Abstract
So far, the majority of in vitro toxicological experiments are conducted after an acute 24 h treatment that does not represent a realistic human chemical exposure. Recently, new in vitro approaches have been proposed to study the chemical toxicological effect over several days in order to be more predictive of a representative exposure scenario. In this study, we investigated the genotoxic potential of chemicals (direct or bioactived clastogen, aneugen and apoptotic inducer) with the γH2AX and pH3 biomarkers, in the human liver-derived HepaRP cell line. We used different treatment durations, with or without a three-day recovery stage (release period), before genotoxicity measurement. Data were analysed with the Benchmark Dose approach. We observed that the detection of clastogenic compounds (notably for DNA damaging agents) was more sensitive after three days of repeated treatment compared to one or three treatments over 24 h. In contrast, aneugenic chemicals were detected as genotoxic in a similar manner whether after a 24 h exposure or a three-day repeated treatment. Globally, the release period decreases the genotoxicity measurement substantially. For DNA damaging agents, after high concentration treatments, γH2AX induction was always observed after a three-day release period. In contrast, for DNA topoisomerase inhibitors, no effect could be observed after the release period. In conclusion, in the HepaRP cell line, there are some important differences between a one-day acute and a three-day repeated treatment protocol, indicating that different cell treatment procedures may differentiate chemical genotoxic mechanisms of action more efficiently.
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Acknowledgements
We acknowledge Biopredic for the use of the HepaRP cell line and specific cell culture media. The authors acknowledge Juliette Cooke for English editing of the manuscript. This work was in part supported by the Genoshift project (ANR-20-CE34-0016).
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CR performed all of the experiments, data analysis and interpretation through discussions with GM and MA. The first draft of the manuscript was written by CR. All the authors discussed the results and contributed to manuscript edition.
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Recoules, C., Mirey, G. & Audebert, M. Effect of cell treatment procedures on in vitro genotoxicity assessment. Arch Toxicol 98, 1225–1236 (2024). https://doi.org/10.1007/s00204-024-03690-w
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DOI: https://doi.org/10.1007/s00204-024-03690-w