Valérie Fessard

Genotoxicity of a freshwater cyanotoxin, cylindrospermopsin, in two human cell lines: Caco-2 and HepaRG.

Cylindrospermopsin (CYN), a cyanotoxin produced by certain freshwater cyanobacteria, causes human intoxications and animal mortalities. CYN is a potent inhibitor of protein‐ and glutathione‐synthesis. Preliminary evidence for in vivo tumor initiation has been found in mice but the mechanism remains unclear. Several in vitro and in vivo studies demonstrate that CYN is genotoxic and requires metabolic activation. In the present study, the genotoxicity of CYN was assessed in human hepatocyte and enterocyte cell lines, which are models for CYN target organs. The cytokinesis‐block micronucleus assay was conducted on liver‐derived HepaRG cells and colon‐derived Caco‐2 cells. Each cell‐type was exposed to CYN in both the differentiated and the undifferentiated states, and both with and without the cytochrome P450 inhibitor, ketoconazole, to determine the involvement of metabolism in CYN genotoxicity. CYN increased the frequency of micronuclei in binucleated cells (MNBNC) in both Caco‐2 and HepaRG cells. Moreover, ketoconazole reduced both the genotoxicity and cytotoxicity caused by CYN. Our results confirm the involvement of metabolic activation of CYN in mediating its toxicity and suggest that CYN is progenotoxic. Environ. Mol. Mutagen. 2010.

High throughput toxicity screening and intracellular detection of nanomaterials

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

DNA Adduct Formation of 4-Aminobiphenyl and Heterocyclic Aromatic Amines in Human Hepatocytes

DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 10(7) DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation.

In vivo DNA damage induced by the cyanotoxin microcystin-LR: comparison of intra-peritoneal and oral administrations by use of the comet assay.

Microcystin-LR (MC-LR), involved in human and animal poisonings by cyanobacteria, has been shown to be both a potent tumour promoter in rat liver and an inhibitor of serine/threonine protein phosphatases, specifically PP1 and PP2A. The research on the genotoxic potential of MC-LR counts only few in vivo studies. In order to determine the target organs for DNA-damage induction by MC-LR, the single-cell gel electrophoresis (SCGE) or comet assay was performed in mice. Following a single oral administration of 2 and 4mg/kg bw of MC-LR, a statistically significant induction of DNA damage in blood cells was obtained after 3h. However, after an intra-peritoneal injection (ip), DNA lesions were mainly induced in the liver, but were also reported in the kidney, the intestine and the colon. The sensitivity of the ip route compared to the oral route suggested a difference in the bio-disponibility of the toxin. In any case, DNA damage was induced by MC-LR irrespective of the administration route. Among the target organs, the DNA damage induced in the intestinal tissues (ileum and colon) may contribute to an increased cancer risk.

Toxicity, genotoxicity and proinflammatory effects of amorphous nanosilica in the human intestinal Caco-2 cell line

Silica (SiO2) in its nanosized form is now used in food applications although the potential risks for human health need to be evaluated in further detail. In the current study, the uptake of 15 and 55nm colloidal SiO2 NPs in the human intestinal Caco-2 cell line was investigated by transmission electron microscopy. The ability of these NPs to induce cytotoxicity (XTT viability test), genotoxicity (γH2Ax and micronucleus assay), apoptosis (caspase 3), oxidative stress (oxidation of 2,7-dichlorodihydrofluorescein diacetate probe) and proinflammatory effects (interleukin IL-8 secretion) was evaluated. Quartz DQ12 was used as particle control. XTT and cytokinesis-block micronucleus assays revealed size- and concentration-dependent effects on cell death and chromosome damage following exposure to SiO2 nanoparticles, concomitantly with generation of reactive oxygen species (ROS), SiO2-15nm particles being the most potent. In the same way, an increased IL-8 secretion was only observed with SiO2-15nm at the highest tested dose (32μg/ml). TEM images showed that both NPs were localized within the cytoplasm but did not enter the nucleus. SiO2-15nm, and to a lower extent SiO2-55nm, exerted toxic effects in Caco-2 cells. The observed genotoxic effects of these NPs are likely to be mediated through oxidative stress rather than a direct interaction with the DNA. Altogether, our results indicate that exposure to SiO2 NPs may induce potential adverse effects on the intestinal epithelium in vivo.

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.

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.

Cytotoxic and genotoxic effects of cylindrospermopsin in mice treated by gavage or intraperitoneal injection

Cylindrospermopsin (CYN), a cyanobacterial hepatotoxin mainly produced by Cylindrospermopsis raciborskii, has been involved in human intoxications and livestock deaths. The widespread occurrence of CYN in the water supplies lead us to investigate its genotoxicity to assess potential chronic effects. This study reports evaluation of CYN‐induced in vivo DNA damage in mice using alkaline comet assay (ACA) and micronucleus assay (MNA) concomittantly. ACA measures DNA breakage from single and double strand breaks as well as alkali labile sites. Conversely, MNA detects chromosome damage events such as chromosomal breakage and numeric alterations. Male Swiss mice were treated with CYN concentrations of 50, 100, and 200 μg/kg by a single intraperitoneal (ip) injection or with 1, 2, and 4 mg/kg by gavage. Methyl methane sulfonate (MMS) was used as positive control at 80 mg/kg. Twenty‐four hours after treatment, samples of liver, blood, bone marrow, kidney, intestine, and colon were taken to perform ACA, the bone marrow and the colon were also used for MNA. Parameters used to quantify DNA damage were % Tail DNA for ACA and both micronucleated immature erythrocytes and epithelial colon cells for MNA. DNA breaks and chromosome damage were significantly increased by MMS in all the organs evaluated. Significant DNA damage was detected within the colon by ACA after ip injection of 100 and 200 μg/kg CYN (P < 0.01). DNA damage was also detected in colon samples after 4 mg/kg oral administration of CYN and in bone marrow after 1 and 2 mg/kg of orally administered CYN. Histological examination showed foci of cell death within the liver and the kidney from mice that received the two highest doses of CYN by either route of administration.

Assessment of the genotoxic potential of indirect chemical mutagens in HepaRG cells by the comet and the cytokinesis-block micronucleus assays

Many chemical carcinogens require metabolic activation to form genotoxic compounds in human. Standard in vitro genotoxicity assays performed with activation systems, such as rat liver S9, are recognised to lead to a high number of false positives. The aim of this study was to evaluate the suitability of differentiated human hepatoma HepaRG cells as an in vitro model system for the detection of DNA damage induced by promutagens using the comet and the cytokinesis-block micronucleus assays. Several promutagens were tested, including aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), acrylamide, N-nitrosodimethylamine (NDMA), cyclophosphamide (CPA), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Cytotoxicity of these compounds was assessed by measuring lactate dehydrogenase leakage. A 24 h exposure was generally needed to obtain an obvious positive response in differentiated HepaRG cells in the comet and in the cytokinesis-block micronucleus assays. Comet formation was observed with all compounds except IQ. B[a]P, CPA and AFB1 showed a dose-dependent increase in micronucleated cells, whereas no increase was observed with PhIP, IQ and acrylamide. These preliminary data on genotoxicity in differentiated HepaRG cells are promising but more chemicals must be tested to determine the ability of HepaRG cells to assess genotoxicity of chemicals in humans.

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.