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Importance of UDP-glucuronosyltransferases 2A2 and 2A3 in tobacco carcinogen metabolism.

ABSTRACT: UDP-glucuronosyltransferase A1 (UGT2A1) is expressed in the lung and exhibits activity against polycyclic aromatic hydrocarbons (PAHs), suggesting UGT2A1 involvement in the local metabolism of PAH tobacco carcinogens. The goal of the present study was to investigate the importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco carcinogen metabolism. Real-time polymerase chain reaction suggested that wild-type UGT2A2 had the highest expression in the breast, followed by trachea > larynx > kidney. A novel splice variant of UGT2A2 lacking exon 3 (termed UGT2A2?exon3) was investigated, with UGT2A2?exon3 expression determined to be 25-50% that of wild-type UGT2A2 in all tissues examined. UGT2A3 was determined to be well expressed in the liver and colon, followed by pancreas > kidney > lung > tonsil > trachea > larynx. Cell homogenates prepared from human embryonic kidney (HEK)293 cells overexpressing wild-type UGT2A2 (termed UGT2A2_i1) exhibited glucuronidation activity, as observed by reverse-phase ultra-pressure liquid chromatography, against 1-hydroxy-(OH)-pyrene, 1-naphthol, and hydroxylated benzo(a)pyrene metabolites, whereas homogenates prepared from HEK293 cells overexpressing UGT2A3 only showed activity against simple PAHs like 1-OH-pyrene and 1-naphthol. Activity assays showed the UGT2A2?exon3 protein (termed UGT2A2_i2) exhibited no detectable glucuronidation activity against all substrates examined; however, coexpression studies suggested that UGT2A2_i2 negatively modulates UGT2A2_i1 activity. Both UGT2A2 and UGT2A3 exhibited no detectable activity against complex PAH proximate carcinogens, tobacco-specific nitrosamines, or heterocyclic amines. These data suggest that, although UGT2A1 is the only UGT2A enzyme active against PAH proximate carcinogens (including PAH diols), both UGTs 2A1 and 2A2 play an important role in the local detoxification of procarcinogenic monohydroxylated PAH metabolites.

SUBMITTER: Bushey RT

PROVIDER: S-EPMC3533432 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Importance of UDP-glucuronosyltransferases 2A2 and 2A3 in tobacco carcinogen metabolism.

Bushey Ryan T RT Dluzen Douglas F DF Lazarus Philip P

Drug metabolism and disposition: the biological fate of chemicals 20121018 1

UDP-glucuronosyltransferase A1 (UGT2A1) is expressed in the lung and exhibits activity against polycyclic aromatic hydrocarbons (PAHs), suggesting UGT2A1 involvement in the local metabolism of PAH tobacco carcinogens. The goal of the present study was to investigate the importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco carcinogen metabolism. Real-time polymerase chain reaction suggested that wild-type UGT2A2 had the highest expression in the breast, followed by trachea > ...[more]

PMID: 23086198

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Project description:2-Amino-9H-pyrido[2,3-b]indole (A?C) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic heterocyclic aromatic amines (HAA) that arise during the burning of tobacco and cooking of meats. UDP-glucuronosyltransferases (UGT) detoxicate many procarcinogens and their metabolites. The genotoxic N-hydroxylated metabolite of A?C, 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-A?C), undergoes glucuronidation to form the isomeric glucuronide (Gluc) conjugates N(2)-(?-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HON(2)-Gluc) and O-(?-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HN(2)-O-Gluc). A?C-HON(2)-Gluc is a stable metabolite but A?C-HN(2)-O-Gluc is a biologically reactive intermediate, which covalently adducts to DNA at levels that are 20-fold higher than HONH-A?C. We measured the rates of formation of A?C-HON(2)-Gluc and A?C-HN(2)-O-Gluc in human organs: highest activity occurred with liver and kidney microsomes, and lesser activity was found with colon and rectum microsomes. A?C-HN(2)-O-Gluc formation was largely diminished in liver and kidney microsomes, by niflumic acid, a selective inhibitor UGT1A9. In contrast, A?C-HON(2)-Gluc formation was less affected and other UGT contribute to N(2)-glucuronidation of HONH-A?C. UGT were reported to catalyze the formation of isomeric Gluc conjugates at the N(2) and N3 atoms of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), the genotoxic metabolite of PhIP. However, we found that the N3-Gluc of HONH-PhIP also covalently bound to DNA at higher levels than HONH-PhIP. The product ion spectra of this Gluc conjugate acquired by ion trap mass spectrometry revealed that the Gluc moiety was linked to the oxygen atom of HONH-PhIP and not the N3 imidazole atom of the oxime tautomer of HONH-PhIP as was originally proposed. UGT1A9, an abundant UGT isoform expressed in human liver and kidney, preferentially forms the O-linked Gluc conjugates of HONH-A?C and HONH-PhIP as opposed to their detoxicated N(2)-Gluc isomers. The regioselective O-glucuronidation of HONH-A?C and HONH-PhIP, by UGT1A9, is a mechanism of bioactivation of these ubiquitous HAAs.

Project description:2-Amino-9H-pyrido[2,3-b]indole (A?C) 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 A?C and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-A?C) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by (1)H NMR and mass spectrometry. A?C and HONH-A?C underwent glucuronidation by UGTs to form, respectively, N(2)-(?-D-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (A?C-N(2)-Gl) and N(2)-(?-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HON(2)-Gl). HONH-A?C also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(?-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HN(2)-O-Gl). A?C-HN(2)-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)-A?C adduct at 20-50-fold higher levels than the adduct levels formed with HONH-A?C. Major UGT isoforms were examined for their capacity to metabolize A?C and HONH-A?C. UGT1A4 was the most catalytically efficient enzyme (V(max)/K(m)) at forming A?C-N(2)-Gl (0.67 ?l·min(-1)·mg of protein(-1)), and UGT1A9 was most catalytically efficient at forming A?C-HN-O-Gl (77.1 ?l·min(-1)·mg of protein(-1)), whereas UGT1A1 was most efficient at forming A?C-HON(2)-Gl (5.0 ?l·min(-1)·mg of protein(-1)). Human hepatocytes produced A?C-N(2)-Gl and A?C-HN(2)-O-Gl in abundant quantities, but A?C-HON(2)-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-A?C.

Dataset Information

Importance of UDP-glucuronosyltransferases 2A2 and 2A3 in tobacco carcinogen metabolism.

Publications

Importance of UDP-glucuronosyltransferases 2A2 and 2A3 in tobacco carcinogen metabolism.

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