Marc Audebert

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.

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.

A central role for heme iron in colon carcinogenesis associated with red meat intake

Epidemiology shows that red and processed meat intake is associated with an increased risk of colorectal cancer. Heme iron, heterocyclic amines, and endogenous N-nitroso compounds (NOC) are proposed to explain this effect, but their relative contribution is unknown. Our study aimed at determining, at nutritional doses, which is the main factor involved and proposing a mechanism of cancer promotion by red meat. The relative part of heme iron (1% in diet), heterocyclic amines (PhIP + MeIQx, 50 + 25 μg/kg in diet), and NOC (induced by NaNO₂+ NaNO₂; 0.17 + 0.23 g/L of drinking water) was determined by a factorial design and preneoplastic endpoints in chemically induced rats and validated on tumors in Min mice. The molecular mechanisms (genotoxicity, cytotoxicity) were analyzed in vitro in normal and Apc-deficient cell lines and confirmed on colon mucosa. Heme iron increased the number of preneoplastic lesions, but dietary heterocyclic amines and NOC had no effect on carcinogenesis in rats. Dietary hemoglobin increased tumor load in Min mice (control diet: 67 ± 39 mm²; 2.5% hemoglobin diet: 114 ± 47 mm², P = 0.004). In vitro, fecal water from rats given hemoglobin was rich in aldehydes and was cytotoxic to normal cells, but not to premalignant cells. The aldehydes 4-hydroxynonenal and 4-hydroxyhexenal were more toxic to normal versus mutated cells and were only genotoxic to normal cells. Genotoxicity was also observed in colon mucosa of mice given hemoglobin. These results highlight the role of heme iron in the promotion of colon cancer by red meat and suggest that heme iron could initiate carcinogenesis through lipid peroxidation. .

Calcium and α-tocopherol suppress cured-meat promotion of chemically induced colon carcinogenesis in rats and reduce associated biomarkers in human volunteers

BACKGROUND Processed meat intake has been associated with increased colorectal cancer risk. We have shown that cured meat promotes carcinogen-induced preneoplastic lesions and increases specific biomarkers in the colon of rats. OBJECTIVES We investigated whether cured meat modulates biomarkers of cancer risk in human volunteers and whether specific agents can suppress cured meat-induced preneoplastic lesions in rats and associated biomarkers in rats and humans. DESIGN Six additives (calcium carbonate, inulin, rutin, carnosol, α-tocopherol, and trisodium pyrophosphate) were added to cured meat given to groups of rats for 14 d, and fecal biomarkers were measured. On the basis of these results, calcium and tocopherol were kept for the following additional experiments: cured meat, with or without calcium or tocopherol, was given to dimethylhydrazine-initiated rats (47% meat diet for 100 d) and to human volunteers in a crossover study (180 g/d for 4 d). Rat colons were scored for mucin-depleted foci, putative precancer lesions. Biomarkers of nitrosation, lipoperoxidation, and cytotoxicity were measured in the urine and feces of rats and volunteers. RESULTS Cured meat increased nitroso compounds and lipoperoxidation in human stools (both P < 0.05). Calcium normalized both biomarkers in rats and human feces, whereas tocopherol only decreased nitro compounds in rats and lipoperoxidation in feces of volunteers (all P < 0.05). Last, calcium and tocopherol reduced the number of mucin-depleted foci per colon in rats compared with nonsupplemented cured meat (P = 0.01). CONCLUSION Data suggest that the addition of calcium carbonate to the diet or α-tocopherol to cured meat may reduce colorectal cancer risk associated with cured-meat intake. This trial was registered at clinicaltrials.gov as NCT00994526.

Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

In vitro genotoxicity tests used in regulatory toxicology studies are sensitive, but the occurrence of irrelevant positive results is high compared with carcinogenicity studies in rodents. Current in vitro genotoxicity tests are also often limited by relatively low throughput. The aim of this study was to validate an in vitro genotoxic assay in a 96‐well plate format that allows the simultaneous examination of cytotoxicity and genotoxicity. The test is based on the quantification of the phosphorylation of the histone H2AX (γH2AX), which reflects a global genotoxic insult, using the In‐Cell Western technique. The assay was evaluated on HepG2 cells by testing a list of 61 compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), whose genotoxic potential has already been characterized. The γH2AX assay on HepG2 cell line was highly sensitive: 75% of the genotoxic compounds gave a positive result, and specific: 90–100% of nongenotoxic compounds gave negative results. Compared with the micronucleus genotoxicity assay using the same cell line and test compounds, the γH2AX assay was more sensitive and specific. In sum, the high‐throughput γH2AX assay described here can accurately detect simultaneously the genotoxic and the cytotoxic potential of compounds with different modes of mutagenic action, notably those who required metabolic activation. The use of this assay in the early discovery phase of drug development may prove to be a valuable way to assess the genotoxic potential of xenobiotics. Environ. Mol. Mutagen. 54:737–746, 2013.

Comparative potency approach based on H2AX assay for estimating the genotoxicity of polycyclic aromatic hydrocarbons

Polycyclic Aromatic Hydrocarbons (PAHs) constitute a family of over one hundred compounds and can generally be found in complex mixtures. PAHs metabolites cause DNA damage which can lead to the development of carcinogenesis. Toxicity assessment of PAH complex mixtures is currently expressed in terms of toxic equivalents, based on Toxicity Equivalent Factors (TEFs). However, the definition of new TEFs for a large number of PAH could overcome some limitations of the current method and improve cancer risk assessment. The current investigation aimed at deriving the relative potency factors of PAHs, based on their genotoxic effect measured in vitro and analyzed with mathematical models. For this purpose, we used a new genotoxic assay (γH2AX) with two human cell lines (HepG2 and LS-174T) to analyze the genotoxic properties of 13 selected PAHs at low doses after 24h treatment. The dose-response for genotoxic effects was modeled with a Hill model; equivalency between PAHs at low dose was assessed by applying constraints to the model parameters. In the two cell lines tested, we observed a clear dose-response for genotoxic effects for 11 tested compounds. LS-174T was on average ten times more sensitive than HepG2 towards PAHs regarding genotoxicity. We developed new TEFs, which we named Genotoxic Equivalent Factor (GEF). Calculated GEF for the tested PAHs were generally higher than the TEF usually used. Our study proposed a new in vitro based method for the establishment of relevant TEFs for PAHs to improve cancer risk assessment.

Genotoxicity of pesticide mixtures present in the diet of the French population

Consumers may be simultaneously exposed to several pesticide residues in their diet. A previous study identified the seven most common pesticide mixtures to which the French population was exposed through food consumption in 2006. The aim of this study was to investigate if the seven mixtures are potentially cytotoxic and genotoxic and if so, whether compounds in a same mixture have a combined effect. The cytotoxicity and genotoxicity of the seven mixtures were investigated with a new assay (γ‐H2AX) using four human cell lines (ACHN, SH‐SY5Y, LS‐174T, and HepG2). Mixtures were tested at equimolar concentrations and also at concentrations reflecting their actual proportion in the diet. Irrespective of the cell line tested, parallel cytotoxicity of the seven mixtures was observed. Only one mixture was genotoxic for the HepG2 cells at concentrations = 3 μM in equimolar proportion and at 30 μM in actual proportion. Caspase 3/7 activity, the comet assay, and reactive oxygen species production were also investigated using the same mixture and HepG2 cells. Our results suggest that pesticide metabolites from the mixture generated by HepG2 cells were responsible for the observed damage to DNA. Among the five compounds in the genotoxic mixture, only fludioxonil and cyprodinil were genotoxic for HepG2 cells alone at concentrations = 4 and 20 μM, respectively. Our data suggest a combined genotoxic effect of the mixture at low concentrations with a significantly higher effect of the mixture of pesticides than would be expected from the response to the individual compounds. Environ. Mol. Mutagen. 2012.

Combined genotoxic effects of a polycyclic aromatic hydrocarbon (B(a)P) and an heterocyclic amine (PhIP) in relation to colorectal carcinogenesis.

Colorectal neoplasia is the third most common cancer worldwide. Environmental factors such as diet are known to be involved in the etiology of this cancer. Several epidemiological studies have suggested that specific neo-formed mutagenic compounds related to meat consumption are an underlying factor involved in the association between diet and colorectal cancer. Heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) are known mutagens and possible human carcinogens formed at the same time in meat during cooking processes. We studied the genotoxicity of the model PAH benzo(a)pyrene (B(a)P) and HCA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), alone or in mixture, using the mouse intestinal cell line ApcMin/+, mimicking the early step of colorectal carcinogenesis, and control Apc+/+ cells. The genotoxicity of B(a)P and PhIP was investigated using both cell lines, through the quantification of B(a)P and PhIP derived DNA adducts, as well as the use of a genotoxic assay based on histone H2AX phosphorylation quantification. Our results demonstrate that heterozygous Apc mutated cells are more effective to metabolize B(a)P. We also established in different experiments that PhIP and B(a)P were more genotoxic on ApcMin/+ cells compared to Apc+/+. Moreover when tested in mixture, we observed a combined genotoxicity of B(a)P and PhIP on the two cell lines, with an increase of PhIP derived DNA adducts in the presence of B(a)P. Because of their genotoxic effects observed on heterozygous Apc mutated cells and their possible combined genotoxic effects, both B(a)P and PhIP, taken together, could be implicated in the observed association between meat consumption and colorectal cancer.

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.

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.