The H2AX Assay for Genotoxic and Nongenotoxic Agents: Comparison

DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phos-phorylated form, H2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objec-tive of this study was to assess if H2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified H2AX foci manually and automatically. In addition, total H2AX activation was deter-mined by flow cytometry. For all chemicals the cytotoxic dose re-sponse was assayed by ametabolic cytotoxicity assay.We show that (1) all genotoxic agents induced H2AXdose-dependently wherea