In vitro application of the comet assay for aquatic genotoxicity: considering a primary culture versus a cell line.

The comet assay, one of the most widely used techniques for the evaluation and detection of DNA strand breaks, is frequently employed in vivo. In vitro assays are usually performed with mammalian cell lines, clearly not the best choice for tests on aquatic genotoxicity. Here we evaluated a fish hepatoma cell line (RTH-149) and a primary blood cell culture from the intertidal colonial tunicate Botryllus schlosseri as possible model targets for comet assays using the genotoxic agent H2O2. We found that DNA strand break levels in RTH-149 fitted dose-dependent responses better than the tunicate cells. Moreover, in B. schlosseri controls, 34% of the cells were already ranked as severely damaged. Assays were then performed on water samples from the polluted Kishon river (Israel) on three different dates, using RTH-149 cells (50% dilutions, 2-h exposures). In all cases, high genotoxicity of the river water was revealed by evaluating comet percentages, average tail lengths and DNA damage levels. This assay was found to be fast and sensitive, appropriate to be employed as a part of a monitoring program. The use of B. schlosseri blood cells should be validated in additional work.