Expression of a human liver cDNA encoding a UDP‐glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture

Fournel‐Gigleux, Sylvie; Jackson, Michael R.; Wooster, Richard; Burchell, Brian

doi:10.1016/0014-5793(89)80111-5

Cited by 81 publications

(26 citation statements)

References 21 publications

(19 reference statements)

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“…A tissue without a visible bar indicates that UGT2A3 mRNA was detected but abundance was less than 1% relative to adult liver. The rates of hyodeoxycholic acid glucuronidation we determined for UGT2A3 (approximately 20 pmol/min/mg protein) are relatively low compared with values reported for other UGT isoforms such as UGT2B7 (Mackenzie et al, 2003) and UGT2B4 (Fournel-Gigleux et al, 1989) that have recombinant enzyme activities as high as 2 nmol/min/mg protein. Consequently, it is possible that UGT2A3 only contributes minimally to hyodeoxycholic acid glucuronidation in human tissues.…”

Section: Downloaded Fromcontrasting

confidence: 70%

Novel Polymorphic Human UDP-glucuronosyltransferase 2A3: Cloning, Functional Characterization of Enzyme Variants, Comparative Tissue Expression, and Gene Induction

et al. 2008

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UDP-glucuronosyltransferases (UGTs) are critical to the detoxification of numerous drugs, environmental pollutants, and endogenous molecules. However, as yet not all of the human UGTs have been cloned and characterized. cDNA clones from the UGT2A3 gene (located on chromosome 4q13) were isolated using pooled human liver RNA. Approximately 10% of clones contained a c.1489AϾG nucleotide substitution, yielding proteins with a residue 497 alanine (UGT2A3.2) instead of a threonine (UGT2A3.1). The allele frequency of this polymorphism (rs13128286) was 0.13 in a European-American population as determined by direct DNA sequencing. Of 81 structurally diverse glucuronidation substrates tested, UGT2A3 expressed by a baculovirus system selectively glucuronidated bile acids, particularly hyodeoxycholic acid at the 6-hydroxy position. Apparent K m values of UGT2A3.1 and UGT2A3.2 for hyodeoxycholic acid 6-glucuronidation were 69 Ϯ 7 and 44 Ϯ 12 M, respectively. Of 29 different extrahepatic tissues evaluated by real-time polymerase chain reaction, UGT2A3 mRNA was most highly expressed in small intestine (160% of liver), colon (78% of liver), and adipose tissue (91% of liver). An in silico scan of the proximal UGT2A3 promoter/5Ј-regulatory region identified transcription factor consensus elements consistent with tissueselective expression in liver (HNF1) and intestine (CXD2), as well as induction by rifampicin (pregnane X receptor). In LS180 human intestinal cells, rifampicin increased UGT2A3 mRNA by more than 4.5-fold compared with vehicle, whereas levels were not significantly affected by the arylhydrocarbon receptor ligand ␤-naphthoflavone. This is the first report establishing UGT2A3 as a functional enzyme, and it represents significant progress toward the goal of having a complete set of recombinant human UGTs for comparative functional analyses.

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Section: Downloaded Fromcontrasting

confidence: 70%

Novel Polymorphic Human UDP-glucuronosyltransferase 2A3: Cloning, Functional Characterization of Enzyme Variants, Comparative Tissue Expression, and Gene Induction

et al. 2008

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“…To assess the biological effect of the identified polymorphic regulation in small intestine catalytic UGT activity assays using HDCA and 4-MU and endoplasmic reticulum protein prepared from the mucosa of small intestine and from liver tissue were performed. HDCA glucuronidation has been identified for UGT2B4, UGT2B7 (30,31), and UGT1A3 (32), whereas 4-MU glucuronidation can be catalyzed by most UGT1A proteins (5). As predicted from the polymorphic expression of UGT2B4 and UGT2B7 transcripts in the duodenum and the jejunum, HDCA glucuronidation varied 7-fold between individuals, whereas 4-MU glucuronidation in the same samples varied little but clearly more than the absence of interindividual variation seen in liver tissue.…”

Section: Discussionmentioning

confidence: 99%

Polymorphic Gene Regulation and Interindividual Variation of UDP-glucuronosyltransferase Activity in Human Small Intestine

Strassburg¹,

Kneip²,

Topp³

et al. 2000

Journal of Biological Chemistry

226

154

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UDP-glucuronosyltransferases (UGTs) convert dietary constituents, drugs, and environmental mutagens to inactive hydrophilic glucuronides. Recent studies have shown that the expression of the UGT1 and UGT2 gene families is regulated in a tissue-specific fashion. Human small intestine represents a major site of resorption of dietary constituents and orally administered drugs and plays an important role in extrahepatic UGT directed metabolism. Expression of 13 UGT1A and UGT2B genes coupled with functional and catalytic analyses were studied using 18 small intestinal and 16 hepatic human tissue samples. Hepatic expression of UGT gene transcripts was without interindividual variation. In contrast, a polymorphic expression pattern of all the UGT genes was demonstrated in duodenal, jejunal, and ileal mucosa, with the exception of UGT1A10. To complement these studies, interindividual expression of UGT proteins and catalytic activities were also demonstrated. Hyodeoxycholic acid glucuronidation, catalyzed primarily by UGT2B4 and UGT2B7, showed a 7-fold interindividual variation in small intestinal duodenal samples, in contrast to limited variation in the presence of 4-methylumbelliferone, a substrate glucuronidated by most UGT1A and UGT2B gene products. Linkage of RNA expression patterns to protein abundance were also made with several mono-specific antibodies to the UGTs. These results are in contrast to a total absence of polymorphic variation in gene expression, protein abundance, and catalytic activity in liver. In addition, the small intestine exhibits considerable catalytic activity toward most of the different classes of substrates accepted for glucuronidation by the UGTs, which is supported by immunofluorescence analysis of UGT1A protein in the mucosal cell layer of the small intestine. Thus, tissue-specific and interindividual polymorphic regulation of UGT1A and UGT2B genes in small intestine is identified and implicated as molecular biological determinant contributing to interindividual prehepatic drug and xenobiotic metabolism in humans.

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“…Microsomes were prepared from frozen human tissue (UK Human Tissue Bank, Leicester, UK) by standard methods (Coughtrie et al, 1991). Cell lines (V79 Chinese hamster lung fibroblasts) expressing human UGTs have been created previously in this laboratory (Jackson et al, 1987;Fournel-Gigleux et al, 1989Wooster et al, 1991) except UGT1A4 expressed in the baculovirus/Sf9 cell system (kindly donated by Robert Tukey, Departments of Chemistry & Biochemistry and Pharmacology, University of California, San Diego, La Jolla, CA). UGT-expressing cells were grown and cell lysates prepared as described previously (Fournel-Gigleux et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

N-Glucuronidation of Carbamazepine in Human Tissues Is Mediated by UGT2B7

Staines¹,

Coughtrie²,

Burchell³

2004

J Pharmacol Exp Ther

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Carbamazepine (CBZ) is one of the most widely prescribed anticonvulsants despite a high incidence of idiosyncratic side effects. Metabolism of CBZ is complex, and of the more than 30 metabolites identified, one of the most abundant is CBZ Nglucuronide. To date the uridine diphosphate glucuronosyltransferase (UGT) isoform responsible for the N-glucuronidation of CBZ has not been identified. We have developed a sensitive liquid chromatography/mass spectrometry assay to quantify CBZ glucuronidation, and we report that CBZ is specifically glucuronidated by human UGT2B7. Kinetics of CBZ glucuronidation in human liver, kidney, and intestine microsomes were consistent with those of recombinant UGT2B7, which displayed a K m value of 214 M and V max value of 0.79 pmol/mg/min. In addition to revealing the isoform responsible for CBZ glucuronidation, this is the first example of primary amine glucuronidation by UGT2B7.Carbamazepine (5H-dibenzo[b,f]azepine-5-carboxamide) is one of the most widely prescribed anticonvulsants and is used to treat a variety of conditions from epilepsy to muscle spasm and trigeminal neuralgia. However its use is associated with a number of idiosyncratic adverse side effects, including skin rash, blood disorders, and hepatitis in onethird to one-half of patients (Ju and Uetrecht 1999). These adverse side effects have been associated with the formation of CBZ metabolites (Shear and Spielberg 1988;Riley et al., 1989); therefore, the study of all CBZ metabolites has important clinical implications.The metabolism of CBZ is complex and has been widely studied in human and in animal models (Madden et al., 1996;Maggs et al., 1997) with over 30 metabolites (Lertratanangkoon and Horning, 1982) identified. The major metabolites are the 10,11-epoxide and its hydrolytic trans-dihydrodiol product. Glucuronidation is also an important detoxification pathway because the CBZ N-glucuronide and glucuronides of the hydroxylated metabolites are significant urinary metabolites; however, to date no glucuronide metabolite has been implicated in the incidence of side effects. Epoxide hydrolase has been the focus of most attention as a potential source of toxic metabolites; however, the available data do not support a major role for this enzyme in causing side effects (Pirmohamed et al., 1992;Green et al., 1995b). One minor metabolite, 2-hydroxy-CBZ, formed through loss of the carboxamide, could be metabolized to an iminoquinone, which due to its potential chemical reactivity might be the metabolite responsible for the idiosyncratic reactions, although this has not been shown to date (Ju and Uetrecht, 1999). In addition CBZ is also a well known enzyme inducer up-regulating cytochrome P450 (Luo et al., 2002) and UGT activity/expression (Tanaka, 1999) Carbamazepine is metabolized to an N-glucuronide (Bauer et al., 1976); in addition, glucuronide metabolites have been demonstrated for all 13 of the hydroxylated metabolites of CBZ (Maggs et al., 1997). Formation of N-glucuronides has been principally studied in nonro...

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Expression of a human liver cDNA encoding a UDP‐glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture

Cited by 81 publications

References 21 publications

Novel Polymorphic Human UDP-glucuronosyltransferase 2A3: Cloning, Functional Characterization of Enzyme Variants, Comparative Tissue Expression, and Gene Induction

Novel Polymorphic Human UDP-glucuronosyltransferase 2A3: Cloning, Functional Characterization of Enzyme Variants, Comparative Tissue Expression, and Gene Induction

Polymorphic Gene Regulation and Interindividual Variation of UDP-glucuronosyltransferase Activity in Human Small Intestine

N-Glucuronidation of Carbamazepine in Human Tissues Is Mediated by UGT2B7

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