Performance of in vitro γH2AX assay in HepG2 cells to predict in vivo genotoxicity

Abstract

The γH2AX assay has recently been suggested as a new in vitro assay for detecting genotoxic (GTX) properties of chemicals. This assay is based on the phosphorylation of H2AX histone in response to DNA damage [i.e. induction of double-strand breaks (DSBs)]. Quantification of γH2AX foci using flow cytometry can rapidly detect DNA damage induced by chemicals that cause DNA DSBs. Up to now, only few compounds have been tested with this assay. The main goal of this study was to compare the performance of this automated γH2AX assay with that of standard in vitro genotoxicity assays in predicting in vivo genotoxicity. HepG2 cells were exposed to 64 selected compounds with known GTX properties and subsequently analysed for induction of γH2AX foci. The results of this assay were compared with public data from standard in vitro genotoxicity tests. Accuracy, sensitivity and specificity in predicting in vivo genotoxicity, using the γH2AX assay alone or in combinations with conventional assays, were calculated. Both the γH2AX assay and the bacterial mutagenicity test (Ames) were highly specific for in vivo GTX, whereas chromosomal aberration/micronucleus test (CA/MN) resulted in highest sensitivity. The currently widely used in vitro genotoxicity test battery-Ames test, mouse lymphoma assay (MLA) and CA/MN test-resulted in low accuracy (55-65%) to predict in vivo genotoxicity. Interestingly, the inclusion of γH2AX assay in the standard battery, instead of MLA assay, resulted in higher accuracy (62-70%) compared with other combinations. Advantage of the γH2AX assay in HepG2 cells is its high sensitivity to detect DNA-reactive GTX compounds, although the reduced sensitivity for compounds that require metabolic activation needs to be improved. In conclusion, the automated γH2AX assay can be a useful, fast and cost-effective human cell-based tool for early screening of compounds for in vivo genotoxicity.