It has been reported that the phosphorylated form of histone variant H2AX (γH2AX) plays an important role in the recruitment of DNA repair and checkpoint proteins to sites of DNA damage, particularly at double strand breaks (DSBs). Using γH2AX foci formation as an indicator for DNA damage, several chemicals/stress factors were chosen to assess their ability to induce γH2AX foci in a 24 h time frame in a human amnion FL cell line. Two direct-acting genotoxins, methyl methanesulfonate (MMS) and N-ethyl-N-nitrosourea (ENU), can induce γH2AX foci formation in a time-and dose-dependent manner. Similarly, an indirect-acting genotoxin, benzo[a]pyrene (BP), also induced the formation of γH2AX foci in a time-and dose-dependent manner. Another indirect genotoxin, 2-acetyl-aminofluorene (AAF), did not induce γH2AX foci formation in FL cells; however, AAF can induce γH2AX foci formation in Chinese hamster CHL cells. Neutral comet assays also revealed the induction of DNA strand breaks by these agents. In contrast, epigenetic carcinogens azathioprine and cyclosporine A, as well as non-carcinogen dimethyl sulfoxide, did not induce γH2AX foci formation in FL cells. In addition, heat shock and hypertonic saline did not induce γH2AX foci. Cell survival analyses indicated that the induction of γH2AX is not correlated with the cytotoxic effects of these agents/factors. Taken together, these results suggest that γH2AX foci formation could be used for evaluating DNA damage; however, the different cell types used may play an important role in determining γH2AX foci formation induced by a specific agent.

Comet assay is a useful technique in the detection of DNA damages, particularly DNA strand breaks; and it has been utilized to show that a potent carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), can induce such damages. Recently, gammaH2AX foci formation has been suggested as another sensitive way to detect DNA double strand breaks (DSBs). However, there is no systematic comparison being conducted to evaluate the consistency of these two methods. Using MNNG as a model chemical, the sensitivity of neutral comet assay and gammaH2AX foci formation in detecting MNNG-induced damage was studied. It was found that at concentrations of 0.1 and 1 microg/ml, both methods can detect MNNG-induced damage in human amnion FL cells. However, at 0.1 microg/ml, comet assay revealed more percentage of cells with DNA damage than gammaH2AX fluorescence revealed. On the other hand, while gammaH2AX foci were readily formed at very early times by 10 microg/ml MNNG treatment, neutral comet assay did not detect any significant DNA damage at the same time points. In addition, 10 microg/ml MNNG induced a distinct whole nuclei staining pattern of gammaH2AX, a type of DNA damage which was not detected by neutral comet assay but could be detected by alkaline comet assay. Therefore, gammaH2AX may be used as a sensitive indicator for DNA damage.