The predominant role of apoptosis in γH2AX formation induced by aneugens is useful for distinguishing aneugens from clastogens

The phosphorylated form of the histone protein H2AX, called gamma H2AX, is recognized as a useful biomarker not only for DNA double-strand breaks but also for a wide range of other DNA damage. An increasing number of publications propose gamma H2AX to be measured when determining genotoxicity, phototoxicity, and the effectiveness of cancer therapy. Because gamma H2AX is also generated by apoptosis, a gamma H2AX-assay might assess genotoxic risk incorrectly. The aim of this study was to elucidate the influence of apoptosis on measurements of gamma H2AX by flow cytometry, with the clastogens mitomycin C (MMC) and etoposide (ETP), and the aneugens vinblastine (VB) and paclitaxel (PT), which do not react directly with DNA. TK6 human lymphoblastoid cells were treated with the clastogens and the aneugens, stained for the apoptotic biomarker caspase-3 and for gamma H2AX, and then analyzed by flow cytometry. All the test compounds caused a dose-dependent increase of gamma H2AX-positive (gamma H2AX+) cells. The gamma H2AX+ cell population included both caspase-3-positive (gamma H2AX+/caspase-3+) and caspase-3-negative (gamma H2AX+/caspase-3) cells. The increase in gamma H2AX+ cells after treatment with the aneugens corresponded to the increase in caspase-3+ cells. The increase in gamma H2AX+/caspase-3- cells after treatment with the clastogens was significant, but there was only a slight increase after treatment with the aneugens. This reflects the fact that the apoptotic pathway of a clastogen starts from DNA damage, whereas that of an aneugen starts from cell-cycle arrest in the M-phase. Therefore, the two pathways contribute differently to apoptosis. Double staining for gamma H2AX and caspase-3 provided helpful information for the different mechanistic effects of aneugens and clastogens that induce gamma H2AX. (C) 2014 Elsevier B.V. All rights reserved.