title: Metabolism of the Tobacco Carcinogen 2-Amino-9H-pyrido[2,3-b]indole (AαC) in Primary Human Hepatocytes.
abstract: 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant carcinogenic heterocyclic aromatic amine (HAA) formed in mainstream tobacco smoke. AαC is a liver carcinogen in rodents, but its carcinogenic potential in humans is not known. To obtain a better understanding of the genotoxicity of AαC in humans, we have investigated its metabolism and its ability to form DNA adducts in human hepatocytes. Primary human hepatocytes were treated with AαC at doses ranging from 0.1-50 μM, and the metabolites were characterized by ultra-performance LC/ion trap multistage mass spectrometry (UPLC/MSn). Six major metabolites were identified: a ring-oxidized doubly conjugated metabolite, N2-acetyl-2-amino-9H-pyrido[2,3-b]indole-6-yl-oxo-(β-d-glucuronic acid) (N2-acetyl-AαC-6-O-Gluc); two ring-oxidized glucuronide (Gluc) conjugates: 2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(β-d-glucuronic acid) (AαC-3-O-Gluc) and 2-amino-9H-pyrido[2,3-b]indol-6-yl-oxo-(β-d-glucuronic acid) (AαC-6-O-Gluc); two sulfate conjugates, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (AαC-3-O-SO3H) and 2-amino-9H-pyrido[2,3-b]indol-6-yl sulfate (AαC-6-O-SO3H); and the Gluc conjugate, N2-(β-d-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N2-Gluc). In addition, four minor metabolites were identified: N2-acetyl-9H-pyrido[2,3-b]indol-3-yl sulfate (N2-acetyl-AαC-3-O-SO3H), N2-acetyl-9H-pyrido[2,3-b]indol-6-yl sulfate (N2-acetyl-AαC-6-O-SO3H), N2-acetyl-2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(β-d-glucuronic acid) (N2-acetyl-AαC-3-O-Gluc), and O-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN2-O-Gluc). The latter metabolite, AαC-HN2-O-Gluc is a reactive intermediate that binds to DNA to form the covalent adduct N-(2'-deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-AαC). Preincubation of hepatocytes with furafylline, a selective mechanism-based inhibitor of P450 1A2, resulted in a strong decrease in the formation of AαC-HN2-O-Gluc and a concomitant decrease in DNA adduct formation. Our findings describe the major pathways of metabolism of AαC in primary human hepatocytes and reveal the importance of N-acetylation and glucuronidation in metabolism of AαC. P450 1A2 is a major isoform involved in the bioactivation of AαC to form the reactive AαC-HN2-O-Gluc conjugate and AαC-DNA adducts.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm), U1085, Institut de Recherche en Santé Environnement et Travail (IRSET), Université de Rennes 1, UMS 3480 Biosit , F-35043 Rennes, France.|ANSES Laboratoire de Fougères, La Haute Marche , Javené BP 90203, 350302 Fougères, France.
authors.forename: Medjda
authors.initials: M
authors.lastname: Bellamri
authors.affiliation: ANSES Laboratoire de Fougères, La Haute Marche , Javené BP 90203, 350302 Fougères, France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Le Hegarat
authors.affiliation: Masonic Cancer Center and Department of Medicinal Chemistry, Cancer and Cardiology Research Building, University of Minnesota , 2231 Sixth Street, Minneapolis, Minnesota 55455, United States.
authors.forename: Robert J
authors.identifier: 0000-0001-7355-9903
authors.initials: RJ
authors.lastname: Turesky
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm), U1085, Institut de Recherche en Santé Environnement et Travail (IRSET), Université de Rennes 1, UMS 3480 Biosit , F-35043 Rennes, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Langouët
mesh_terms: D002243:Carbolines / Q000378:metabolism*; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
chemical_list: D002243:Carbolines; C017822:2-amino-9H-pyrido(2,3-b)indole