A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue.

Glucuronidation is a major pathway of drug and xenobiotic metabolism that is catalyzed by members of the UDP-glucuronosyltransferase (UGT) family. Predicting the contribution of individual UGTs to drug metabolism would be of considerable value in drug development and would be greatly aided by the availability of detailed absolute expression levels of these proteins; this is hampered by the lack of purified protein standards because of the hydrophobic membrane-associated nature of UGTs and the consequential difficulties in expression and purification. Here we describe a novel solution to this problem by expressing UGTs in Escherichia coli as fusion proteins with ribonuclease S-peptide, targeted to the periplasm with the pelB leader sequence.

Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and 2B15 associated with substrate selectivity and autoactivation.

Despite the important role of UDP-glucuronosyltransferases (UGT) in the metabolism of drugs, environmental chemicals and endogenous compounds, the structural features of these enzymes responsible for substrate binding and selectivity remain poorly understood. Since UGT2B7 and UGT2B15 exhibit distinct, but overlapping, substrate selectivities, UGT2B7-UGT2B15 chimeras were constructed here to identify substrate binding domains. A UGT2B7-15-7 chimera that incorporated amino acids 61-194 of UGT2B15 glucuronidated the UGT2B15 substrates testosterone and phenolphthalein, but not the UGT2B7 substrates zidovudine and 11alpha-hydroxyprogesterone.

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family.

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE.

Identification of the human liver UDP-glucuronosyltransferase involved in the metabolism of p-ethoxyphenylurea (dulcin).

Dulcin (DL), now banned, was once a widely used artificial sweetener. DL possesses an ureido group that is metabolized by direct glucuronidation in rabbit liver microsomes. Dulcin N-glucuronide (DNG) is the only type of ureido N-glucuronide known to date; ureido glucuronidation in humans has not been previously reported.

Glucuronidation of SN-38 and NU/ICRF 505 in human colon cancer and adjacent normal colon.

Background: Glucuronidation represents a novel mechanism of intrinsic drug resistance in colon cancer cells. To safely reverse this mechanism in vivo, it is essential to identify which isoforms of UDP-glucuronosyltransferases are responsible for catalysing this drug metabolism in tumour tissue.

Materials And Methods: LC-MS was applied to measure rates of glucuronidation of two anticancer compounds (SN-38 and NU/ICRF 505) by patient colon cancer biopsies and paired normal colon.

Substrate specificity of human hepatic udp-glucuronosyltransferases.

Five human hepatic UDP-glucuronosyltransferases (UGTs) catalyze the facilitated excretion of more than 90% of drugs eliminated by glucuronidation. The substrate specificity of these UGTs has been examined using cloned expressed enzymes and liquid chromatography-mass spectrometry assays to determine the intrinsic clearance of drug glucuronidation in vitro. Specific substrates for the five individual UGTs have been identified.

Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily.

Several novel UDP glycosyltransferase (UGT) genes, mainly UDP glucuronosyltransferases, have been identified in the human, mouse and rat genomes and in other mammalian species. This review provides an update of the UGT nomenclature to include these new genes and prevent the confusion that arises when the same gene is given different names. The new genes are named following previously established recommendations, taking into consideration evolutionary relatedness and the names already in general usage in the literature.

Application of high-performance liquid chromatography-electrospray ionization-mass spectrometry to measure microsomal membrane transport of glucuronides.

A primary reason for poor characterization of microsomal transport to date is the limitations of the measurement techniques used. Radiodetection provides sufficient sensitivity, but it can be applied only when labeled analogue is available. In this article, we report the novel application of high-performance liquid chromatography and electrospray tandem mass spectrometry (LC-MS/MS) in "rapid filtration" transport assays.

Identification of the rabbit liver UDP-glucuronosyltransferase catalyzing the glucuronidation of 4-ethoxyphenylurea (dulcin).

Dulcin (DL), 4-ethoxyphenylurea, a synthetic chemical about 200 times as sweet as sucrose, has been proposed for use as an artificial sweetener. DL is excreted as a urinary ureido-N-glucuronide after oral administration to rabbits. The phenylurea N-glucuronide is the only ureido conjugate with glucuronic acid known at present; therefore, DL is interesting as a probe to search for new functions of UDP-glucuronosyltransferases (UGTs).

Assessment of catechol induction and glucuronidation in rat liver microsomes.

Catechols are substances with a 1,2-dihydroxybenzene group from natural or synthetic origin. The aim of this study was to determine whether catechols (4-methylcatechol, 4-nitrocatechol, 2,3-dihydroxynaphthalene) and the antiparkinsonian drugs, entacapone and tolcapone, at doses 150 to 300 mg/kg/day, for 3 days, are able to enhance their own glucuronidation. The induction potency of catechols on rat liver UDP-glucuronosyltransferases (UGTs) was compared with that of a standard polychlorinated biphenyl (PCB) inducer, Aroclor 1254.

Evidence for multiple glucuronide transporters in rat liver microsomes.

The transport of glucuronides across the endoplasmic reticulum membrane is an important step in the overall process of biotransformation, although the mechanism remains unclear and the participating transporters are unidentified. Using a rapid filtration assay in combination with liquid chromatography-mass spectrometry, we measured the transport of a variety of beta-D-glucuronides in rat liver microsomes and investigated the substrate specificity of the participating transporter(s) by inhibition studies. Time-dependent and bi-directional transport of phenolphthalein glucuronide was detected and the kinetic parameters for transport were determined.

Farnesol is glucuronidated in human liver, kidney and intestine in vitro, and is a novel substrate for UGT2B7 and UGT1A1.

Farnesol is an isoprenoid found in many aromatic plants and is also produced in humans, where it acts on numerous nuclear receptors and has received considerable attention due to its apparent anticancer properties. Although farnesol has been studied for over 30 years, its metabolism has not been well characterized. Recently, farnesol was shown to be metabolized by cytochromes P450 in rabbit; however, neither farnesol hydroxylation nor glucuronidation in humans have been reported to date.

N-glucuronidation of carbamazepine in human tissues is mediated by UGT2B7.

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 N-glucuronide. To date the uridine diphosphate glucuronosyltransferase (UGT) isoform responsible for the N-glucuronidation of CBZ has not been identified.

Conjugation of catechols by recombinant human sulfotransferases, UDP-glucuronosyltransferases, and soluble catechol O-methyltransferase: structure-conjugation relationships and predictive models.

Conjugation of a structurally diverse set of 53 catechol compounds was studied in vitro using six recombinant human sulfotransferases (SULTs), five UDP-glucuronosyltransferases (UGT) and the soluble form of catechol O-methyltransferase (S-COMT) as catalyst. The catechol set comprised endogenous compounds, such as catecholamines and catecholestrogens, drugs, natural plant constituents, and other catechols with diverse substituent properties and substitution patterns. Most of the catechols studied were substrates of S-COMT and four SULT isoforms (1A1, 1A2, 1A3, and 1B1), but the rates of conjugation varied considerably, depending on the substrate structure and the enzyme form.

Glucuronidation of HMR1098 in human microsomes: evidence for the involvement of UGT1A1 in the formation of S-glucuronides.

HMR1098, a novel KATP-blocking agent, is metabolized to form an S-glucuronide in rat and dog bile. Synthesis of the S-glucuronide metabolite was studied in human liver and kidney microsomes. Recombinant UPD-glucuronosyltransferases (UGTs) were screened for activity, and kinetic analysis was performed to identify the isoform or isoforms responsible for the formation of this novel S-glucuronide in humans.

Cloning and characterisation of the first drug-metabolising canine UDP-glucuronosyltransferase of the 2B subfamily.

Glucuronidation is a major route of clearance for a diverse set of both drug and endogenous substrates. The present study was undertaken to redress the lack of molecular information currently available on drug glucuronidation by the dog, a species widely used in metabolism studies by the pharmaceutical industry. A novel dog uridine diphosphate glucuronosyltransferase (UGT), designated UGT2B31 (GenBank Accession Number: AY135176), has been isolated from a dog cDNA library, expressed in V79 cells and characterised using various methods: (i) UGT2B31 sequence has been compared with mammalian UGT sequences using both sequence alignments and phylogenetic analysis; and (ii) the substrate specificity of UGT2B31 has been determined using functional analysis and compared with that obtained using UGT2B7 and dog liver microsomes.

MRP1-transfected cells do not show increased resistance against oxidative stress.

Sensitivity of V79 Chinese hamster cells and V79 cells transfected with human MRP1 gene to several agents inducing oxidative stress was compared. Cells overexpressing MRP1 did not show increased resistance to tert-butyl hydroperoxide, diamide, paraquat, menadione, dichromate and carmustine as estimated by cell survival and DNA damage assessed by comet assay. These findings suggest that overexpression of MRP1 does not confer increased resistance to oxidative stress.

Glucuronidation of catechols by human hepatic, gastric, and intestinal microsomal UDP-glucuronosyltransferases (UGT) and recombinant UGT1A6, UGT1A9, and UGT2B7.

The substrate specificity of human gastric and intestinal UDP-glucuronosyltransferases (UGTs) toward catechols was investigated and compared to that of liver UGTs. Small catechols were efficiently glucuronidated by stomach (0.8-10.

Genetic variation of human UDP-glucuronosyltransferase: implications in disease and drug glucuronidation.

The uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) are key enzymes in human detoxication of xeno- and endobiotics. Potentially toxic endogenous compounds such as bilirubin, or exogenous compounds such as drugs, pesticides, and carcinogens, are generally transformed into water-soluble glucuronides for excretion in bile and urine. The UGTs are encoded by a multigene family in humans.

Glucuronidation of dihydroartemisinin in vivo and by human liver microsomes and expressed UDP-glucuronosyltransferases.

The aim of this study was to elucidate the metabolic pathways for dihydroartemisinin (DHA), the active metabolite of the artemisinin derivative artesunate (ARTS). Urine was collected from 17 Vietnamese adults with falciparum malaria who had received 120 mg of ARTS i.v.

Histone 2A stimulates glucose-6-phosphatase activity by permeabilization of liver microsomes.

Histone 2A increases glucose-6-phosphatase activity in liver microsomes. The effect has been attributed either to the conformational change of the enzyme, or to the permeabilization of microsomal membrane that allows the free access of substrate to the intraluminal glucose-6-phosphatase catalytic site. The aim of the present study was the critical reinvestigation of the mechanism of action of histone 2A.

Quantitative structure activity relationships for the glucuronidation of simple phenols by expressed human UGT1A6 and UGT1A9.

UGT1A6 and UGT1A9 have both been demonstrated to rapidly glucuronidate simple phenolic compounds. A series of simple phenols were selected and screened with both isoforms and then used as model substrates for the generation of V(max) and K(m) values. UGT1A6 showed a more restricted acceptance of phenolic substrates compared with UGT1A9.

The importance of cysteine 126 in the human liver UDP-glucuronosyltransferase UGT1A6.

The human UDP-glucuronosyltransferase 1A6 (UGT1A6) isoform is actively involved in the detoxication of phenolic compounds. In an effort to gain insight on active-site amino acids, we investigated the functional relevance of cysteinyl residues in the glucuronidation process. The enzyme was irreversibly inactivated upon exposure to thiol-specific reagents, especially N-phenylmaleimide.