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Chemical risk assessment: Historical perspectives and current trends
Abstracts / Toxicology Letters 196S (2010) S1–S36
shown that neurospheres from human and mouse origin mimic the very basic processes of brain development: proliferation, migration and differentiation of premature brain cells in vitro. They are also able to undergo caspase-dependent and -independent apoptosis (Fritsche et al. EHP 2005; Moors et al. TAAP 2007; Moors et al. EHP 2009; Schreiber et al. EHP 2010). Establishment of endpoint-specific controls for stimulation and inhibition of proliferation (titration/withdrawal of growth factors), migration (Phosphokinase C inhibition/stimulation) and differentiation (cAMP/methylmercury/thyroid hormone) indicate that these endpoints can be modulated with model compounds. Thus, the neurosphere system is able to assess developmental neurotoxicity (DNT). Reproducibility of the endpoints was shown by different investigators in the laboratory. Thus, human and mouse neurospheres might be suitable in vitro systems for assessing toxic potentials of chemicals or pharmaceuticals on neurodevelopment. At the moment, we are testing the effects of methylmercury, methylazoxymethanol, valproic acid, lead, polychlorinated biphenyls, polybrominated diphenyl ethers (PBDEs), TCDD, benzo(a)pyrene (B(a)P), chlorpyrifos, parathion as well as the negative compounds glutamate and paracetamol on proliferation, migration, differentiation and viability of NPCs. To increase the throughput, we have developed semi-automated image analyses for assessing proliferation and differentiation of neurospheres. The data from these endpoints are compared to basic cytotoxicity data of the compounds. First results indicate that human neurospheres can distinguish between positive and negative compounds. They also distinguish between general cytotoxicity and specific DNT (e.g. inhibition of migration). Moreover, studies comparing effects of selected compounds on human vs. mouse NPCs indicate that there are species-specific differences in susceptibility towards DNT compounds. Besides compound screening, we investigate mechanisms of action of selected chemicals. Our work shows that PBDEs inhibit human neural migration and differentiation by inhibiting cellular thyroid hormone signaling. These substances exert little effects on mouse NPCs due to dissimilar thyroid hormone receptor expression. Moreover, polycyclic aromatic hydrocarbons like B(a)P selectively inhibit mouse neural migration by Arylhydrocarbon receptor (AhR) activation, while human neurospheres remain unaffected. AhR inhibition by 3-methoxy-4-nitroflavone again only inhibits mouse NPC proliferation. Lacking effects on human cells is thereby due to an absent AhR expression. Our data shows that neurospheres can be used for DNT testing in a medium-throughput system. Moreover, they are useful for identifying chemical modes of action. In this regard, species comparisons are of great value for hazard assessment of humans. doi:10.1016/j.toxlet.2010.03.107
S18-5 Present state analysis and policy direction of high-throughput methods for developmental neurotoxicity R. Tsuji Sumitomo Chemical, Japan Developmental disorders such as learning disabilities, ADHD, autism, etc., are increasing causes of concern. Although diverse hypotheses have proposed, the causes underlying such disorders have not been identified. However, chemical substances, used for various purposes, have been suggested to be involved in the etiology of developmental disorders. More than 80,000 chemical substances are currently in use, and most of their toxicological
S23
characteristics have not sufficiently been evaluated. Current in vivo testing guideline studies for developmental neurotoxicity (DNT) are time and cost intensive and use large numbers of animals. In addition, laboratories that can conduct the guideline DNT study are limited. To address these problems, high throughput (HTP) in vitro assays need to be developed to screen and prioritize chemicals having DNT potential, followed by additional assays to confirm substantial adverse effects and dose–response relationship for hazard assessments. These assays and testing strategies need to be developed and accepted by regulatory authorities. As the first screening assay for DNT, a battery of alternative in vitro high-throughput methods, rather than a single one, is desired, because of complexity of brain development. Various HTP model system evaluating differentiation, viability, neurite outgrowth, electrical activity, mRNA expression, etc. have been reported, but they are all in the development phase and have not been validated. Selecting sensitive type of cell/ tissue or methods and building a battery to detect various compounds that affect a variety of target sites in developing brain is essential. High accuracy and low false negatives are key issues in such battery. Both positive and negative controls should be identified. Simplified measurement and steady supply of cell/tissue are also important to disseminate methods. New technologies, such as the various “omics” approaches should also be considered. doi:10.1016/j.toxlet.2010.03.108
S19 New Trends in Food Safety S19-1 Chemical risk assessment: Historical perspectives and current trends J. Dorne 1 , B. Amzal 1 , G. Frampton 2 , D. Liem 1 , B. Bottex 1 , C. Heppner 1 , C. Svendsen 3 , D. Spurgeon 3 1 Unit on Contaminants in the Food chain European Food Safety Authority, UK, 2 School of Medicine University of Southampton, Southampton, UK, 3 Centre for Ecology and Hydrology, Wallingford, UK
Over the last 30 years, chemical risk assessment (RA) has evolved as a multidisciplinary and integrated science at the crossroad between human and ecological RA (HRA and ERA) by slowly bringing together these different and historically separated disciplines. Intuitively, the four pillars of RA namely hazard identification and characterisation, exposure assessment and risk characterisation apply to both fields although with slight variations. In practice, both start from the dose–response of a chemical (toxicity), to derive either a health-based guidance value or environmental standard. Using validated analytical techniques, the occurrence of the chemical is measured in a matrix (water, food or media), which in combination with knowledge of consumption and behaviour patterns of the organism (human, ecological species) lead to exposure estimates. Exposure is then related to the health-based guidance value (HRA) or environmental standards (ERA) to characterise the potential risk of adverse effects in humans (individuals) or the environment (generally populations). A historical perspective of chemical RA is given together with examples of tools and methods to move towards more mechanistic and quantitative approaches for different levels of biological complexity (molecular, individual and populations). The use of meta-analysis in HRA is illustrated by the recent RA of the European Food Safety Authority on cadmium relating human biomarkers of renal function and urinary cadmium concentrations. Current general trends and future directions in HRA
S24
Abstracts / Toxicology Letters 196S (2010) S1–S36
and ERA are presented with particular emphasis on the applicability of mechanistic data, systems biology and physiology-based modelling and their associated uncertainties, in the assessment of chemical risk for both single compounds and chemical mixtures. doi:10.1016/j.toxlet.2010.03.110
S19-2 Molecular mechanistic basis for risk assessment of biotoxins in foods E. Creppy University Bordeaux 2, France Mycotoxins are secondary metabolites of several species of fungi contaminating foods and feedstuffs. Similarly phycotoxins are produced by microalgae in seafood and contaminate consumers following ingestion. They may cause organ toxicity and cancer, for some of them, when repeatedly ingested. They are of increasing concern for human and animal health, especially for combinations of toxins in the same commodity. Although well regulated for each individual toxin by international food safety authorities, their combinations are increasingly regarded as being of high toxicological risk and therefore deserve thorough evaluation even though there is a lack of data. Examples from studies on both mycotoxins and marine toxins illustrate the difficult problem of the risk assessment of mixtures of toxins. Preliminary data indicate that prediction of combined effects from existing data on each individual toxin is uncertain and that experimental in vitro and in vivo data will be needed which in conditions of ingestion very similar to those encountered in our environment. For example marine toxins may be combined and synergise each other and also be synergised by toxic metals additionally. Mixtures of mycotoxins may act by antagonism or mostly by synergism as shown by several studies, some of which have been conducted on more than two toxins such as in the following example. Considering the effects of DON (45 g/kg B.W./day), ZEA (50 g/kg B.W./day) and FB1 (110 g/kg B.W./day) and the tolerable daily intake (TDI-EU 1881, 19/12/2006) for FB1 (2 g/kg B.W./day); DON (1 g/kg B.W./day); ZEA (0.2 g/kg B.W./day), respectively and a safety coefficient of 100, our in vivo data show that these TDI values are too high for at least two of them. Similar conclusions are reached for some phycotoxins indicating the need of new data and regulation taking the mixtures of toxins into account as for the mixture of aflatoxins. doi:10.1016/j.toxlet.2010.03.111
S19-3 Mycotoxicological risks of main European food/feed chains
of plant products also contributes significantly to the spread of toxigenic fungi worldwide and represents an important source of inoculum for new plant diseases in Europe and for broadening the genetic diversity of already existing local populations. The major problems associated with mycotoxin contamination of agricultural products in Europe are focusing on three crops and their relevant diseases: (i) wheat with Fusarium head blight and Penicillum infections, (ii) maize for Fusarium ear rot and Aspergillus infections and (iii) grapes with black Aspergillus rot. In order to comply with the needs of EU and address global strategies for mycotoxin reduction, a large collaborative project for a four year duration on “Novel integrated strategies for worldwide mycotoxin reduction in food and feed chains”, MYCORED as acronym, has been recently approved within the European FP7-“Food, Agriculture and Biotechnologies’ Work Programmes” (www.mycored.eu). doi:10.1016/j.toxlet.2010.03.112
S19-4 Protective effect of antioxidants against mycotoxins induced citotoxicity in cells M.J. Ruiz Faculty of Pharmacy, University of Valencia, Spain Beauvericin (BEA), deoxynivalenol (DON) and T-2 toxin are important food-borne mycotoxins that have been implicated in human health. The aims of this study were: firstly, to investigate the effect of BEA, DON and T-2 toxin on cell viability in Chinese hamster ovary (CHO-K1) cells and secondly, to determine whether the presence of vitamins and flavonoids have protective effects against mycotoxins-induced cytotoxicity in vitro. CHO-K1 cells were supplemented with either 0–100 M vitamin C or 0–50 M vitamin E and flavonoids (quercetin and p-terostilbene). Cytotoxicity was measured by two end points, the neutral red (NR) and the MTT assay. The IC50 values obtained on the MTT and NR assays ranged from 0.017 to 5.25 M and 0.041–17.22 M, respectively. CHO-K1 cells were exposed to medium containing mycotoxins (1/10, 1/5 and 1/2 their IC50 ) in the presence or absence of the vitamins or flavonoids for a period of 24 h. The addition of the vitamins and flavonoids to CHO-K1 cells exposed to mycotoxins exerted a significant protective effect against the cytotoxicity of mycotoxins in ovarian cells. Generally, there is a positive correlation between antioxidant concentration and the protective cell effects. However, cells exposed to an intermediate concentration of vitamin E were partially protected against the effects of mycotoxins, but the highest vitamin E exposure did not suppress mycotoxin cytotoxicities. These results suggest that certain flavonoid and vitamin concentrations may play a role in reducing mycotoxin-induced cytoxicities in CHO-K1 cells. This work was supported by the Science and Education Spanish Ministry (AGL2007-61493). doi:10.1016/j.toxlet.2010.03.113
A.F. Logrieco Institute of Sciences of Food Production, Research, Italy Contamination by toxigenic fungi and their mycotoxins of agricultural products grown in Europe is a problem and the dramatic differences in environmental conditions in various European countries significantly influences the distribution of toxigenic fungi and related mycotoxicological risks. Emerging problems due to climate change and new mycotoxin/commodity combinations increase these concerns. Trans-global transposition and trade exchanges
shown that neurospheres from human and mouse origin mimic the very basic processes of brain development: proliferation, migration and differentiation of premature brain cells in vitro. They are also able to undergo caspase-dependent and -independent apoptosis (Fritsche et al. EHP 2005; Moors et al. TAAP 2007; Moors et al. EHP 2009; Schreiber et al. EHP 2010). Establishment of endpoint-specific controls for stimulation and inhibition of proliferation (titration/withdrawal of growth factors), migration (Phosphokinase C inhibition/stimulation) and differentiation (cAMP/methylmercury/thyroid hormone) indicate that these endpoints can be modulated with model compounds. Thus, the neurosphere system is able to assess developmental neurotoxicity (DNT). Reproducibility of the endpoints was shown by different investigators in the laboratory. Thus, human and mouse neurospheres might be suitable in vitro systems for assessing toxic potentials of chemicals or pharmaceuticals on neurodevelopment. At the moment, we are testing the effects of methylmercury, methylazoxymethanol, valproic acid, lead, polychlorinated biphenyls, polybrominated diphenyl ethers (PBDEs), TCDD, benzo(a)pyrene (B(a)P), chlorpyrifos, parathion as well as the negative compounds glutamate and paracetamol on proliferation, migration, differentiation and viability of NPCs. To increase the throughput, we have developed semi-automated image analyses for assessing proliferation and differentiation of neurospheres. The data from these endpoints are compared to basic cytotoxicity data of the compounds. First results indicate that human neurospheres can distinguish between positive and negative compounds. They also distinguish between general cytotoxicity and specific DNT (e.g. inhibition of migration). Moreover, studies comparing effects of selected compounds on human vs. mouse NPCs indicate that there are species-specific differences in susceptibility towards DNT compounds. Besides compound screening, we investigate mechanisms of action of selected chemicals. Our work shows that PBDEs inhibit human neural migration and differentiation by inhibiting cellular thyroid hormone signaling. These substances exert little effects on mouse NPCs due to dissimilar thyroid hormone receptor expression. Moreover, polycyclic aromatic hydrocarbons like B(a)P selectively inhibit mouse neural migration by Arylhydrocarbon receptor (AhR) activation, while human neurospheres remain unaffected. AhR inhibition by 3-methoxy-4-nitroflavone again only inhibits mouse NPC proliferation. Lacking effects on human cells is thereby due to an absent AhR expression. Our data shows that neurospheres can be used for DNT testing in a medium-throughput system. Moreover, they are useful for identifying chemical modes of action. In this regard, species comparisons are of great value for hazard assessment of humans. doi:10.1016/j.toxlet.2010.03.107
S18-5 Present state analysis and policy direction of high-throughput methods for developmental neurotoxicity R. Tsuji Sumitomo Chemical, Japan Developmental disorders such as learning disabilities, ADHD, autism, etc., are increasing causes of concern. Although diverse hypotheses have proposed, the causes underlying such disorders have not been identified. However, chemical substances, used for various purposes, have been suggested to be involved in the etiology of developmental disorders. More than 80,000 chemical substances are currently in use, and most of their toxicological
S23
characteristics have not sufficiently been evaluated. Current in vivo testing guideline studies for developmental neurotoxicity (DNT) are time and cost intensive and use large numbers of animals. In addition, laboratories that can conduct the guideline DNT study are limited. To address these problems, high throughput (HTP) in vitro assays need to be developed to screen and prioritize chemicals having DNT potential, followed by additional assays to confirm substantial adverse effects and dose–response relationship for hazard assessments. These assays and testing strategies need to be developed and accepted by regulatory authorities. As the first screening assay for DNT, a battery of alternative in vitro high-throughput methods, rather than a single one, is desired, because of complexity of brain development. Various HTP model system evaluating differentiation, viability, neurite outgrowth, electrical activity, mRNA expression, etc. have been reported, but they are all in the development phase and have not been validated. Selecting sensitive type of cell/ tissue or methods and building a battery to detect various compounds that affect a variety of target sites in developing brain is essential. High accuracy and low false negatives are key issues in such battery. Both positive and negative controls should be identified. Simplified measurement and steady supply of cell/tissue are also important to disseminate methods. New technologies, such as the various “omics” approaches should also be considered. doi:10.1016/j.toxlet.2010.03.108
S19 New Trends in Food Safety S19-1 Chemical risk assessment: Historical perspectives and current trends J. Dorne 1 , B. Amzal 1 , G. Frampton 2 , D. Liem 1 , B. Bottex 1 , C. Heppner 1 , C. Svendsen 3 , D. Spurgeon 3 1 Unit on Contaminants in the Food chain European Food Safety Authority, UK, 2 School of Medicine University of Southampton, Southampton, UK, 3 Centre for Ecology and Hydrology, Wallingford, UK
Over the last 30 years, chemical risk assessment (RA) has evolved as a multidisciplinary and integrated science at the crossroad between human and ecological RA (HRA and ERA) by slowly bringing together these different and historically separated disciplines. Intuitively, the four pillars of RA namely hazard identification and characterisation, exposure assessment and risk characterisation apply to both fields although with slight variations. In practice, both start from the dose–response of a chemical (toxicity), to derive either a health-based guidance value or environmental standard. Using validated analytical techniques, the occurrence of the chemical is measured in a matrix (water, food or media), which in combination with knowledge of consumption and behaviour patterns of the organism (human, ecological species) lead to exposure estimates. Exposure is then related to the health-based guidance value (HRA) or environmental standards (ERA) to characterise the potential risk of adverse effects in humans (individuals) or the environment (generally populations). A historical perspective of chemical RA is given together with examples of tools and methods to move towards more mechanistic and quantitative approaches for different levels of biological complexity (molecular, individual and populations). The use of meta-analysis in HRA is illustrated by the recent RA of the European Food Safety Authority on cadmium relating human biomarkers of renal function and urinary cadmium concentrations. Current general trends and future directions in HRA
S24
Abstracts / Toxicology Letters 196S (2010) S1–S36
and ERA are presented with particular emphasis on the applicability of mechanistic data, systems biology and physiology-based modelling and their associated uncertainties, in the assessment of chemical risk for both single compounds and chemical mixtures. doi:10.1016/j.toxlet.2010.03.110
S19-2 Molecular mechanistic basis for risk assessment of biotoxins in foods E. Creppy University Bordeaux 2, France Mycotoxins are secondary metabolites of several species of fungi contaminating foods and feedstuffs. Similarly phycotoxins are produced by microalgae in seafood and contaminate consumers following ingestion. They may cause organ toxicity and cancer, for some of them, when repeatedly ingested. They are of increasing concern for human and animal health, especially for combinations of toxins in the same commodity. Although well regulated for each individual toxin by international food safety authorities, their combinations are increasingly regarded as being of high toxicological risk and therefore deserve thorough evaluation even though there is a lack of data. Examples from studies on both mycotoxins and marine toxins illustrate the difficult problem of the risk assessment of mixtures of toxins. Preliminary data indicate that prediction of combined effects from existing data on each individual toxin is uncertain and that experimental in vitro and in vivo data will be needed which in conditions of ingestion very similar to those encountered in our environment. For example marine toxins may be combined and synergise each other and also be synergised by toxic metals additionally. Mixtures of mycotoxins may act by antagonism or mostly by synergism as shown by several studies, some of which have been conducted on more than two toxins such as in the following example. Considering the effects of DON (45 g/kg B.W./day), ZEA (50 g/kg B.W./day) and FB1 (110 g/kg B.W./day) and the tolerable daily intake (TDI-EU 1881, 19/12/2006) for FB1 (2 g/kg B.W./day); DON (1 g/kg B.W./day); ZEA (0.2 g/kg B.W./day), respectively and a safety coefficient of 100, our in vivo data show that these TDI values are too high for at least two of them. Similar conclusions are reached for some phycotoxins indicating the need of new data and regulation taking the mixtures of toxins into account as for the mixture of aflatoxins. doi:10.1016/j.toxlet.2010.03.111
S19-3 Mycotoxicological risks of main European food/feed chains
of plant products also contributes significantly to the spread of toxigenic fungi worldwide and represents an important source of inoculum for new plant diseases in Europe and for broadening the genetic diversity of already existing local populations. The major problems associated with mycotoxin contamination of agricultural products in Europe are focusing on three crops and their relevant diseases: (i) wheat with Fusarium head blight and Penicillum infections, (ii) maize for Fusarium ear rot and Aspergillus infections and (iii) grapes with black Aspergillus rot. In order to comply with the needs of EU and address global strategies for mycotoxin reduction, a large collaborative project for a four year duration on “Novel integrated strategies for worldwide mycotoxin reduction in food and feed chains”, MYCORED as acronym, has been recently approved within the European FP7-“Food, Agriculture and Biotechnologies’ Work Programmes” (www.mycored.eu). doi:10.1016/j.toxlet.2010.03.112
S19-4 Protective effect of antioxidants against mycotoxins induced citotoxicity in cells M.J. Ruiz Faculty of Pharmacy, University of Valencia, Spain Beauvericin (BEA), deoxynivalenol (DON) and T-2 toxin are important food-borne mycotoxins that have been implicated in human health. The aims of this study were: firstly, to investigate the effect of BEA, DON and T-2 toxin on cell viability in Chinese hamster ovary (CHO-K1) cells and secondly, to determine whether the presence of vitamins and flavonoids have protective effects against mycotoxins-induced cytotoxicity in vitro. CHO-K1 cells were supplemented with either 0–100 M vitamin C or 0–50 M vitamin E and flavonoids (quercetin and p-terostilbene). Cytotoxicity was measured by two end points, the neutral red (NR) and the MTT assay. The IC50 values obtained on the MTT and NR assays ranged from 0.017 to 5.25 M and 0.041–17.22 M, respectively. CHO-K1 cells were exposed to medium containing mycotoxins (1/10, 1/5 and 1/2 their IC50 ) in the presence or absence of the vitamins or flavonoids for a period of 24 h. The addition of the vitamins and flavonoids to CHO-K1 cells exposed to mycotoxins exerted a significant protective effect against the cytotoxicity of mycotoxins in ovarian cells. Generally, there is a positive correlation between antioxidant concentration and the protective cell effects. However, cells exposed to an intermediate concentration of vitamin E were partially protected against the effects of mycotoxins, but the highest vitamin E exposure did not suppress mycotoxin cytotoxicities. These results suggest that certain flavonoid and vitamin concentrations may play a role in reducing mycotoxin-induced cytoxicities in CHO-K1 cells. This work was supported by the Science and Education Spanish Ministry (AGL2007-61493). doi:10.1016/j.toxlet.2010.03.113
A.F. Logrieco Institute of Sciences of Food Production, Research, Italy Contamination by toxigenic fungi and their mycotoxins of agricultural products grown in Europe is a problem and the dramatic differences in environmental conditions in various European countries significantly influences the distribution of toxigenic fungi and related mycotoxicological risks. Emerging problems due to climate change and new mycotoxin/commodity combinations increase these concerns. Trans-global transposition and trade exchanges