ABSTRACT:The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K m or S 50 (sigmoidal kinetics) and V max values varied 525-(8-4204 M) and 5535-fold, respectively, for 4MU glucuronidation, and 1360-(1.3-1768 M) and 560-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-MichaelisMenten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 1A6, 2B15, and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K i values, determined for selected isoforms, ranged from 11 to 60 M and 96 to 2790 M, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.
This article is available online at http://dmd.aspetjournals.org ABSTRACT:The pregnane X receptor (PXR) has three known major transcript variants resulting from alternative splicing. The less well characterized variants T2 and T3 are identical to the well described variant T1 except for a 39-amino acid N-terminal extension in T2 and an internal 37-amino acid deletion in T3. We have developed reverse transcription-polymerase chain reaction (RT-PCR) methods to detect and quantify each human PXR (hPXR) in human liver and intestinal tissues and HepG2 and Caco-2 cell lines. All three isoforms were expressed in hepatic cells, whereas only T1 transcripts were found in Caco-2 cells. In general, most normal human liver and intestinal mucosa contained all three hPXR variants, but considerable interindividual variation in expression levels was found. The effect of each hPXR variant on expression of UDPglucuronosyltransferase (UGT) UGT1A and UGT2B family isoforms was investigated in transiently transfected HepG2 and Caco-2 cells. As a family, UGT1A transcripts were up-regulated by T1 and T2 but not T3. Isoform-specific RT-PCR revealed that UGT1A1, 1A3, and 1A4 were the major isoforms induced in both cell lines. The levels of several UGT1A isoforms were also examined in human liver samples from a number of donors with characterized PXR expression. The data suggest that individual variation in PXR expression may account for differential expression of some UGT isoforms between subjects.
The UDP glucuronosyltransferase (UGT) content of cells and tissues is a major determinant of our response to those chemicals that are primarily eliminated by conjugation with glucuronic acid. There are marked interindividual differences in the content of UGTs in the liver and other organs. The mechanisms that lead to these differences are unknown but are most likely the result of differential UGT gene expression. Several transcription factors involved in the regulation of UGT genes have been identified. These include factors such as Hepatocyte Nuclear Factor 1, CAAT-Enhancer Binding Protein, Octamer transcription Factor 1 and Pbx2, which appear to control the constitutive levels of UGTs in tissues and organs. In addition, UGT gene expression is also modulated by hormones, drugs and other foreign chemicals through the action of proteins that bind and/or sense the presence of these chemicals. These proteins include the Ah receptor, members of the nuclear receptor superfamily, such as CAR and PXR and transcription factors that respond to stress.
The human UDP-glucuronosyltransferases (UGT) -1A8 and -1A10 are exclusively expressed in extrahepatic tissues and primarily in the gastrointestinal tract, whereas UGT1A9 is expressed mainly in the liver and kidneys. We have demonstrated previously that the UGT1A8 and UGT1A10 genes, in contrast to the UGT1A9 gene, are regulated via an initiator-like element in their proximal promoters. To determine the elements that contribute to the gastrointestinal expression of UGT1A8 and -1A10, we conducted deletion analysis of the UGT1A8, -1A9, and -1A10 promoters in the colon-derived cell line Caco2. DNA elements contributing significantly to UGT1A8, -1A9, and -1A10 promoter activity were found to reside primarily within 140 base pairs of the transcription start site. Within this region, putative binding sites for the intestine-specific transcription factor, caudal-related homeodomain protein 2 (Cdx2), and hepatocyte nuclear factor 1 (HNF1) were identified. Using gel shift and functional assays, HNF1␣ was demonstrated to bind to and activate the UGT1A8, -1A9, and -1A10 promoters. In contrast, Cdx2 bound to and activated the UGT1A8 and -1A10 promoters but could not activate the UGT1A9 promoter. A single base pair difference between the UGT1A8 and -1A10 promoters, three base pairs downstream of the consensus Cdx2 site, contributed to the observed difference in Cdx2 binding and Cdx2-mediated promoter activation of these two promoters. In addition, Cdx2 was shown to cooperate with HNF1␣ to synergistically activate the UGT1A8, -1A9, and -1A10 promoters. These studies provide insight into the mechanisms controlling the extrahepatic expression of the UGT1A8, -1A9, and -1A10 genes.
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