Activation of 3‐nitrobenzanthrone and its metabolites by human acetyltransferases, sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells

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Abstract
3‐Nitrobenzanthrone (3‐NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and ambient air pollution. 3‐Aminobenzanthrone (3‐ABA), 3‐acetylaminobenzanthrone (3‐Ac‐ABA) and N‐acetyl‐N‐hydroxy‐3‐aminobenzanthrone (N‐Ac‐N‐OH‐ABA) have been identified as 3‐NBA metabolites. Recently we found that 3‐NBA and its metabolites (3‐ABA, 3‐Ac‐ABA and N‐Ac‐N‐OH‐ABA) form the same DNA adducts in vivo in rats. In order to investigate whether human cytochrome P450 (CYP) enzymes (i.e., CYP1A2), human N,O‐acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3‐NBA and its metabolites, we developed a panel of Chinese hamster V79MZ‐h1A2 derived cell lines expressing human CYP1A2 in conjunction with human NAT1, NAT2, SULT1A1 or SULT1A2, respectively. Cells were treated with 0.01, 0.1 or 1 μM 3‐NBA, or its metabolites (3‐ABA, 3‐Ac‐ABA and N‐Ac‐N‐OH‐ABA). Using both enrichment versions of the 32P‐postlabeling assay, nuclease P1 digestion and butanol extraction, essentially 4 major and 2 minor DNA adducts were detected in the appropriate cell lines with all 4 compounds. The major ones were identical to those detected in rat tissue; the adducts lack an N‐acetyl group. Human CYP1A2 was required for the metabolic activation of 3‐ABA and 3‐Ac‐ABA (probably viaN‐oxidation) and enhanced the activity of 3‐NBA (probably via nitroreduction). The lack of acetylated adducts suggests N‐deacetylation of 3‐Ac‐ABA and N‐Ac‐N‐OH‐ABA. Thus, N‐hydroxy‐3‐aminobenzanthrone (N‐OH‐ABA) appears to be a common intermediate for the formation of the electrophilic arylnitrenium ions capable of reacting with DNA. Human NAT1 and NAT2 as well as human SULT1A1 and SULT1A2 strongly contributed to the high genotoxicity of 3‐NBA and its metabolites. Moreover, N,O‐acetyltransfer reactions catalyzed by human NATs leading to the corresponding N‐acetoxyester may be important in the bioactivation of N‐Ac‐N‐OH‐ABA. As human exposure to 3‐NBA is likely to occur primarily via the respiratory tract, expression of CYPs, NATs and SULTs in respiratory tissues may contribute significantly and specifically to the metabolic activation of 3‐NBA and its metabolites. Consequently, polymorphisms in these genes could be important determinants of lung cancer risk from 3‐NBA.