A comparative study of using comet assay and γH2AX foci formation in the detection of N-methyl- N′-nitro- N-nitrosoguanidine-induced DNA damage

Abstract

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, γH2AX 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 γH2AX foci formation in detecting MNNG-induced damage was studied. It was found that at concentrations of 0.1 and 1 μg/ml, both methods can detect MNNG-induced damage in human amnion FL cells. However, at 0.1 μg/ml, comet assay revealed more percentage of cells with DNA damage than γH2AX fluorescence revealed. On the other hand, while γH2AX foci were readily formed at very early times by 10 μg/ml MNNG treatment, neutral comet assay did not detect any significant DNA damage at the same time points. In addition, 10 μg/ml MNNG induced a distinct whole nuclei staining pattern of γH2AX, a type of DNA damage which was not detected by neutral comet assay but could be detected by alkaline comet assay. Therefore, γH2AX may be used as a sensitive indicator for DNA damage.