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Updated: Apr 5, 2026
An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes
Published on: December 25, 2021
Evaluation of genotoxicity using automated detection of γH2AX in metabolically competent HepaRG cells
Nicolas Quesnot1, Karine Rondel1, Marc Audebert2
1Liver, Metabolisms and Cancer, INSERM, UMR991, CHU Pontchaillou, F-35033 Rennes, France, Université de Rennes 1, F-35043 Rennes, France and.
Abstract:
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.
Insights
Automated detection of gamma-H2AX (a DNA damage marker) in HepaRG cells offers a promising high-throughput method for assessing genotoxicity. This approach effectively identifies damage from various contaminants, even after long-term exposure.
Area of Science:
- Toxicology
- Cell Biology
- Biomarker Discovery
Background:
- Gamma-H2AX (γH2AX) is a recognized biomarker for genotoxicity.
- Human HepaRG cells are suitable for studying liver genotoxicity due to drug-metabolizing enzymes and wild-type p53.
- Micronucleus assays are a standard method for evaluating genotoxicity.
Purpose of the Study:
- To evaluate the relevance of automated in situ γH2AX detection in HepaRG cells for genotoxicity assessment.
- To compare γH2AX detection with the micronucleus assay after single and repeated in vitro exposures.
- To investigate the genotoxic effects of environmental contaminants on HepaRG cells over time.
Main Methods:
- Metabolically competent HepaRG cells were exposed daily to environmental contaminants.
- Genotoxicity was assessed at 1, 7, and 14 days using automated in situ γH2AX detection.
- Results were compared with established genotoxicity assays like the micronucleus assay.
Main Results:
- The study confirmed the genotoxicity of known agents like aflatoxin B1 and benzo(a)pyrene.
- Dimethylbenzanthracene, fipronil, and endosulfan induced γH2AX phosphorylation, consistent with prior findings.
- Fluoranthene and bisphenol A induced γH2AX, with some compounds showing effects only after 7 days of exposure.
- Automated γH2AX detection identified genotoxicity in HepaRG cells, including effects not previously reported in HepG2 cells.
Conclusions:
- Automated γH2AX detection in HepaRG cells is a viable high-throughput screening assay for genotoxicity.
- This method is effective for evaluating both single and long-term repeated exposure genotoxicity.
- The study highlights the importance of assessing long-term effects of low-dose contaminant exposure.
