Inhibition of human UGT2B7 gene expression in transgenic mice by the constitutive androstane receptor.

The xenobiotic receptors, constitutive androstane receptor (CAR), and pregnane X receptor (PXR) regulate and alter the metabolism of xenobiotic substrates. Among the 19 functional UDP-glucuronosyltransferases (UGTs) in humans, UGT2B7 is involved in the metabolism of many structurally diverse xenobiotics and plays an important role in the clearance and detoxification of many therapeutic drugs. To examine whether this gene is regulated by CAR and PXR in vivo, transgenic mice expressing the entire UGT2B7 gene (TgUGT2B7) were created. Gene expression profiles revealed that UGT2B7 is differentially expressed in liver, kidney, adipocytes, brain, and estrogen-sensitive tissues, such as ovary and uterus. Liver UGT2B7 expression levels were decreased when TgUGT2B7 mice were treated with the CAR ligand 1,4-b-s-[2-(3,5,-dichloropyridyloxy)] (TCPOBOP) but not the PXR ligand pregnenolone 16α-carbonitrile. Although TCPOBOP decreased the levels of UGT2B7 mRNA in TgUGT2B7 mice, it had no affect on Tg(UGT2B7)Car(-/-) mice, adding support for a CAR-dependent mechanism contributing toward UGT2B7 gene suppression. Expression of promoter constructs in HepG2 cells showed the CAR-dependent inhibition was linked to hepatocyte nuclear factor-4α (HNF4α)-mediated transactivation of the UGT2B7 promoter. The inhibitory effect of CAR on UGT2B7 gene expression was validated in chromatin immunoprecipitation assays in which TCPOBOP treatment blocked HNF4α binding to the UGT2B7 promoter. These results suggest that HNF4α plays an important role in the constitutive expression of hepatic UGT2B7, and CAR acts as a negative regulator by interfering with HNF4α binding activity.

Studies on induction of lamotrigine metabolism in transgenic UGT1 mice.

A transgenic 'knock-in' mouse model expressing a human UGT1 locus (Tg-UGT1) was recently developed and validated. Although these animals express mouse UGT1A proteins, UGT1A4 is a pseudo-gene in mice. Therefore, Tg-UGT1 mice serve as a 'humanized' UGT1A4 animal model. Lamotrigine (LTG) is primarily metabolized to its N-glucuronide (LTGG) by hUGT1A4. This investigation aimed at examining the impact of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPAR) activators on LTG glucuronidation in vivo and in vitro. Tg-UGT1 mice were administered the inducers phenobarbital (CAR), pregnenolone-16alpha-carbonitrile (PXR), WY-14643 (PPAR-alpha), ciglitazone (PPAR-gamma), or L-165041 (PPAR-beta), once daily for 3 or 4 days. Thereafter, LTG was administered orally and blood samples were collected over 24 h. LTG was measured in blood and formation of LTGG was measured in pooled microsomes made from the livers of treated animals. A three-fold increase in in vivo LTG clearance was seen after phenobarbital administration. In microsomes prepared from phenobarbital-treated Tg-UGT1 animals, 13-fold higher CL(int) (Vmax/K(m)) value was observed as compared with the untreated transgenic mice. A trend toward induction of catalytic activity in vitro and in vivo was also observed following pregnenolone-16alpha-carbonitrile and WY-14643 treatment. This study demonstrates the successful application of Tg-UGT1 mice as a novel tool to study the impact of induction and regulation on metabolism of UGT1A4 substrates.

Polymorphisms of the carcinogen detoxifying UDP-glucuronosyltransferase UGT1A7 in proximal digestive tract cancer.

Cancer of the proximal digestive tract is associated with tobacco smoke and ethanol exposure. The UDP-glucuronosyltransferase (UGT) 1A7 is a detoxifying enzyme capable of tobacco-borne carcinogen detoxification and cellular protection and has been implicated as a cancer risk gene. In this study, UGT1A7 expression is demonstrated in oral, esophageal, and gastric tissue, which are the principle sites of proximal digestive tract cancer. Genomic DNA from the blood of 76 patients with esophageal, orolaryngeal and gastric cancer as well as from 210 healthy blood donors was analysed for the presence of UGT1A7 polymorphisms by sequencing and temperature gradient gel electrophoresis. Wild type UGT1A7 alleles were equally distributed between controls (19 %) and cancer patients (22 %). However, the UGT1A7*3 allele combining W208R, N129K and R131K missense mutations and exhibiting substantially reduced carcinogen detoxification activity was significantly associated with proximal gastrointestinal cancer and identified as a risk allele present in 32 % of cancer patients and 19 % of controls (P = 0.0008, OR 2,02 (95 %-CI 1.33-3.07)). We identify the significant association of the UGT1A7*3 allele encoding a low catalytic activity protein as a risk gene in proximal digestive tract cancer and as a potential marker for cancer susceptibility.

Polymorphisms of the human UDP-glucuronosyltransferase (UGT) 1A7 gene in colorectal cancer.

BACKGROUND: Genetic polymorphisms in the human UDP-glucuronosyltransferase-1A7 (UGT1A7) gene are detected and significantly correlated with sporadic colorectal carcinoma. UGT1A7, which has recently been demonstrated to glucuronidate environmental carcinogens, is now implicated as a cancer risk gene. A silent mutation at codon 11 and missense mutations at codons 129, 131, and 208 lead to the description of three polymorphic alleles designated UGT1A7*2, UGT1A7*3, and UGT1A7*4. METHODS: UGT1A7 polymorphisms were analysed by polymerase chain reaction amplification and sequencing, as well as temperature gradient gel electrophoresis in 210 healthy blood donors and 78 subjects with colorectal cancer. RESULTS: Homozygous wild-type UGT1A7 alleles were present in 20% of normal controls but were only detected in 9% of patients with colorectal carcinoma (odds ratio (OR) 0.39 (95% confidence interval (CI) 0.17-0.92); p=0.03). Analysis of individual polymorphic alleles identified a highly significant association between the presence of UGT1A7*3 alleles and colorectal cancer (OR 2.75 (95% CI 1.6 - 4.71); p<0.001). Recombinant expression of UGT1A7 polymorphic cDNA in eukaryotic cell culture showed reduced carcinogen glucuronidation activity in comparison with wild-type UGT1A7. UGT1A7 may therefore represent a modifier gene in colorectal carcinogenesis. CONCLUSION: We have identified a potential novel risk factor in sporadic colorectal cancer which may contribute to the identification of risk groups and to the elucidation of factors involved in colon carcinogenesis.

Developmental aspects of human hepatic drug glucuronidation in young children and adults.

BACKGROUND AND AIMS: The liver represents one of the major sites of human glucuronidation. Many therapeutic drugs are substrates for UDP-glucuronosyltransferases (UGT) leading to the formation of usually inactive glucuronides. Hepatic glucuronidation undergoes significant changes during fetal and neonatal development requiring age adapted drug therapy. Regulation of individual UGT genes during hepatic development has not been defined. SUBJECTS AND METHODS: Expression of 13 UGT genes and glucuronidation activities were analysed in 16 paediatric liver samples (aged 7-24 months), two fetal samples, and 12 adult liver samples (aged 25-75 years) using duplex reverse transcription-polymerase chain reaction, western blot, and specific catalytic UGT activity assays. RESULTS: No UGT transcripts were detected in fetal liver at 20 weeks' gestation. In contrast, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, and UGT2B15 transcripts were present without variation in all 28 hepatic samples after six months of age. Significantly lower expression of UGT1A9 and UGT2B4 mRNA was identified in paediatric liver. Hepatic glucuronidation activity in children aged 13-24 months was found to be lower than in adults for ibuprofen (24-fold), amitriptyline (16-fold), 4-tert-butylphenol (40-fold), estrone (15-fold), and buprenorphine (12-fold). CONCLUSIONS: An early phase characterised by the appearance of UGT gene transcripts and a later phase characterised by upregulation of UGT expression is demonstrated during human hepatic development. The differential regulation of UGT1A9 and UGT2B4 expression extends beyond two years of age and is capable of influencing hepatic glucuronidation of common therapeutic drugs in children. The development of hepatic UGT activities is significant for paediatric drug therapy and the prevention of adverse drug effects.

Genetic link of hepatocellular carcinoma with polymorphisms of the UDP-glucuronosyltransferase UGT1A7 gene.

BACKGROUND & AIMS: Hepatocellular carcinoma is associated with risk factors including hepatitis C, hepatitis B, cirrhosis, genetic liver diseases, and environmental carcinogens. Uridine 5'-diphosphate-glucuronosyltransferases are a superfamily of detoxifying enzymes capable of tobacco-borne carcinogen detoxification and cellular protection. This study examines the association of UGT1A7 and UGT1A9 gene polymorphisms with hepatocellular carcinoma. METHODS: Genomic DNA from the blood of 59 patients with hepatocellular carcinoma and 70 control subjects without evidence of cancer was analyzed by UGT1A7- and UGT1A9-specific PCR, sequencing analysis, and temperature gradient gel electrophoresis. RESULTS: Three UGT1A7 missense mutations were detected defining the UGT1A7*2, UGT1A7*3, and UGT1A7*4 alleles. Wild-type UGT1A7 alleles were present in 41.4% of controls but only in 6.8% of cancer patients (P < 0.001; odds ratio [OR], 9.73; 95% confidence interval [CI], 3.17-29.83). UGT1A7 polymorphisms were present in 93.2% of hepatocellular cancer patients, 74.5% carried the UGT1A7*3 allele (P < 0.001; OR, 10.76; 95% CI, 4.75-24.38), which combines the W208R, N129K, and R131K mutations and encodes a protein with low carcinogen detoxification activity. No UGT1A9 polymorphisms were detected. CONCLUSIONS: The significant association of hepatocellular carcinoma with the UGT1A7*3 allele encoding a low detoxification activity protein is identified and implicates UGT1A7 as a risk gene of hepatocarcinogenesis in addition to a role as potential marker for cancer risk assessment in chronic liver disease.

Identification of cyclosporine A and tacrolimus glucuronidation in human liver and the gastrointestinal tract by a differentially expressed UDP-glucuronosyltransferase: UGT2B7.

BACKGROUND/AIMS: The oral administration of the major transplant immunosuppressants cyclosporine A and tacrolimus leads to unpredictable drug levels requiring drug monitoring. Hepatic and extrahepatic metabolism of cyclosporine A and tacrolimus by cytochrome P450 proteins has been analyzed but metabolism and inactivation by glucuronidation has not been investigated. METHODS: Cyclosporine A and tacrolimus glucuronidation was measured in hepatic and gastrointestinal microsomal protein, and with 11 recombinant hepatic and extrahepatic family 1 and 2 UDP-glucuronosyltransferases. UDP-glucuronosyltransferase transcripts were determined by polymerase chain reaction. RESULTS: Significant cyclosporine and tacrolimus glucuronidation activity was present in endoplasmic reticulum from liver, duodenum, jejunum, ileum and colon, but was absent in stomach. Specific cyclosporine A glucuronidation activity was highest in liver and colon, tacrolimus glucuronidation was highest in liver. Analyses using recombinant UDPglucuronosyltransferases identified UGT2B7 as a human UDP-glucuronosyltransferase with specific activity toward cyclosporine A and tacrolimus. The hepato-gastrointestinal distribution of immunosuppressant glucuronidation activity corresponded to the differential expression pattern of UGT2B7 mRNA. CONCLUSIONS: This study provides conclusive evidence of hepatic and extrahepatic immunosuppressant glucuronidation by human UGT2B7 which was identified to be differentially expressed in the human hepatogastrointestinal tract. Hepatic and extrahepatic glucuronidation may influence the therapeutic efficacy of transplant immunosuppressants.

The contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines.

The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.

Glucuronide and glucoside conjugation of mycophenolic acid by human liver, kidney and intestinal microsomes.

Mycophenolic acid (MPA) is primarily metabolized to a phenolic glucuronide (MPAG) as well as to two further minor metabolites: an acyl glucuronide (AcMPAG) and a phenolic glucoside (MPAG1s). This study presents investigations of the formation of these metabolites by human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as by recombinant UDP-glucuronosyltransferases. HLM (n=5), HKM (n=6), HIM (n=5) and recombinant UGTs were incubated in the presence of either UDP-glucuronic acid or UDP-glucose and various concentrations of MPA. Metabolite formation was followed by h.p.l.c. All microsomes investigated formed both MPAG and AcMPAG. Whereas the efficiency of MPAG formation was greater with HKM compared to HLM, AcMPAG formation was greater with HLM than HKM. HIM showed the lowest glucuronidation efficiency and the greatest interindividual variation. The capacity for MPAGls formation was highest in HKM, while no glucoside was detected with HIM. HKM produced a second metabolite when incubated with MPA and UDP-glucose, which was labile to alkaline treatment. Mass spectrometry of this metabolite in the negative ion mode revealed a molecular ion of m/z 481 compatible with an acyl glucoside conjugate of MPA. All recombinant UGTs investigated were able to glucuronidate MPA with K:(M:) values ranging from 115.3 to 275.7 microM l(-1) and V(max) values between 29 and 106 pM min(-1) mg protein(-1). Even though the liver is the most important site of MPA glucuronidation, extrahepatic tissues particularly the kidney may play a significant role in the overall biotransformation of MPA in man. Only kidney microsomes formed a putative acyl glucoside of MPA.

Genetic multiplicity of the human UDP-glucuronosyltransferases and regulation in the gastrointestinal tract.

The metabolism of ingested foods and orally administered drugs occurs in the hepato-gastrointestinal tract. This process is facilitated by several supergene families that catalyze oxidative metabolism as well as conjugation of the small molecular weight substances that enter the systemic circulation through resorption in the gastrointestinal tract. The catalytic action carried out by one of several conjugation reactions leads to the eventual elimination of the resultant metabolites from the cell. As early as 1959 (R. T. Williams, Detoxification Mechanisms) it was suggested that the detoxification of most agents is efficiently performed by the phase II conjugation reactions, because the addition of bulky, water-soluble groups to the target substrates facilitates the partitioning of these metabolites from the lipid into the aqueous compartments of the cell. The combined efforts of the phase II reactions provides remarkable redundancy in a biological system that seems to be designed to assure that many endogenously generated catabolic products as well as exogenous agents introduced through the surface tissues of the digestive tracts are efficiently removed through excretion to the bile or urine. In this review, we focus on recent findings that highlight the genetic multiplicity and regulatory patterns of the phase II superfamily UDP-glucuronosyltransferases (UGTs). Although much is known regarding the number of UGTs that make up the UGT1 and UGT2 gene families, as demonstrated after the characterization of expressed cDNAs, examples are also presented in which information obtained from the human genome project will aid in the final characterization of the genetic multiplicity. In addition, tools have now been developed and examples presented to identify the expression patterns of the UGTs in human tissues, paying particular attention to expression patterns of these genes in the hepato-gastrointestinal tract.

Human UDP-glucuronosyltransferases: metabolism, expression, and disease.

In vertebrates, the glucuronidation of small lipophilic agents is catalyzed by the endoplasmic reticulum UDP-glucuronosyltransferases (UGTs). This metabolic pathway leads to the formation of water-soluble metabolites originating from normal dietary processes, cellular catabolism, or exposure to drugs and xenobiotics. This classic detoxification process, which led to the discovery nearly 50 years ago of the cosubstrate UDP-glucuronic acid (19), is now known to be carried out by 15 human UGTs. Characterization of the individual gene products using cDNA expression experiments has led to the identification of over 350 individual compounds that serve as substrates for this superfamily of proteins. This data, coupled with the introduction of sophisticated RNA detection techniques designed to elucidate patterns of gene expression of the UGT superfamily in human liver and extrahepatic tissues of the gastrointestinal tract, has aided in understanding the contribution of glucuronidation toward epithelial first-pass metabolism. In addition, characterization of the UGT1A locus and genetic studies directed at understanding the role of bilirubin glucuronidation and the biochemical basis of the clinical symptoms found in unconjugated hyperbilirubinemia have uncovered the structural gene polymorphisms associated with Crigler-Najjar's and Gilbert's syndrome. The role of the UGTs in metabolism and different disease states in humans is the topic of this review.

Characterization of the UDP-glucuronosyltransferase 1A locus in lagomorphs: evidence for duplication of the UGT1A6 gene.

The UGT1 locus is felt to be highly conserved between species, as is evident from the characterization of the locus in rodents and humans. In rabbits, cDNAs encoding proteins homologous to human UGT1A4, UGT1A6, and UGT1A7 have previously been identified. Here we demonstrate by Southern blot analysis, using exon 1 divergent 5' segments from rabbit UGT1A4 and UGT1A6 cDNAs, the existence of a cluster of highly related genes that are homologous to each of these exon 1 sequences. In comparing rabbit and human, it is evident that the UGT1A4 and UGT1A6 gene clusters in rabbit have undergone gene duplication. This is particularly evident with rabbit UGT1A6. The human UGT1A6 cDNA anneals to only a single gene fragment, as displayed by Southern blot analysis, indicating that the UGT1A6 exon 1 sequence is highly conserved. However, up to six rabbit UGT1A6 genes could be predicted from Southern blot analysis. To examine the potential linkage of the rabbit UGT1A6 genes, multiple UGT1A6 exons were identified from genomic DNA by extended polymerase chain reaction techniques and cloning of the UGT1A6 exon 1 sequences. Five unique UGT1A6 exon 1 gene sequences were characterized that could be predicted to encode proteins that are 98% similar in amino acid structure. Using a conserved region of the rabbit UGT1A6 cDNA as a probe to screen cDNA libraries, we identified a second UGT1A6 cDNA, termed UGT1A6alpha. In addition, a cDNA that encodes a protein similar to human UGT1A3 was also cloned. Characterization of UGT1A6alpha demonstrated the protein to be 98.9% identical to UGT1A6. The expression of rabbit UGT1A3, UGT1A4, and UGT1A6 displayed catalytic activities similar to their human orthologs. However, UGT1A6alpha was catalytically divergent from UGT1A6, indicating that UGT1A6 and UGT1A6alpha do not arise from allelic polymorphism. These results demonstrate that lagomorphs have evolved at least five additional UGT1A6 genes, an event that is not duplicated in rodents or humans.

Polymorphic gene regulation and interindividual variation of UDP-glucuronosyltransferase activity in human small intestine.

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.

Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a heterocyclic aromatic amine found in cooked meats and dietary exposure to PhIP has been implicated in the etiology of colon cancer in humans. PhIP, along with other heterocyclic aromatic amines, requires metabolic activation to exhibit genotoxic effects. PhIP is initially oxidized by the activity of cytochrome P4501A2 to produce 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a reaction occurring primarily in the liver. Whereas subsequent biotransformation of N-OH-PhIP via acetylation or sulfation can produce reactive electrophiles that readily bind to DNA, N-glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), functions as a detoxification mechanism. Although hepatic glucuronidation of N-OH-PhIP has been well characterized, the extrahepatic metabolism of this compound is poorly understood. Studies in our laboratory now indicate that the intestinal tract, and particularly the colon, is a significant site of glucuronidation of N-OH-PhIP. When assays were performed with microsomes prepared from the mucosa of the intestinal tract, it was determined that glucuronidation of N-OH-PhIP occurs throughout the intestinal tract, with activity approximately three times higher in the colon as that found in the upper intestine. Glucuronidation rates from colon microsomes showed considerable interindividual variability and incubation with N-OH-PhIP yielded two glucuronides. HPLC analysis showed that the predominant product formed is the N-OH-PhIP-N2-glucuronide, while the N3-glucuronide accounts for <10% of the total glucuronidation product. These rates approach the rates found in human liver microsomes, demonstrating the significance of extrahepatic metabolism of this food-borne carcinogen. Subsequent assays with human recombinant UGTs demonstrated that at least four human UGT isoforms, all from the UGT1A subfamily, are capable of catalyzing the biotransformation of N-OH-PhIP. Members of the UGT2B family available for this study did not conjugate N-OH-PhIP, although immunoinhibition studies in human liver microsomes strongly suggest the involvement of a UGT2B isoform(s) in this organ.

Retigabine N-glucuronidation and its potential role in enterohepatic circulation.

The metabolism of retigabine in humans and dogs is dominated by N-glucuronidation (), whereas in rats, a multitude of metabolites of this new anticonvulsant is observed (). The comparison of the in vivo and in vitro kinetics of retigabine N-glucuronidation in these species identified a constant ratio between retigabine and retigabine N-glucuronide in vivo in humans and dog. An enterohepatic circulation of retigabine in these species is likely to be the result of reversible glucuronidation-deglucuronidation reactions. Rats did not show such a phenomenon, indicating that enterohepatic circulation of retigabine via retigabine N-glucuronide does not occur in this species. In the rat, 90% of retigabine N-glucuronidation is catalyzed by UDP-glucuronosyltransferase (UGT)1A1 and UGT1A2, whereas family 2 UGT enzymes contribute also. Of ten recombinant human UGTs, only UGTs 1A1, 1A3, 1A4, and 1A9 catalyzed the N-glucuronidation of retigabine. From the known substrate specificities of UGT1A4 toward lamotrigine and bilirubin and our activity and inhibition data, we conclude that UGT1A4 is a major retigabine N-glucuronosyl transferase in vivo and significantly contributes to the enterohepatic cycling of the drug.

Regulation and function of family 1 and family 2 UDP-glucuronosyltransferase genes (UGT1A, UGT2B) in human oesophagus.

Human UDP-glucuronosyltransferases (UGTs) are expressed in a tissue-specific fashion in hepatic and extrahepatic tissues [Strassburg, Manns and Tukey (1998) J. Biol. Chem. 273, 8719-8726]. Previous work suggests that these enzymes play a protective role in chemical carcinogenesis [Strassburg, Manns and Tukey (1997) Cancer Res. 57, 2979-2985]. In this study, UGT1 and UGT2 gene expression was investigated in human oesophageal epithelium and squamous-cell carcinoma in addition to the characterization of individual UGT isoforms using recombinant protein. UGT mRNA expression was characterized by duplex reverse transcriptase-PCR analysis and revealed the expression of UGT1A7, UGT1A8, UGT1A9 and UGT1A10 mRNAs. UGT1A1, UGT1A3, UGT1A4, UGT1A5 and UGT1A6 transcripts were not detected. UGT2 expression included UGT2B7, UGT2B10 and UGT2B15, but UGT2B4 mRNA was absent. UGT2 mRNA was present at significantly lower levels than UGT1 transcripts. This observation was in agreement with the analysis of catalytic activities in oesophageal microsomal protein, which was characterized by high glucuronidation rates for phenolic xenobiotics, all of which are classical UGT1 substrates. Whereas UGT1A9 was not regulated, differential regulation of UGT1A7 and UGT1A10 mRNA was observed between normal oesophageal epithelium and squamous-cell carcinoma. Expression and analysis in vitro of recombinant UGT1A7, UGT1A9, UGT1A10, UGT2B7 and UGT2B15 demonstrated that UGT1A7, UGT1A9 and UGT1A10 catalysed the glucuronidation of 7-hydroxybenzo(alpha)pyrene, as well as other environmental carcinogens, such as 2-hydroxyamino-1-methyl-6-phenylimidazo-(4, 5-beta)-pyridine. Although UGT1A9 was not regulated in the carcinoma tissue, the five-fold reduction in 7-hydroxybenzo(alpha)pyrene glucuronidation could be attributed to regulation of UGT1A7 and UGT1A10. These data elucidate an individual regulation of human UGT1A and UGT2B genes in human oesophagus and provide evidence for specific catalytic activities of individual human UGT isoforms towards environmental carcinogens that have been implicated in cellular carcinogenesis.

UDP-glucuronosyltransferase activity in human liver and colon.

BACKGROUND & AIMS: The contribution of glucuronidation toward human drug metabolism is carried out by the Super gene family of UDP-glucuronosyltransferases (UGTs). Regulation of the human UGT1A locus is tissue specific, resulting in the unique expression of multiple hepatic and extrahepatic gene products. Studies were undertaken to examine UGT1A expression in human hepatic and colonic tissues. METHODS: UGT1A messenger RNA, protein, catalytic activity, and substrate kinetics were studied in 5 samples of normal hepatic and sigmoid colon tissue using duplex reverse-transcription polymerase chain reaction (RT-PCR), enzymatic and Western blot analysis, and indirect immunofluorescence analysis. RESULTS: Specific patterns of UGT1A gene expression occur in the liver and colon, which were consistent with different banding patterns as detected by Western blot analysis using a UGT1A-specific antibody. However, microsomal UGT activities in colon were up to 96-fold lower for many phenolic substrates, a finding that was not concordant with RT-PCR and Western blot analysis. Interestingly, UGT activity toward tertiary amines and some steroid hormones was equal. CONCLUSIONS: Differences of glucuronidation activity between human liver and colon suggest that UGT1A activity may be regulated as a result of the relative presence of individual isoforms with differing catalytic activities or by tissue-specific modulators after gene expression.

Physiological and pathophysiological regulation of cytochrome P450.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 1998 Experimental Biology '98 meeting in San Francisco. The presentations focused on the mechanisms of regulation of cytochrome P450 gene expression by developmental factors and by hormones and cytokines, as well as on the interplay between physiological and chemical regulation. Approaches and systems used to address these questions included conditional gene knockouts in mice, primary hepatocyte cultures, immunofluorescence imaging of cells, and cell lines stably expressing reporter gene constructs.

Polymorphic expression of the UDP-glucuronosyltransferase UGT1A gene locus in human gastric epithelium.

The human UDP-glucuronosyltransferase (UGT) 1A (UGT1A) locus is regulated in a tissue specific fashion in liver and extrahepatic tissues. Three extrahepatic UGT1A proteins, UGT1A7, UGT1A8, and UGT1A10, have been discovered and are believed to contribute to the diversity of extrahepatic glucuronidation. UGTs eliminate by glucuronidation a broad variety of endobiotic and xenobiotic substrates, which include bilirubin, therapeutic drugs, and carcinogens. Human gastric mucosa represents a primary location of tissue contact with dietary constituents, pharmaceutical drugs, and environmental carcinogens. To study the role and regulation of UGT1A gene products in stomach UGT1A mRNA expression and UGT catalytic activities were investigated in a panel of 14 normal gastric mucosa/adenocarcinoma sample pairs. UGT1A mRNA levels were differentially regulated in stomach, a feature not found in hepatic tissue. Normal gastric epithelium consistently expressed extrahepatic UGT1A7 and UGT1A10. However, polymorphic expression of UGT1A1 (29%), UGT1A3 (21%), and UGT1A6 (36%) was detected. Polymorphic UGT1A regulation was confirmed in adenocarcinoma samples with the additional observation of differential down-regulation of UGT1A1, UGT1A3, UGT1A6, and UGT1A10 and up-regulation of UGT1A7 mRNA. The polymorphic UGT1A regulation in stomach contrasts the homogeneous regulation of UGT1A gene products in human liver. Activity assays demonstrated 2- to 4-fold interindividual differences in UGT activity and qualitative differences between individuals. The polymorphic regulation of UGT1A gene products in gastric tissue may be the biological basis that determines interindividual differences in extrahepatic microsomal drug metabolism.

Expression of the UDP-glucuronosyltransferase 1A locus in human colon. Identification and characterization of the novel extrahepatic UGT1A8.

UDP-glucuronosyltransferases (UGT) catalyze the conjugation of lipophilic exobiotic and endobiotic compounds, which leads to the excretion of hydrophilic glucuronides via bile or urine. By a mechanism of exon sharing, the transcripts of individual first exon cassettes located at the 5' end of the human UGT1A locus are spliced to exons 2-5, leading to the expression of at least nine individual UGT genes. Recently, the tissue-specific expression of the UGT1A locus has been demonstrated in extrahepatic tissue, leading to the identification of UGT1A7 and UGT1A10 mRNA (Strassburg, C. P., Oldhafer, K., Manns, M. P., and Tukey, R. H. (1997) Mol. Pharmacol. 52, 212). However, UGT1A expression has not been defined in human colon, which is a metabolically active, external surface organ and a common route of drug administration. UGT1A expression was analyzed in 5 colonic, 16 hepatic, 4 biliary, and 13 gastric human tissue specimens by quantitative duplex reverse transcription-polymerase chain reaction and Western blot analysis, demonstrating lower UGT1A mRNA in the extrahepatic tissues. The precise analysis of unique UGT1A transcripts by exon 1-specific duplex reverse transcription-polymerase chain reaction revealed the expression of UGT1A1, UGT1A3, UGT1A4, UGT1A6, and UGT1A9 in the colon, which are also present in human liver. In addition, the expression of extrahepatic UGT1A10 and UGT1A8 was demonstrated. UGT1A8 was found to be closely related to gastric UGT1A7 with a 93.8% identity of first exon sequences. Expressed UGT1A7 and UGT1A10 protein showed unique catalytic activity profiles, while UGT1A8 was not active with the substrates tested. The ability of UGT1A10 to glucuronidate estrone represents only the second example of a human estrone UGT. The highly related human UGT1A7-1A10 cluster is expressed in a tissue-specific fashion and underlines the role and diversity of physiological glucuronidation at the distal end of the digestive tract.