Appréciation quantitative de l'exposition alimentaire à des mélanges de pesticides et mécanismes de génotoxicité

Les consommateurs sont exposés à plusieurs résidus de pesticides présents simultanément via leur alimentation. L'exposition réelle des consommateurs aux mélanges de pesticides est mal connue, et l'évaluation des risques prend rarement en compte les effets possibles d'une multi-exposition à ces composés. L'objectif de ce travail de thèse était d'étudier in vitro le potentiel génotoxique de mélanges de pesticides, déterminés à partir de l'exposition alimentaire de la population française. Pour déterminer ces mélanges de pesticides, la co-exposition a été évaluée à partir de données provenant des plans nationaux de surveillance et de contrôle des résidus de pesticides dans les aliments, couplées aux données de consommation individuelle française (enquête INCA2). Un modèle statistique développé à l'ANSES a comparé ces co-expositions aux pesticides, et a défini des groupes d'individus aux niveaux d'exposition semblables. A partir de ces données, sept mélanges de pesticides ont été déterminés et leur potentiel génotoxique a été étudié sur quatre lignées cellulaires humaines (ACHN, HepG2, LS-174T, SH-SY5Y), à l'aide du test de génotoxicité H2AX. Ces mélanges ont été testés en proportions équimolaires et aux proportions reflétant l'exposition de la population. Les résultats obtenus ont été comparés avec l'effet des composés individuels. Seul un mélange, constitué de 5 composés, est génotoxique et uniquement sur la lignée HepG2. Par ailleurs, les résultats ont montrés que seul, deux composés du mélange, fludioxonil cyprodinil, sont génotoxiques. Il a également été montré un effet du mélange à faibles doses. De plus, l'analyse du potentiel génotoxique de 14 autres pesticides seuls, également sélectionnés à partir de la co-exposition, a permis d'identifier une famille de pesticides (méthyl-pyrazoles) capable d'induire des cassures double brin d'ADN sur deux lignées cellulaires humaines (Jurkat/ SH-SY5Y).Consumers are exposed to several pesticide residues present simultaneously in food. This multi-exposure is insufficiently characterized and risk assessment procedures do not take into account appropriately the possible effects of contaminant mixtures. The purpose of this study was to investigate in vitro the genotoxic potential of pesticide mixtures relevant to the dietary exposure of the French population. In order to determine these mixtures, the French population co-exposure was estimated considering both food residue patterns originating from French food monitoring programs and French consumption survey (INCA2). Then, a statistic model was developed by ANSES to compare co-exposures, in order to define groups of individuals with similar pesticide exposure patterns. Based on these results, 7 relevant mixtures were determined and their genotoxic potential was screened in 4 different human cell lines (ACHN, HepG2, LS-174T, SHSY-5Y), using the genotoxic H2AX assay. Mixtures were tested at both equimolar concentrations and proportions reflecting the real diet contamination. The results were compared with the effects of individual compounds. One mixture, consisting of 5 compounds, was found to be genotoxic, only for HepG2. Data obtained with individual compounds showed that only two compounds from the mixture, namely fludioxonil and cyprodinil, were genotoxic. A mixture effect was observed at low doses. The screening of additional pesticides selected from the co-exposure characterization allowed to identify a family of pesticides (methyl-pyrazoles) able to induce DNA double strand break on two human cell lines (Jurkat/SH-SY5Y)

Evidence of the in vitro genotoxicity of methyl-pyrazole pesticides in human cells.

Consumers are exposed daily to several pesticide residues in food, which can be of potential concern for human health. Based on a previous study dealing with exposure of the French population to pesticide residues via the food, we selected 14 pesticides frequently found in foodstuffs, on the basis of their persistence in the environment or their bioaccumulation in the food chain. In a first step, the objective of this study was to investigate if the 14 selected pesticides were potentially cytotoxic and genotoxic. For this purpose, we used a new and sensitive genotoxicity assay (the γH2AX test, involving phosphorylation of histone H2AX) with four human cell lines (ACHN, SH-SY5Y, LS-174T and HepG2), each originating from a potential target tissue of food contaminants (kidney, nervous system, colon, and liver, respectively). Tebufenpyrad was the only compound identified as genotoxic and the effect was only observed in the SH-SY5Y neuroblastoma cell-line. A time-course study showed that DNA damage appeared early after treatment (1h), suggesting that oxidative stress could be responsible for the induction of γH2AX. In a second step, three other pesticides were studied, i.e. bixafen, fenpyroximate and tolfenpyrad, which - like tebufenpad - also had a methyl-pyrazole structure. All these compounds demonstrated genotoxic activity in SH-SY5Y cells at low concentration (nanomolar range). Complementary experiments demonstrated that the same compounds show genotoxicity in a human T-cell leukemia cell line (Jurkat). Moreover, we observed an increased production of reactive oxygen species in Jurkat cells in the presence of the four methyl-pyrazoles. These results demonstrate that tebufenpyrad, bixafen, fenpyroximat and tolfenpyrad induce DNA damage in human cell lines, very likely by a mode of action that involves oxidative stress. Nonetheless, additional in vivo data are required before a definitive conclusion can be drawn regarding hazard prediction to humans

Use of the γH2AX assay for assessing the genotoxic effects of pesticide mixtures present in the French diet

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Genotoxicity of cytolethal distending toxin (cdt) on isogenic human colorectal cell lines: potential promoting effects for colorectal carcinogenesis

The composition of the human microbiota influences tumorigenesis, notably in colorectal cancer (CRC). Pathogenic Escherichlacoli possesses a variety of virulent factors, among them the Cytolethal Distending Toxin (CDT). CDT displays dual DNase and phosphatase activities and induces DNA double strand breaks, cell cycle arrest and apoptosis in a broad range of mammalian cells. As CDT could promote malignant transformation, we investigated the cellular outcomes induced by acute and chronic exposures to E. coli CDT in normal human colon epithelial cells (HCECs). Moreover, we conducted a comparative study between isogenic derivatives cell lines of the normal HCECs in order to mimic the mutation of three major genes found in CRC genetic models: APC, KRAS, and TP53. Our results demonstrate that APC and p53 deficient cells showed impaired DNA damage response after CDT exposure, whereas HCECs expressing oncogenic KRAS(V12) were more resistant to CDT. Compared to normal HCECs, the precancerous derivatives exhibit hallmarks of malignant transformation after a chronic exposure to CDT. HCECs defective in APC and p53 showed enhanced anchorage independent growth and genetic instability, assessed by the micronucleus formation assay. In contrast, the ability to grow independently of anchorage was not impacted by CDT chronic exposure in KRAS(V12) HCECs, but micronucleus formation is dramatically increased. Thus, CDT does not initiate CRC by itself, but may have promoting effects in premalignant HCECs, involving different mechanisms in function of the genetic alterations associated to CRC

Genotoxic activities of the food contaminant 5-hydroxymethylfurfural using different in vitro bioassays

WOS: 000274743300016International audience5-Hydroxymethylfurfural (5-HMF) is known as an indicator of quality deterioration in a wide range of foods. 5-HMF is formed as an intermediate in the Maillard reaction and has been identified in a wide variety of heat-processed foods. In recent years, the presence of 5-HMF in foods has raised toxicological concerns: data have shown cytotoxic, genotoxic and tumoral effects but further studies suggest that 5-HMF does not pose a serious health risk. However the subject is still a matter of debate. We investigated the genotoxicity of the food-borne contaminant 5-HMF using the Ames test, the micronucleus (MN) and the single-cell gel electrophoresis (SCGE) assays in the human metabolically active HepG2 cell line. Cytotoxic effect of 5-HMF was first assessed using Alamar Blue (TM) as a sensitive sub-lethal assay. 5-HMF did not induce any genic mutation in bacteria whatever the concentration in the Ames test. Furthermore, it does not induce clastogenic or aneugenic effects in the HepG2 cells. In contrast, 5-HMF induced HepG2 DNA damage at concentrations from 7.87 to 25 mM in the comet assay suggesting a weak genotoxic effect of 5-HMF in the HepG2 cells probably repaired. (C) 2009 Elsevier Ireland Ltd. All rights reserved

Identification of the main pesticide residue mixtures to which the French population is exposed

Owing to the intensive use of pesticides and their potential persistence in the environment, various pesticide residues can be found in the diet. Consumers are therefore exposed to complex pesticide mixtures which may have combined adverse effects on human health. By modelling food exposure to multiple pesticides, this paper aims to determine the main mixtures to which the general population is exposed in France. Dietary exposure of 3337 individuals from the INCA2 French national consumption survey was assessed for 79 pesticide residues, based on results of the 2006 French food monitoring programmes. Individuals were divided into groups with similar patterns of co-exposure using the clustering ability of a Bayesian nonparametric model. In the 5 groups of individuals with the highest exposure, mixtures are formed by pairs of pesticides with correlations above 0.7. Seven mixtures of 2-6 pesticides each were characterised. We identified the commodities that contributed the most to exposure. Pesticide mixtures can either be components of a single plant protection product applied together on the same crop or be from separate products that are consumed together during a meal. Of the 25 pesticides forming the mixtures, two-DDT and Dieldrin are known persistent organic pollutants. The approach developed is generic and can be applied to all types of substances found in the diet in order to characterise the mixtures that should be studied first because of their adverse effects on health. (C) 2013 Elsevier Inc. All rights reserved

Comparative genotoxic potential of 27 polycyclic aromatic hydrocarbons in three human cell lines

International audiencePolycyclic Aromatic Hydrocarbons (PAHs) form a family of compounds that are generally found in complex mixtures. PAHs can lead to the development of carcinogenesis. The Toxicity Equivalent Factor (TEF) approach has been suggested for estimating the toxicity of PAHs, however, due to the relative weakness of available data, TEF have not been applied for the risk characterization of PAHs as food contaminants in Europe. The determination of new TEFs for a large number of PAHs could overcome some limitations of the current method and improve cancer risk assessment. The present investigation aimed at deriving new TEFs for PAHs, based on their genotoxic effect measured in vitro and analyzed with mathematical models. For this purpose, we used a genotoxicity assay (gamma H2AX) with three human cell lines to analyze the genotoxic properties of 27 selected PAHs after 24 h treatment. For 11 compounds, we did not detect any genotoxic potential. For the remaining 16 PAHs, the concentration-response for genotoxic effect was modelled with the Hill equation; equivalency between PAHs at low dose was assessed by applying constraints to the model parameters. We developed for each compound, in each cell line, Genotoxic Equivalent Factor (GEF). Calculated GEF for the tested PAHs were similar in all cell lines and generally higher than the TEF usually used. These new equivalent factors for PAHs should improve cancer risk assessment

Genotoxic effect of polycyclic aromatic hydrocarbons alone or in mixture

Polycyclic Aromatic Hydrocarbons (PAHs) are a family of more than one hundred compounds and are potentially carcinogenic to humans. Hazard characterization of PAH complex mixtures is currently based on Toxicity Equivalent Factors (TEFs). However, this method present some limits and it is relevant to define new TEFs for a large number of PAH in order to improve cancer risk assessment for humans. In this study, we used a new genotoxic assay (based on gH2AX quantification) to analyze the genotoxicity of 27 PAHs on three human cell lines representative of major target organs of PAHs: liver (Hep3B), colon (LS-174T) and lung (NCI-H358). Mathematical models were used to determine new TEFs values for PAHs. Dose-genotoxic response modelling was performed using a Hill model, with a focus on effects equivalency between PAHs at low dose. We observed a significant dose-response of genotoxicity in the three cell lines tested for 16 tested compounds and developed new TEFs that we named GEF for Genotoxic Equivalent Factor. Calculated GEFs for the tested PAHs were generally higher than usually utilized TEFs. We further explored the effect of PAH mixtures by testing 16 binary mixtures on LS-174T cells. In some cases, antagonistic, potentiation or synergic actions, or a variation of the maximum response were observed in relation to concentration addition. To confirm these interactions, DNA adducts quantification and metabolic studies are under investigation. Finally, more complex mixtures of PAHs based on 4, 8 or 16 compounds were tested and the genotoxic effect of these mixtures was predicted with concentration addition. In this study, we proposed new TEFs for PAHs based on their genotoxic effects on human cells investigated at low doses. Moreover, we developed mathematical models to predict PAH mixtures genotoxic effect. This method will be useful to improve cancer risk assessment for humans exposed to PAHs

The PERICLES research program : an original approach to characterize thecombined effects of the main pesticide residue mixtures to which the Frenchpopulation is exposed

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The PERICLES research program: An integrated approach to characterize the combined effects of mixtures of pesticide residues to which the French population is exposed.

Due to the broad spectrum of pesticide usages, consumers are exposed to mixtures of residues, which may have combined effects on human health. The PERICLES research program aims to test the potential combined effects of pesticide mixtures, which are likely to occur through dietary exposure. The co-exposure of the French general population to 79 pesticide residues present in the diet was first assessed. A Bayesian nonparametric model was then applied to define the main mixtures to which the French general population is simultaneously and most heavily exposed. Seven mixtures made of two to six pesticides were identified from the exposure assessment. An in vitro approach was used for investigating the toxicological effects of these mixtures and their corresponding individual compounds, using a panel of cellular models, i.e. primary rat and human hepatocytes, liver, intestine, kidney, colon and brain human cell lines. A set of cell functions and corresponding end-points were monitored such as cytotoxicity, real-time cell impedance, genotoxicity, oxidative stress, apoptosis and PXR nuclear receptor transactivation. The mixtures were tested in equimolar concentrations. Among the seven mixtures, two appeared highly cytotoxic, five activated PXR and depending on the assay one or two were genotoxic. In some experiments, the mixture effect was quantitatively different from the effect expected from the addition concept. The PERICLES program shows that, for the most pesticides mixtures to which the French general population is exposed, the toxic effects observed on human cells cannot be easily predicted based on the toxic potential of each compound. Consequently, additional studies should be carried on in order to more accurately define the mixtures of chemicals to which the consumers are exposed, as well as to improve the investigation, prediction and monitoring of their potential human health effects