivity of this method. However, these preliminary data suggest that CSFE incorporation may be applied to provide a supplementary nonradioisotopic endpoint for the LLNA, particularly for the identification of potent contact allergens.

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Table 1 Percentage of CD4+ and CD8+ proliferating cells in the total viable LNC population following topical exposure to DNCB or methyl salicylate CD4+ proliferating cells (%)

0.5% DNCB 0.25% DNCB 20% MS 10% MS AOO (mean ± S.D.)

CD8+ proliferating cells (%)

Divison 1

Divison 2

Divison 3

Divison 4

Divison 1

Divison 2

Divison 3

Divison 4

4.9 3.3 0.5 0.6 0.68 ± 0.16

4.1 2.6 0.2 0.2 0.23 ± 0.16

3.4 2.1 0.1 0.1 0.14 ± 0.05

2.8 1.6 0.1 0.0 0.08 ± 0.04

3.5 2.1 0.1 0.2 0.15 ± 0.06

3.1 1.8 0.1 0.1 0.08 ± 0.05

2.8 1.6 0.0 0.0 0.05 ± 0.4

2.4 1.4 0.0 0.0 0.03 ± 0.03

Reference Basketter, D.A., Evans, P., Fielder, R.J., Gerberick, G.F., Dearman, R.J., Kimber, I., 2002. Food Chem. Toxicol. 40, 593–598. Keywords: Contact allergy; Local lymph node assay; Flow cytometry; Proliferation Inter-Laboratory Comparisons of the Allergenic Potential of Proteins: Studies in Mice R.J. Dearman1 , R. Skinner1 , C Herouet2 , E Debruyne2 and I Kimber1 . 1 Syngenta

Central Toxicology Laboratory, Macclesfield, UK; 2 Bayer CropScience, Sophia Antipolis, France

E-mail address: [email protected] There is a growing interest in the development and application of methods for the evaluation of the allergenic potential of novel proteins. One approach that shows some promise is the measurement of specific IgE antibody production stimulated following systemic (intraperitoneal; i.p.) exposure of BALB/c strain mice. Inherent sensitizing potential is measured as a function of induced IgE responses under conditions where proteins are immunogenic (provoke vigorous IgG antibody responses). Inter-laboratory comparisons have been performed of the ability of two food proteins, of differing allergenic potential, to induce IgE and IgG antibody. Female BALB/c strain mice were exposed to 2% ovalbumin (OVA), a major allergenic constituent of hens’ egg, or to a protein considered to lack significant allergenicity, potato agglutinin (5%). Mice (n = 5) received an i.p. injection of protein (0.25 ml) on days 0 and 7, with serum samples prepared for analysis 7 days later. Specific IgE antibody was measured by homologous passive cutaneous anaphylaxis assay (PCA) and specific IgG antibody production was analyzed by enzyme-linked immunosorbant assay. Two experiments were conducted in each laboratory. The number of IgE responder animals was determined using individual (undiluted) serum samples and the titre derived by analysis of serial doubling dilutions of serum samples pooled by treatment group for each experiment. Serum samples were analyzed for IgG titre using doubling dilutions of serum; the number of IgG responders and the mean IgG titre (log2 ) is displayed for each experiment. Table 1 IgG and IgE antibody responses stimulated by OVA and potato lectin (PL) IgG

IgE

Responders

Bayer experiment 1 Bayer experiment 2 CTL experiment 1 CTL experiment 2

Reciprocal titre (log2 )

Responders

OVA

PL

OVA

PL

OVA

PL

OVA

PL

4/4 5/5 5/5 5/5

4/4 5/5 5/5 5/5

10 10 12.4 10.2

10.5 10.5 12 10.6

4/4 5/5 4/5 5/5

3/4 4/5 3/5 2/5

8 16 32 32

1 1 −ne −ne

−ve: negative PCA with undiluted serum.

Reciprocal titre

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In each experiment conducted in both laboratories, vigorous IgG responses were induced by OVA and potato lectin and there were no significant differences in IgG titres either between proteins or between laboratories (Students’ t-test). Under these conditions where OVA and potato lectin were of equivalent immunogenicity, marked differences in their ability to stimulate specific IgE antibody responses were observed. These data demonstrate that the induction of IgE antibody by food proteins of differing allergenic potential is a relatively robust phenomenon and transferable between laboratories. Furthermore, these results suggest that the measurement of antibody (IgE) responses in BALB/c mice may allow discrimination between allergens and those materials that apparently lack allergenicity. Keywords: Food allergy; IgG; IgE Peanut Lectin and Purified Protein Derivative (PPD) Provoke Divergent Cytokine Responses in C57BL/6 Mice 1 Catherine

J. Betts, 1 Helen T. Caddick, 2 Brian F. Flanagan, 1 Rebecca J. Dearman, 1 Ian Kimber

1 Syngenta

Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK; 2 Department of Immunology, Duncan Building, University of Liverpool, Liverpool L69 3GA, UK E-mail address: [email protected] In the United Kingdom, peanuts are an important cause of food-related anaphylactic reactions. Allergic sensitisation is associated usually with high titre IgE antibody, consistent with the preferential activation of T helper (Th) 2 type cells. We have characterised cytokine secretion profiles induced by exposure of C57BL/6 strain mice to purified peanut lectin, an allergenic constituent of peanuts. Groups of mice (n = 10) received an intradermal injection of 30 ␮l peanut lectin (1 mg/ml) or purified protein derivative (PPD, 0.5 mg/ml), a non-allergenic mycobacterial extract that is associated with a Th1 response in both mice and humans, in the dorsum of both ears on days 0 and 7. Fourteen days following initiation of exposure the draining auricular lymph nodes were excised and a single cell suspension prepared by mechanical disaggregation through sterile gauze. Lymph node cells (LNC) were cultured in the presence or absence of peanut lectin (200 ␮g/ml), PPD (25 ␮g/ml), the T cell mitogen concanavalin A (con A, 2 ␮g/ml), or medium alone for 24 to 120 h. Cell proliferation was assessed by incorporation of tritiated thymidine over the final 24 of a 72 h culture period. Supernatants were harvested for cytokine protein analysis by cytokine-specific enzyme-linked immunosorbent assays (ELISAs). Restimulation of peanut lectin- or PPD-primed cells with peanut lectin or PPD, respectively, induced marked antigen-specific proliferative responses of similar magnitudes (data not shown). Low level proliferative responses were observed following culture of primed LNC with irrelevant antigen. Culture of both PPD- and peanut lectin-

Table 1 Cytokine protein levels in pg/ml (mean ± sem for three independent experiments) for peanut- and PPD-primed LNC cultured in the presence of peanut lectin, PPD or concanavalin A for 120 h Cytokine

+Peanut lectin Peanut-primed

IFN␥ IL-4 IL-5 IL-10 IL-13

1887 ± 883 66 ± 45 1262 ± 379 1017 ± 491 4695 ± 1705

+PPD PPD-primed 1551 ± 1051 11 ± 1 22 ± 8 214 ± 147 243 ± 146

Peanut-primed 8387 ± 1653 10 ± 1 17 ± 4 299 ± 119 314 ± 171

+Con A PPD-primed 3345∗∗

30167 ± 14 ± 1 279 ± 84 6240 ± 1431∗∗ 739 ± 258

Peanut-primed

PPD-primed

4359 ± 1499 11 ± 1 180 ± 64 269 ± 13 701 ± 385

3507 ± 1133 12 ± 2 123 ± 20 236 ± 94 584 ± 246

Statistical analyses between groups were conducted using two sided Student’s t-test and significance of P ≤ 0.01 is denoted by ‘∗∗ ’.

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255

primed LNC with con A induced vigorous proliferative responses. Restimulation of cells from peanut-primed mice with peanut lectin or PPD induced similar levels of the type 1 cytokine interferon ␥ (IFN␥), whereas higher levels of the type 2 cytokines interleukin (IL)-4, IL-5, IL-10 and IL-13 were recorded following antigenic-specific restimulation, although these values did not reach statistical significance. In contrast, cells from PPD exposed mice restimulated with PPD exhibited a more Th1-like profile with significantly higher levels of IFN␥ production compared with PPD-primed LNC cultured in the presence of irrelevant antigen (peanut lectin). There were no significant differences in the cytokine expression profiles observed following culture of peanut lectin- or PPD-primed LNC in the presence of con A. Culture of LNC from both treatment groups with medium alone did not induce detectable levels of cytokine secretion. Interestingly, IL-10 protein secretion was associated with IFN␥ production in these studies. IL-10 is regarded as a Th2 cytokine in most mouse strains, however, in C57BL/6 strain mice expression appears to reflect observations in human T cell clones where IL-10 can associate with either Type 1 or Type 2 responses. These data indicate that systemic exposure of mice to purified peanut allergen stimulates a more Th2-like cytokine protein secretion profile when compared with an immunogenic protein that is regarded as being non-allergenic. Keywords: Peanut allergen; Cytokine expression; T helper cell subsets Diagnosis of Anticonvulsant Hypersensitivity Syndrome Using An In Vitro Lymphocyte Proliferation Assay John Farrell, Dean J. Naisbitt, Munir Pirmohamed, B. Kevin Park Department of Pharmacology, The University of Liverpool, Sherrington Building, Ashton St., Liverpool L69 3GE, UK E-mail address: [email protected] Anticonvulsant hypersensitivity syndrome is a cause of patient morbidity and mortality and is characterised by skin rash, eosinophilia and development of systemic symptoms. Skin rashes occur in up to 5% of patients, while more severe reactions are less common, with a frequency of ca. 1 in 1000. Laboratory investigations have shown that blood lymphocytes from anticonvulsant hypersensitive patients but not from drug-na¨ıve patients or patients administered the drug without detectable adverse effects proliferate following in vitro drug exposure (Mauri-Hellweg et al., 1995). We have cloned T-cells from carbamazepine and lamotrigine hypersensitive patients (Naisbitt et al. in press). These data characterized T-cells that were phenotypically different to T-cells involved in other serious adverse drug reactions and showed that IFN-␥ secreting, skin homing and cytotoxic CD4+ T-cells mediate anticonvulsant hypersensitivity. Despite this, a reliable in vitro assay for the diagnosis of anticonvulsant hypersensitivity has not been forthcoming. In these studies, we measured in vitro drug-specific proliferation of lymphocytes from 18 hypersensitive patients (13 carbamazepine, 3 lamotrigine and 2 phenytoin—all patients had maculopapular-type skin reactions) and 10 drug exposed healthy controls. Blood lymphocytes (1.5 × 105 ; total volume 0.2ml) were incubated with carbamazepine (1–100 ␮g ml−1 ), lamotrigine (1–100 ␮g ml−1 ), phenytoin (1–100 ␮g ml−1 ) and tetanus toxoid (1 ␮g ml−1 ; positive control) in 96-well plates for 6 days (37 ◦ C; 5% CO2 ). Culture media consisted of RPMI-1640 supplemented with 10% pooled heat-inactivated human AB serum, HEPES buffer (25 mM), l-glutamine (2 mM), transferrin (25 ␮g ml−1 ), streptomycin (100 ␮g ml−1 ) and penicillin (100 U ml−1 ). Proliferation was determined by the addition of [3 H] thymidine (0.5 ␮Ci) for the final 16h of the incubation period. Proliferative responses were calculated as stimulation indices (SI; cpm in drug-treated cultures/cpm in cultures with solvent alone). Lymphocytes from patients hypersensitive to carbamazepine (11/13), lamotrigine (3/3) and phenytoin (2/2) proliferated when stimulated with the suspect drug in vitro (Fig. 1). Lymphocyte proliferation was concentration-dependent (1–50 ␮g ml−1 ); 75 ␮g ml−1 and above inhibited proliferation. No proliferation was seen when lymphocytes were stimulated with anticonvulsants that they had not been previously exposed to or were not exposed to at the time of the

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Fig. 1. Demonstration of drug-specific proliferation of lymphocytes from anticonvulsant hypersensitive patients.

reaction (i.e., lamotrigine and phenytoin in carbamazepine hypersensitive patients and vice versa; SI < 2). Control patient lymphocytes did not proliferate in the presence of carbamazepine, Lamotrigine or phenytoin. Hypersensitive patient and control patient lymphocytes proliferated following in vitro stimulation with tetanus toxoid (range SI 5.1–48.6). Our study shows that measurement of in vitro drug-specific lymphocyte proliferation is a highly specific and sensitive assay that can be used to diagnose anticonvulsant hypersensitivity syndrome in patients with maculopapular-type skin reactions. References Mauri-Hellweg, D., Bettens, F., Mauri, D., Brander, C., Hunziker, T., Pichler, W.J., 1995. J. Immunol. 155, 462–472. Naisbitt, D.J., Britschgi, M., Wong, G., Farrell, J., Depta, J.P.H., Chadwick, D.W., Pichler, W.J., Pirmohamed, M., Park, B.K., in press. Mol. Pharmacol. Naisbitt, D.J., Farrell, J., Wong, G., Depta, J.P.H., Dodd, C.C., Hopkins, J.E., Gibney, C.A., Chadwick, D.W., Pichler, W.J. Pirmohamed, M., Park, B.K., in press. J. Allergy Clin. Immunol. Keywords: Anticonvulsant hypersensitivity; Lymphocyte proliferation; T-cells Development of a Method for the Analysis of the Rat Spleen by Flow Cytometry Using CD45-gating to Avoid the Need for Erythrocyte Lysis Emma L. Hunter, David F. Lanham, Jo M. Bidgood, Mark G. Wing Experimental Biology Department, Huntingdon Life Sciences Ltd., Huntingdon, Cambridgeshire PE28 4HS, UK There is an increasing regulatory requirement to incorporate immunotoxicology endpoints into toxicology studies. Flow cytometry is commonly requested on such studies to enumerate changes in lymphocyte numbers in the peripheral blood or secondary lymphoid tissues such as the spleen. To improve the accuracy of analysis, a CD45 gating method was developed to distinguish the white from the red blood cells thereby avoiding the need to lyse the erythrocytes prior to analysis, a process associated with significant cell losses and lymphocyte gate contamination if the erythrocyte lysis is incomplete. A single cell suspension of splenocytes was prepared and two antibody tubes set up for each animal and stained with a four-colour combination of commercial antibodies conjugated to different fluorochrome dyes, to allow detection and discrimination of the different cell lineages. The four fluorochromes used were fluorescein isothiocyanate (FITC), phycoerythrin (PE), Energy Coupled Dye (ECD) and Cy-chromeTM (Cy). Tube 1 contained CD3-PE/CD45RA-Cy/NKR-P1-FITC to detect total T lymphocytes, total B lymphocytes and Natural Killer (NK) cells, respectively. Tube 2 contained CD3-PE/CD4-Cy Chr/CD8b-FITC to differentiate the T lymphocyte subsets. Biotinylated anti-CD45 antibody was added to both tubes which was visualised using streptavidin-ECD. The sam-

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257

Table 1 Cell counts CD3+ T lymphocytes

CD45RA+ B lymphocytes

a Percent

b Cells/spleen

a Percent

b Cells/spleen

Han Wistarc Male Female

44.4 ± 6.6 45.4 ± 6.6

0.518 ± 0.17 0.425 ± 0.19

43.8 ± 6.5 42.6 ± 7.6

0.508 ± 0.19 0.395 ± 0.18

CDd Male Female

44.1 ± 5.1 48.1 ± 5.0

1.03 ± 0.45 0.83 ± 0.33

47.0 ± 5.6 43.5 ± 6.4

1.111 ± 0.54 0.742 ± 0.33

a Mean

± S.D., b mean × 108 ± S.D., c n = 55, d n = 30.

ples were then analysed using a CD45 primary acquisition gate to identify the white blood cells followed by a forward and side scatter gate to remove any remaining erythrocyte contamination. Analysis of the lineage specific markers was then used to distinguish the lymphocyte subsets. Using a CD45-gate it was possible to distinguish the white from the red blood cells, with the remaining erythrocyte contamination removed using a forward and side scatter gate. The white blood cells were then distinguished by the expression of lineage specific markers, revealing approximately equal numbers of B and T lymphocytes in the spleen, with a CD4:CD8 ration of ∼2:1 and 3–4% NK cells in both the Han Wistar and CD rats. The female absolute counts were not surprisingly lower than the males, with the outbreed CD rats having more splenocytes than the Han Wistar. Of note was the fact that the absolute number of T and B lymphocytes varied considerably between individuals in both rat strains, where as the ratio of these cell types was rather more consistent. This observation of higher absolute count variability was supported by the fact that the weight of the spleen also displays high variability having a CV (coefficient of variance) of between 25 and 40% compared for example with the CV of the brain and liver which is between 5 and 15%. Given the inherent inter-animal variability of the immune system due to genetic and environmental factors, any methodology which reduces data variability will increase the likelihood of detecting toxicological changes to the immune system. Principal Components Time Trend Analysis of Organochlorine Contaminants Detected in Omega-3 Rich Fish and Vegetable Oil Dietary Supplements from UK outlets Miriam N. Jacobs School of Biomedical and Life Sciences, University of Surrey, Guildford, GU2 7XH, UK; Department of Pathology & Infectious Diseases, Royal Veterinary College, Hawkeshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UK E-mail address: [email protected], [email protected] Selected organochlorine (OC) contaminant levels have been reported in two sets of omega-3 rich fish and vegetable oil dietary supplements (n = 22; old samples, n = 21; recent samples). The majority obtained from the same outlets and suppliers in late 1994/early 1995 (Jacobs et al., 1997, 1998) and late 2001/early 2002 (Jacobs et al., 2002). The 8-year time trend differences in selected organochlorine contaminant level data sets and patterns for these dietary supplements have been examined using principal component analysis (PCA) (SIMCA-P version 10.0). As the datasets are multivariate (13 measured PCBs and organochlorine contaminants) and there is a large variation within some of the variables.

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Abstracts / Toxicology 194 (2004) 197–272 Time trend analysis: OC contaminants in omega 3 rich supplements, Primary variables plot (PCA-X) R2=estimate of goodness of fit Q2=estimate of goodness of prediction

R2VX[3](cum) Q2VX[3](cum)

1.00 0.80 0.60 0.40 0.20 0.00 -0.20 PC B 52

PC B 10 1

PC B 11 8

PC B 12 8

PC B 13 8

PC B 14 9

PC B 15 3

PC B 17 0

PC B 18 0

HC B

aHC H

gHC H

p,p' DD E

p,p' DD T

Var ID (Primary)

OC contaminant data for omega-3 rich dietary oils, reported in Jacobs et al. (1997, 2002) were as follows: polychlorinated biphenyls (PCBs) 52, 101, 118, 138, 149, 153, 170, 180, HCB, alpha-HCH, gamma-HCH, p,p
DDE and p,p
DDT. Sample descriptions have been previously reported, and while the samples cover the leading brands available on the London/UK market, both studies were not comprehensive surveys of all brands available. The data were converted to ng/g where necessary. A 3 component PCA model was constructed (R2 X = 0.76, Q2 = 0.44), the primary variables plot is shown above. While the recent oil samples, particularly the fish oils showed less contamination compared with past reports, the cod liver oils had the greatest levels of contaminants compared to the other supplement groups. Fish and vegetable oil mixtures had lower levels than the whole fish body oils, while no PCBs were detected in the vegetable oils as observed previously, but HCHs were detected (plots not shown). With most PCBs detected, a clear reduction in PCB contaminants were observed, but still at similar magnitudes, except for PCB 52 (a high detection limit of 18 ␮g/l for the old samples) and the pesticides HCHs and DDT, which were dissimilar in pattern. A part explanation for the comparative increase in DDT in the recent samples may be the poor recoveries for DDT for the old samples (Jacobs et al., 1997). The difference in the HCH patterns may be partly due to the greater proportion of vegetable oils analysed in the recent study, which were not subjected to the same treatment processes as observed in the fish oils. In terms of risk assessment, the potential contribution to the human diet of OC contaminants from omega-3 fatty acid dietary supplements still increases if sourced from fish, particularly cod liver, and further investigation of contaminants such as DDT is needed. References Jacobs, M.N., Johnston, P.A., Wyatt, C.L., Santillo, D., French, M., 1997. Int. J. Environ. Pollut. 8, 74–93. Jacobs, M.N., Johnston, P.A., Santillo, D., Wyatt, C.L., 1998. Chemosphere 37, 1709–1731. Jacobs, M.N., Covaci, A., Gheorge, A., Schepens, P., 2002. Organohalogen Compd. 57, 225–228. Keywords: Risk assessment; Fish oils; Omega-3 rich oils; Time trends

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Recombinant Expression of the Ah Receptor Ligand Binding Domain Tao Jiang, David R. Bell School of Life and Environmental Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK E-mail address: [email protected] Polycyclic aromatic compounds and dioxins are present as environmental toxicants in the atmosphere, soil, water and food, of which, TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is the most potent congener. TCDD can cause toxicity in animals, including tumour promotion, embryotoxicity/teratogenesis and a severe wasting syndrome followed ultimately by death (Schmidt and Bradfield, 1996; Gu et al., 2000). The toxic effects of TCDD are mediated by binding to a cytosolic protein, aryl hydrocarbon receptor (AhR). When AhR binds the ligand, it initiates the dioxin-signalling pathway through transcriptional activation. Cytochromes P450 1A1, 1A2 and 1B1, and many phase II detoxification genes are induced through this pathway (Gu et al., 2000). The functional AhR requires molecular chaperones, such as Hsp90, p23 and AhR-interacting protein (Bell and Poland, 2000). However, the mechanisms controlling production of the functional, ligand-binding AhR have yet to be determined. Therefore, defining the structural basis of the interactions between ligands, both agonists and antagonists, and the AhR ligand binding domain (LBD) is essential for understanding mechanisms of toxicity. In the effort to facilitate the structural and biochemical analysis of the functional AhR ligand binding domain, we have recently succeeded in subcloning four distinct constructs of the murine AhR LBD, containing amino acids 228–416, into pFASTBAC transfer vector. These four deletion constructs contain amino acids 228–416; 265–416; 285–416 and 285–410 amino acids of the murine AhR LBD, respectively. The recombinant virus was generated by site-specific transposition with Tn7 to insert foreign genes into bacmid DNA. To express the AhR protein, Sf9 cells were infected with high titer recombinant baculovirus ((1–7) × 108 pfu/ml). The soluble form of AhR protein was recovered from the cytosol of baculovirus-infected cell extract, and shown to be present by Western blotting. The data show that high level expression of cytosolic AhR LBD was achieved (>0.1% of cytosolic protein), and the protein specifically binds to the ligand TCDD. The specific binding of recombinant AhR construct for ligand TCDD determined is 116 f mols/mg cytosol protein. These constructs may be useful for structural and binding studies in the future. References Bell, D.R., Poland, A., 2000. J. Biol. Chem. 275 (46), 36407–36414. Gu, Y., Hogenesch, J.B., Bradford, C.A., 2000. Annu. Rev. Pharmacol. Toxicol. 40, 519–561. Schmidt, J.V., Bradford, C.A., 1996. Annu. Rev. Cell Dev. Biol. 12, 55–89. Keywords: Ah Receptor; TCDD; Ligand binding domain; Expression Initial Characterisation of the Control Gene Expression of the Human Pregnane X-Receptor Sihem Aoubadi, G. Gordon Gibson, Nick Plant School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK E-mail address: [email protected] The pregnane-X-receptor (PXR) has been identified in a number of species including man, and has emerged as a major controller of a number of biologically important enzymes including CYP3A4, CYP2B6, GST-A2 as well as being implicated in bile acid homeostasis. PXR gene expression has been shown to be regulated by several different xenobiotics in both primary human hepatocytes (dexamethasone) and rats (clofibrate, perfuorodecanoic acid, isoniazid and troleandomycin), although the molecular mechanisms underlying this activation has not yet been elucidated.

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Table 1 Induction of reporter gene expression Xenobiotic

Induction (relative to control)

Rifampicin Dexamethasone Hydrocortisone

4.8 2.1 2.2

To begin to understand control of PXR gene expression we have used bioinformatic analysis to predict potential binding sites within the PXR proximal promoter. A computer search of 2000 bp of DNA immediately upstream of the PXR transcription start site was carried out, using MatInspector Professional to interrogate the Transfac 4.0 database (http://transfac.gbf.de/TRANSFAC/). Putative binding sites for numerous transcription factors were identified. These include both general transcription factors (e.g. Oct1, C/EBP␣, COUPTF, HNF1, 3, 4 and CCAAT) and ligand-activated receptors already implicated in regulation of xenobiotic-inducible gene expression (e.g. progesterone receptor, Vitamin D receptor, PPAR␣ and barbitone response (Barbie) box). To further investigate the control of PXR gene expression in vitro we have constructed vectors containing 1000 bp of PXR proximal promoter linked to a secretory alkaline phosphatase reporter gene (pSEAP Basic; Clontech). HuH7 cells were transiently transfected with these constructs, or control plasmids lacking the insert and exposed to 10 ␮M rifampicin, 10 ␮M dexamethasone or 10 ␮M hydrocortisone for 48 h. Reporter gene expression was significantly increased, relative to solvent control in all cases (Table 1), confirming the ability of PXR gene expression to be regulated by a number of chemically distinct xenobiotics. In summary, we have used both in silico and in vitro approaches to characterise potential molecular mechanisms underlying control of PXR gene expression. Computer analysis suggests that the PXR proximal promoter is highly complex, containing binding sites for several ligand-activated transcription factors. This complexity probably underpins the ability of PXR gene expression to be activated by a wide variety of xenobiotics, as shown by the reporter gene assay experiments. Further experiments are now required to conclusively link specific transcription factor binding sites and the activation of PXR gene expression by xenobiotics. Developing Chemiluminescent Assays for Gene Expression Profiling Ceri Morris1 , Kirsty Roberts1,2 , Rhian Morgan1 , Ian Weeks1 , Stuart Woodhead1 1 Molecular

Light Technology Research Ltd., Cardiff Industrial Park, Cardiff, UK; 2 School of Biosciences, Cardiff University, P.O. Box 911, Cardiff E-mail address: [email protected] There is an increasing interest in the use of gene transcription as a means of investigating mechanisms involved in either adaptive or adverse effects. We have developed a novel approach based on the hybridisation protection assay (HPA) (Arnold et al., 1989; Thomas-Jones et al., 2003). The HPA assay consists of two steps; the probe labelled with acridinium ester (AE) is hybridised to its target and then addition of the alkaline reagent causes preferential hydrolysis of unhybridised probe. The AE molecule reacts rapidly with hydrogen peroxide under alkaline conditions to produce light at 430 nm—this is the basis of the chemiluminescent reaction. The intensity of emitted light is a function of the amount of target present. As little as 0.1 fmol of mRNA can be accurately quantified by this method. We have used this technique to develop chemiluminescent assays to measure the expression of cytochrome p450 isoforms CYP1A1 and CYP1A2. Isoform specific probes were designed against the rat sequences and labelled with acridinium ester. The characteristics of the probes were analysed using a complementary oligonucleotide target. The differential rate of hydrolysis of the bound and unbound probe at 60 ◦ C was characterised over a range of time points. In the absence of target, the probe was degraded rapidly allowing a low background to the assay whereas in the presence of target, the probe was protected from hydrolysis. Following measurement of chemiluminescence

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at each time point, an optimum time (15 min) was selected that gave the greatest range of chemiluminescence intensity between hybridised and unhybridised states. To investigate the dynamic range of the assay, standard curves with oligonucleotide target (0.01–100 fmol) were performed with different concentrations of probe (0.1, 0.075 and 0.05 pmol). The optimal amount of CYP1A1 and 1A2 probe was determined (0.1 pmol) that gave a linear response between 0.01 and 100 fmol target (R2 = 0.988 and 0.987). An HPA assay for ␤-actin mRNA, an invariant housekeeping gene, was also developed to act as an internal control for RNA quantity and quality. The HPA is a sensitive assay for measurement of CYP1A1/1A2 mRNA transcripts. To validate the system, it will be used to investigate the time and dose dependent responses of primary hepatocytes and cell lines exposed to known CYP1A inducers. References Arnold, L.J., Hammond, P.W., Wiese, W.A., Nelson, N.C., 1989. Clin. Chem. 35, 1588–1594. Thomas-Jones, E., Walkley, N., Morris, C., Kille, P., Cryer, J., Weeks, I., Woodhead, J.S., 2003. Environ. Toxicol. Chem. 22 (5), in press. Keywords: Chemiluminescence; Hybridisation protection assay; Cytochrome p450 Metabolism of Chlorpyrifos by Human Liver Microsomes Craig Sams1 , Martin S. Lennard 2 1 Health

and Safety Laboratory, Broad Lane, Sheffield S3 7HQ, UK; 2 Academic Unit of Molecular Pharmacology and Pharmacogenetics, University of Sheffield, Sheffield S10 2JF, UK E-mail address: [email protected]

Chlorpyrifos (CPS) is an extensively used organophosphate insecticide (OP). Its established toxic effects arise from bioactivation to the potent acetylcholinesterase inhibitor chlorpyrifos-oxon (CPO). The individual cytochromes P450 (CYP) mediating this bioactivation have been recently identified (Tang et al., 2001). Variation in rates of bioactivation may help to explain why some individuals appear particularly sensitive to the toxic effects of CPS, and OPs in general. Therefore, the kinetics of CPS bioactivation to CPO and detoxification to trichloropyridinol (TCP) have been determined in vitro using liver microsomes from individual human donors. The role of individual CYPs in this reaction was also investigated using chemical inhibitors and cDNA-expressed CYPs. Incubations of CPS with liver microsomes (HLM) from individual human donors (n = 5) and pooled HLM (21 mixed gender donors) were conducted in duplicate under conditions of linearity over a range of concentrations (3–100 ␮M). The metabolites CPO and TCP were quantified by HPLC and kinetic constants were determined by conventional techniques. The role of individual CYPs in CPS metabolism was investigated using the selective inhibitors furafylline, sulfaphenazole, ketoconazole, quinidine, and diethyldithiocarbamate. Metabolite production by individual cDNA-expressed CYPs was quantified at 12.5 and 100 ␮M CPS. Linear Eadie–Hofstee plots were obtained for CPO and TCP production by HLM. Kinetic constants are presented in Table 1. None of the selective inhibitors greatly inhibited metabolism of 20 ␮M CPS by pooled HLM. Ketoconazole (1 ␮M) was the most potent inhibitor, causing 27% and 24% decreases in CPO and TCP production, respectively. Recombinant CYP2B6 was at least 2-fold more active in CPO production than other CYPs, whereas CYP2C19 was over five-fold more active in TCP production than any other isoform. The kinetics of CPS bioactivation to CPO and detoxification to TCP by HLM presented here are similar to those published by Tang et al. (2001). Metabolic intrinsic clearance values (Vmax /Km ) indicate that TCP is more readily produced by HLM than CPO, suggesting that CPS is more readily detoxified than bioactivated. The present study has found that CYP2B6 is the major CYP involved in CPS bioactivation to CPO, and that CYP2C19 is the major CYP in its detoxification to TCP, findings which are in agreement with those of Tang et al. (2001). The activities of both of these CYPs are highly variable in humans, and moreover CYP2C19 exhibits an important polymorphism.

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Table 1 Kinetic data for metabolism of chlorpyrifos by HLM CPO production

Mean Range S.D. Pooled HLM

TCP production

Km (␮M)

Vmax (pmol/min mg)

Km (␮M)

Vmax (pmol/min mg)

29 16–52 15 16

342 255–399 62 379

12 5–17 5 8

653 507–813 123 367

Hence, individuals possessing the CYP2C19 poor metaboliser phenotype may be less able to detoxify CPS, and thus could be more susceptible to its toxic effects. Reference Tang, J., Cao, Y., Rose, R.L., Brimfield, A.A., Dai, D., Goldstein, J.A., Hodgson, E., 2001. Drug Metab. Dispos. 29, 1201–1204. Keywords: Cytochrome P450; Chlorpyrifos; Human liver microsomes; Variation Differential Gene Expression Of DNA Repair Genes in Response to DNA Damaging Agents Catherine C. Smith1 , Anthony M. Lynch2 , Nigel J. Gooderham1 1 Molecular Toxicology, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London SW7

2AZ, UK; 2 GlaxoSmithKline, Genetic Toxicology, Ware, Hertfordshire SG12 0DP, UK E-mail address: [email protected] The differential expression of a number of DNA repair genes was examined to identify potential biomarkers for DNA damage. The selection of DNA repair genes was based on the hypothesis that DNA damage, including strand breaks and insertional mutagenesis would be expected to induce biological pathways associated with the repair. We have used human HepG2 cells exposed to chemical DNA-damaging agents as positive controls (etoposide and methylmethansulphonate—MMS) and also biological agents (exogenous DNA). Whilst there is a considerable literature regarding chemical-induced DNA damage, the ability of exogenous DNA to damage DNA is often overlooked. For the latter, cells were transfected with pL3112BSKS, containing a GFP reporter gene, using non-viral transfection facilitators (TFs) Effectene or ExGen 500 and viral vector TFs, retrovirus or adeno-associated virus (AAV). Transient transfection efficiencies, determined by flow cytometry, were 35–45% with non-viral TFs and 58–85% with viral TFs. Following transfection (6–72 h), the cells were harvested for RNA and differential gene expression was determined by quantitative real-time PCR (qRT-PCR). The threshold for significant gene expression changes was set at the 99% confidence interval and calculated using ANOVA for each gene. The expression of genes involved in the repair of double strand breaks (DSB) were significantly increased after MMS (3–5-fold) or Etoposide (3–4-fold) treatment. These results are concordant with the DNA damaging ability of these chemicals and subsequent DNA repair via homologous recombination. In parallel to these studies a known genotoxic endpoint, the in vitro micronucleus assay was performed and showed a significant 4-fold (P < 0.01) induction of micronuclei following treatment with MMS (50 ␮g/ml) for 24 h. Using the non-DNA integrating, non-viral transfection of pL3112BSKS with Effectene and ExGen 500 showed no significant change in the expression of DSB repair genes. However, transduction with viral vectors potently increased DSB repair gene expression (3–11-fold) (Table 1). Increase in genes associated with DSB repair following treatment with Retrovirus is concordant with the level of GFP expression (Table 1). Retrovirus is an integrating vector only capable of expressing reporter and transgenes upon insertion into a cell.

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Table 1 Fold changes in gene expression following transduction of HepG2 cells with retrovirus Time (h)

Transduction efficiency (%) REV3L gene RAD50 gene RAD52 gene ∗ Significant

6

24

48

72

24

24

0.6 ± 0.5 1.0 ± 0.1 1.6 ± 0.1 1.9 ± 0.1

11.3 ± 1.3 3.6 ± 0.2∗ 2.6 ± 0.1∗ 5.7 ± 0.2∗

36.8 ± 1.6 3.2 ± 0.1∗ 2.5 ± 0.1∗ 3.2 ± 0.1∗

57.0 ± 1.6 −1.9 ± 0.1 1.2 ± 0.1 −1.8 ± 0.1

MMS (50 ug/ml) 4.4 ± 1.6∗ 1.9 ± 1.2 5.3 ± 1.9∗

Etoposide (50 ng/ml) 2.1 ± 0.6 2.3 ± 0.6 4.1 ± 0.8∗

fold changes (P < 0.01); values are mean ± S.E.M. for triplicate determinations of at least three separate experiments.

Our data confirm proof of principle for the approach and provide the basis for a sensitive and novel method to rapidly detect DNA damage at the level of the genome rather than at a selected gene. In Vitro Assessment of the Ocular Irritancy Potential of Isothiazolinone-based Preservatives Using the BCOP Assay Colin Smith1, Bruce Alexander2 1 Thor

Specialities (UK) Ltd., Wincham Avenue, Northwich, Cheshire CW9 6GB, UK; 2 Thor Group Management, Ramsgate Road, Margate, Kent CT9 4JY, UK E-mail address: [email protected]

Isothiazolinone-based preservative systems are used to prevent microbiological spoilage in a wide range of consumer products and are effective at much lower concentrations than several other preservative systems. A common problem associated with the use of products containing 2-methyl-5-chloroisothiazolin-3-one (CIT) is their potential for causing allergic contact dermatitis. Other isothiazolinones (2-methyl-isothiazolin-3-one, MIT, and 1,2-benzisothiazolin-3-one, BIT) are now under consideration for regulatory acceptance within the EU. These have a much-reduced contact sensitisation potential, (Alexander, 2002) however several in vitro toxicological endpoints such as eye irritation potential have not previously been determined. The BCOP assay (INVITOX Method 124) has been used to compare the ocular irritancy potential of CIT/MIT, MIT and MIT/BIT formulations. These have been tested at in-use concentrations, 100 x in-use, and neat concentrations. Bovine corneas were excised and mounted in corneal holders, containing MEM + 1% FCS (cMEM). After a 1 h period of equilibration at 32 ◦ C the corneas were washed with cMEM and initial opacity measurements taken using an opacitometer (StagBIO). The anterior surface of the cornea was then exposed for 10 min to either a control solution of 0.9% NaCl, absolute ethanol, or dilutions of the test compound (n = 3 or 4 per treatment). The corneas were rinsed and incubated in cMEM for a further 2 h. The final opacity readings were then taken. Corneal permeability was assessed by applying 1 ml of fluorescein solution in cMEM (4 mg/ml) to the anterior surface of the cornea and incubating for a further 1.5 h. 200 ␮l samples were removed from the posterior compartments into a microplate and the fluorescein concentration was estimated at 490nm against cMEM blanks using a Molecular Devices Vmax platereader. The absorbance values obtained were multiplied by a correction factor of 1.65 to give an equivalent absorbance to a 1 cm pathlength. The in vitro score (IVS) was calculated from the opacity and absorbance measurements as follows: Corrected corneal opacity = test opacity (t120 − t0) − mean (negative control opacity (t120 − t0)) Corrected OD490 = test OD490 value − mean (negative control OD490) In vitro score = corrected opacity value + (15 × corrected OD490 value) The prediction model established by Gautheron et al. (1992) was used to place the formulations on an in vitro irritation scale according to their scores and to predict a potential Draize classification. The mean IVS for the positive

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control (ethanol, 50.4 ± 7.5, n = 36) fell within the limits set for a valid test according to the INVITOX protocol. The prediction model classifies ethanol as a moderate, tending to marked, irritant. Only the neat test formulations had a mean IVS greater than 3, the threshold score for irritation. The relative ranking of potential eye irritation for the neat formulations was: MIT/BIT (21.8±3.2, n = 9) > CIT/MIT (16.8±7.3, n = 9)>MIT (9.3 ± 5.3, n = 7). According to the prediction model, all isothiazolinone formulations tested are all mild eye irritants, with an equivalent Draize classification of minimal/slight. The results obtained for dilutions of CIT/MIT are in accordance with results of previous work carried out using the Draize test (Anon, 1992), which found that concentrations of 560 ppm were non-irritant. However, the neat formulation of CIT/MIT, which was predicted to be a minimal/slight irritant from the IVS was found to be corrosive by the Draize method. There is no evidence in the literature that the other formulations tested here, MIT/BIT and MIT, have been assessed using the Draize method. From the prediction model, none of the formulations will cause eye irritation at “in use” concentrations, however caution should be exercised when using the prediction model to assess the severity of a positive irritant reaction, given the disparities between the data described here and the Draize results published elsewhere. References Alexander, B.R., 2002. Contact Dermat. 46 (4), 191–196. Gautheron, P., Dukic, M., Alix, D., Sina, J.F., 1992. Fundam. Appl. Toxicol. 18, 442–449. Anon., 1992. J. Am. Coll. Toxicol. 11 (1), 75–128. The Role of Nerve Growth Factor in the Development of Adenomyosis in CD-1 mice Caused by Tamoxifen Andrew, R. Green1 , Jerry A. Styles1 , Emma Parrott1 , Suzanne Johnson1 , Richard E. Edwards2 , Andrew G. Smith2 , Timothy W. Gant2 , Peter Greaves2 , Ian N.H. White1 1 MRC

Molecular Endocrinology Group, Department of Obstetrics & Gynaecology, Robert Kilpatrick Building, University of Leicester, Leicester LE2 7LX, UK; 2 MRC Toxicology Unit, University of Leicester, Leicester LE1 9HN, UK E-mail address: [email protected] Adenomyosis is a common uterine disorder of women where there is the haphazard development of endometrial glands and stroma in regions of the myometrium. We showed in a mouse model, the early onset of adenomyosis Table 1 Genes over-expressed in mouse uterus after neonatal tamoxifen treatment (fold change:log ratio) Gene

Actin, beta, cytoplasmic Actin, gamma, cytoplasmic Alcohol dehydrogenase 4 (class II), pi polypeptide Annexin 6 Brain acyl-coA hydrolase Procollagen, type I, alpha Procollagen, type II, alpha 1 Myosin heavy chain 11, smooth muscle Nerve growth factor alpha Protein tyrosine phosphatase, receptor type, C Ribosomal protein L9 Ribosomal protein S29 Transforming growth factor, beta induced, 68 kDa

Time (months) 1.5

3

6

9

12

0.61 0.75 0.81 1.17 0.64 1.09 1.08 0.78 0.86 0.60 0.72 0.62 0.59

1.98 2.10 1.98 1.83 2.77 1.83 1.97 2.07 2.92 1.63 1.59 1.54 2.46

0.89 0.62 0.66 1.13 1.04 1.13 1.06 1.04 0.53 0.79 0.59 0.67 1.06

1.16 0.63 0.86 1.52 1.26 0.97 1.10 0.56 1.34 0.68 1.19 0.72 1.24

1.02 0.88 1.23 0.91 0.78 0.83 0.99 0.92 2.41 1.58 0.81 1.36 0.93

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following 4 day oral dosing with the antiestrogen tamoxifen in the neonatal period (Parrott et al., 2001). The aim of this study was to establish if pathological changes during the development of adenomyosis over 12 months could be related to changes in uterine gene expression. Newborn pups from CD-1 mice were dosed orally on days 2–5 after birth with 1 mg/kg tamoxifen. Controls received vehicle only. At 1.5, 3, 6, 9 and 12 months after dosing, uterine tissues from 12 animals were either snap frozen in liquid N2 (n = 8) or fixed in neutral buffered formalin (n = 4). Pooled RNA from four groups of two mice was labelled with Cy3 or Cy5 and hybridised using cDNA microarrays containing approximately 4700 mouse ESTs (Gant, 2002). Advanced adenomyosis was characterised by a myometrium that was thickened by bulky, enlarged, disorganised fascicles of smooth muscle with increased amounts of interstitial collagen deposition, particularly in regions penetrated by abnormal endometrial tissue. At 3 months uterine weights expressed as a percentage of body weight were significantly lower than those of controls. (0.37 ± 0.04% versus 0.54 ± 0.05%, mean ± S.E., n = 10, P < 0.05 by ANOVAR for dosed and controls, respectively). At that time there was no overall increase in cell proliferation although Ki-67 labelling was marked in both stroma and glands in zones of adenomyosis. cDNA array data showed the greatest numerical and general magnitude of change in both up and down regulated genes was at 3 months after dosing when about 3400 of the 4500 genes on the microarray showed >3-fold change in expression. There was evidence for tissue remodelling with upregulation of genes involved in proteolysis and protein synthesis. Remodelling did not appear to involve increased apoptosis. Over 12 months, 13 genes were continuously over-expressed by neonatal tamoxifen treatment compared to controls, while none were continuously down regulated. Over expressed genes included those for actin (acta ␥) myosin (myh11) and collagen (col1a1, col2a1), consistent with myometrial reorganisation and collagen deposition, respectively. Nerve growth factor alpha (ngfa) was also in this group. Ngfa and its low affinity p75NTR receptor is involved in the differentiation of mouse C2C12 myocytes (Seidl et al., 1998). The key gene array changes were corroborated by RT-PCR. The data indicate that the constant over expression of ngfa suppresses uterine myometrial differentiation and permits the continued development of adenomyosis in these animals. References Gant, T., 2002. Trends Pharmacol. Sci. 23, 388–393. Parrott, E., Butterworth, M., Green, A., White, I.N.H., Greaves, P., 2001. Am. J. Pathol. 159, 623–630. Seidl, K., Erck, C., Buchberger, A., 1998. J. Cell Physiol. 176, 10–21. Cytochrome P450 3A and Transcription Factor mRNA Expression in HuH7 Cells and Human Liver Anna L. Phillips1 , Steve R. Hood2 , G. Gordon Gibson1 , Nick J. Plant1 1 School

of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK; 2 DMPK, GlaxoSmithKline, Park Road, Ware, Hertfordshire, SG12 0DP E-mail address: [email protected] Cytochrome P450 3A represents the most abundant P450 subfamily in human liver, and is responsible for the metabolism of >50% of drugs on the market today, which undergo oxidative metabolism (Cholerton et al., 1992). An area of intense study has been the xenobiotic regulation of CYP3A gene expression; this phenomenon potentially leads to adverse drug reactions and altered pharmacokinetics/pharmacodynamics of substrates. The use of human hepatoma cell lines, such as HuH7 cells, is common in drug metabolism studies, but detailed information about these cell lines is not available. We determined basal mRNA expression of CYP3A isoforms, in total RNA extracted from HuH7 cells and also from total RNA from human adult and foetal livers, by quantitative RT-PCR. In addition, expression levels of transcription factors believed to be involved in the CYP3A induction mechanism were also determined. Finally, changes in the levels of these transcripts were examined following exposure of HuH7 cells to pregnenolone-16a-carbonitrile (PCN), rifampicin, dexamethasone and phenobarbital. Table 1 shows mRNA expression levels of the CYP3As. HuH7 cells appear to express a foetal phenotype, as levels of CYP3A4 mRNA are significantly lower (P < 0.001) than CYP3A7. In HuH7 cells, expression of

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Table 1 Basal expression levels of CYP3As in various liver cell types Cell type

Adult human liver Foetal human liver HuH7 cells

Copies mRNA/ng total RNA ± S.E.M. CYP3A4

CYP3A5

CYP3A7

CYP3A43

3532 ± 315 20.4 ± 4.0 3.3 ± 0.5

1135 ± 162 827.8 ± 168.1 26.3 ± 3.3

3473 ± 436 34762 ± 1369 24.0 ± 4.7

13.2 ± 0.6 40.4 ± 7.9 4.3 ± 0.7

all CYP3As is significantly lower than in human adult liver. In comparison to human foetal liver, significantly lower levels of all but CYP3A4 are observed in HuH7 cells. Principal component analysis (PCA) of basal expression levels of transcription factors was used to identify the determinants of variance between the cell types. With respect to relative receptor abundance, ratios of PXR to its heterodimerisation partner RXR␣ and also to the transcriptional regulator COUP-TF1, were the major variables, with percentage variability of 38 and 21%, respectively. Following exposure of HuH7 cells to a range of concentrations of xenobiotics, no changes in expression of the enzymes or factors measured were observed, indicating that regulatory pathways within HuH7 cells have been disrupted. PCA analysis indicated that RXR␣:PXR was a major variable for all xenobiotic treatments, and COUP-TF1:PXR was also a major variable for all treatments except rifampicin. This indicates a difference in response between rifampicin treated cells and those exposed to the other xenobiotics, as previously proposed by El-Sankary et al., 2002. Coupled with the lowered expression of PXR in these cells we hypothesise that the interaction of these factors is a key determinant in the non−responsiveness of this cell line to xenobiotics. References Cholerton, S., Daly A.K., Idle, J. 1992. Trends Pharmacol. Sci. 13 (12), 434–439. El-Sankary, W., Bombail, V., Gibson, G., Plant, N., 2002. Drug Metab. Dispos. 30 (9), 1029–1034. Keywords: QRT-PCR, Cytochrome P450 3A; HuH7 cells; Principal component analysis Measurement of DNA Damage from Organophosphate Pesticides Elaine Mutch, Lindsey Ferrie, Christopher Pedder, Faith M. Williams The Toxicology Unit, Environmental Medicine, The Medical School, University of Newcastle, NE2 4HH, UK E-mail address: [email protected] Many chemical carcinogens require metabolic activation to become capable of causing DNA damage. Although the organophosphate pesticides (OPs) diazinon and malathion have well recognised anti-cholinergic effects, it is unknown whether metabolism plays a role in their potential genotoxicity. This study determined DNA damage by diazinon, malathion and it’s oxon, maloxon using the alkaline single cell gel electrophoresis (COMET) assay (Singh et al., 1988). Investigations were carried out in the human hepatoma cell line, HepG2, which are more metabolically competent than human lymphocytes which have minimal metabolic capacity. HepG2 cells (150 × 103 ) or freshly isolated human lymphocytes (150 × 103 ) were incubated with DMSO (2 ␮l, control), diazinon (100 ␮M), malathion (75 ␮M and 200 ␮M) or maloxon (5, 25, 75 and 200 ␮M) for 1 h at 37 ◦ C. In parallel, HepG2 cells which had been pre-incubated for 24 h with ␤-napthoflavone (2 ␮M) to induce CYP1A1/2, were also incubated with diazinon and malathion. Harvested cells were then subjected to COMET and confocal microscopy was used to capture the images of 50 cells per parameter. Komet software determined each tail distributed moment (TDM), a measure of DNA damage. A spectrophotometric assay was used to determine phenylvalerate (1.4 mM) hydrolysis by HepG2 cells, which assessed carboxylesterase activity. Comparisons between OP concentrations and vehicle controls were made by ANOVA.

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Non-induced and induced cells incubated with diazinon had mean TDMs (±S.E.M.) of 27.0±0.90 and 44.9±2.2, respectively. Both parameters were significantly different from control (17.6 ± 1.0; 18.1 ± 0.74, P < 0.01). Conversely, non-induced cells incubated with malathion (75 and 200 ␮M) had mean TDMs of 30.0 ± 2.0 and 26.3 ± 1.8, respectively, which were not different from control (27.0 ± 2.2). However, induced cells incubated with 200 ␮M malathion (31.2 ± 2.0) had significantly more DNA damage than control (22.5 ± 1.8, P < 0.01), but not at the lower (75 ␮M) malathion concentration (26.7 ± 1.6). Although HepG2 cells did not show DNA damage with any maloxon concentration, lymphocytes incubated with the highest maloxon concentration (200 ␮M) had significantly more damage than control (23.1 ± 1.1 and 18.7 ± 0.8, respectively, P < 0.01). Lymphocytes did not show DNA damage at the lower maloxon concentrations. The carboxylesterase activity of HepG2 cells was 323.3 nmol/mg protein/min, which was four-fold greater than previously determined for lymphocytes (79.2 ± 4.0, Mutch et al., 1992). The HepG2 data suggest that metabolism is involved in the genotoxicity of both diazinon and malathion. However, the mechanism(s) underlying the DNA damage may be different for diazinon, since this OP was also genotoxic in non-induced cells. The study also indicates that, although neither malathion or maloxon were genotoxic to non-induced HepG2 cells, maloxon was capable of causing DNA damage to lymphocytes. The high carboxylesterase activity seen in HepG2s compared to lymphocytes would have enhanced removal of the oxon, thus sustaining protection from DNA damage. References Singh, N.P., McCoy, R.R., Tice, R.R., Schneider, E.L., 1988. Exp. Cell Res. 175, 184–191. Mutch, E., Blain, P.G., Williams, F.M., 1992. Hum. Exp. Toxicol. 11, 109–116. Keywords: Organophosphates; Genotoxicity; Human; HepG2 cells; Lymphocytes; Metabolism Haemotoxic Effects of Mitomycin C (MMC) in the CD-1 Mouse After Repeat Dose Administration Gemma Molyneux1,2 , Frances M. Gibson2 , Sue Sulsh3 , Andrew M. Pilling4 , C. Michael Andrews5 , Sian Rizzo2 , Edward C. Gordon-Smith2 , John A. Turton1 1 School

of Pharmacy, London, UK; 2 St. George’s Hospital Medical School, London, UK; 3 BIBRA International, Surrey, UK; 4 HLS, Huntingdon, UK; 5 Syngenta, Macclesfield, UK

E-mail address: [email protected] Aplastic anaemia (AA) is a potentially life threatening disorder characterised by hypoplastic bone marrow and peripheral blood pancytopenia. The causes of AA are varied and include viruses, irradiation, chemicals and drugs (Young and Alter, 1994). Recently, Gibson et al. (2003) reported a new model of chronic bone marrow aplasia (CBMA) in the busulphan-treated female mouse. This model of ‘late stage’ bone marrow aplasia shared many similarities with the human condition (AA), however at the dose level of busulphan used, there was significant mortality. In order to develop a model of CBMA without significant toxicity we assessed the ability of mitomycin C (MMC) to induce late stage bone marrow aplasia as suggested in a report by Morley (1980). Female CD-1 mice (312) were given eight doses of MMC (intraperitoneal 2.5 mg/kg) or vehicle. Animals were dosed three times per week (Monday, Wednesday, Friday) over a total of 18 days. On days 1, 7, 14, 28, 42 and 50 post-dosing, animals (n = 13–18) were sacrificed for blood and marrow investigations. A full blood count was performed and serum prepared to measure the levels of the cytokine FL (FMS-like tyrosine kinase 3 receptor ligand). Femoral marrow suspensions were prepared to assess the total femoral nucleated cell count (FNCC), the number of committed progenitor cells (CFU-C) and using flow cytometry levels of apoptosis were determined. Immediately post-dosing, MMC induced a ’predictable’ bone marrow depression. The FNCC and number of CFU-C were significantly reduced to below 5% of control values (P < 0.001). The percentage of marrow cells undergoing apoptosis was significantly increased (P < 0.001). In the peripheral blood a severe anaemia was

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observed with reductions in erythrocyte count (RBC), haemoglobin (Hb), haematocrit (HCT), mean cell volume (MCV), reticulocytes (retic), platelets (PLT) and leucocytes (WBC). The concentration of FLT3 in MMC-treated mice was greatly increased (P < 0.001). On day 7 post-dosing, the FNCC of MMC-treated mice continued to be hypocellular. RBC, Hb, HCT, PLT and WBC remained low, and there were significant reductions in CFU-C. Levels of apoptotic cells in the marrow and serum levels of FLT3 remained significantly elevated (P < 0.01 and P < 0.001, respectively). On day 14 and 28, RBC, HCT and WBC remained significantly reduced in MMC-treated animals, although there were clear signs of haematological recovery and a return towards normal; a rebound reticulocytosis occurred from day 14. The FNCC and CFU-C were still significantly reduced but returning towards control values. Apoptosis and FLT3 concentrations remained greatly elevated. On day 42/52 post-dosing, many parameters had returned to normal but residual (late stage) effects of MMC treatment were seen. However, these were confined to FNCC and RBC parameters, both of which showed significant reductions. In conclusion, MMC does not induce late stage bone marrow aplasia in the mouse equivalent to that seen in the busulphan model of Gibson et al. (2003). Furthermore, evidence was found of significant MMC toxicity to other target organs (liver, kidneys) and there was resulting mortality. References Gibson, F., Andrews, M., Diamanti, P., Macharia, G., Williams, T., Gordon-Smith, E.C., Turton, J.A., 2003. Int. J. Exp. Pathol., in press. Morley, A., 1980. Aust. N. Z. J. Med. 10, 569–571. Young, N., Alter, B.P., 1994. Aplastic Anaemia: Acquired and Inherited. Saunders, Philadelphia. Keywords: Mitomycin C; Haemotoxicity; Mouse; Toxicity Probabilistic Assessment of Consumer Exposure to Ingredients Used in Hair Care Products Robert J. Safford, David Briggs, Louise N. Conway, Garrett F. Moran, Christopher R. Jones, Anita J.E. Irwin Safety and Environmental Assurance Centre, Unilever Colworth, Sharnbrook, Bedshire MK44 1LQ, UK E-mail address: [email protected] Accurate estimation of consumer exposure is a fundamental step in the assessment of the toxicological safety of ingredients in personal care products. Traditional methods of doing this usually involve the use of single point, often worst case, estimates of parameters such as amount of product used, skin penetration and body weight. In reality these parameters will vary greatly, dependant on such factors as consumer habits, which we know will differ across the world, and even within single population groups. When worst case assumptions are used and multiplied together, this can often lead the production of totally unrealistic exposure values. In addition to this, estimating skin exposure to, and hence penetration of ingredients used in hair care products is further complicated by the presence of hair. One approach to this is to calculate the percentage of product that comes into contact with the skin based on the distribution of product between hair, scalp and hands. Again there will be a wide variation in hair length, and thus hair surface area, both within and between populations, and this also needs to be taken into account. The model described uses a decision tree approach to account for variation in the parameters used to assess consumer exposure to ingredients in hair care products. The parameters used in the model are scalp, hand and hair surface areas, amount of product used, frequency of washing, body weight and skin penetration. Variation within each parameter is represented by taking the 10th, 50th and 90th percentile points of the distribution. The model has been written as a macro in Microsoft Excel, and provides estimates of exposure at pre-determined percentile points. Comparison of the model to the more commonly used Monte Carlo approach shows it to give comparable outputs.

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269

Table 1 Typical output from the model for an ingredient included in a shampoo at 0.5% Percentile

Exposure (␮g/kg bw per day)

5th

10th

50th

90th

95th

0.394

0.574

3.353

11.959

16.164

Such output provides a more realistic assessment of consumer exposure than single point worst case estimations (which in this case would give a figure of 39 ␮g/kg bw per day) leading to more refined risk assessments. Input data can easily be changed to reflect different consumer habits, physical attributes and skin penetration ranges. In addition, the model is being developed further to consider different exposure scenarios for other personal care products. Keywords: Exposure assessment; Probabilistic assessment; Personal care products; Risk assessment

The Adequacy of Toxicokinetic Default Safety Factors Used in the Risk Assessment of Food Additives Sara C. Tullberg, Warren E. Keene, Manjit Toor, Andrew G. Renwick Clinical Pharmacology Group, Allergy and Inflammation Sciences Research Division, Biomedical Sciences Building, Bassett Crescent East, Southampton, SO16 7PX, UK E-mail address: [email protected] The objective of the current work is to assess the validity of the default toxicokinetic safety factors using experimentally derived data following oral dosing of food additives to the test species and human subjects. Since the 1950s, a 100-fold safety factor has been used in the risk assessment of food additives to allow for differences between the test species and humans and for human variability. This factor represents two 10-fold factors, one to allow for inter-species differences and the other inter-individual differences, (WHO, 1987). More recently pharmaceutical data has been used to propose a further breakdown of these 10-fold factors (Renwick, 1993), with default factors of 4.0 and 3.16 for inter-species and inter-individual variations due to toxicokinetic differences and 2.5 and 3.16, respectively for toxicodynamic differences (WHO, 1999). Food additives were chosen because they were discrete, single chemical entities with diverse metabolic pathways, and had numerical NOAEL (no-observed adverse effects level) and ADI (acceptable daily intake) values. The food additive was administered as a gavage dose to the test species at the NOAEL (n = 12), whilst human volunteers (n = minimum of 6) received a capsule containing a dose equivalent to the ADI. Serial blood samples were taken for up to 7 h, and the resulting plasma analysed to obtain plasma-concentration time curves. Pharmacokinetic parameters were calculated for the animal and human data sets using the modelling program WinNonlinTM (Scientific Consulting Inc.). After correcting for dose, these values were used to calculate chemical specific pharmacokinetic factors for inter-species differences for the food additives under investigation. The preliminary results are shown below in table 1; the ratios are calculated such that a value >1 indicates a higher plasma concentration in humans than in the test species for the same dose on a mg/kg body weight basis. Indicators of chronic exposure, i.e. AUC and clearance/bioavailability (CL/F) suggest that the inter-species toxicokinetic default of 4.0-fold is adequate for the compounds studied to date. Curcumin was undetectable in human plasma following oral dosing at the ADI, therefore “worse-case scenario” factors have been calculated, based on the limit of detection of the assay. Only the Cmax ratio for butylated hydroxytoluene (BHT) had a ratio that exceeds the default safety factor.Further human data will allow the calculation of chemical-specific inter-individual toxicokinetic factors for these food additives, and permit an overall assessment of the toxicokinetic defaults factors.

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Table 1 Chemical specific inter-species toxicokinetic factors for a range of pharmacokinetic parameters Substrate

Test species

Propyl gallate BHT Thiabendazole Curcumin

Rat Rat Rat Mouse

Cmax ratio maximal plasma concentration 0.49 21 0.02 <1.7

AUC ratio area under the concentration–time curve

CL/F ratio clearance/bioavailability

1.6 2.7 0.01 <4.5

2.0 3.2 0.01 –

References Renwick, A.G., 1993. Food Additives Contam. 10, 275–305. WHO, 1987. Environ. Health Criteria, 70. WHO, 1999. Environ. Health Criteria, 210. Keywords: Default safety factors; Toxicokinetic; Inter-species; ADI; NOAEL; Food additives Acknowledgement We would like to acknowledge the Food Standards Agency who are funding the project. Urinary 19-Norsteroid Excretion after Ingestion of Dietary Supplements Kenneth C. Muir, Aoife B. MacManus, Lisa Gregory, Peter Mackie, Ron Maughan and Gabrielle Hawksworth Departments of Medicine and Therapeutics and Biomedical Sciences, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, UK E-mail address: [email protected] There has been a recent increase in the number of athletes testing positive for nandrolone abuse. This is surprising since the steroid has been available for many years and athletes are aware that its metabolites, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE), can be detected in urine. Several positives have been found in sports which are not usually associated with anabolic steroid misuse, such as football and judo, and it has been suggested that dietary supplement contamination could be responsible. Two precursors of nandrolone, 19-norandrostenedione and 19-norandrostenediol, have been identified in three nutritional supplements purchased in the UK at levels ranging from 0.03 to 6.4 ␮g/g. Geyer et al. (2000) analysed three supplements produced in the USA and found 19-norsteroid concentrations ranging from 0.04 to 1336 ␮g per capsule. Similarly, De Cock et al. (2001) analysed a supplement from the USA and found capsules to contain 4.8 mg 19-norandrostenedione. Based on the manufacturers recommendation of 7 capsules per day, a daily intake of 19-norandrostenedione in excess of 30mg would therefore be possible. To determine whether this could be a source of positives, 1mg of 19-norandrostenedione was administered to 9 male volunteers. Complete urine collections were made at 1.75, 4, 8, 12, 16 and 24 h and samples analysed by GC-MS. Both 19-NA and 19-NE were detected in samples from all individuals following ingestion of the steroid with the highest concentrations at approximately 2 h post-ingestion. Samples from subjects A and H had the lowest and highest metabolite concentrations respectively. Subject E had average levels. Male athletes are deemed guilty of 19-norsteroid abuse if they provide a urine sample with a 19-NA concentration exceeding 2 ng/ml. All volunteers in this study therefore provided positive samples. The highest concentration found was 7.9 ␮g/ml. More than 90% of the steroid was excreted in the first 8 h. Mean 19-NA concentrations for the 9 subjects at 12 and 24 h were 41.0 and 3.3 ng/ml. At the same time points, mean 19-NE concentrations were 7.4 and 0.3ng/ml. Based on these data, it is possible that consumption of dietary supplements contaminated with nandrolone precursors at the levels observed could result in a positive test for nandrolone.

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271

Metabolite Excretion 9000 8000 7000 6000 5000 4000 3000 2000 1000 0

Subject A 19-NA Subject A 19-NE Subject E 19-NA Subject E 19-NE Subject H 19-NA Subject H 19-NE

0

2

4

6

8

10 12 14 16 18 20 22 24 Time (hours)

Fig. 1. Urinary metabolite excretion profile in three subjects.

References De Cock, K.J., Delbecke, F.T., Van Eenoo, P., Desmet, N., Roels, K., De Backer, P., 2001. J. Pharmaceut. Biomed. Anal. 25, 843–852. Geyer, H., Mareck-Engelke, U., Reinhart, U., Thevis, M., Schänzer, W., 2000. Deutsche Zeitschrift Für Sportsmedizin 51 (11), 378–382. Keywords: Nandrolone; Dietary supplements; Norandrostenedione The Identification of Superoxide Dismutase in Rat Urine Following Carbon Tetrachloride-Induced Hepatotoxicity Rosemary Smyth1 , John. A. Turton1 , Christopher J. Clarke2 , Malcolm J. York2 , Theo O. Dare2 , Catherine S. Lane1 , Kevin J. Welham1 , Michael R. Munday1 1 School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX, UK; 2 GlaxoSmithKline

Research and Development, Park Road, Ware, Hertfordshire, SG12 ODP, UK E-mail address: [email protected] Carbon tetrachloride (CCl4 ) is commonly used to induce hepatic injury. Damage may be induced in a number of organs but the liver is the main target. CCl4 is metabolised by the cytochrome P450 system, and induces fatty liver, hydropic degeneration and centrilobular necrosis. The present project is concerned with the identification of novel protein markers in the urine following a hepatic insult, as a means of identifying hepatoxicity using non-invasive methods. In the first experiment, female Wistar Han rats were dosed with CCl4 by gavage at 0, 0.4, 0.8 and 1.2 ml/kg (n = 5) and urine collected for 24 h, as described by Dare et al. (2002). The second experiment was a time course study where rats were dosed with 0.8 ml/kg CCl4 and urine was collected at 12, 24, 36, 48, 60 and 72 h and then at days 4, 7, 11 and 17 post-dosing (n = 5). Thirdly a repeat dose experiment was carried out. Rats were dosed with CCl4 at 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 ml/kg (n = 5) twice a week for six weeks; urine samples were collected after 3 weeks and after 6 weeks of dosing. The fourth experiment involved dosing rats with CCl4 at 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 and 0.8 ml/kg (n = 5) and collecting urine samples 36 h after dosing. Urine samples were analysed using Surface Enhanced Laser Desorption/Ionisation (SELDI) ProteinChip® technology. In all experiments a protein peak was identified using SELDI at 15.7 KDa in the CCl4 -treated urine that was not present in the control urine samples. An 18.4 KDa band was identified in the CCl4 -treated urine when the samples

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were run on sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE). An in-gel digest of the 18.4 KDa band was carried out and using tandem mass spectrometry (ms/ms) by static nanospray ionisation on a ThermoFinnigan LCQ duo, the protein was identified as Cu/Zn superoxide dismutase (SOD). SOD levels were measured in the serum and urine samples obtained in the repeat dose study. In CCl4 -treated urine samples SOD was found to be 5-fold increased over control levels, whereas serum SOD levels were 1.5- to 2-fold greater than control. Hepatotoxicity was assessed by histopathology in all experiments. Serum enzymes were measured as indicators of hepatic injury. ALP, AST, ALT and GLDH levels were elevated in CCl4 -treated rats, confirming histological findings of hepatocyte degeneration and necrosis, and possible subsequent inflammatory changes. The enzyme SOD catalyses the destruction of the superoxide anion (O2 − ) and therefore acts as a defence against oxidative damage. SOD exists as a homodimer (molecular weight of 32.5 KDa). This would correlate with a 15.7 KDa subunit observed by SELDI but presumably means that the SOD subunit runs anomalously on SDS PAGE at 18.4 KDa. The importance of identifying SOD in the urine following hepatic injury has yet to be determined. Reference Dare, T.O., Davies, H.A., Turton, J.A., Lomas, L., Williams, T.C., York, M.J., 2002. Electrophoresis 23, 3241– 3251. Keywords: Carbon tetrachloride; Superoxide dismutase; Urine; Liver; Rat