Quesnot Nicolas, Rondel Karine, Audebert Marc, Martinais Sophie, Glaise Denise, Morel Fabrice, Loyer Pascal, Robin Marie-Anne
Liver, Metabolisms and Cancer, INSERM, UMR991, CHU Pontchaillou, F-35033 Rennes, France, Université de Rennes 1, F-35043 Rennes, France and.
Research Centre in Food Toxicology, INRA, UMR1331, Toxalim, F-31027 Toulouse, France.
Mutagenesis. 2016 Jan;31(1):43-50. doi: 10.1093/mutage/gev059. Epub 2015 Aug 17.
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.
最近有报道称，γH2AX的原位检测有望成为遗传毒性的生物标志物。此外，人源HepaRG肝癌细胞似乎与研究肝脏遗传毒性相关，因为它们表达大多数药物代谢酶和野生型p53。本研究的目的是确定与微核试验相比，γH2AX阳性HepaRG细胞的自动原位检测是否可用于评估单次或长期重复体外暴露后的遗传毒性。将具有代谢活性的HepaRG细胞每日用环境污染物处理，并在1、7和14天后评估遗传毒性。利用这些细胞，我们证实了黄曲霉毒素B1和苯并(a)芘的遗传毒性，并证明了之前通过彗星试验或微核试验发现具有遗传毒性的二甲基苯并蒽、氟虫腈和硫丹也可诱导γH2AX磷酸化。此外，我们发现荧蒽和双酚A可诱导γH2AX，而之前在HepG2细胞中未报道有此效应。另外，一些化合物仅在7天后才观察到γH2AX的诱导，这突出了研究低剂量污染物长期效应的重要性。总之，我们的数据表明，在具有代谢活性的HepaRG细胞中进行自动γH2AX检测是一种合适的高通量遗传毒性筛选试验。
