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Glucocorticoids precociously induce fetal rat UGT1A6 and potentiate polycyclic aromatic hydrocarbon (PAH)-dependent induction of this enzyme in vivo and in isolated rat hepatocytes. To establish whether induction was due to glucocorticoid receptor (GR), luciferase reporter vectors were tested in transfection assays with HepG2 cells. Using a reporter construct containing approximately 2.26 kilobases of the 5'-flanking region of the UGT1A6-noncoding leader exon (A1*), dexamethasone increased basal activity 3- to 7-fold in cells cotransfected with an expression plasmid for GR. PAH increased gene expression 23-fold, but the presence of dexamethasone only induced PAH-dependent expression by 1.5-fold, suggesting interaction between GR and the aryl hydrocarbon (Ah) receptor. Furthermore, the GR antagonist RU 38486 [17beta-hydroxy-11beta-(4-dimethylamino-phenyl)-17alpha-(prop-1-ynyl)-estra-4,9-dien-3-one] was a partial agonist that increased, rather than inhibited, basal activity 3-fold. 5'-deletion analysis defined the 5'-boundary for a functional glucocorticoid-responsive unit between base pairs -141 and -118 relative to the transcription start site. This region contains the Ah receptor response element (AhRE), and both PAH and glucocorticoid-dependent gene activation were lost when this area was deleted. Mutation of a single base pair located in the AhRE region simultaneously reduced induction by PAH and increased glucocorticoid induction. Thus, the sequences of both the AhRE and glucocorticoid response elements seem to overlap, suggesting that Ah receptor binding may decrease glucocorticoid-dependent induction due to interactions of these two cis-acting elements. Mutation of a putative GRE located between base pair -81 and -95 reduced, but did not completely eliminate, glucocorticoid-dependent induction of the reporter, suggesting that a nonclassic mechanism of induction is involved in this response.

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UDP-glucuronosyltransferase 1A6 (UGT1A6), a key enzyme catalyzing the glucuronidation of small planar phenols and amines, is expressed in a tissue- and inducer-dependent manner. Expression is high in kidney, gastrointestinal tract, and induced liver, with low expression in spleen, lung, and ovary. Exposure to certain chemicals, such as 3-methylcholanthrene, benzo[a]pyrene, beta-naphthoflavone, and oltipraz elevates UGT1A6 mRNA in liver and to a lesser extent gastrointestinal tract and kidney, but not in other tissues. The mechanisms underlying this complex pattern of expression have been elusive. We have identified a new type of UGT1A6 mRNA (class 2) that differs in its 5' untranslated sequence. The class 2 transcript is the more abundant type expressed in liver, gastrointestinal tract, and kidney. Transcription of the class 2 mRNA is initiated 107 bases 5' of the UGT1A6 coding exon. The promoter region flanking the transcription start site contains an HNF1-like binding site identical to that in the human UGT1A6 gene. Both class 1 and class 2 mRNAs were elevated in liver by 3-methylcholanthrene, benzo[a]pyrene, beta-naphthoflavone, and oltipraz, with preferential elevation of class 1 occurring after 3-methylcholanthrene and benzo[a]pyrene treatment. These data suggest that transcription from a second promoter contributes to tissue- and inducer-specific expression of rat UGT1A6.

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One of the most important mechanisms involved in host defense against xenobiotic chemicals and endogenous toxins is the glucuronidation catalysed by UDP-glucuronosyltransferase enzymes (UGT). The role of genetic factors in determining variable rates of glucuronidation is not well understood, but phenotypic evidence in support of such variation has been reported. In the present study, six single nucleotide polymorphisms were discovered in the first exon of the UGT1A7 gene, which codes for the putative substrate-binding domain, revealing a high structural heterogeneity at the UGT1 gene locus. The new UGT1A7 proteins differ in their primary structure at amino acid positions 129, 131 and 208, creating four distinct UGT1A7 allelic variants in the human population: UGT1A7*1 (N129 R131 W208), *2 (K129 K131 W208), *3 (K129 K131 R208), and *4 (N129 R131 R208). In functional studies, HEK cells stably transfected to express the four allelic UGT1A7 variants exhibited significant differences in catalytic activity towards 3-, 7-, and 9-hydroxy-benzo(a)pyrene. UGT1A7*3 exhibited a 5.8-fold lower relative Vmax compared to wild-type *1, whereas *2 and *4 had a 2.6- and 2.8-fold lower relative Vmax than *1, respectively, suggesting that these mutations confer slow glucuronidation phenotype. Kinetic characterization suggested that these differences were primarily attributable to altered Vmax. Additionally, it suggested that each amino acid substitutions can independently affect the UGT1A7 catalytic activity, and that their effects are additive. The expression pattern of UGT1A7 studied herein and its catalytic activity profile suggest a possible role of UGT1A7 in the detoxification and elimination of carcinogenic products in lung. A population study demonstrated that a considerable proportion of the population (15.3%) was found homozygous for the low activity allele containing all three missense mutations, UGT1A7*3. These findings suggest that further studies are needed to investigate the impact of the low UGT1A7 conjugator genotype on individual susceptibility to chemical-induced diseases and responses to therapeutic drugs.

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The UDP-glucuronosyltransferases (UGTs) have long been known to be inducible by various chemicals, including drugs, although the extent of induction in general has been modest. In the present study, we determined the ability of the dietary flavonoid chrysin to induce UGT activity, protein and mRNA. When pretreating human hepatoma Hep G2 cells with 25 microM chrysin, the glucuronidation of chrysin itself increased 4.2-fold when measured in the intact cell and 14-fold in the cell homogenate, i.e., autoinduction. Microsomes from chrysin-treated cells probed with specific antibodies in Western analyses showed marked induction of the UGT1A family of proteins. Isoform-specific induction of the important hepatic UGT1A1 protein was observed but not of UGT1A6 or UGT2B7. The strong induction of UGT1A1 was confirmed by Northern analyses of total RNA as well as mRNA, using a specific probe. UGT1A1 message as well as protein was detectable also in untreated Hep G2 cells. In catalytic activity assays with recombinant UGT1A1, 1A4, 1A6 and 1A9, chrysin was found to be a high affinity substrate for UGT1A1 (K(m) 0.35 microM). Catalytic activity was also found for UGT1A9 and 1A6 but not for 1A4. Further studies demonstrated a 20-fold induction of the glucuronidation of bilirubin by the chrysin-treated cells and a 7. 9-fold induction of the glucuronidation of the oral contraceptive drug ethinylestradiol, two of the best known and specific UGT1A1 substrates, demonstrating the potential importance of this induction. In view of these findings, it will be important to extend these studies to other dietary flavonoids.

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UDP-glucuronosyltransferase 1A7 (UGT1A7) is a major UGT contributing to the glucuronidation of xenobiotic phenols in rats. Its expression in rat liver is tightly regulated, with low constitutive and high inducible expression in response to aryl hydrocarbon receptor ligands and oltipraz. Previously, we reported the absence of 3-methylcholanthrene- or oltipraz-responsive elements in the 1.6-kbp region flanking the UGT1A7 promoter. However, potential binding sites were noted for several liver-enriched transcription factors. Here we show that deletion of the hepatic nuclear factor (HNF)3, HNF4, and CCAAT-enhancer binding protein-like binding sites had no effect on the expression of a UGT1A7 reporter plasmid, p(-965/+56)1A7-Luc, in primary rat hepatocytes. The full activity of the promoter was contained in the region between bases -157 and +76. Two sites of binding by rat liver nuclear proteins were detected in this region by DNase footprinting. PR-1 corresponded to the HNF1-like binding site between bases -52 and -38, whereas PR-2 was located between -30 to -6. Gel retardation studies supported the presence of HNF1alpha in the PR-1 DNA-liver nuclear protein complex. Mutation of PR-1 inhibited binding in the gel shift assay, prevented activation by overexpressed HNF1 in human embryonic kidney cells, and reduced by >80% the maximal luciferase activities expressed from basal and 3-methylcholanthrene-responsive UGT1A7 gene reporter constructs in primary rat hepatocytes. These data provide evidence for an important stimulatory role of HNF1 in promoting UGT1A7 gene expression in rat liver.

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UDP-glucuronosyltransferase 1A7 (UGT1A7) is a polyaromatic hydrocarbon (PAH)-inducible UGT with activity toward various benzo[a]pyrene (B[a]P) metabolites. To investigate the influence of rat UGT1A7 on B[a]P-induced cytotoxicity, human lymphoblastoid L3 cells were transfected with pMF6 (control expression vector), p167Dtk2 (microsomal epoxide hydrolase expression vector), or p167Dtk2-1A7 (epoxide hydrolase/UGT1A7 coexpression vector), and the cell populations were compared for sensitivity to B[a]P-induced effects. B[a]P inhibited cell proliferation and decreased relative cell survival of p167Dtk2 and p167Dtk2-1A7 cells to a similar extent. Metabolism studies using [(3)H]B[a]P revealed increased formation of glucuronide conjugates of B[a]P-4,5-diol, 3-OH-, or 9-OH-B[a]P and an unidentified metabolite by p167Dtk2-1A7 cells, but the presence of unconjugated metabolites suggested that glucuronidation capacity may be limited. No differences between p167Dtk2 and p167Dtk2-1A7 L3 cells were observed in the growth inhibitory effects of 3-OH-B[a]P or B[a]P-7,8-diol, but p167Dtk2-1A7-expressing cells were found to be less sensitive to B[a]P-3,6-quinone-induced effects on cell proliferation and relative cell survival. The effect was also observed in AHH-1 lymphoblastoid cells expressing UGT1A7 without epoxide hydrolase. The UGT1A7-expressing AHH-1 cells were also less sensitive to growth inhibition by B[a]P-1,6-quinone and B[a]P-6,12-quinone. Flow cytometric analysis of vehicle and B[a]P-3, 6-quinone-exposed cell populations showed an association between UGT1A7 expression and resistance to B[a]P-3,6-quinone-induced apoptosis and loss of cell viability. These data suggest that UGT1A7 may be preferentially active toward B[a]P-quinones and that UGT1A7 may represent the PAH-inducible UGT activity previously implicated in protection against toxic redox cycling by B[a]P-3,6-quinone.

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Glucuronide conjugates represent one of the major types of naturally occurring phase 2 metabolites of xenobiotics and endobiotics. The process underlying their formation, glucuronidation, is normally considered detoxifying, because glucuronides usually possess less intrinsic biological or chemical activity than their parent aglycones and they are rapid excreted. However, a number of glucuronide conjugates are known that are active and may contribute to pharmacological activities or toxicities associated with their parent compounds. These include two classes of glucuronides with electrophilic chemical reactivity (N-O-glucuronides of hydroxamic acids and acyl glucuronides of carboxylic acids) and several types of glucuronides that impart biological effects through non-covalent interactions (morphine 6-O-glucuronide, retinoid glucuronides, and D-ring glucuronides of estrogens). Glucuronides may thus contribute to clinically significant effects, including environmental arylamine-induced carcinogenesis, drug hypersensitivity and other toxicities associated with carboxylic acid drugs, morphine analgesia, and cholestasis from estrogens. This review summarizes the rat and human UDP-glucuronosyltransferases that may be involved in the formation of bioactive glucuronides, including their substrate- and tissue-specificity and genetic and environmental influences on their activity. This knowledge may be useful for enhancing the therapeutic efficacy and minimizing the risk of adverse effects associated with xenobiotics that undergo bioactivating glucuronidation reactions.

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In Crigler-Najjar type II patients and, recently, in Crigler-Najjar type I patients treated with human hepatocyte cell therapy, phenobarbital has been used for reducing the serum bilirubin load. Its effect is attributed to induction of the enzyme required for hepatic bilirubin elimination, UDP-glucuronosyltransferase, UGT1A1. This study investigated the expression and inducibility of UGT1A1 in human donor livers and their corresponding primary hepatocyte cultures. Immunoblot analysis using a specific antibody directed against the amino terminal of the human UGT1A1 isoform showed that 5 hepatocyte donors exhibited a >50-fold difference in UGT1A1 level. UGT1A1 protein level correlated strongly with both liver microsomal bilirubin UGT activity and liver UGT1A1 mRNA level (r(2) =.82 and.72, respectively). Of the 4 patients with the lowest UGT1A1 levels, 3 were homozygotes for the UGT1A1 promoter variant sequence associated with Gilbert's syndrome, and the fourth was a heterozygote. The 3 donors with the highest levels had a history of phenytoin exposure. Hepatocytes isolated from the phenytoin-exposed donors exhibited marked declines in UGT1A1 mRNA levels during culturing. Induction studies using hepatocytes treated for 48 hours with phenobarbital (2 mmol/L), oltipraz (50 micromol/L), or 3-methylcholanthrene (2.5 micromol/L) revealed UGT1A1-inducing effects of phenobarbital, oltipraz, and, in particular, 3-methylcholanthrene. Our data suggest that both genetic and environmental factors play an important role in the marked interindividual variability in UGT1A1 expression. An understanding of these mechanisms could lead to advances in the pharmacological therapy of life-threatening unconjugated hyperbilirubinemia.

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Polycyclic aromatic hydrocarbon (PAH)-type compounds induce at least two rat UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT1A7. Among the glucuronidation reactions of PAH metabolites studied, mono- and diglucuronide formation of benzo[a]pyrene and chrysene-3,6-diphenol showed the highest induction factors in rat liver microsomes. Availability of AHH-1 cells stably expressing UGT1A7 allowed us to study whether this PAH-inducible isoform could catalyze benzo[a]pyrene and chrysene-3,6-diphenol glucuronidation. It was found that UGT1A7 indeed catalyzed mono- and diglucuronide formation of both benzo[a]pyrene and chrysene 3,6-diphenols. V79 cell-expressed rat UGT1A6 also catalyzed these reactions, except for chrysene diphenol diglucronide formation (Bock et al., Mol Pharmacol 42: 613-618, 1992). Enzyme kinetic studies of the glucuronidation of 6-hydroxychrysene (used as a stable PAH phenol) indicated that UGT1A7 conjugated this compound with a lower apparent Km value (0.1 microM) than UGT1A6 (10 microM). The results suggest that the two PAH-inducible UGTs may cooperate in conjugating PAH metabolites, but that UGT1A7 is more efficient.

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OBJECTIVE: We compared the efficacy of in vivo and ex vivo liposome transfection in rat lung transplantation. METHODS: (1) Chloramphenicol acetyltransferase group: Fischer rats underwent isogeneic transplantation (n = 4 per group). Recipients were put to death on postoperative day 2 for chloramphenicol acetyltransferase activity. Ex vivo setting: Grafts received cDNA complexed or not with liposomes and were transplanted after 1.5 or 10 hours at 10 degreesC. In vivo setting: Donors were intravenously injected with cDNA complexed or not with liposomes. Lungs were harvested after 1.5 or 10 hours, preserved at 10 degreesC, and transplanted. (2) Transforming growth factor-beta1 group: Brown-Norway rats served as donors and Fischer rats as recipients. All grafts were preserved for 3 hours at 10 degreesC. On postoperative day 5, arterial oxygenation and histologic rejection scores were assessed. Ex vivo setting: Grafts received transforming growth factor-beta1 sense (n = 8) or antisense (n = 7) complexed with liposomes or cDNA alone (n = 5). In vivo setting: Donors were intravenously injected with liposome:transforming growth factor-beta1 sense cDNA (n = 7). Exposure time was 3 hours. RESULTS: (1) Chloramphenicol acetyltransferase-transfection was superior in the ex vivo group but was not statistically different for longer exposure times. (2) Transforming growth factor-beta1-arterial oxygenation was superior in the ex vivo liposome:sense group. cDNA alone was inefficient. Rejection scores were not statistically different between ex vivo and in vivo liposome:sense groups but were better when the ex vivo liposome:sense group was compared with the cDNA alone or the antisense groups. CONCLUSIONS: (1) With current liposome technology, the ex vivo route is superior to the in vivo approach; (2) cDNA alone does not provide transgene expression at levels to produce a functional effect.

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UDP-glucuronosyltransferase UGT1A7 catalyzes the glucuronidation of benzo(a)pyrene metabolites and other bulky aromatic compounds. Both UGT1A7 mRNA and an associated enzyme activity (benzo(a)pyrene7, 8-dihydrodioltransferase activity) are markedly increased in livers of rats treated with beta-naphthoflavone or 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz). Nuclear runoff assays show that the effects of both inducers are primarily due to transcriptional activation. A 27-kilobase region that included the UGT1A7/UGT1A6 promoter regions was cloned. Primer extension and RNase protection studies indicated >/=30 transcription start sites in five clusters between bases -85 and -40 respective to the translation start codon. There was no recognizable TATA box, but the promoter region is TA-rich. Sequence analysis revealed potential binding sites for CCAAT enhancer-binding protein, activator protein 1, and hepatic nuclear factors 1, 3, and 4, but no xenobiotic response elements or antioxidant response elements, implicated in the regulation of other genes by beta-naphthoflavone or oltipraz, were found. A UGT1A7 gene reporter plasmid directed strong constitutive expression in transient transfection assays using primary rat hepatocytes. Treatment with 3-methylcholanthrene or oltipraz had no effect compared with similarly treated pGL3-Basic-transfected cells. These results suggest that the regulatory elements controlling xenobiotic inducibility of UGT1A7 transcription are located either 5' or 3' of bases -1600 to +54. One possibility is that the polycyclic aromatic-mediated regulation of UGT1A7 occurs via the xenobiotic response element flanking the UGT1A6 locus 7 kilobase pairs downstream.

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Transcriptional regulation of human UGT1A6, a UDP glucuronosyltransferase isoform conjugating a wide variety of planar phenols, has been studied using transfection experiments with plasmids containing its 3-kb 5' upstream region and chloramphenicol acetyltransferase as reporter gene. Previously, two modes of expression of the isoform have been described: in colon carcinoma Caco-2 cells UGT1A6 was found to be TCDD-inducible, whereas in lung carcinoma A549 cells it was constitutively expressed. Therefore functional analysis of UGT1A6 regulation was carried out using these two cell lines. In the upstream region of human UGT1A6 one xenobiotic-responsive element (XRE) was found between-1498 and -1502 bp. In Caco-2 cells the reporter gene activity of the entire plasmid and of deletion mutants containing the XRE were TCDD-inducible, in contrast to experiments with a deletion mutant which did not contain the XRE. TCDD induction was marginal in transfection studies with A549 cells. Gel mobility shift analysis indicated that the aryl hydrocarbon receptor and its partner Arnt bind to the XRE. Furthermore, primer extension studies suggest cell-specific use of multiple TATA boxes. Hence, regulation of human UGT1A6 appears to be cell-specific including both constitutive and aryl hydrocarbon receptor-controlled expression.

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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.

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We report here that rats possess a hitherto unrecognized xenobiotic-inducible hepatic 7,8-dihydro-7,8-diol-benzo[a]pyrene (BPD) UDP-glucuronosyltransferase (UGT) activity. BPD UGT activity is induced in female F344 rat liver by treatment with the selective Phase 2 conjugation enzyme inducer oltipraz [4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione at 75-450 mg/kg per day for 3 days] and also by a polycyclic aromatic hydrocarbon-type inducer, beta-naphthoflavone (80 mg/kg per day for 3 days). Incubations of oltipraz-treated rat liver microsomes with racemic trans BPD (100 microM) resulted in formation of two fluorescent glucuronides that were resolved by silica thin layer chromatography (Rf 0.5 and 0.6). Incubations with either the (-) or (+) trans BPD isomers resulted in selective formation of the Rf 0.5 [designated -DS, for (-) diol specific] or Rf 0.6 [designated +DS, for (+) diol specific] glucuronide, respectively. The -DS and +DS BPD glucuronides were fluorescent under long wave ultraviolet irradiation, dependent on the presence of UDP-glucuronic acid in the incubation, and were beta-glucuronidase-sensitive. The inducing effect of oltipraz on BPD UGT activity was dose-dependent. The mean BPD UGT activity of the vehicle-treated control group was 0.05 +/- 0.02 nmol/mg per min compared with 0.53 +/- 0.07 nmol/mg per min in the group treated with oltipraz (450 mg/kg per day for 3 days) (P < 0.001). The apparent Km of the induced BPD UGT for BPD was 20 microM, suggesting that the enzyme has the capacity to bind and turnover BPD under physiological conditions. Pretreatment with beta-naphthoflavone, but not phenobarbital, induced BPD UGT activity to approximately the same extent as oltipraz. Neither oltipraz nor beta-naphthoflavone exhibited induction of BPD UGT in livers of homozygous Gunn rats, which lack functional UGT1-encoded isozymes. We conclude that the oltipraz- and polycyclic hydrocarbonresponsive BPD UGT is a member of the UGT1 family. The role of this isoform as a modifier of susceptibility to carcinogenesis elicited by B[a]P remains to be determined.

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Previous work has shown that polycyclic aromatic hydrocarbons and oltipraz both induce an unidentified rat liver UDP-glucuronosyltransferase with activity toward benzo(a)pyrene-7, 8-diol, the proximate carcinogenic form of benzo(a)pyrene. Here we report the isolation of a benzo(a)pyrene-7,8-diol transferase-encoding cDNA, LC14, from an adult rat hepatocyte-derived cell line (RALA255-10G LCS-3). The predicted amino acid sequence of LC14 is nearly identical (5 differences out of 531 residues) to that deduced from UGT1A7, recently cloned at the genomic DNA level (Emi, Y., Ikushiro, S., and Kyanagi, T. (1995) J. Biochem. (Tokyo) 117, 392-399). Northern analysis of RNA from female F344 rat liver and LCS-3 cells revealed over a 40-fold and 4.4-fold enhancement by oltipraz treatment, respectively. Benzo(a)pyrene-7, 8-diol glucuronidating activity was detected (0.4 nmol/10(6) cells/16 h) in AHH-1 cells transfected with the LC14 expression vector, pMF6-LC14-3. The LC14-encoded transferase exhibited even higher activity toward certain benzo(a)pyrene phenols, including the major 3- and 9-phenol metabolites (4.1 and 2.8 nmol/10(6) cells/16 h, respectively). The Km of the enzyme for (-)-trans benzo(a)pyrene-7, 8-diol and 3-OH-BP was 15.5 and 12.3 microM, respectively. Northern analyses of total RNA revealed expression of LC14 or LC14-like RNA in all extrahepatic tissues tested. Marked inducibility by oltipraz was observed only in liver and (to a lesser extent) intestine. The results suggest that induction of UGT1A7 may explain the increased glucuronidating activities toward benzo(a)pyrene-7,8-diol and other metabolites that occur following treatment with polycyclic aromatic hydrocarbon-type inducing agents and oltipraz. UGT1A7 appears to represent an important cellular chemoprotective enzyme which mediates conjugation and elimination of toxic benzo(a)pyrene metabolites.

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The UDP-glucuronosyltransferase system (transferase) plays an important role in the pharmacokinetics of clearance of endogenous metabolites, therapeutic drugs, and xenobiotics. The human bilirubin and phenol transferases are encoded by the same gene complex which we designate UGT1. The gene arrangement indicates there are 6 exon 1s each with a promoter and each of which can predictably undergo differential splicing to the 4 common exons (2 through 5) to generate possibly 6 different mRNAs. The entire unique amino acid terminus of each isoform is encoded by an exon 1, and the common carboxyl terminus is encoded by the 4 common exons. Evidence supports the existence of other exon 1s upstream of the currently described locus. The 13-bp deletion in exon 2 represents the most common defect, to date, in the Crigler-Najjar, Type I individuals. Different point mutations in the 4 common exons and in exon 1 of UGT1A, however, also account for defective bilirubin transferase activity. The gene arrangement, in conjunction with the toxicity data from the Gunn rat, leads to the prediction that detoxification of bilirubin, xenobiotics, and therapeutic drugs is linked to the UGT1 locus. The Crigler-Najjar syndromes are uncommon, but the Gilbert individuals are commonly represented in 6% of the population. It is expected that, similar to the deleterious mutations in the common region of the UGT1 locus in Crigler-Najjar, Type I individuals, there is a range of moderate to intermediate deleterious mutations in this region of the gene of at least some Gilbert's individuals. Linkages, therefore, at this locus could signal that these individuals are at risk for certain drug toxicities and/or idiosyncratic drug reactions.

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BACKGROUND & AIMS: Heterogeneity in uridine 5'-diphosphate (UDP) glucuronosyltransferase expression across the human hepatic acinus may be important in the manifestation of certain zone-specific chemical hepatotoxicities. Previous immunohistochemical studies suggested that a phenol transferase induced by polycyclic aromatic hydrocarbons may be differentially expressed in centrilobular hepatocytes of rats. The aim of this study was to assess the distribution of the phenol and bilirubin transferases in human liver at the RNA level. METHODS: In situ RNA hybridization was used with two human liver samples and specific probes for the phenol transferase RNA, HLUG P1, and the bilirubin transferase RNAs, HUG-Br1 and HUG-Br2. RESULTS: The highest density signals were observed for the bilirubin transferase RNAs, both appearing to be evenly expressed in hepatocytes across the liver lobule. Slightly higher density of HUG-Br1 message was observed in some centrilobular hepatocytes surrounding larger central vein structures. HLUG P1 RNA was expressed at low levels (approximately fivefold greater than background signal) and was evenly distributed. CONCLUSIONS: The data suggest that a species difference exists in the distribution of the human and rat phenol transferase. No evidence was found for significant zonation in the pattern of expression of either the phenol or bilirubin transferase genes in human liver.

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Accumulating evidence indicates that mutations in the human UGT1 gene locus abolish hepatic bilirubin UDP-glucuronosyltransferase activity and cause the subsequent accumulation of bilirubin to toxic levels in patients with Crigler-Najjar type 1 (CN-I). Genetic and biochemical criteria are required to link CN-I with mutations in UGT1. Here we present analysis of mutations at the UGT1 locus in three individuals that were clinically diagnosed with CN-I (two related and one unrelated). Each patient carries a single base substitution that alters conserved residues in the transferase enzyme molecule, serine to phenylalanine at codon 376 and glycine to glutamic acid at codon 309. Each was homozygous for the defect as demonstrated by sequencing and RFLPs. Mutant cDNAs, constructed by site-directed mutagenesis, inserted into expression vectors, and transfected into COS-1 cells, supported the synthesis of the bilirubin transferase protein but only cells transfected with the wild-type cDNA expressed bilirubin UDP-glucuronosyltransferase activity. The data provide conclusive evidence that alterations at Gly 309 and Ser 376 are the genetic basis for CN-I in these families. These results suggest that the two codons, located in conserved regions of the molecule, are part of the active site of the bilirubin enzyme.

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