Comparison of different techniques for γH2AX/pH3 biomarkers quantification for chemical genotoxicity assessment

Chemical risk assessment relies on in vitro genotoxicity tests. Histone modifications (γH2AX and pH3) have emerged as valuable biomarkers for genotoxicity detection. In this study, we compared three parameters (global intensity, nuclear intensity, and foci number) for the γH2AX biomarker and two parameters (global intensity and % cell in mitosis) for the pH3 biomarker. These analyzes were performed in three cell lines: human osteosarcoma U2OS cells, human hepatocellular carcinoma HepG2 cells and rat intestinal epithelial IEC-6 cells. Cells were exposed for 24 h to four well-characterized hazardous substances: nocodazole (aneugen), etoposide (topoisomerase inhibitor), benzo[a]pyrene (DNA adducts inducer), and tunicamycin (apoptosis inducer). The Benchmark Concentration (BMC) approach indicated that the sensitivity of the technics varied depending on both the chemical compounds and the tested cell line. The γH2AX foci analysis provided the higher sensitivity for clastogenic compounds. For the aneugenic compound, the global intensity and the proportion of mitotic cells showed similar sensitivity. Following tunicamycin treatment, we only detected increase in γH2AX nuclear intensity in U2OS cell model, indicating that apoptosis does not interfere with γH2AX global intensity or foci number, thereby minimizing the risk of false positive results. Finally, we observed that compared to the other methods, global intensity permitted to monitor weaker fold inductions of the biomarkers. By comparing the different quantification methods of histone modifications used as genotoxicity biomarkers, this study highlights the most suitable parameters to be used.