Use of the Comet Assay for Studying Environmental Genotoxicity: Comparisons between Visual and Image Analyses

In order to evaluate the applicability of different measurement parameters employed in the comet assay for analyzing environmental samples, fish hepatoma (RTH-149) cells were exposed to concentrations of the model genotoxic agent hydrogen peroxide (H₂O₂; 1, 5, and 10 μM) and to five water samples from sites along the Kishan River, the most polluted river in Israel. DNA damage was scared in parallel by visual and computer-image (Viscamet) analyses using 12 different parameters. Each parameter exhibited a different profile of responses. The four visual parameters were highly sensitive to the lowest (1 μM) H₂O₂ concentration (1.8-7.0-fold of the control). At 10 μM H₂O₂ exposure, the visual parameter, percentage severe damage, showed the highest (40.3-fold) response while four other parameters, tail area, tail extent moment (Viscomet), mean actual tail length and cumulative tail length (visual analysis), also had substantially elevated responses (8-11-fold). We found that the DNA damage induced by field samples was similar in magnitude to the damage induced by 1 μM H₂O₂, with only some of the parameters being highly sensitive to the damage. Only about one-half of the parameters could distinguish four significant levels of genotoxicity among the five sampling sites, while the remaining parameters detected only three levels. It is concluded that the choice of parameters for analyzing genotoxicity in ecotoxicological studies should be made in accordance with the characteristics of each parameter.