³²P-postlabelling analysis of DNA adducts in fish as a biomarker of genotoxic exposure

Abstract: The 32P-postlabelling assay was used for analysis of hydrophobic DNA adducts in fish as a biomarker of genotoxic exposure. DNA adducts were analysed in perch (Perca fluviatilis) and northern pike (Esox lucius), two relatively stationary fish species which are common in Swedish freshwater systems and along the Baltic coast. Experimental studies on time-course, dose-response and persistence of DNA adducts in liver and extrahepatic tissues of pike were performed in the laboratory. DNA adducts were readily formed in pike exposed to carcinogenic model substances. Oral exposure gave rise to higher adduct levels in both liver and intestine compared to intraperitoneal exposure. Following repeated oral exposure, adduct levels increased in a dose-related manner in liver, gills, brain and intestine, with highest levels in the intestine. No significant decrease in total adduct levels was observed in liver, gills and brain during a 78-day period after the last exposure, while adduct levels in intestine decreased to one third of the maximum value. DNA adducts in the intestine are probably removed due to a high cell turnover rate in this tissue, and may thus represent ongoing or relatively recent exposure.Perch and pike from minimally polluted sites were analysed at several occasions, including during the reproductive season for perch, with the results showing no detectable adducts, or very low levels. No adducts related to spawning season were detected in unexposed female or male perch.In a field investigation carried out in a pollution gradient of polycyclic aromatic hydrocarbons leading away from an aluminium smelter, adduct levels in liver and kidney were correlated to distance from the suspected point source and to contaminant levels in the sediment. Adduct levels in gills, spleen, blood and brain were also elevated in perch from the site closest to the smelter compared to the outermost site. Increased adduct levels in these tissues were correlated to effects on higher biological organisation levels, i.e. hepatocellular degeneration and decreased growth rate at the two innermost sites, and decreased spleen somatic index at the innermost site. Adduct levels were also increased in perch from a river with creosote-contaminated bottom sediments, indicating that potentially genotoxic compounds in the sediment were bioavailable to the fish. Exposure of perch in the laboratory to a solvent extract prepared from the contaminated sediment resulted in adduct patterns which closely resembled those obtained from field captured perch, thus verifying the origin of the genotoxic substances. Furthermore, increased DNA adduct levels in liver and intestine of feral fish from coastal waters receiving bleached kraft pulp mill effluents were detected.The results show that 32P-postlabelling analysis of hydrophobic DNA adducts in feral perch and pike can be used as a sensitive biomarker of exposure to potentially genotoxic compounds in the aquatic environment. Adduct levels in liver and several extrahepatic tissues were positively correlated to exposure concentrations. Levels of aromatic/hydrophobic DNA adducts in unexposed perch and pike can be considered as practically zero. DNA adducts thus exhibit a wide range in response, from almost zero in unexposed fish to high levels in exposed fish, which is desirable for a good biomarker.