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Detection of deoxyadenosine-4-aminobiphenyl adduct in DNA of human uroepithelial cells treated with N-hydroxy-4-aminobiphenyl following nuclease P1 enrichment and 32P-postlabeling analysis
University of Wisconsin, Comprehensive Cancer Center, K41548 Clinical Science Center 600 Highland Avenue, Madison, WI 53792, USA
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To characterize the DNA adducts in human uroepithelial cells (HUC) exposed to 4-aminobiphenyl and its proximate N-hydroxy metabolites, we used 32P-analyses following butanol extraction of the DNA hydrolysates. Using this method, we identified N-(deoxyguanosin-3', 5'-bisphospho-8-yl)-4-aminobiphenyl (pdGp-ABP) as a major adduct and N-(deoxyguanosin-3', 5'-bisphospho-8-yl)-4- aminobiphenyl (pdAp-ABP) as a minor adduct in an immortalized non-tumorigenic cell line of HUC following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP). Towards characterization of pdAp-ABP, we postlabeled the synthetic N-(deoxyguanosin-3', 5'-bisphospho-8-yl)-4-aminobi- phenyl (dAp-ABP) adduct to generate pdAp-ABP and determined its chromatographic (TLC and HPLC) proper ties and sensitivity to nuclease P1 digestion. In contrast to pdGp-ABP, which was cleaved to the corresponding 5' monophosphate by nuclease P1, the pdAp-ABP adduct was unaffected when incubated with nuclease P1 under similar conditions. To test whether nuclease P1 digestion could be adopted for enrichment of the dAp-ABP adduct in HUC samples, postlabeling analyses were carried out after but anol extraction following nuclease P1 digestion of the DNA hydrolysate. Under these conditions, the pdAp-ABP adduct was detected in DNA from HUC E7 cells treated with N OH-ABP and in calf thymus DNA reacted with N-OH ABP under acidic (pH 5.0) conditions. These data indicate that pdGp-ABP and pdAp-ABP adducts are generated in HUC E7 on treatment with N-OH-ABP and that nuclease P1 enrichment may provide a method for qualitative and quantitative analyses of the pdAp-ABP adduct in DNA.
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