Liver-enriched transcription factors and their role in regulating UDP glucuronosyltransferase gene expression.

Variations in the capacity to detoxify carcinogens and other environmental toxins, and to eliminate drugs and waste products of metabolism, are likely to have significant effects on health and drug efficacy. As the UDP glucuronosyltransferases metabolize many of these substances to less toxic glucuronides, variations in UGT expression are likely to be important in maintenance of health and therapeutic outcomes. The factors that regulate UGT gene expression are beginning to be identified. From among these factors, the Liver-Enriched Transcription Factors (LETFs), including Hepatocyte Nuclear Factors 1 and 4alpha, have a major role in UGT regulation in the major sites of drug metabolism, the liver and gastrointestinal tract. This review will describe what is currently known about these LETFs and their role in UGT gene expression. It is likely that polymorphisms in LETFs and the sites to which they bind in UGT genes, may impact on drug induced disease and drug therapy.

Comparative homology modeling of human cytochrome P4501A1 (CYP1A1) and confirmation of residues involved in 7-ethoxyresorufin O-deethylation by site-directed mutagenesis and enzyme kinetic analysis.

CYP1A1 homology models based on the CYP2C5 and a composite of CYP2C5, CYP2C8, and CYP2C9 X-ray crystal structures were compared to a model generated using the recently published coordinates of CYP1A2. The model using the CYP1A2 coordinates, CYP1A1-(1A2), gave near ideal stereochemical quality and was favored energetically. Docking studies identified the active-site residues potentially involved in binding of the prototypic CYP1A1 substrate 7-ethoxyresorufin. CYP1A1 mutants S122A, F123A, F224A, A317Y, T321G, and I386G were generated to explore the roles of these residues in 7-ethoxyresorufin binding and turnover, and generally confirmed the importance of aromatic interactions over hydrogen bonding in orientating 7-ethoxyresrufin in a catalytically favorable orientation. Although 7-ethoxyresorufin O-deethylation by CYP1A1 and several mutants exhibited substrate inhibition, it is unlikely that inhibition arises from the simultaneous binding of two substrates within the active-site given the geometry of the active site-cavity.

Glucuronidation of carcinogen metabolites by complementary DNA-expressed uridine 5'-diphosphate glucuronosyltransferases.

Five UDP glucuronosyltransferases (UGT) were synthesized from complementary DNAs expressed in COS 7 cells and were tested for their capacities to glucuronidate a range of 2-acetylaminofluorene and benzo(a)pyrene-hydroxylated metabolites. Three forms, UGT1*06, UGT2B1, and UGT2B2 [names of UGT forms follow recommended nomenclature (B. B. Burchell et al., DNA Cell Biol., 10: 487-494, 1991)], had similar capacities to glucuronidate the reactive metabolite, N-hydroxy-2-acetylaminofluorene. The less reactive 1-, 3-, 5-, and 8-hydroxy derivatives of this aromatic amine were glucuronidated by UGT1*06 and UGT2B2 to varying degrees, but these were not substrates of UGT2B1. The three isozymes also glucuronidated phenolic metabolites of benzo(a)pyrene. UGT1*06 was more active toward 2- and 5-hydroxybenzo(a)pyrene, whereas UGT2B1 preferentially glucuronidated the 4- and 11-hydroxy derivatives and UGT2B2 preferentially glucuronidated the 1-, 2-, 8-, and 9-hydroxy metabolites. Two other UDP glucuronosyltransferases, UGT2B3 and UGT2B6, that glucuronidated testosterone when expressed in COS 7 cells were both inactive toward all the carcinogen metabolites tested. These results demonstrate that the glucuronidation of metabolites of 2-acetylaminofluorene and benzo(a)pyrene is mediated by at least three UDP glucuronosyltransferases and that each form glucuronidates a unique spectrum of metabolites.

Preclinical prediction of factors influencing the elimination of 5,6-dimethylxanthenone-4-acetic acid, a new anticancer drug.

The glucuronidation of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a newly developed anticancer drug, was investigated in vitro to determine factors likely to affect the elimination of this compound in patients. Human liver microsomal DMXAA glucuronidation followed Michaelis-Menten kinetics, with a mean apparent Km of approximately 100 microM. Two cDNA-expressed UGT isoforms, UGT1*02 and UGT2B7, had the capacity to glucuronidate DMXAA, although comparative kinetic and inhibitor studies were more consistent with a greater contribution of UGT2B7 to the human hepatic reaction. Microsomal DMXAA glucuronide formation was screened for inhibition by drugs known to be eliminated by glucuronidation. Of the drugs screened, significant inhibition was observed with diclofenac, epirubicin, indomethacin, R,S-ketoprofen, lorazepam, S-naproxen, oxazepam, and temazepam; apparent Ki values ranged from 9.5-318 microM. These values are substantially above unbound concentrations of the individual drugs achieved in vivo. DMXAA glucuronide was found to be unstable at physiological pH values, and the rate of degradation was marginally increased in the presence of albumin. Taken together, these data indicate that the kinetics of DMXAA glucuronidation in vivo are likely to be linear and unaffected by the coadministration of most glucuronidated drugs, but plasma DMXAA clearance may be decreased in patients with renal dysfunction. This study illustrates the utility of in vitro techniques for the prediction of potential drug interactions and other dispositional characteristics of newly developed anticancer drugs before their administration to patients.

Bile acid glucuronidation by rat liver microsomes and cDNA-expressed UDP-glucuronosyltransferases.

Four rat UDP-glucuronosyltransferases (UGTs), UGT2B1, UGT2B2, UGT2B3 and UGT2B6, synthesized in COS-7 cells from appropriate cDNA clones were screened for activity towards a range of bile acids, neutral steroids and retinoic acid. For comparison, as well as optimization of enzymatic assays and product identification, rat liver microsomal preparations from Sprague-Dawley, Fischer 344 and phenobarbital-induced Fischer 344 male rats were also used as enzyme sources. Only two of the expressed proteins, UGT2B1 and UGT2B2, were active in bile acid glucuronidation. UGT2B1 exhibited a high substrate specificity for the carboxyl function of bile acids, whereas UGT2B2 demonstrated less specificity, accepting both hydroxyl and carboxyl functions of bile acids. The preferred substrates for both cloned enzymes were mono-hydroxylated bile acids, followed by di-hydroxylated 6-OH compounds. The levels of UGT activity were sufficient to allow for the identification of the biosynthesized products. The data presented here demonstrate that bile acid glucuronidation is carried out, at least in part, by members of the UGT2B subfamily. Similar results have been obtained previously for neutral steroid glucuronidation. UGT2B3 and UGT2B6 was not involved in BA glucuronidation; none of the cloned enzymes was active toward retinoic acid.

Drug glucuronidation in humans.

Glucuronidation is a major metabolic pathway for a large number of drugs in humans. Conjugation of drugs and other chemicals with glucuronic acid is catalyzed by the multigene UDP-glucuronosyltransferase family. It is believed that a number (unspecified at present) of glucuronosyltransferase isozymes, which probably differ in terms of substrate specificity and regulation, contribute to drug glucuronidation. Factors known to influence the pharmacokinetics of glucuronidated drugs in man, presumably via an effect on specific glucuronosyltransferases, include age (especially the neonatal period), cigarette smoking, diet, certain disease states, coadministered drugs, ethnicity, genetics and hormonal effects.

Polymorphic variations in the expression of the chemical detoxifying UDP glucuronosyltransferases.

The UDP glucuronosyltransferases (UGT) are expressed predominantly in the liver and gastrointestinal tract in humans. Their expression varies widely between individuals, due in part to coding region polymorphisms that alter catalytic function and in part, to differences in the regulation of UGT genes. The latter differences are most likely the result of polymorphisms in the regulatory elements of UGT genes and in the transcription factors that bind to these elements. Several frequent polymorphisms in the promoters of UGT genes have been described; however, few of these fall within critical regulatory elements and alter UGT expression. Some rare mutations alter UGT promoter activity in in vitro systems but their effect in the clinic is still to be confirmed. Several transcription factors that regulate UGT gene expression in cells of hepatic and intestinal origin have been identified. These include positive regulators of UGT gene expression such as hepatocyte nuclear factor 1 alpha (HNF1 alpha), octamer transcription factor-1 (Oct-1) and the intestine-specific transcription factor, caudal-related homeodomain protein 2 (Cdx2). Negative regulators include the Pre B cell homeobox factor (Pbx2) and its dimerization partner, Pbx regulating protein 1 (Prep1). Polymorphisms in these transcription factors may cause differences in their interaction and binding to UGT promoters. Current work describing the effects of these transcription factor polymorphisms on UGT expression will be described. Knowledge of UGT promoter elements and the proteins that bind to these elements, as well as knowledge of polymorphisms that alter their function, may aid in the prediction of an individual's response to chemicals and in the prediction of chemical toxicities.

Tissue specific differences in the regulation of the UDP glucuronosyltransferase 2B17 gene promoter.

The human UDP glucuronosyltransferase UGT2B17, glucuronidates androgens and is expressed in the liver and the prostate. Although evidence suggests that variations in UGT2B17 expression between tissues may be a critical determinant of androgen response, the factors that regulate UGT2B17 expression in the liver and prostate are unknown. In this study, we have isolated a 596 bp promoter of the UGT2B17 gene and studied its regulation in the liver cell line, HepG2 and the prostate cell line, LNCaP. The transcription start site of UGT2B17 was mapped and proteins that bound to the proximal promoter were detected by DNase1 footprint analysis. A region (-40 to -52 bp) which resembled a hepatocyte nuclear factor 1 (HNF1) binding site bound proteins in nuclear extracts from HepG2 cells, but did not bind proteins from LNCaP nuclear extracts. In HepG2 cells, HNF1alpha bound to this region and activated the UGT2B17 promoter, as assessed by functional and gel shift assays. HNF1alpha activation of the promoter was prevented by mutation or deletion of the putative HNF1 site. The related transcription factor HNF1beta, which is present in HepG2 cells, did not activate the promoter. The UGT2B17 promoter could also be activated by exogenous HNF1alpha in LNCaP cells. However, because these cells do not contain HNF1alpha, other transcription factors must regulate the UGT2B17 promoter. Cotransfection experiments showed that HNF1beta, elevates promoter activity in LNCaP cells. This activation did not involve the putative HNF1 region (-40 to -52 bp) since mutation of this region did not affect promoter activation by HNF1beta. These results suggest that the UGT2B17 promoter is regulated by different factors in liver-derived HepG2 and prostate-derived LNCaP cells.

The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence.

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

Regulation of UDP glucuronosyltransferase genes.

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.

Isolation and characterization of full-length mouse cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome P1-450 and P3-450.

Polysome immuno-adsorption, with immunoglobulin G directed against two 3-methylcholanthrene-induced mouse liver cytochrome P-450 proteins, was used to enrich mRNA from 3-methylcholanthrene-treated C57BL/6N mouse liver. cDNA transcribed from the P-450-enriched mRNA was then cloned into the Okayama-Berg vector. Two cDNA classes were detected upon differential screening of the clone bank with [32P]cDNA derived from 3-methylcholanthrene-induced immuno-enriched versus control mRNA. Several representatives of these two classes were judged to be near full length by comparison with their corresponding mRNA mobilities on denaturing agarose gels. A continuous reading-frame near the 5' end of one cDNA class (P1-450) corresponds to a protein having 15 of 17 residues the same as the published N-terminal sequence of rat P-450c. A continuous reading frame near the 5' end of the other class (P3-450) corresponds exactly to the first 25 amino acids of the published N-terminal sequence of rat P-450d. The P1-450 cDNA is at least 700 bp longer than the P3-450 cDNA. Heteroduplex analysis and Southern blot hybridization demonstrate that these mRNAs share approx. 1100 bp of sequence homology. Genomic P1-450 and P3-450 clones were isolated from a gene library constructed from C57BL/6N mouse liver DNA. By heteroduplex analysis with the corresponding cDNA, the P1-450 gene spans about 6 kb and the P3-450 gene about 7 kb. The intron-exon patterns are very similar, with the second and seventh exons being much larger than the other five. The 3' terminal exon of P1-450 is about 500 bp longer than that of P3-450. These data suggest that both P1-450 and P3-450 have diverged from a common ancestral gene.

Steroid UDP glucuronosyltransferases: characterization and regulation.

Under normal physiological conditions, glucuronidation generally terminates the biological activities of steroids and leads to their elimination in the bile and urine. This process is postulated to play a role in homeostasis by regulating the intracellular steady-state levels of these effector ligands. Indeed, the duration of response to specific steroid signals may be partly determined by the capacity of the cell or tissue to eliminate the steroids as unreactive glucuronides. Under pathophysiological conditions or during steroid therapies, glucuronidation may sometimes result in the formation of more biologically active or toxic metabolites as exemplified by the steroid D ring glucuronides. The degree of toxicity or biological effect in the cell exposed to these steroids will also depend on its complement of UGTs. To investigate these processes in more detail, the steroid specificities and distribution of individual UGTs in various target organs require elucidation. In this review, our current knowledge of the steroid specificities of various rat and human UGTs is described and preliminary investigations on the mechanisms governing tissue specificity are presented.

Differences in UDP-glucuronosyltransferase activities in congenic inbred rats homozygous and heterozygous for the jaundice locus.

The genic transfer of the jaundice locus (jj) from the Gunn rat into the inbred RHA/++ rat produced congenic inbred homozygous RHA/jj rats which lacked detectable bilirubin UDP-glucuronosyltransferase activity. Congenic inbred RHA/j+ rats contained half the activity for bilirubin of the RHA/++ strain. Constitutive activities for glucuronidation of sixteen substrates of twenty-one tested were inherited additively. Approximately seven groups were discernible based on the defect in activity for these substrates in the RHA/jj strain. Activity for 1-hydroxybenzo[a]pyrene was, after that for bilirubin, the most severely reduced (188-fold), while no differences in the glucuronidation of three androgens and of the 6-hydroxy-, 10-hydroxy-, and 11-hydroxybenzo[a]pyrenes were observed. The conjugation of other substrates was affected to an intermediate extent. Most of the twenty-one glucuronidating activities were induced by phenobarbital in the RHA/jj strain as well as in the RHA/++ and RHA/j+ strains. Activities for 9-hydroxybenzo[a]pyrene and for the 2-hydroxy- and 4-hydroxybiphenyls were induced such that the defect was overcome, and the RHA/jj had the same level of activity as the RHA/++ strain. Cytochrome p-450 content and cytochrome c reductase and aminopyrine demethylase activities were unaffected in the congenic strains. Cytochrome p-450 content and cytochrome c reductase activity were induced approximately 2.5- and 2.0-fold, respectively, by phenobarbital while aminopyrine demethylase activity was induced about 30% in each strain. The congenic inbred rats should provide a stable and reproducible genetic model for studying defective UDP-glucuronosyltransferase specified by the jaundice (jj) locus.

Separation of different UDP glucuronosyltransferase activities according to charge heterogeneity by chromatofocusing using mouse liver microsomes. Three major types of aglycones.

Hepatic UDP glucuronosyltransferase (EC 2.4.1.17) (GT) enzymes in control, phenobarbital- and 3-methylcholanthrene-induced microsomes from C57BL/6N mice have been fractionated according to charge heterogeneity on a chromatofocusing system using a pH 9.5 to 6 gradient. Transferase activities for eleven different substrates were determined on column fractions. Activities toward 3-hydroxybenzo[a]pyrene, phenolphthalein and estrone (type 1 substrates) were enhanced by both effector compounds and always eluted primarily at pH 8.5. In control and phenobarbital-induced microsomes, activities toward testosterone, 4-hydroxybiphenyl, morphine, naphthol and 9-hydroxybenzo[a]pyrene (type 2 substrates) eluted primarily at about pH 6.7. Activities toward p-nitrophenol, 4-methylumbelliferone and 2-hydroxybiphenyl (type 3 substrates) in control and phenobarbital-induced microsomes exhibited two peaks which eluted at pH 8.5 and 6.7. 3-Methylcholanthrene treatment increased almost exclusively activities which eluted at pH 8.5 for each of the three types of substrates. The pH value of elution corresponds to the approximate isoelectric point of the eluted protein. Immunoabsorption studies with an antibody preparation raised against a purified low pI form confirmed that a 51,000-dalton transferase form, GTM1, eluted primarily at pH 6.7 and that a 54,000-dalton form, GTM2, eluted at pH 8.5. A mathematical treatment of the ratios of activity after 3-methylcholanthrene treatment to that after phenobarbital treatment versus pH produced six patterns of activity. A minimum of two enzymes at the low pH region and one enzyme at the high pH region, all with broad-substrate specificity, could account for these patterns.

Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7.

In this work, UDP-glucuronosyltransferases (UGTs), UGT1A3, 2B7(H268) and 2B7(Y268), stably expressed in human embryonic kidney cells (HK293) were used to assess glucuronidation activities with a variety of steroid hormone and bile acid substrates. The rate of synthesis of carboxyl- and hydroxyl-linked glucuronides was determined under optimal reaction conditions. Expressed UGT1A3 catalyzed bile acid glucuronidation at high rates exclusively at the carboxyl moiety for all compounds tested. In contrast, UGT1A4 catalyzed bile acid glucuronidation at very low rates exclusively at the 3alpha-hydroxyl function. Both UGT2B7 allelic variants glucuronidated the bile acid substrates at both carboxyl and hydroxyl moieties, however, the 3alpha-hydroxyl position was preferentially conjugated compared to the carboxyl function. Similarly, androsterone, a 3alpha-hydroxylated androgenic steroid, was glucuronidated at very high rates by expressed UGT2B7. Of the estrogenic compounds tested, UGT2B7 catalyzed the glucuronidation of estriol at rates comparable to those determined for androsterone. Other structural discrimination was found with UGT2B7 which had activity toward estriol and estradiol exclusively at the 17beta-OH position, yielding the cholestatic steroid D-ring glucuronides.

Mutational analysis of the carboxy-terminal region of UDP-glucuronosyltransferase 2B1.

UDP-glucuronosyltransferases (UGTs) are membrane-bound glycoproteins that are resident in the endoplasmic reticulum with a type I topology. The roles of the membrane-spanning and membrane-proximal cytoplasmic domains in UGT activity were investigated. Site-directed and deletional mutagenesis techniques were used to generate truncated forms of the enzyme, forms with altered residues, or forms with heterologous tails appended to the carboxyl terminus. The presence of the transmembrane domain was a critical requirement for UGT activity whereas the cytoplasmic domain seemed to be a modulator of activity but was not essential. Truncation of the protein did not appear to lead to scavenging and degradation, although appending long heterologous tails to the cytoplasmic domain did seem to trigger proteolysis. Analysis of enzyme kinetic parameters and enzyme latency allowed us to discount substrate binding or substrate transport defects as the cause of ameliorated UGT activity in the mutants.

Isolation, sequence, and developmental expression of rat UGT2B2: the gene encoding a constitutive UDP glucuronosyltransferase that metabolizes etiocholanolone and androsterone.

The UDP glucuronosyltransferase gene UGT2B2 is constitutively expressed in rat liver, and the enzyme has been shown to conjugate glucuronic acid with various endogenous steroids, especially etiocholanolone and androsterone. We have cloned and sequenced much of the UGT2B2 gene and 5'-flanking (247 bp) and 3'-flanking (734 bp) regions. The gene contains six exons spanning about 15.3 kb. Translation begins at nucleotide 36 of exon 1 and terminates with 280 coding nucleotides into exon 6, encoding a protein of 530 amino acids (calculated Mr of the unmodified chain = 60,913). We have determined that the UGT2B2 full-length cDNA is 1,974 bp. Northern hybridization revealed that the hepatic UGT2B2 transcript is detectable 4 days before birth, becomes markedly elevated in the neonate, and is even further increased at 3 and 12 weeks of age in the liver of both male and female rats.

HNF1 alpha activates the rat UDP glucuronosyltransferase UGT2B1 gene promoter.

The rat UDP glucuronosyltransferase UGT2B1 is expressed mainly in the liver where it glucuronidates steroids and environmental toxins and carcinogens. A region between -42 and -55 bp upstream from the UGT2B1 gene transcription start site was previously identified as sharing sequence similarity with the hepatocyte nuclear factor 1 (HNF1) consensus binding site. In this study, the importance of this region in the regulation of the UGT2B1 gene was confirmed by functional and DNA binding assays. A minimal UGT2B1 gene promoter containing the putative HNF1 binding site was fused to the CAT reporter gene and transfected into HepG2 cells. Only low levels of CAT activity were detected. This activity was increased 50-fold when an HNF1 alpha expression vector was co-transfected with the UGT2B1 promoter CAT construct but was not altered when a HNF1 beta expression vector was used. A UGT2B1 promoter construct with the HNF1-like region deleted was not activated by either co-transfected HNF1 expression vector. DNase 1 footprinting and gel-shift analysis demonstrated that nuclear proteins present in both HepG2 cells and rat liver bind to the HNF1-like element. The presence of HNF1 alpha in these nuclear proteins that bind to the HNF1-like element was confirmed by supershift analysis with antisera to HNF1 alpha. Specific binding of nuclear proteins to the HNF1-like element was not seen in extracts from three cell lines derived from nonhepatic tissues. These data strongly suggest that the liver-enriched factor HNF1 alpha binds to, and activates, the UGT2B1 gene promoter

Glucuronidation of catechol estrogens by expressed human UDP-glucuronosyltransferases (UGTs) 1A1, 1A3, and 2B7.

Catechol estrogens are major estrogen metabolites in mammals and are the most potent naturally occurring inhibitors of catecholamine metabolism. These estrogen compounds have been implicated in carcinogenic activity and the 4/2-hydroxyestradiol concentration has been shown to be elevated in neoplastic human mammary tissue compared to normal human breast tissue. Three human liver UDP-glucuronosyltransferases, UGT2B7, UGT1A1, and UGT1A3, have been shown to catalyze the glucuronidation of catechol estrogens and lead to their enhanced elimination via urine or bile. The present study was designed to study the kinetic interaction of expressed human UGT2B7(Y) or (H), UGT1A1, and UGT1A3 toward 2- and 4-hydroxycatechol estrogens. cDNAs encoding UGT2B7(Y) or (H), UGT1A1, and UGT1A3 were expressed in HK293 cells, and cell homogenates or membrane preparations were used to determine their glucuronidation ability. UGT2B7(Y) reacted with higher efficiency toward 4-hydroxyestrogenic catechols, whereas UGT1A1 and UGT1A3 showed higher activities toward 2-hydroxyestrogens. UGT2B7(H) catalyzed estrogen catechol glucuronidation with efficiencies similar to UGT2B7(Y). Flunitrazepam (FNZ), a competitive inhibitor of morphine glucuronidation in hepatic microsomes, competitively inhibited catechol estrogen glucuronidation catalyzed by UGT2B7(Y), UGT1A1, and UGT1A3. Buprenorphine, an opioid substrate that reacts at high efficiency with each of these UGTs, was also studied. FNZ competitively inhibited buprenorphine glucuronidation with UGT1A1 and UGT2B7 but had no inhibitory activity toward UGT1A3. This suggests that buprenorphine and 2-hydroxycatechol estrogens react with separate active sites of UGT1A3. A catecholamine, norepinephrine, did not inhibit UGT2B7(Y)-, UGT1A1-, and UGT1A3-catalyzed glucuronidation of catechol estrogens. These results also suggest that drug-endobiotic interactions are possible in humans and may have implication in carcinogenesis.

Variability of human hepatic UDP-glucuronosyltransferase activity.

The availability of a unique series of liver samples from human subjects, both control patients (9) and those with liver disease (6; biliary atresia (2), retransplant, chronic tyrosinemia type I, tyrosinemia, Wilson's disease) allowed us to characterize human hepatic UDP-glucuronosyltransferases using photoaffinity labeling, immunoblotting and enzymatic assays. There was wide inter-individual variation in photoincorporation of the photoaffinity analogs, [32P]5-azido-UDP-glucuronic acid and [32P]5-azido-UDP-glucose and enzymatic glucuronidation of substrates specific to the two subfamilies of UDP-glucuronosyltransferases. However, the largest differences were between subjects with liver disease. Glucuronidation activities toward one substrate from each of the UDP-glucuronosyltransferases subfamilies, 1A and 2B, for control and liver disease, respectively, were 1.7-4.5 vs 0.4-4.7 nmol/mg x min for hyodeoxycholic acid (2B substrate) and 9.2-27.9 vs 8.1-75 nmol/mg x min for pchloro-m-xylenol (1A substrate). Microsomes from a patient with chronic tyrosinemia (HL32) photoincorporated [32P]5-azido-UDP-glucuronic acid at a level 1.5 times higher than the other samples, was intensely photolabeled by [32P]5-azido-UDP-glucose and had significantly higher enzymatic activity toward p-chloro-m-xylenol. Immunoblot analysis using anti-UDP-glucuronosyltransferase antibodies demonstrated wide inter-individual variations in UDP-glucuronosyltransferase protein with increased UDP-glucuronosyltransferase protein in HL32 microsomes, corresponding to one of the bands photolabeled by both probes. Detailed investigation of substrate specificity, using substrates representative of both the 1A (bilirubin, 4-nitrophenol) and 2B (androsterone, testosterone) families was carried out with HL32, HL38 (age and sex matched control) and HL18 (older control). Strikingly increased (5-8-fold) glucuronidation activity was seen in comparison to HL18 only with the phenolic substrates. The results indicate that one or more phenol-specific UDP-glucuronosyltransferase 1A isoforms are expressed at above normal levels in this tyrosinemic subject.