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32P-Postlabeling analysis of adducts generated by peroxidase-mediated binding of N-hydroxy-4-acetylaminobiphenyl to DNA
University of Wisconsin Comprehensive Cancer Center K4/548 CSC, 600 Highland Avenue, Madison, WI 53792, USA
1To whom all correspondence should be addressed
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'-dichloro-benzoyloxy-4-acetylaminobiphenyl with the 3'-monophosphate of deoxyguanosine, and (ii) N-acetoxy-4-acetylamino-biphenyl (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-(deoxyguanosin-N2-yl)-4-acetylaminobiphenyl (dG-N2-AABP) and N-(deoxyguanosin-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-(deoxyguanosin-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.
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