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 N(2)-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HON(2)-Gluc) and O-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN(2)-O-Gluc). AαC-HON(2)-Gluc is a stable metabolite but AαC-HN(2)-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-HON(2)-Gluc and AαC-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. AαC-HN(2)-O-Gluc formation was largely diminished in liver and kidney microsomes, by niflumic acid, a selective inhibitor UGT1A9. In contrast, AαC-HON(2)-Gluc formation was less affected and other UGT contribute to N(2)-glucuronidation of HONH-AαC. 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-AαC and HONH-PhIP as opposed to their detoxicated N(2)-Gluc isomers. The regioselective O-glucuronidation of HONH-AαC and HONH-PhIP, by UGT1A9, is a mechanism of bioactivation of these ubiquitous HAAs.

Figure 1

Histogram showing the mean and standard deviation of the rates of HONH-AαC-Gluc conjugate formation catalyzed by human microsomes (liver, n=13; colon, n=4; rectum, n=4; kidney, n=6, each sample was measured in triplicate).

Figure 2

Aligned dot plot depicting the mean and range in glucuronidation activities of UGT1A1 and UGT1A9 in human microsomes with specific substrates. A) Estradiol-3-O-Gluc formation as a measure of UGT1A1 activity; B) Propofol-O-Gluc as a measure of UGT1A9 activity (human liver, n=13; human colon, n=4; human rectum, n=13; human kidney, n=6; each sample was measured in triplicate).

Figure 3

Aligned dot plot showing the mean and the range in levels of protein expression of UGT1A1 and UGT1A9 in human liver (n=13), colon (n=4), rectum (n=4) and kidney (n=6) microsomes.

Figure 4

Linear regression analysis and correlations among of HONH-AαC-Gluc conjugates with UGT1A1 and UGT1A9 activities and protein expression levels in human liver microsomes (n=13).

Figure 5

The inhibition of UGT1A9 activity by niflumic acid. A) In liver, propofol-O-Gluc is carried out by UGT1A9, and niflumic acid leads to a concentration-dependent decrease in propofol-O-Gluc formation; B) In liver, estradiol-3-O-Gluc formation is largely carried out by UGT1A1 and niflumic acid has no effect on estradiol-3-O-Gluc levels; C) The rate of AαC-HN²-O-Gluc formation by liver and kidney microsomes with niflumic acid; and D) The rate of AαC-HON²-Gluc formation in liver and kidney microsomes with niflumic acid. The inhibition studies in liver and kidney were carried out for 20 min. (ANOVA with Dunnett’s multiple comparison test: *, p<0.05; **, p<0.01).

Figure 6

The characterization of Gluc conjugates of HONH-PhIP. A) The HPLC and UV spectra of HONH-PhIP Gluc catalyzed by human liver and kidney microsomes; B) Product ion spectra of HONH-PhIP-Gluc conjugates in positive ion mode (MS² at m/z 417.1405 >; MS³ at m/z 417.1405 > 241.1084 >; and m/z 417.1405 > 225.1135 >); C) Product ion spectra of HONH-PhIP-Gluc conjugates in negative ion mode (MS² at m/z 415.1259 >; MS³ at 415.1259 > 239.0938 >; m/z 415.1259 > 223.0989 >; and m/z 415.1259 > 191.0197 >).

Figure 7

The proposed structures of HONH-PhIP-Gluc conjugates.

Figure 8

Reactivity of HONH-PhIP, HONH-AαC and their Gluc conjugates with calf thymus DNA to form dG-C8 adducts.

Scheme 1

Metabolism pathways of AαC and HONH-AαC by P450 and UGT.

Scheme 2

UGT-mediated bioactivation of the N-hydroxy metabolite of 2-acetylaminofluorene, AαC and PhIP.