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Improvement in the diagnostic potential of 32P-postlabeling analysis demonstrated by the selective formation and comparative analysis of nitrated-PAH-derived adducts arising from diesel particle extracts
Health Effects Research Laboratory, Genetic Toxicology Division, US Environmental Protection Agency Research Triangle Park, NC 27711
1Environmental Health Research and Testing Inc. Research Tnangle Park, NC 27709, USA
Two versions of the 32P-postlabeling assay (nuclease P1 and butanol extraction) enhance the detection limit of polycyclic aromatic hydrocarbon (PAH)-modified DNA. Previously published studies suggest that DNA adducts derived from N-substituted aryl compounds are poorly recovered in the nuclease P1 version. In this study, both versions were employed to ascertain whether the apparent differences in sensitivity could be used to select diagnostically for nitroaromatic-DNA adducts derived by treating calf thymus DNA with organic extracts isolated from four diesel and one gasoline vehicle emission particles. We enhanced the formation of nitrated-PAH-derived adducts through xanthine oxidase (XO)-catalyzed nitroreduction of nitrated-PAHs, constituents previously detected in the diesel emissions. Chromatographic mobilities of the XO-derived DNA adducts were compared to adducts detected in calf thymus DNA resulting from rat liver S9-mediated metabolism. All four diesel organic extracts treated with XO resulted in the formation of one major DNA adduct, chromatographically distinct from the multiple DNA adducts detected in the rat liver S9-treated incubations. This adduct was detectable with the butanol extraction but not the nuclease P1 version of the 32P-postlabeling assay and was chromatographically similar to DNA adducts formed following XO nitroreduction of 1-nitropyrew or ascorbic add treatment of 1-nitroso-8-pyrene and 1-nitro-6-nitrosopyrene. In contrast, when S9 activation was used, multiple DNA adducts were detected along a diagonal zone of radioactivity and were radioactively labeled with equivalent efficiency irrespedive of the assay version employed. The in vitro calf thymus DNA model described in this study enhances the diagnostic potential of the 32P-postlabeling assay through the selective formation of nuclease P1-sensitive N-subsbituted aryl-derived DNA adducts.
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