QUANTITATION OF CHEMICALLY-INDUCED DAMAGE TO DNA OF AQUATIC ORGANISMS BY ALKALINE UNWINDING ASSAY

The effect of chronic exposure to the genotoxic chemical benzo[a]pyrene (BaP) on the integrity of DNA, as measured by the level of double-strandedness of this macromolecule, for two aquatic organisms was investigated. In order to facilitate these studies, a method for the rapid and facile isolation of DNA from either the liver of bluegill sunfish (Lepomis macrochirus) or the entire fathead minnow (Pimephales promelas) was developed. DNA was examined for chemically-induced strand breaks using an alkaline unwinding assay where strand separation is achieved under defined conditions of pH and temperature, and the amounts of double-stranded and single-stranded DNA present upon termination of alkaline unwinding were quantified by fluorescence measurements using Hoechst dye 33258. The kinetics of DNA strand separation under the alkaline denaturation conditions employed demonstrated the suitability of the technique for estimating strand breaks. It was observed that BaP-induced dmage to liver DNA of the bluegill sunfish during chronic exposure via their water proceeds through two distinct phases. There is an initial phase of decline in the level of double-strandedness of the DNA, which is immediate and rapid. By day 16 the level is 10% of that observed in nonexposed fish. During the next phase, which occurs subsequent to day 16, the level of double-strandedness increases and by day 30 is approximately that of the control population. Exposure of fathead minnows to BaP, under conditions identical to that for the bluegill sunfish, results in similar DNA damage during the initial phase of exposure.