UDP-Glucuronosyltransferase-mediated Metabolic Activation of the Tobacco Carcinogen 2-Amino-9H-pyrido[2,3-b]indole

2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of AαC and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by 1H NMR and mass spectrometry. AαC and HONH-AαC underwent glucuronidation by UGTs to form, respectively, N2-(β-d-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N2-Gl) and N2-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HON2-Gl). HONH-AαC also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN2-O-Gl). AαC-HN2-O-Gl is a biologically reactive metabolite and binds to calf thymus DNA (pH 5.0 or 7.0) to form the N-(deoxyguanosin-8-yl)-AαC adduct at 20–50-fold higher levels than the adduct levels formed with HONH-AαC. Major UGT isoforms were examined for their capacity to metabolize AαC and HONH-AαC. UGT1A4 was the most catalytically efficient enzyme (Vmax/Km) at forming AαC-N2-Gl (0.67 μl·min−1·mg of protein−1), and UGT1A9 was most catalytically efficient at forming AαC-HN-O-Gl (77.1 μl·min−1·mg of protein−1), whereas UGT1A1 was most efficient at forming AαC-HON2-Gl (5.0 μl·min−1·mg of protein−1). Human hepatocytes produced AαC-N2-Gl and AαC-HN2-O-Gl in abundant quantities, but AαC-HON2-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-AαC. Background: 2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogen formed in tobacco smoke, but little is known about its metabolism in humans. Results: UDP-Glucuronosyltransferases catalyze the binding of N-oxidized-AαC to DNA. Conclusion: Glucuronidation, normally a detoxication pathway, contributes to the genotoxicity of AαC. Significance: The exposure to and UGT bioactivation of AαC provides a biochemical mechanism for the elevated risk of liver and digestive tract cancers in smokers.