Identification of new DNA adducts in human bladder epithelia exposed to the proximate metabolite of 4-aminobiphenyl using 32P-postlabeling method

The DNA adducts were analyzed by 32P-postlabeling method following exposure of human uroepithelial cells (HUC) to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). TLC of the postlabeled products on the first dimension revealed several products, the majority of which stayed close to the origin and were earlier identified as the 3',5' -bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (Carcinogenesis 13 (1993) 955; Carcinogenesis 16 (1995) 295). Here we report characterization of two additional adducts that amounted to less than 5% of the total adducts. Autoradiography of D1 chromatogram of the postlabeled products of calf thymus DNA chemically interacted with N-OH-ABP under acidic conditions revealed two adducts, #1 and #2, with R(f) values of about 0.2 and 0.3, respectively. Two adducts with D1 thin layer chromatographic properties similar to those of adducts #1 and #2 were obtained on postlabeling analyses of products generated by chemical interaction of N-acetoxy-4-aminobiphenyl (N-OAc-ABP) with deoxyguanosine-3' -monophosphate (dGp). Based on proton NMR and mass spectroscopic analyses of the synthetic products derived from N-OAc-ABP, the chemical structures of adducts #1 and #2 have been identified as 3-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, and N-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, respectively. Both of these adducts were insensitive to digestion with nuclease P1. 32P-Postlabeling analysis of the nuclease P1 enriched DNA hydrolysate of HUC cells treated with N-OH-ABP showed the presence of adduct #2 but not adduct #1. Adduct #2 was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl CoA. These results suggest that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP is bioactivated by acetyl CoA-dependent acyltransferases to reactive arylnitrenium ions that covalently interact at N(2)-position of deoxyguanosine in DNA.