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. © 2012 Wiley Periodicals, Inc.

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Many efforts have been made in the last 30 years to develop more relevant in vitro models to study genotoxic responses of drugs and environmental contaminants. While 2D HepaRG cells are one of the most promising models for liver toxicology, a switch to 3D cultures that integrate both in vivo architecture and cell-cell interactions has occurred to achieve even more predictive models. Preliminary studies have indicated that 3D HepaRG cells are suitable for liver toxicity screening. Our study aimed to evaluate the response of HepaRG spheroids exposed to various genotoxic compounds using the single cell gel electrophoresis assay. HepaRG spheroids were used at 10 days after seeding and exposed for 24 and 48 hours to certain selected chemical compounds (methylmethansulfonate (MMS), etoposide, benzo[a]pyrene (B[a]P), cyclophosphamide (CPA), 7,12-dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), 4-nitroquinoline (4-NQO), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), acrylamide, and 2-4-diaminotoluene (2,4-DAT)). After treatment, the comet assay was performed on single cell suspensions and cytotoxicity was determined by the ATP assay. Comet formation was observed for all compounds except IQ, etoposide and 2,4-DAT. Treatment of spheroids with rifampicin increased CYP3A4 activity, demonstrating the metabolic capacity of HepaRG spheroids. These data on genotoxicity in 3D HepaRG spheroids are promising, but further experiments are required to prove that this model can improve the predictivity of in vitro models to detect human carcinogens.

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Micronucleus induction by the diarrhetic shellfish toxin okadaic acid (OA) was investigated in two intestinal models, cultured human Caco-2 cells and colon epithelial cells of mice treated in vivo. Exposure to OA for 4 and 24 h induced dose-responsive increases in the frequency of micronucleated Caco-2 cells; the minimum OA doses increasing micronucleus frequency were 20 nM for the 4 h treatment and 5 nM for the 24 h treatment. OA treatment of Caco-2 cells also resulted in dose- and time-dependent increases in mitotic arrest and multinucleated cells. Two experiments were conducted in which mice were treated with single oral gavages of 435-610 and 115-1341 microg/kg OA. In the first experiment, samples were taken 24 h after the treatment, and the frequencies of both micronucleated and mitotic gut cells were increased after treatment with 525 microg/kg OA. In the second experiment, no increases in micronucleus frequency were detected at 24, 36, or 48 h following OA doses of 230 and 115 microg/kg; however, an increase in the mitotic index was observed 36 h after a gavage with 115 microg/kg OA. In this experiment, doses higher than 230 microg/kg were rapidly lethal to the mice. Immunohistology with monoclonal OA antibodies showed that OA was distributed into the liver at all the sampling times and in the small intestine at 24 and 36 h; OA was not detected in the colon. In addition, the TUNEL assay indicated that OA induced apoptosis in mouse ileum, liver, and kidney. The results of our investigations suggest that OA is aneugenic in Caco-2 cells, whereas the in vivo data were inconclusive. Further studies should be performed in mice using intragastric doses of 230-525 microg/kg OA. Moreover, the apoptosis and cell proliferation results indicate that OA can reach organs other than colon, indicating further evaluation of the genotoxic potential of OA in these organs is warranted.