DNA damage in human transitional cell carcinoma cells after exposure to the proximate metabolite of the bladder carcinogen 4-aminobiphenyl

The DNA damage induced by N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobi phenyl (ABP), was examined in human transitional cell carcinoma (TCC) cells after exposure to the chemical in vitro. P-32-postlabeling analysis of TCC cultures exposed to N-OH-AABP revealed a minor adduct identified as 3-(deo xyguanosin-N-2-yl)-4-acetylaminobiphenyl (dG-N2-AABP) based on comparison o f the HPLC and TLC mobility of the product with the synthetic standard. An adduct with the same chromatographic properties was also detected on postla beling analyses of calf thymus DNA bound to N-OH-AABP by incubation with ho rseradish peroxidase and hydrogen peroxide. Detection of dG-N2-AABP, which contains the acetyl moiety, suggests that N-acetoxy-4-acetylamino-biphenyl might be formed as a reactive intermediate and could conceivably arise by a free-radical-mediated reaction of N-OH-AABP with endogenous peroxidases. T he radical intermediates could also form reactive oxygen species (ROS). To test this possibility, TCC cultures were exposed to N-OH-AABP and the forma tion of ROS was measured using 2,7-dichlorofluorescein (DCF) fluorescence a ssay. TCC cultures exposed to N-OH-AABP showed a dose-dependent increase in the ratio of DCF/DNA fluorescence compared to the untreated controls. Form ation of ROS was inhibited by butylated hydroxyanisole (BHA). Furthermore, oxidative DNA damage resulting from ROS was monitored by measurement of 8-o xoguanine products by immunochemical staining and the TCC cells treated wit h N-OH-AABP revealed a characteristic staining. These results suggest that N-OH-AABP caused oxidative DNA damage as well as bulky covalent adducts in urothelial DNA, possibly involving endogenous peroxidases. These findings s how that human uroepithelial cells, which are the target cell types in vivo for arylamine-induced cancers, are metabolically capable of activating the se proximate carcinogenic metabolites of arylamines, and these reactions mi ght play a determinate role in the genotoxicity of these environmental carc inogens. (C) 2001 Wiley-Liss, Inc.