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Genotoxicity and genetic changes in RTG-2 fish cell line upon exposure to benzo[a]pyrene and ethyl methanesulfonate detected by the Comet assay and AFLP marker analysis

Genotoxicity is often one of the earliest signs of toxicant impact and is frequently assessed using in vitro models in biomonitoring programmes. In this study Comet assay and amplified fragment length polymorphism (AFLP) were applied to assess DNA damage in RTG-2 fish cell line after 3 days of exposure to a concentration range of model genotoxic agents (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)). Quantitative modifications arising in AFLP profiles as a measure of DNA effects were analyzed in order to establish permanent genetic changes arising from mutation events (e.g. rearrangements, point mutations, small inserts or deletions of DNA) induced by toxicant exposure. Significant induction of DNA damage measured by the Comet assay was noticed in RTG-2 cells after 3 days of B[a]P treatment at all concentrations used (0.1µM, 1µM, 5µM, 10µM). After 3 days of recovery all the values returned to the control level. In contrast, 3 days exposure to EMS (10µM, 50µM, 100µM, 500µM, 1000µM) induced significant DNA damage only at the highest concentration used and 3 days of recovery resulted in more pronounced genotoxic effect. The changes occurring in AFLP profiles of RTG-2 cells following both toxicant treatment included loss of normal bands and appearance of new bands at higher toxicant concentrations in comparison to the band profile of the control. Our results indicate that AFLP method could be useful biomarker for detection of permanent genotoxic influence of toxicant exposure and encourage the use of Comet assay on fish cell lines as a versatile tool for genotoxicity assessment.

RTG-2 cell line; Comet assay; AFLP; B[a]P; EMS

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