Identification of N-(deoxyguanosin-8-yl)-4-azobiphenyl by (32)P-postlabeling analyses of DNA in human uroepithelial cells exposed to proximate metabolites of the environmental carcinogen 4-aminobiphenyl.
DNA adducts formed in human uroepithelial cells (HUC) following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), were analyzed by the (32)P-postlabeling method. Two adducts detected by (32)P-postlabeling were previously identified as the 3',5'-bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-C8-ABP) (Frederickson S et al. [1992] Carcinogenesis 13: 955-961; Hatcher and Swaminathan [1995b] Carcinogenesis 16: 295-301). In contrast to the dG-C8-ABP adduct, which was 3'-dephosphorylated by nuclease P1, dA-C8-ABP was resistant to nuclease P1, thus providing an enrichment step before postlabeling. Autoradiography of the two-dimensional thin-layer chromatogram of the postlabeled products obtained following nuclease P1 digestion revealed several minor adducts, one of which has been identified in the present study. Postlabeling analyses following nuclease P1 digestion of the products obtained from the reaction of N-acetoxy-4-aminobiphenyl with deoxyguanosine-3'-monophosphate (dGp) demonstrated the presence of this minor adduct. The 3'-monophosphate derivative of the adduct was subsequently chromatographically purified and subjected to spectroscopic analyses. Based on proton NMR and mass spectroscopic analyses of the synthetic product, the chemical structure of the adduct has been identified as N-(deoxyguanosin-N(2)-yl)-4-azobiphenyl (dG-N==N-ABP). (32)P-Postlabeling analysis of the nuclease P1-enriched DNA hydrolysate of HUCs treated with N-OH-ABP or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) showed the presence of the dG-N==N-ABP adduct. It was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl-CoA, or incubated with HUC microsomes and N-OH-AABP. These results demonstrate that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP and N-OH-AABP are bioactivated by acyltransferases to reactive arylnitrenium ions that covalently interact at the N2 position of deoxyguanosine in DNA.
Hatcher
James F
JF
Department of Pharmacology and Environmental Health Science Center, University of Wisconsin, Madison, Wisconsin 53706, USA.
Swaminathan
Santhanam
S
D000611:Aminobiphenyl Compounds / Q000633:toxicity*; D001391:Azo Compounds / Q000032:analysis*; D001713:Biphenyl Compounds / Q000032:analysis*; D002274:Carcinogens, Environmental / Q000633:toxicity*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis*; D003854:Deoxyribonucleotides / Q000032:analysis*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010761:Phosphorus Radioisotopes; D001749:Urinary Bladder Neoplasms / Q000139:chemically induced; D019459:Urothelium / Q000737:chemistry / Q000187:drug effects*
D000611:Aminobiphenyl Compounds; D001391:Azo Compounds; D001713:Biphenyl Compounds; D002274:Carcinogens, Environmental; D018736:DNA Adducts; D003854:Deoxyribonucleotides; C464359:N-(deoxyguanosin-8-yl)-4-azobiphenyl; D010761:Phosphorus Radioisotopes; C006757:4-biphenylamine; D004247:DNA