Glucuronidation of PhIP and N-OH-PhIP by UDP-glucuronosyltransferase 1A10

Abstract

The UDP-glucuronosyltransferase (UGT) 1A10 is an extra-hepatic enzyme that plays an important role in the glucuronidation of a variety of endogenous and exogenous substances and is expressed throughout the aerodigestive and digestive tracts. Two classes of carcinogens that target the colon, heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons, are known to be detoxified by the UGT family of enzymes. Recently, our laboratory demonstrated that UGT1A10 has considerably more activity against polycyclic aromatic hydrocarbons in vitro than any other UGT family member. In this study, we focused on the glucuronidation of the HCA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and its bioactivated metabolite, N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). We demonstrated that UGT1A10 exhibited a significantly higher glucuronidation rate against PhIP and N-OH-PhIP than any other UGT family member in vitro using whole-cell homogenates of HEK293 cells over-expressing individual UGTs. Kinetic analysis revealed a 9- and 22-fold higher level of activity for UGT1A10 homogenates as compared with the next most active UGT, UGT1A1, against N-OH-PhIP as determined by maximum rate/apparent Michaelis constant (V(max)/K(M)) at the N3 and N2 positions, respectively. The polymorphic UGT1A10(139Lys) variant exhibited a 2- to 16-fold decrease in glucuronidation activity against PhIP and N-OH-PhIP, as compared with the wild-type UGT1A10(139Glu) isoform. These data suggest that UGT1A10 is the most active UGT against PhIP and N-OH-PhIP and that UGT1A10 may play an important role in susceptibility to HCA-induced colon cancer.

Key Amino Acid Residues Responsible for the Differences in Substrate Specificity of Human UDP-Glucuronosyltransferase (UGT)1A9 and UGT1A8

“…Such higher activities in the HEK293 system than the Supersomes were also observed for the glucuronidations of 5-(4Ј-hydroxyphenyl)-5-phenylhydantoin (Nakajima et al, 2007) and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (Malfatti and Felton, 2004;Dellinger et al, 2007). The differences in the membrane environment or lipid components and/or the differences in the post-translational modulation of UGT such as glycosylation or phosphorylation between the host cells (Barbier et al, 2000;Basu et al, 2005) may be involved in the inconsistency.…”

Glucuronidation of Active Tamoxifen Metabolites by the Human UDP Glucuronosyltransferases

“…Levels of UGT1A protein were determined as described previously (Dellinger et al, 2007) by our laboratory. UGT2B expression in UGT-overexpressing cell lines was measured by Western blot analysis using a newly synthesized affinity-purified chicken anti-UGT2B antibody generated against the peptide CKWDQFYSEVLGRPTTL, which is common to all human UGT2B family members (Pocono Rabbit Farm, Canadensis, PA).…”

“…UGTs 1A6, 2B4, 2B10, and 2B11 did not exhibit detectable glucuronidating activity against either 4-OH-TAM isomer. After normalizing for UGT1A protein expression as determined by Western blot analysis (Dellinger et al, 2007), the order of O-glucuronidation activity of the trans-4-OH-TAM isomer based upon V max /K M was 1A8 Ͼ 1A10 Ͼ 1A1 Ͼ 1A3 Ͼ 1A9 Ն 1A7. A newly designed anti-UGT2B antibody (described under Materials and Methods) was reactive against UGTs 2B7, 2B15, and 2B17 (Fig.…”

Functional Characterization of Low-Prevalence Missense Polymorphisms in the UDP-Glucuronosyltransferase 1A9 Gene

“…The rate of glucuronidation by UGT1A9-overexpressing cell microsomes was determined essentially as described previously (Fang et al, 2002;Wiener et al, 2004;Al-Zoughool and Talaska, 2005;Dellinger et al, 2006Dellinger et al, , 2007. For glucuronidation rate determinations, substrate concentrations, microsomal protein levels, and incubation times for individual assays were chosen to maximize levels of detection within a linear range of uptake and were similar to established protocols (Fang et al, 2002;Wiener et al, 2004;Dellinger et al, 2007;Sun et al, 2007). For O-glucuronidated substrates, kinetic analysis against 3-OH-B[a]P was performed using UGT1A9-overexpressing cell microsomes (1 g of protein) preincubated with alamethicin (50 g/mg protein) for 10 min on ice.…”