Bioactivation of Heterocyclic Aromatic Amines by UDP Glucuronosyltransferases

Abstract

2-Amino-9H-pyrido[2,3-b]indole (AαC) 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 AαC, 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC), undergoes glucuronidation to form the isomeric glucuronide (Gluc) conjugates N2-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HON2-Gluc) and O-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN2-O-Gluc). AαC-HON2-Gluc is a stable metabolite but AαC-HN2-O-Gluc is a biologically reactive intermediate, which covalently adducts to DNA at levels that are 20-fold higher than HONH-AαC. We measured the rates of formation of AαC-HON2-Gluc and AαC-HN2-O-Gluc in human organs: highest activity occurred with liver and kidney microsomes, and lesser activity was found with colon and rectum microsomes. AαC-HN2-O-Gluc formation was largely diminished in liver and kidney microsomes, by niflumic acid, a selective inhibitor UGT1A9. In contrast, AαC-HON2-Gluc formation was less affected and other UGT contribute to N2-glucuronidation of HONH-AαC. UGT were reported to catalyze the formation of isomeric Gluc conjugates at the N2 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-AαC and HONH-PhIP as opposed to their detoxicated N2-Gluc isomers. The regioselective O-glucuronidation of HONH-AαC and HONH-PhIP, by UGT1A9, is a mechanism of bioactivation of these ubiquitous HAAs.

The development and prevalidation of anin vitromutagenicity assay based on MutaMouse primary hepatocytes, Part II: Assay performance for the identification of mutagenic chemicals

“…The heterocyclic amine, PhIP, is a by-product of the Maillard, or browning, reaction that occurs during the cooking of meat (Jägerstad et al, 1983). Aromatic amines, such as 2-AAF and PhIP, require both Phase I enzymes, such as CYP 1A1 and 1A2, as well as Phase II enzymes, such as, SULT, NAT, and/or UGT to generate DNA-reactive nitrenium or carbenium ions (Heflich & Neft, 1994;Schut & Snyderwine, 1999;Cai et al, 2016). Both 2-AAF and PhIP are positive in the MLA and HPRT tests with the addition of S9 (Oberly et al, 1990;Morgenthaler & Holzhäuser, 1995;Mitchell et al, 1997;Kirkland et al, 2008;Kirkland et al, 2016).…”

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part I: Isolation, structural, genetic, and biochemical characterization

“…The Phase I metabolic enzyme NQO1 has been postulated to have both azoreductase and nitroreductase activity and has been implicated in the activation of azo compounds and nitro‐PAHs (Huang et al, ; Møller and Wallin, ; Arlt et al, ). Aromatic amines (AAs), including 2‐acetylaminofluorene, and heterocyclic amines (HCAs), such as PhIP, generally require both Phase I (e.g., CYPs 1A1 and 1A2) and Phase II (e.g., SULT, NAT, and/or UGT) enzymatic reactions to generate DNA‐reactive nitrenium or carbenium ions (Heflich and Neft, ; Schut and Snyderwine, ; Cai et al, ). Although GSTs are well‐known for their detoxification and antioxidant functions, they have also been implicated in the mutagenic activation of halogenated hydrocarbons, such as 1,2‐dibromo‐3‐chloropropane (van Bladeren et al, ; Miller et al, ).…”

Metabolism and biomarkers of heterocyclic aromatic amines in humans

“…Gluc conjugation of HONH-HAAs occurs at the exocyclic amine group to form proposed detoxication metabolites [ 158 ]. Human liver microsomes catalyze Gluc formation of N -hydroxy-HAAs of IQ, MeIQx, PhIP, and AαC [ 142 , 158 – 160 ]. Many of these metabolites are excreted in urine of meat-eaters [ 49 , 120 , 122 , 144 , 161 ].…”