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Epigenetic changes after prolonged exposure to alkenylbenzenes – An important signature of potential toxicological effects
S86
Abstracts / Toxicology Letters 238S (2015) S56–S383
P02-063 Reproductive safety evaluation of l-Ergothioneine P. Singh 1,∗ , F. Spézia 1 , D. Papineau 1 , C. Sabadie 1 , R. Forster 1 , I. Erdelmeier 2 , M. Moutet 2 , J.-C. Yadan 2 1
CiToxLAB, Evreux Cedex, France 2 Tetrahedron, Vincennes, France l-Ergothioneine is a naturally occurring histidine-derived betaine (CAS No: 497-30-3), synthesized by bacteria and fungi. It is found ubiquitously in plants and animals and is present in many foodstuffs. We have evaluated the potential reproductive toxicity of l-Ergothioneine in a 13-week reproductive toxicity study in Sprague–Dawley rats. l-Ergothioneine was administered at concentrations of 0.1, 0.3 or 0.9% in diet to F0 males (for 10 weeks before pairing and 3 weeks during pairing) and F0 females (for 13 weeks before pairing, during pairing and gestation, and until day 5 of lactation). Toxicokinetic analysis showed that systemic exposure to l-Ergothioneine increased with dose-level. There were no clinical signs of toxicity in any group. There were no effects on mating or fertility data. All animals mated within a similar number of days and all females were pregnant (except one low dose animal). There were no effects on the duration of gestation, pre-and post-implantation losses, number of pups delivered or viability index. There was no evidence of an effect of treatment with l-Ergothioneine on litter parameters (litter size, clinical signs, body weight and sex ratio) or on the repartition of found dead/cannibalized pups. In conclusion l-Ergothioneine was well tolerated and did not elicit any adverse effects on the reproductive parameters evaluated. http://dx.doi.org/10.1016/j.toxlet.2015.08.287
P02-064 Epigenetic changes after prolonged exposure to alkenylbenzenes – An important signature of potential toxicological effects C. Martins ∗ , I. Silva, J. Rueff, A. Rodrigues NOVA Medical School – Faculdade de Ciências Médicas, Genetics, Lisboa, Portugal Genotoxicity studies with chemicals, either natural or manmade, focus essentially on genetic alterations such as mutations or chromosomes alterations leading to visible changes such as DNA breaks. In this regard, our past results have shown that some members of the alkenylbenzene family are genotoxic whereas others are apoptotic but not genotoxic. Eugenol, present in clove oil, was positive in assays that measured DNA damage and myristicin altered gene expression of various genes involved in the DNA damage response pathways. In this work we attempted to go a step further and understand the impact of these compounds at the gene regulation level, namely studying their impact on the methylation status of several genes. We exposed human breast cancer MCF-7 cells, to prolonged (15 days) low dose (10 M) of a group of selected alkenylbenzenes. First we analyzed alterations in ␥H2AX expression, a known double strand break signaling protein, then as our main objective, we studied alterations in methylation patterns. We choose CpGs islands of promoter regions of genes usually methylated in MCF-7 cells and in most tumours. Eugenol enhanced ␥-H2AX protein in nuclear extracts and demethylated the RASSF-1 gene with a concomitant increase in RASSF-1 expression, as measured by RT-PCR. In summary, our study shows that
eugenol, belonging to a family of natural compounds, considered as safe, can induce the DNA damage response and cause alteration in gene methylation status at low doses. http://dx.doi.org/10.1016/j.toxlet.2015.08.288
P02-065 The re-evaluation programme of food additives authorised for use in Europe P.A. Colombo ∗ , A. Christodoulidou, F. Lodi, C. Smeraldi, A.M. Rincon, A. Tard, S. Tasiopoulou, C. Roncancio Pena EFSA, REPRO/FIP, Parma, Italy Food additives are substances that are intentionally added to food for a specific technological purpose. Only food additives that are included in a positive list may be used under the specified conditions set by the Annex II of Regulation (EC) No. 1333/2008. An identification system is used in Europe, which includes an “E” number (e.g. sorbic acid (E200)). A common authorisation procedure for food additives, food enzyme and food flavouring is established by Regulation (EC) No. 1331/2008. Food additives are evaluated by the European Food Safety Authority (EFSA) Panel on Food Additives and Nutrient Sources added to food (ANS) with the support of the Food Ingredient and Packaging (FIP) Unit. The risk assessment paradigm is applied to identify and characterise the hazard, to estimate the exposure (dietary intake) and to characterise the risk. In the European food safety system, risk assessment is done independently from risk management. As the risk assessor, EFSA produces scientific opinions and advice to provide a sound foundation for European policies and legislation and to support the European Commission, European Parliament and EU Member States in taking effective and timely risk management decisions. The most common additives are antioxidants, colours, emulsifiers, stabilisers, gelling agents, thickeners, preservatives and sweeteners. Commission Regulation (EU) No. 257/2010 has set up a programme for the re-evaluation of food additives that were approved before January 2009; in particular according to the type of food additive, different deadlines are set from April 2010 (some food colours) until December 2020 (all approved sweeteners). These priorities are defined according to the time since the last evaluation, extent of use of the food additive, potential human exposure and availability of new scientific evidence. A new re-evaluation can start at any time in case a possible risk for human health is identified. Priorities, deadlines, procedures to be followed, main features and challenges encountered and future programme are shown and discussed. In the 2015–2016 time frame a total of more than 50 scientific opinions referring to the re-evaluation of food additives are expected to be finalized. http://dx.doi.org/10.1016/j.toxlet.2015.08.289
P02-066 Effects of food chemical contaminants in human HepaRG and Caco-2 cells using an automated microscopy and high content analysis based approach P.-J. Ferron, S. Huet, K. Hogeveen, V. Fessard, L. Le Hegarat Anses ∗ Fougeres-Laboratory, FOUGERES, France The aim of this study was to investigate the in vitro toxicity of major chemicals commonly found in food by using cellular imag-
Abstracts / Toxicology Letters 238S (2015) S56–S383
P02-063 Reproductive safety evaluation of l-Ergothioneine P. Singh 1,∗ , F. Spézia 1 , D. Papineau 1 , C. Sabadie 1 , R. Forster 1 , I. Erdelmeier 2 , M. Moutet 2 , J.-C. Yadan 2 1
CiToxLAB, Evreux Cedex, France 2 Tetrahedron, Vincennes, France l-Ergothioneine is a naturally occurring histidine-derived betaine (CAS No: 497-30-3), synthesized by bacteria and fungi. It is found ubiquitously in plants and animals and is present in many foodstuffs. We have evaluated the potential reproductive toxicity of l-Ergothioneine in a 13-week reproductive toxicity study in Sprague–Dawley rats. l-Ergothioneine was administered at concentrations of 0.1, 0.3 or 0.9% in diet to F0 males (for 10 weeks before pairing and 3 weeks during pairing) and F0 females (for 13 weeks before pairing, during pairing and gestation, and until day 5 of lactation). Toxicokinetic analysis showed that systemic exposure to l-Ergothioneine increased with dose-level. There were no clinical signs of toxicity in any group. There were no effects on mating or fertility data. All animals mated within a similar number of days and all females were pregnant (except one low dose animal). There were no effects on the duration of gestation, pre-and post-implantation losses, number of pups delivered or viability index. There was no evidence of an effect of treatment with l-Ergothioneine on litter parameters (litter size, clinical signs, body weight and sex ratio) or on the repartition of found dead/cannibalized pups. In conclusion l-Ergothioneine was well tolerated and did not elicit any adverse effects on the reproductive parameters evaluated. http://dx.doi.org/10.1016/j.toxlet.2015.08.287
P02-064 Epigenetic changes after prolonged exposure to alkenylbenzenes – An important signature of potential toxicological effects C. Martins ∗ , I. Silva, J. Rueff, A. Rodrigues NOVA Medical School – Faculdade de Ciências Médicas, Genetics, Lisboa, Portugal Genotoxicity studies with chemicals, either natural or manmade, focus essentially on genetic alterations such as mutations or chromosomes alterations leading to visible changes such as DNA breaks. In this regard, our past results have shown that some members of the alkenylbenzene family are genotoxic whereas others are apoptotic but not genotoxic. Eugenol, present in clove oil, was positive in assays that measured DNA damage and myristicin altered gene expression of various genes involved in the DNA damage response pathways. In this work we attempted to go a step further and understand the impact of these compounds at the gene regulation level, namely studying their impact on the methylation status of several genes. We exposed human breast cancer MCF-7 cells, to prolonged (15 days) low dose (10 M) of a group of selected alkenylbenzenes. First we analyzed alterations in ␥H2AX expression, a known double strand break signaling protein, then as our main objective, we studied alterations in methylation patterns. We choose CpGs islands of promoter regions of genes usually methylated in MCF-7 cells and in most tumours. Eugenol enhanced ␥-H2AX protein in nuclear extracts and demethylated the RASSF-1 gene with a concomitant increase in RASSF-1 expression, as measured by RT-PCR. In summary, our study shows that
eugenol, belonging to a family of natural compounds, considered as safe, can induce the DNA damage response and cause alteration in gene methylation status at low doses. http://dx.doi.org/10.1016/j.toxlet.2015.08.288
P02-065 The re-evaluation programme of food additives authorised for use in Europe P.A. Colombo ∗ , A. Christodoulidou, F. Lodi, C. Smeraldi, A.M. Rincon, A. Tard, S. Tasiopoulou, C. Roncancio Pena EFSA, REPRO/FIP, Parma, Italy Food additives are substances that are intentionally added to food for a specific technological purpose. Only food additives that are included in a positive list may be used under the specified conditions set by the Annex II of Regulation (EC) No. 1333/2008. An identification system is used in Europe, which includes an “E” number (e.g. sorbic acid (E200)). A common authorisation procedure for food additives, food enzyme and food flavouring is established by Regulation (EC) No. 1331/2008. Food additives are evaluated by the European Food Safety Authority (EFSA) Panel on Food Additives and Nutrient Sources added to food (ANS) with the support of the Food Ingredient and Packaging (FIP) Unit. The risk assessment paradigm is applied to identify and characterise the hazard, to estimate the exposure (dietary intake) and to characterise the risk. In the European food safety system, risk assessment is done independently from risk management. As the risk assessor, EFSA produces scientific opinions and advice to provide a sound foundation for European policies and legislation and to support the European Commission, European Parliament and EU Member States in taking effective and timely risk management decisions. The most common additives are antioxidants, colours, emulsifiers, stabilisers, gelling agents, thickeners, preservatives and sweeteners. Commission Regulation (EU) No. 257/2010 has set up a programme for the re-evaluation of food additives that were approved before January 2009; in particular according to the type of food additive, different deadlines are set from April 2010 (some food colours) until December 2020 (all approved sweeteners). These priorities are defined according to the time since the last evaluation, extent of use of the food additive, potential human exposure and availability of new scientific evidence. A new re-evaluation can start at any time in case a possible risk for human health is identified. Priorities, deadlines, procedures to be followed, main features and challenges encountered and future programme are shown and discussed. In the 2015–2016 time frame a total of more than 50 scientific opinions referring to the re-evaluation of food additives are expected to be finalized. http://dx.doi.org/10.1016/j.toxlet.2015.08.289
P02-066 Effects of food chemical contaminants in human HepaRG and Caco-2 cells using an automated microscopy and high content analysis based approach P.-J. Ferron, S. Huet, K. Hogeveen, V. Fessard, L. Le Hegarat Anses ∗ Fougeres-Laboratory, FOUGERES, France The aim of this study was to investigate the in vitro toxicity of major chemicals commonly found in food by using cellular imag-