32P-postlabeling analysis of adducts generated by peroxidase-mediated binding of N-hydroxy-4-acetylaminobiphenyl to DNA.
32P-Postlabeling analysis of the bisphosphate derivatives was conducted to characterize the DNA adducts generated from the peroxidase-mediated activation of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP). Autoradiography of the D1 chromatogram of the postlabeled DNA hydrolysate revealed a major adduct (adduct 1) that migrated at Rf 0.15. An adduct with similar chromatographic characteristics was also obtained by postlabeling the products generated by chemical interaction of: (i) 2',6'-dichlorobenzoyloxy-4-acetylaminobiphenyl with the 3'-monophosphate of deoxyguanosine, and (ii) N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP) with calf thymus DNA. The adduct derived from chemical reaction exhibited the same mobilities on two-dimensional TLC as that obtained from the peroxidase-mediated DNA binding of N-OH-AABP. Moreover, on HPLC analyses, these bisphosphate derivatives exhibited identical retention times, suggesting that structurally they might be the same. Furthermore, adduct 1 was insensitive to digestion with nuclease P1. In addition to adduct 1, another minor adduct (adduct 2) was also detected in the peroxidase-mediated DNA binding of N-OH-AABP. The adduct 2 in D1 exhibited an Rf of 0.66. Adduct 2 was also observed in the DNA sample chemically interacted with N-OAc-AABP. Both these adducts retained the acetyl moiety, which was confirmed by the presence of radioactivity in the hydrolysate of DNA derived by interaction with N-OAc-[14C-acetyl]AABP (labeled at the N-acetyl group). Based on proton NMR and MS analyses of the 5'-phospho analogs of adducts 1 and 2, the structures of these have been identified as 3-(deoxyguanosine-N2-yl)-4-acetylaminobiphenyl (dG-N2-AABP) and N-(deoxyguanosine-8-yl)-4-acetylaminobiphenyl (dG-C8-AABP). Analyses of the DNA samples obtained from human uroepithelial cells following exposure to N-OH-AABP revealed primarily the non-acetylated derivative N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-ABP) with trace amounts of dG-N2-AABP. These results suggest that in the target cells for 4-aminobiphenyl carcinogenesis, the prevalence of the peroxidase mediated activation reaction of N-OH-AABP is relatively minor compared to the acetyltransferase pathway.
Hatcher
J F
JF
University of Wisconsin Comprehensive Cancer Center, Madison 53792, USA.
Swaminathan
S
S
D000107:Acetylation; D000611:Aminobiphenyl Compounds / Q000032:analysis / Q000378:metabolism* / Q000493:pharmacokinetics / Q000633:toxicity; D000818:Animals; D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism* / Q000493:pharmacokinetics / Q000633:toxicity; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis* / Q000096:biosynthesis; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D007553:Isotope Labeling; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D014551:Urinary Tract / Q000187:drug effects / Q000378:metabolism
D000611:Aminobiphenyl Compounds; D002273:Carcinogens; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; C001365:N-hydroxy-4-acetylaminobiphenyl; C053420:N-(deoxyguanosin-8-yl)-4-aminobiphenyl; D004247:DNA; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D003849:Deoxyguanosine