Flow cytometric detection of micronuclei and cell cycle alterations in fish-derived cells after exposure to three model genotoxic agents: mitomycin C, vincristine sulfate and benzo(a)pyrene

The measurement of cytogenetic alterations in vitro is considered an initial step in the risk assessment procedures for genotoxic agents. The concern about genotoxic pollutants in natural fish population makes the use of fish-derived cells an useful tool for these purposes. The technological improvements in well-established cytogenetic endpoints, such as micronuclei (MN) estimations by means of flow cytometry, have been proposed in the later years using mammalian cells. In this work, we test the capability of flow cytometry to evaluate MN induction and cell cycle alterations in an established fish cell line (RTG-2) using three agent-inductor models at different concentrations and exposure periods. For mitomycin C, an inverse relationship between length of exposure period and concentrations was observed. A dose-response relationship was observed after exposing RTG-2 cells to vincristine sulfate and benzo(a)pyrene. As this study shows, RTG-2 cells respond to clastogenic and aneugenic effects of the tested chemicals through the induction of MN at similar doses to mammalian cells and without the addition of exogenous metabolic activity. The possibility to check cell cycle alterations, in the same sample, gives the opportunity to evaluate early signals of cytotoxicity. The use of flow cytometry improves the assay by means of its speed and objectivity, which makes the assay very useful for genotoxicity assessment of aquatic chemicals.