Evaluation of the comet assay as a method for the detection of DNA damage in the cells of a marine invertebrate, Mytilus edulis L. (Mollusca: Pelecypoda).

摘要

The potential application of the comet assay for monitoring the effect of DNA damaging agents on the marine mussel, Mytilus edulis (an important pollution indicator organism), was explored. A detailed investigation of the baseline levels of single-strand breaks in isolated gill cells, and how they were affected by age/size of animal, time since collection, feeding regime, in vivo vs. in vitro exposure conditions, and by antioxidant supplementation was undertaken. The level of cometing in untreated controls was found to be highly variable over time (fluctuations between low and very high DNA damage occurred over just 14 days post collection). No difference was observed between age/size and feeding regime of the animals. On exposure to 0, 100, 500 and 1000 microM H2O2, it was observed that the in vitro exposure produced a markedly more homogeneous dose response compared to the in vivo studies (where gill cells were exposed as a tissue). An important finding of our research was the effect of prior supplementation of the animals' diet with 1 mg/ml alpha-tocopherol acetate (vitamin E compound), which resulted in a marked reduction in the levels of DNA damage expressed by the negative controls, without influencing the actual response to H2O2 (0, 5, 25, and 100 microM) and N-nitrosodimethylamine, NDMA (0, 5, 25, and 100 mM). The effect of vitamin E supplementation was to increase the sensitivity of the comet assay at the lower end of the dose range. This study demonstrated the potential application of the comet assay to the gill cells of the mussel, M. edulis. Although preliminary findings suggest that antioxidant supplementation can improve the sensitivity of the assay by lowering the baseline damage in untreated animals, our conclusion is that the assay has more potential for use in an in vitro context for the screening of agents destined for release or disposal into the marine environment.