Effect of cell treatment procedures on in vitro genotoxicity assessment.

So far, the majority of in vitro toxicological experiments are conducted after an acute 24 h treatment that does not represent a realistic human chemical exposure. Recently, new in vitro approaches have been proposed to study the chemical toxicological effect over several days in order to be more predictive of a representative exposure scenario. In this study, we investigated the genotoxic potential of chemicals (direct or bioactived clastogen, aneugen and apoptotic inducer) with the γH2AX and pH3 biomarkers, in the human liver-derived HepaRP cell line. We used different treatment durations, with or without a three-day recovery stage (release period), before genotoxicity measurement. Data were analysed with the Benchmark Dose approach. We observed that the detection of clastogenic compounds (notably for DNA damaging agents) was more sensitive after three days of repeated treatment compared to one or three treatments over 24 h. In contrast, aneugenic chemicals were detected as genotoxic in a similar manner whether after a 24 h exposure or a three-day repeated treatment. Globally, the release period decreases the genotoxicity measurement substantially. For DNA damaging agents, after high concentration treatments, γH2AX induction was always observed after a three-day release period. In contrast, for DNA topoisomerase inhibitors, no effect could be observed after the release period. In conclusion, in the HepaRP cell line, there are some important differences between a one-day acute and a three-day repeated treatment protocol, indicating that different cell treatment procedures may differentiate chemical genotoxic mechanisms of action more efficiently.

A 90-day oral exposure to food-grade gold at relevant human doses impacts the gut microbiota and the local immune system in a sex-dependent manner in mice.

BACKGROUND: Edible gold (Au) is commonly used as a food additive (E175 in EU) for confectionery and cake decorations, coatings and in beverages. Food-grade gold is most often composed of thin Au sheets or flakes exhibiting micro- and nanometric dimensions in their thickness. Concerns about the impact of mineral particles used as food additives on human health are increasing with respect to the particular physico-chemical properties of nanosized particles, which enable them to cross biological barriers and interact with various body cell compartments. In this study, male and female mice were exposed daily to E175 or an Au nanomaterial (Ref-Au) incorporated into food at relevant human dose for 90 days in order to determine the potential toxicity of edible gold. RESULTS: E175 or Ref-Au exposure in mice did not induce any histomorphological damage of the liver, spleen or intestine, nor any genotoxic effects in the colon and liver despite an apparent higher intestinal absorption level of Au particles in mice exposed to Ref-Au compared to the E175 food additive. No changes in the intestinal microbiota were reported after treatment with Ref-Au, regardless of sex. In contrast, after E175 exposure, an increase in the Firmicutes/Bacteroidetes ratio and in the abundance of Proteobacteria were observed in females, while a decrease in the production of short-chain fatty acids occurred in both sexes. Moreover, increased production of IL-6, TNFα and IL-1ß was observed in the colon of female mice at the end of the 90-day exposure to E175, whereas, decreased IL-6, IL-1ß, IL-17 and TGFß levels were found in the male colon. CONCLUSIONS: These results revealed that a 90-day exposure to E175 added to the diet alters the gut microbiota and intestinal immune response in a sex-dependent manner in mice. Within the dose range of human exposure to E175, these alterations remained low in both sexes and mostly appeared to be nontoxic. However, at the higher dose, the observed gut dysbiosis and the intestinal low-grade inflammation in female mice could favour the occurrence of metabolic disorders supporting the establishment of toxic reference values for the safe use of gold as food additive.

Synergic toxic effects of food contaminant mixtures in human cells.

Humans are exposed to multiple exogenous substances, notably through food consumption. Many of these compounds are suspected to impact human health, and their combination could exacerbate their harmful effects. We previously observed in human cells that, among the six most prevalent food contaminant complex mixtures identified in the French diet, synergistic interactions between component appeared in two mixtures compared with the response with the chemicals alone. In the present study, we demonstrated in human cells that these properties are driven only by two heavy metals in each mixture: tellurium (Te) with cadmium (Cd) and Cd with inorganic arsenic (As), respectively. It appeared that the predicted effects for these binary mixtures using the mathematical model of Chou and Talalay confirmed synergism between these heavy metals. Based on different cell biology experiments (cytotoxicity, genotoxicity, mutagenesis and DNA repair inhibition experiments), a detailed mechanistic analysis of these two mixtures suggests that concomitant induction of oxidative DNA damage and decrease of their repair capacity contribute to the synergistic toxic effect of these chemical mixtures. Overall, these results may have broad implications for the fields of environmental toxicology and chemical mixture risk assessment.

Validation of the γH2AX biomarker for genotoxicity assessment: a review.

The H2AX histone protein is rapidly phosphorylated at the serine-139 position (γH2AX) in response to a broad range of DNA lesions. γH2AX induction is one of the earliest events in the DNA damage response (DDR) and plays a central role in sensing and repairing DNA damage. Since its discovery, measuring γH2AX formation using numerous methods in in vitro and in vivo experiments has been an attractive endpoint for the detection of genotoxic agents. Our review focuses on validation studies performed using this biomarker to detect the genotoxicity of model chemicals using different methods. To date, nearly two hundred genotoxic and carcinogenic model chemicals have been shown to induce in vitro γH2AX in different cell lines by numerous laboratories. Based on 27 published reports comprising 329 tested chemicals, we compared the performance of the γH2AX assay with other genotoxic endpoints (Ames assay, micronucleus, HPRT and comet) regularly used for in vitro genotoxicity assessment. Notably, the γH2AX assay performs well (91% predictivity) and efficiently differentiates aneugenic and clastogenic compounds when coupled with the pH3 biomarker. Currently, no formal guidelines have been approved for the γH2AX assay for regular genotoxicity studies, but we suggest the γH2AX biomarker could be used as a new standard genotoxicity assay and discuss its future role in genotoxicity risk assessment.

Role of human sulfotransferase 1A1 and N-acetyltransferase 2 in the metabolic activation of 16 heterocyclic amines and related heterocyclics to genotoxicants in recombinant V79 cells.

Heterocyclic aromatic amines (HAAs) are primarily produced during the heating of meat or fish. HAAs are mutagenic and carcinogenic, and their toxicity in model systems depend on metabolic activation. This activation is mediated by cytochrome P450 (CYP) enzymes, in particular CYP1A2. Some studies have indicated a role of human sulfotransferase (SULT) 1A1 and N-acetyltransferase (NAT) 2 in the terminal activation of HAAs. In this study, we conducted a metabolism/genotoxicity relationship analysis for 16 HAAs and related heterocyclics. We used the γH2AX genotoxicity assay in V79 cells (deficient in CYP, SULT and NAT) and V79-derived cell lines genetically engineered to express human CYP1A2 alone or in combination with human SULT1A1 or NAT2. Our data demonstrated genotoxic properties for 13 out of the 16 compounds tested. A clear relationship between metabolic bioactivation and genotoxicity allowed to distinguish four groups: (1) Trp-P-1 genotoxicity was linked to CYP1A2 bioactivation only-with negligible effects of phase II enzymes; (2) Glu-P-2, Glu-P-1, Trp-P-2, APNH, MeAαC and AαC were bioactivated by CYP1A2 in combination with either phase II enzyme tested (NAT2 or SULT1A1); (3) IQ, 4-MeIQ, IQx, 8-MeIQx, and 4,8-DiMeIQx required CYP1A2 in combination with NAT2 to be genotoxic, whereas SULT1A1 did not enhance their genotoxicity; (4) PhIP became genotoxic after CYP1A2 and SULT1A1 bioactivation-NAT2 had not effect. Our results corroborate some previous data regarding the genotoxic potency of seven HAAs and established the genotoxicity mechanism for five others HAAs. This study also permits to compare efficiently the genotoxic potential of these 13 HAAs.

Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening.

In a previous study, we validated an in vitro genotoxicity assay based on γH2AX quantification using the In-Cell Western (ICW) method in HepG2 cells. The assay demonstrated high sensitivity and specificity but failed to detect genotoxicity for few compounds that require specific metabolic bioactivation not sufficiently covered by HepG2 cells. The aim of the present study was to assess γH2AX ICW sensitivity using a broader range of genotoxic molecules with HepG2 cells and three additional human cell lines with distinct biotransformation properties: two cell lines expressing some phase I and II bioactivation capabilities (LS-174T and Hep3B), and one with poor general bioactivation properties (ACHN). We evaluated the four cell lines by testing 24 compounds recommended by European Centre for the Validation of Alternative Methods and a set of 24 additional chemicals with different mode of genotoxic action (MOA) (aneugenicity, DNA adducts formation, induction of oxidative stress), including some known to require specific cytochrome P450 metabolic bioactivation. Results for the 48 compounds tested showed that the γH2AX ICW assay was more sensitive with LS-174T and HepG2 cells than with Hep3B or ACHN cell lines. Among the 38 compounds tested with positive or equivocal carcinogenicity data, 36 (95%) showed a positive genotoxic response with the γH2AX ICW assay compared to only 27 (71%) using the Ames assay. We confirm that the γH2AX ICW assay on HepG2 cells, without an exogenous metabolic activation system, may be a suitable test to predict the in vivo genotoxicity of chemicals with different genotoxic MOA. Moreover, the use of the ACHN cell line in combination with LS-174T and HepG2 cells may permit in many cases to discriminate direct from bioactivated genotoxins. Overall, our results confirm the high sensitivity of the γH2AX ICW assay which, in turn, should reduce the number of animals used for genotoxicity assessment.

Evaluation of genotoxicity using automated detection of γH2AX in metabolically competent HepaRG cells.

The in situ detection of γH2AX was recently reported to be a promising biomarker of genotoxicity. In addition, the human HepaRG hepatoma cells appear to be relevant for investigating hepatic genotoxicity since they express most of drug metabolizing enzymes and a wild type p53. The aim of this study was to determine whether the automated in situ detection of γH2AX positive HepaRG cells could be relevant for evaluation of genotoxicity after single or long-term repeated in vitro exposure compared to micronucleus assay. Metabolically competent HepaRG cells were treated daily with environmental contaminants and genotoxicity was evaluated after 1, 7 and 14 days. Using these cells, we confirmed the genotoxicity of aflatoxin B1 and benzo(a)pyrene and demonstrated that dimethylbenzanthracene, fipronil and endosulfan previously found genotoxic with comet or micronucleus assays also induced γH2AX phosphorylation. Furthermore, we showed that fluoranthene and bisphenol A induced γH2AX while no effect had been previously reported in HepG2 cells. In addition, induction of γH2AX was observed with some compounds only after 7 days, highlighting the importance of studying long-term effects of low doses of contaminants. Together, our data demonstrate that automated γH2AX detection in metabolically competent HepaRG cells is a suitable high-through put genotoxicity screening assay.

Complementarity of phosphorylated histones H2AX and H3 quantification in different cell lines for genotoxicity screening.

The in vitro micronucleus assay is broadly used, but is not per se able to discriminate aneugenic from clastogenic compounds, and cytotoxicity can be a confounding factor. In vitro genotoxicity assays generally rely on cell lines with limited metabolic capabilities. Recently, the use of histone H2AX and H3 phosphorylation markers (γH2AX and p-H3) was proposed to discriminate aneugenic from clastogenic chemicals. The aim of the present study was to develop a new genotoxic screening strategy based on the use of the γH2AX and p-H3 biomarkers in combination with cell lines with distinct biotransformation properties. First, we tested a training set of 20 model chemicals comprised of 10 aneugens, five clastogens and five cytotoxics on three human cell lines (HepG2, LS-174T and ACHN). Our data confirm the robustness of these two biomarkers to discriminate efficiently clastogens, aneugens and misleading cytotoxic chemicals in HepG2 cells. Aneugenic compounds induced either an increase or a decrease in p-H3 depending on their mode of action. Clastogens induced γH2AX, and cytotoxic compounds generated a marked decrease in these two biomarkers. Moreover, the use of different cell lines permits to discriminate direct from bioactivated genotoxins without the need of an exogenous metabolic activation system. Finally, we further evaluated this strategy using a test set of 13 chemicals with controversial genotoxic potential. The resulting data demonstrate that the combined analysis of γH2AX and p-H3 is an efficient strategy. Notably, we demonstrated that three compounds (fisetin, hydroquinone and okadaic acid) display both aneugenic and clastogenic properties.

Interplay between siderophores and colibactin genotoxin biosynthetic pathways in Escherichia coli.

In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4'-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies.

Human Next-Generation Risk Assessment of Trichothecene Toxicity.

Trichothecenes are naturally occurring chemicals, produced by fungi, that can be found in contaminated crops. Trichothecenes have the potential to indirectly damage DNA and exacerbate genotoxic effects of genotoxicants. However, genotoxicity data for most trichothecenes are limited and data gaps remain. Here we use the γH2AX/pH3 assay to evaluate DNA damage in vitro of 13 trichothecenes. Three human cell lines (SH-SY5Y, ACHN, and HepG2) were exposed to each trichothecene (0.001-100 µM) to assess toxicity as models for the brain, kidney, and liver, respectively. Concentration-dependent induction of DNA damage, illustrated by γH2AX induction, was observed for all trichothecenes. In vitro-in vivo extrapolation (IVIVE) modeling was employed to support in vivo equivalent potency ranking and screen for risk potential. Diacetoxyscirpenol, T-2, and HT-2 had the highest genotoxic potency, notably in SH-SY5Y cells. Administered equivalent doses (AEDs) derived from IVIVE were compared against exposure data from French total diet studies to assess risk potential. AEDs derived for T-2 and HT-2 from the SH-SY5Y model were within 100-fold of exposure levels for infants aged one year or less. Overall, the potential for trichothecenes to damage DNA and higher exposures in infants highlights the need to investigate the cumulative effects across the broader trichothecene family.

Assessment of the performance of the Ames MPF™ assay: A multicenter collaborative study with six coded chemicals.

The Ames MPF™ is a miniaturized, microplate fluctuation format of the Ames test. It is a standardized, commercially available product which can be used to assess mutagenicity in Salmonella and E. coli strains in 384-well plates using a color change-based readout. Several peer-reviewed comparisons of the Ames MPF™ to the Ames test in Petri dishes confirmed its suitability to evaluate the mutagenic potential of a variety of test items. An international multicenter study involving seven laboratories tested six coded chemicals with this assay using five bacterial strains, as recommended by the OECD test guideline 471. The data generated by the participating laboratories was in excellent agreement (93%), and the similarity of their dose response curves, as analyzed with sophisticated statistical approaches further confirmed the suitability of the Ames MPF™ assay as an alternative to the Ames test on agar plates, but with advantages with respect to significantly reduced amount of test substance and S9 requirements, speed, hands-on time and, potentially automation.

Quantitative in vitro to in vivo extrapolation of genotoxicity data provides protective estimates of in vivo dose.

Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, and aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points-of-departures (PODs) for setting exposure limits. The more recent higher-throughput in vitro genotoxicity assays, many of which also provide mechanistic information, offer a powerful approach for determining defined PODs for potency ranking and risk assessment. In order to obtain relevant human dose context from the in vitro assays, in vitro to in vivo extrapolation (IVIVE) models are required to determine what dose would elicit a concentration in the body demonstrated to be genotoxic using in vitro assays. Previous work has demonstrated that application of IVIVE models to in vitro bioactivity data can provide PODs that are protective of human health, but there has been no evaluation of how these models perform with in vitro genotoxicity data. Thus, the Genetic Toxicology Technical Committee, under the Health and Environmental Sciences Institute, conducted a case study on 31 reference chemicals to evaluate the performance of IVIVE application to genotoxicity data. The results demonstrate that for most chemicals considered here (20/31), the PODs derived from in vitro data and IVIVE are health protective relative to in vivo PODs from animal studies. PODs were also protective by assay target: mutations (8/13 chemicals), micronuclei (9/12), and aneugenicity markers (4/4). It is envisioned that this novel testing strategy could enhance prioritization, rapid screening, and risk assessment of genotoxic chemicals.

Innovative tools and methods for toxicity testing within PARC work package 5 on hazard assessment.

New approach methodologies (NAMs) have the potential to become a major component of regulatory risk assessment, however, their actual implementation is challenging. The European Partnership for the Assessment of Risks from Chemicals (PARC) was designed to address many of the challenges that exist for the development and implementation of NAMs in modern chemical risk assessment. PARC's proximity to national and European regulatory agencies is envisioned to ensure that all the research and innovation projects that are initiated within PARC agree with actual regulatory needs. One of the main aims of PARC is to develop innovative methodologies that will directly aid chemical hazard identification, risk assessment, and regulation/policy. This will facilitate the development of NAMs for use in risk assessment, as well as the transition from an endpoint-based animal testing strategy to a more mechanistic-based NAMs testing strategy, as foreseen by the Tox21 and the EU Chemical's Strategy for Sustainability. This work falls under work package 5 (WP5) of the PARC initiative. There are three different tasks within WP5, and this paper is a general overview of the five main projects in the Task 5.2 'Innovative Tools and methods for Toxicity Testing,' with a focus on Human Health. This task will bridge essential regulatory data gaps pertaining to the assessment of toxicological prioritized endpoints such as non-genotoxic carcinogenicity, immunotoxicity, endocrine disruption (mainly thyroid), metabolic disruption, and (developmental and adult) neurotoxicity, thereby leveraging OECD's and PARC's AOP frameworks. This is intended to provide regulatory risk assessors and industry stakeholders with relevant, affordable and reliable assessment tools that will ultimately contribute to the application of next-generation risk assessment (NGRA) in Europe and worldwide.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

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.

AOP report: Development of an adverse outcome pathway for oxidative DNA damage leading to mutations and chromosomal aberrations.

The Genetic Toxicology Technical Committee (GTTC) of the Health and Environmental Sciences Institute (HESI) is developing adverse outcome pathways (AOPs) that describe modes of action leading to potentially heritable genomic damage. The goal was to enhance the use of mechanistic information in genotoxicity assessment by building empirical support for the relationships between relevant molecular initiating events (MIEs) and regulatory endpoints in genetic toxicology. Herein, we present an AOP network that links oxidative DNA damage to two adverse outcomes (AOs): mutations and chromosomal aberrations. We collected empirical evidence from the literature to evaluate the key event relationships between the MIE and the AOs, and assessed the weight of evidence using the modified Bradford-Hill criteria for causality. Oxidative DNA damage is constantly induced and repaired in cells given the ubiquitous presence of reactive oxygen species and free radicals. However, xenobiotic exposures may increase damage above baseline levels through a variety of mechanisms and overwhelm DNA repair and endogenous antioxidant capacity. Unrepaired oxidative DNA base damage can lead to base substitutions during replication and, along with repair intermediates, can also cause DNA strand breaks that can lead to mutations and chromosomal aberrations if not repaired adequately. This AOP network identifies knowledge gaps that could be filled by targeted studies designed to better define the quantitative relationships between key events, which could be leveraged for quantitative chemical safety assessment. We anticipate that this AOP network will provide the building blocks for additional genotoxicity-associated AOPs and aid in designing novel integrated testing approaches for genotoxicity.

Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project.

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union's Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising 'real-life' chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children's development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.

Use of the γH2AX assay for assessing the genotoxicity of bisphenol A and bisphenol F in human cell lines.

Bisphenol A (BPA) and bisphenol F (BPF) are widely used to manufacture plastics and epoxy resins. Both compounds have been shown to be present in the environment and are food contaminants, with, as a result, a low but chronic exposure of humans. However, the fate and possible bioactivation of these compounds at the level of human cell lines was not completely elucidated yet. In this study, we investigated the ability of human cells (intestinal cell line: LS174T, hepatoma cell line: HepG2, and renal cell line: ACHN) to biotransform BPA and BPF, and focused on the cytotoxicity and genotoxicity of these two bisphenols, through the use of a novel and efficient genotoxic assay based on the detection of histone H2AX phosphorylation. BPA and BPF were extensively metabolized in HepG2 and LS174T cell lines, with stronger biotransformation capabilities in intestinal cells than observed in liver cells. Both cell lines produced the glucuronide as well as the sulfate conjugates of BPA. Conversely, the ACHN cell line was found to be devoid of any metabolic capabilities for the two examined bisphenols. Cytotoxicity was tested for BPA, BPF, as well as one metabolite of BPF produced in vivo in rat, namely dihydroxybenzophenone (DHB). In the three cell lines used, we observed similar ranges of toxicity, with DHB being weakly cytotoxic, BPF exhibiting an intermediary cytotoxicity, and BPA being the most cytotoxic compound tested. BPA and DHB were not found to be genotoxic, whatever the cell line examined. BPF was clearly genotoxic in HepG2 cells. These results demonstrate that some human cell lines extensively metabolize bisphenols and establish the genotoxic potential of bisphenol F.

Cutting edge: Multiple sclerosis-like lesions induced by effector CD8 T cells recognizing a sequestered antigen on oligodendrocytes.

CD8 T cells are emerging as important players in multiple sclerosis (MS) pathogenesis, although their direct contribution to tissue damage is still debated. To assess whether autoreactive CD8 T cells can contribute to the pronounced loss of oligodendrocytes observed in MS plaques, we generated mice in which the model Ag influenza hemagglutinin is selectively expressed in oligodendrocytes. Transfer of preactivated hemagglutinin-specific CD8 T cells led to inflammatory lesions in the optic nerve, spinal cord, and brain. These lesions, associating CD8 T cell infiltration with focal loss of oligodendrocytes, demyelination, and microglia activation, were very reminiscent of active MS lesions. Thus, our study demonstrates the potential of CD8 T cells to induce oligodendrocyte lysis in vivo as a likely consequence of direct Ag-recognition. These results provide new insights with regard to CNS tissue damage mediated by CD8 T cells and for understanding the role of CD8 T cells in MS.

Evaluation of the genotoxic potential of apoptosis inducers with the γH2AX assay in human cells.

Human risk assessment of genotoxic chemicals is an important area of research. However, the specificity of in vitro mammalian genotoxicity assays is sometime low, as they yield to misleading positive results that are not observe in in vivo studies. Apoptosis can be a confounding factor in the interpretation of the results. Recently, a new strategy for genotoxicity screening, based on the combined analysis of phosphorylated histones H2AX (γH2AX) and H3 (pH3), was proposed to discriminate efficiently aneugenic from clastogenic compounds. However, γH2AX biomarker could also be induce by apoptosis. The aim of the present study was to investigate the specificity of this genotoxic biomarker. For this purpose, we analyzed 26 compounds inducing apoptosis by different mechanism of action, with the γH2AX assay in three human cell lines after 24 h treatment. Most of the tested chemicals were negative in the assay, whatever the cell line tested. The few compounds that generated positive data have also been report positive in other genotoxicity assays. The data presented here demonstrate that the γH2AX assay is not vulnerable to the generation of misleading positive results by apoptosis inducers. Currently, no formal guidelines have been approve for the γH2AX assay for regular genotoxicity studies, but we suggest that this biomarker could be used as a new standard genotoxicity assay.