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-aminobiphenyl (ABP), was examined in human transitional cell carcinoma (TCC) cells after exposure to the chemical in vitro. 32P-postlabeling analysis of TCC cultures exposed to N-OH-AABP revealed a minor adduct identified as 3-(deoxyguanosin-N2-yl)-4-acetylaminobiphenyl (dG-N2-AABP) based on comparison of the HPLC and TLC mobility of the product with the synthetic standard. An adduct with the same chromatographic properties was also detected on postlabeling analyses of calf thymus DNA bound to N-OH-AABP by incubation with horseradish 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. The radical intermediates could also form reactive oxygen species (ROS). To test this possibility, TCC cultures were exposed to N-OH-AABP and the formation of ROS was measured using 2,7-dichlorofluorescein (DCF) fluorescence assay. TCC cultures exposed to N-OH-AABP showed a dose-dependent increase in the ratio of DCF/DNA fluorescence compared to the untreated controls. Formation of ROS was inhibited by butylated hydroxyanisole (BHA). Furthermore, oxidative DNA damage resulting from ROS was monitored by measurement of 8-oxoguanine products by immunochemical staining and the TCC cells treated with 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 show that human uroepithelial cells, which are the target cell types in vivo for arylamine-induced cancers, are metabolically capable of activating these proximate carcinogenic metabolites of arylamines, and these reactions might play a determinate role in the genotoxicity of these environmental carcinogens. Copyright 2001 Wiley-Liss, Inc.