In vitro assessment of DNA damage after short- and long-term exposure to benzo(a)pyrene using RAPD and the RTG-2 fish cell line

Abstract

Genotoxins present in the aquatic environment are often associated with the decline or disappearance of many wild populations. The hazard assessment of chemicals requires sensitive and specific tests to study the genotoxic effects in order to establish the maximum allowable chemical concentrations prior to the release to the environment. We have previously shown that an established fish cell line (RTG-2) together with the random amplified polymorphic DNA (RAPD) technique, can be used to detect alterations in the DNA caused by direct acting genotoxins. The current study takes this a step further and examines in the same system the effect of a pro-mutagen benzo(a)pyrene (B(a)P) at different concentrations (0.05, 0.1, and 0.5 μg/ml) and at different exposure periods (1, 2, 3, 15, and 30 days). After comparing DNA fingerprints from control and exposed cells, both qualitative and quantitative analysis show an increase in the instability in the DNA fingerprint of exposed cells over a time- and concentration-dependent manner. At the higher concentration (0.5 μg/ml) three out the four primers showed altered bands after 1 day of exposure, while after 3 days all used primers showed an altered pattern. At the lower concentration of B(a)P (0.05 μg/ml) the appearance of new bands was observed with a 100% level of reproducibility after 30 days of exposure suggesting an inheritance of the altered DNA. We conclude that this in vitro system is useful to evaluate genotoxic effects, both after acute and chronic exposures and of direct and non-direct acting genotoxins. Cultured cells can be considered as genetically homogenous populations. Therefore, in vitro systems permits us to undertake mechanistic studies avoiding the interference of polymorphisms inherent in the in vivo systems. Furthermore, the RTG-2 fish cell line combined with a RAPD assay could be used in studies of hazard identification in risk assessment protocols of chemicals.