Influences of DNA isolation and RNA contamination on carcinogen-DNA adductanalysis by P-32-postlabeling

P-32-Postlabeling is a widely applied assay For the analysis of carcinogen- DNA adducts. Optimization of most steps in this assay has been given attent ion, but influences of DNA isolation and DNA purity on adduct quantitation have not been investigated systematically. In this study, DNA was isolated from human lymphocytes exposed to benzo[a]pyrene (B[a]P, 10 mu M) for 18 hr and From liver of rats i.p.-treated with B[a]P (10 mg/kg body weight) usin g two different DNA isolation methods: a phenol-extraction and a salting-ou t procedure. Subsequently, DNA was analysed by nuclease P1 (NP1) or butanol -enriched P-32-postlabeling. influences of RNA contamination were studied b y labeling RNA isolated from in vitro exposed lymphocytes. In the in vitro experiment, DNA adduct levels were significantly higher using the salting-o ut procedure (63.2 +/- 13.7 adducts per 10(8) nucleotides, n = 9) as compar ed with the phenol-extraction (14.3 +/- 0.8). RNA was similar to 4 times le ss efficiently labeled as compared to DNA. Nonetheless, RNA contamination o f DNA samples may result in an overestimation of DNA adduct levels when but anol enrichment is used, because RNA adduct levels seemed to be substantial ly higher than DNA adduct levels in the same cells. DNA adduct analysis by nuclease P1 enrichment is probably less effected, since RNA adducts appeare d to be NP1 sensitive. In vivo, three different adducts were found by NP1 e nriched P-32-postlabeling in the liver of B[a]P-exposed rats. Again, DNA ad duct levels were significantly higher using salting out as compared to phen ol extraction for the adduct which comigrated with the BPDE-DNA adduct stan dard (adduct 1) and on unknown adduct (adduct 2). However, the results were the opposite for another B[a]P-derived DNA adduct (adduct 3). Our results suggest that differences in DNA isolation procedures as well as RNA contami nation influence quantitative DNA adduct analysis by P-32-postlabeling. Env iron. Mol. Mutagen. 32:344-350, 1998 (C) 1998 Wiley-Liss, Inc.