UDP-Glucuronosyltransferase 1A1 gene polymorphisms and total bilirubin levels in an ethnically diverse cohort of women.

The objective of this study was to investigate variations in UGT1A1 polymorphisms and haplotypes among African-American and Caucasian women and to assess whether variants other than UGT1A1*28 are associated with total serum bilirubin levels. The (TA)(n) repeats and 14 single nucleotide polymorphisms (SNPs) in the UGT1A1 gene were genotyped in 335 African Americans and 181 Caucasians. Total serum bilirubin levels were available in a subset of 125 women. Allele frequencies of all SNPs and (TA)(n) repeats were significantly different between African Americans and Caucasians. In Caucasians, three common haplotypes accounted for 71.8% of chromosomes, whereas five common haplotypes accounted for only 46.6% of chromosomes in African Americans. Mean total serum bilirubin levels were significantly lower (p = 0.005) in African Americans (0.36 mg/dl) than in Caucasians (0.44 mg/dl). The (TA)(n) repeats explained a significant amount of variation in total bilirubin levels (R(2) = 0.27, p < 0.0001), whereas other SNPs were less correlative. Thus, significant variations in UGT1A1 haplotype structure exist between African Americans and Caucasians in this relatively large cohort of women. The correlation of UGT1A1 with total bilirubin levels was mainly due to (TA)(n) repeats in Caucasians but a clear correlation was not observed in African Americans because of the high diversity of haplotypes and the small sample size. These data have implications for the design of epidemiologic studies of cancer susceptibility and pharmacogenetic studies for adverse drug reactions in populations of African ancestry.     

Identification and characterization of a functional TATA box polymorphism of the UDP glucuronosyltransferase 1A7 gene

UDP glucuronosyltransferases (UGT) detoxify bilirubin and therapeutic drugs, a process influenced by single nucleotide polymorphisms (SNPs) in their structural genes and promoter elements. UGT1A1*28 is a functional UGT promoter polymorphism associated with Gilbert's disease and severe irinotecan toxicity, which also occurs in the absence of UGT1A1*28. The aim of this study was to identify and characterize UGT promoter variants relevant for irinotecan detoxification. Recombinant UGT1A proteins were analyzed for irinotecan metabolite glucuronidation by UGT activity assays. In 427 healthy blood donors and 71 homozygous UGT1A1*28 carriers, the 5'-untranslated region of the UGT1A7 gene locus was studied. An SNP was detected by allelic discrimination and characterized by reporter gene experiments. A novel -57 T--> G SNP with a gene frequency of 0.39 in healthy blood donors was identified in the putative TATA box of the UGT1A7 gene, reducing promoter activity to 30%. It is in linkage dysequilibrium with a variant of the UGT1A7 first exon that is present in the reduced-activity UGT1A7*3 and UGT1A7*4 alleles. Homozygous UGT1A1*28 carriers simultaneously carried this variant in 97%. We identified a novel reduced-function TATA box SNP of the UGT1A7 gene that catalyzes irinotecan metabolite detoxification. Its association with variants of the UGT1A1 promoter and UGT1A7 gene may influence irinotecan metabolism. Our finding emphasizes the importance of combinations of structural and regulatory gene polymorphisms that may be useful as markers of drug toxicity.     

Human UGT1A6 pharmacogenetics: identification of a novel SNP, characterization of allele frequencies and functional analysis of recombinant allozymes in human liver tissue and in cultured cells

BACKGROUND: UDP-glucuronosyltransferase (UGT) enzymes catalyze the glucuronidation and typically inactivation of endogenous and exogenous molecules including steroid hormones, bilirubin and many drugs. The UGT1A6 protein is expressed predominantly in liver and metabolizes small phenolic drugs including acetaminophen, salicylates and many beta-blockers. Interindividual variation in the capacity of humans to glucuronidate drugs has been observed. RESULTS: We have identified a novel common single nucleotide polymorphism (SNP) in the human UGT1A6 gene resulting in a Ser7Ala change in encoded amino acid. We have further functionally characterized that polymorphism in the context of two previously reported polymorphisms, Thr181Ala and Arg184Ser. These non-synonymous cSNPs define four common haplotypes. Alleles appear with similar frequencies in Caucasian and African-American populations with distributions adhering to Hardy-Weinberg equilibrium. UGT1A6 genotype, rate of substrate glucuronidation and level of immunoreactive UGT1A6 protein was determined. A 25-fold variation in the rate of substrate glucuronidation and an 85-fold variation in level of immunoreactive protein were measured. Liver tissue samples that were homozygous for UGT1A6*2 exhibited a high rate of glucuronidation relative to tissues with other genotypes. Biochemical kinetic studies of recombinant UGT1A6 expressed in HEK293 cells indicated that the UGT1A6*2 allozyme, expressed homozygously, had almost two-fold greater activity toward p-nitrophenol than UGT1A6*1 and when expressed heterozygously (UGT1A6*1/*2) it was associated with low enzyme activity. CONCLUSIONS: These data suggest that common genetic variation in human UGT1A6 confers functionally significant differences in biochemical phenotype as assessed in human tissue and cultured cells expressing recombinant allozymes. This genetic variation might impact clinical efficacy or toxicity of drugs metabolized by UGT1A6.     

Frequent co-occurrence of the TATA box mutation associated with Gilbert's syndrome (UGT1A1*28) with other polymorphisms of the UDP-glucuronosyltransferase-1 locus (UGT1A6*2 and UGT1A7*3) in Caucasians and Egyptians

Polymorphisms of drug metabolizing enzymes are frequently associated with diseases and side effects of drugs. Recently, a TATA box mutation of UGT1A1 (UGT1A1*28), a common genotype leading to Gilbert's syndrome, and several missense mutations of other UDP-glucuronosyltransferase 1 (UGT1) family members have been described. Furthermore, co-occurrence of UGT1A1*28 and UGT1A6*2 has been observed. In order to elucidate the basis for co-occurrence of UGT1 mutations, fluorescence resonance energy transfer techniques were developed for rapid determination of polymorphisms of three UGT isoforms (UGT1A1*28, 1A6*2, and 1A7*2/*3). Hundred healthy Caucasians and 50 Egyptians were genotyped. All genotypes followed the Hardy-Weinberg equilibrium. Only three major haplotypes were found, including a haplotype consisting of allelic variants of all three isoforms (29% in Caucasians and 22% in Egyptians), all leading to reduced UGT activity. Frequent haplotypes containing several UGT1 allelic variants should be taken into account in studies on the association between diseases, abnormal drug reactions, and UGT1 family polymorphisms.     

Structural heterogeneity at the UDP-glucuronosyltransferase 1 locus: functional consequences of three novel missense mutations in the human UGT1A7 gene

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.     

Functional impact and prevalence of polymorphisms involved in the hepatic glucuronidation of valproic acid

Metabolism of valproic acid, a widely used drug, is only partially understood. It is mainly metabolized through glucuronidation and acts as a substrate for various UDP-glucuronosyltransferases (UGTs). UGTs metabolizing valproic acid in the liver are UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, with UGT1A6 and UGT2B7 being the most prominent. Polymorphisms in genes expressing these enzymes may have clinical consequences, regarding dosing, blood levels of the drug and adverse reactions. Not all genes are well studied and studies, where they exist, report conflicting results. Prevalence of polymorphisms and various haplotypes is also of great importance, as it may suggest different therapeutic approaches in various populations. Presented here is a review of currently known polymorphisms, their functional impact, when known, and their prevalence in different populations, highlighting the current state of understanding and areas where there is a lack of data and suggesting new perspectives for further research.     

Influence of mutations associated with Gilbert and Crigler-Najjar type II syndromes on the glucuronidation kinetics of bilirubin and other UDP-glucuronosyltransferase 1A substrates

OBJECTIVES: UGT1A1 coding region mutations, including UGT1A1*6 (G71R), UGT1A1*7 (Y486D), UGT1A1*27 (P229Q) and UGT1A1*62 (F83L), have been linked to Gilbert syndrome in Asian populations, whereas homozygosity for UGT1A1*7 is associated with the Crigler-Najjar syndrome type II. This work compared the effects of (a) the individual UGT1A1 mutations on the glucuronidation kinetics bilirubin, beta-estradiol, 4-methylumbelliferone (4MU) and 1-naphthol (1NP), and (b) the Y486 mutation, which occurs in the conserved carboxyl terminal domain of UGT1A enzymes, on 4MU, 1NP and naproxen glucuronidation by UGT1A3, UGT1A6 and UGT1A10. METHODS: Mutant UGT1A cDNAs were generated by site-directed mutagenesis and the encoded proteins were expressed in HEK293 cells. The glucuronidation kinetics of each substrate with each enzyme were characterized using specific high-performance liquid chromatography (HPLC) methods. RESULTS: Compared with wild-type UGT1A1, in-vitro clearances for bilirubin, beta-estradiol, 4MU and 1NP glucuronidation by UGT1A1*6 and UGT1A1*27 were reduced by 34-74%, most commonly as a result of a reduction in Vmax. However, the magnitude of the decrease in the in-vitro clearances varied from substrate to substrate with each mutant. The glucuronidation activities of UGT1A1*7 and UGT1A1*62 were reduced by >95%. Introduction of the Y486D mutation essentially abolished UGT1A6 and UGT1A10 activities, and resulted in 60-90% reductions in UGT1A3 in-vitro clearances. CONCLUSIONS: The glucuronidation of all UGT1A1 substrates is likely to be impaired in subjects carrying the UGT1A1*6 and UGT1A1*62 alleles, although the reduction in metabolic clearance might vary with the substrate. The Y486D mutation appears to greatly reduce most, but not all, UGT1A activities.     

Common human UGT1A polymorphisms and the altered metabolism of irinotecan active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38)

7-Ethyl-10-hydroxycamptothecin (SN-38) is the pharmacologically active metabolite of irinotecan, in addition to being responsible for severe toxicity. Glucuronidation is the main metabolic pathway of SN-38 and has been shown to protect against irinotecan-induced gastrointestinal toxicity. The purpose of this study was to determine whether common polymorphic UDP-glucuronosyltransferase (UGT) affects SN-38 glucuronidation. First, kinetic characterization of SN-38-glucuronide (SN-38-G) formation was assessed for all known human UGT1A and UGT2B overexpressed in human embryonic kidney 293 cells. To assess the relative activity of UGT isoenzymes for SN-38, rates of formation of SN-38-G were monitored by liquid chromatography/mass spectrometry analysis and normalized by level of UGT cellular expression. Determination of intrinsic clearances predicts that hepatic UGT1A1 and UGT1A9 and the extrahepatic UGT1A7 are major components in SN-38-G formation, whereas a minor role is suggested for UGT1A6, UGT1A8, and UGT1A10. In support of the involvement of UGT1A9, a strong coefficient of correlation was observed in the glucuronidation of SN-38 and a substrate, mainly glucuronidate, by UGT1A9 (flavopiridol) by human liver microsomes (coefficient of correlation, 0.905; p = 0.002). In vitro functional experiments revealed a negative impact of the UGT1A1 allelic variants. Residual activities of 49, 7, 8, and 11% were observed for UGT1A1*6 (G(71)R), UGT1A1*27 (P(229)Q), UGT1A1*35 (L(233)R), and UGT1A1*7 (Y(486)D), respectively. Common variants of UGT1A7, UGT1A7*3 (N(129)K;R(131)K;W(208)R), and UGT1A7*4 (W(208)R), displayed residual activities of 41 and 28% compared with the UGT1A7*1 allele. Taken together, these data provide the evidence that molecular determinants of irinotecan response may include the UGT1A polymorphisms studied herein and common genetic variants of the hepatic UGT1A9 isoenzyme yet to be described.     

Genetic variations and haplotypes of UDP-glucuronosyltransferase 1A locus in a Korean population

Glucuronidation catalyzed by UDP-glucuronosyltransferases (UGTs) is one of the most important mechanisms for host defense against xenobiotics and endobiotics. Although genetic polymorphisms of several UGT1A isoforms have been reported separately, the haplotypes in all functional exons have not been identified, and little information is available regarding single nucleotide polymorphisms in Koreans. We analyzed genetic polymorphisms in all functional exons of the UGT1A locus by direct sequencing of genomic DNA from 50 healthy Korean subjects, and their haplotypes were inferred from genotype data using an expectation-maximization algorithm. We identified 67 polymorphisms, including three novel single nucleotide polymorphisms, 233C>T in UGT1A1 (T78M), 292C>T in 1A4 (Q98Stop), and 701T>C in 1A7 (I234T). Two amino acid substitutions, 1A4 Q98Stop and 1A7 I234T, were each associated with a decrease of enzymatic activity, whereas UGT1A1 T78M had no significant influence on catalytic function. The frequencies of the known variants in Koreans differed significantly from those reported in other ethnic groups. Haplotype analysis was performed within the polymorphisms in each UGT1A isoform as well as across the isoforms. Based on strong linkage disequilibrium within UGT1A7, between 1A5 and 1A4, and within 1A3, the complex was divided into three blocks, Block 7, Block 5/4, and Block 3. The haplotypes for each block were subsequently determined, revealing a profile that differed from those of other ethnic groups. These results suggest that genetic polymorphisms of the UGT1A locus differ between Koreans and other ethnic populations. Such differences should be considered in pharmacogenetic studies.     

Limited influence of UGT1A1*28 and no effect of UGT2B7*2 polymorphisms on UGT1A1 or UGT2B7 activities and protein expression in human liver microsomes

AIMS: UGT1A1 and UGT2B7 are enzymes that commonly contribute to drug glucuronidation. Since genetic factors have been suggested to contribute to variability in activities and expression levels of these enzymes, a quantitative assessment of the influence of the major genotypes (UGT1A1*28 or UGT2B7*2) on enzyme activities was conducted. METHODS: Using a bank of microsomal samples from 59 human livers, the effect of UGT1A1*28 or UGT2B7*2 polymorphisms were investigated on rates of estradiol 3-glucuronidation (a marker of UGT1A1 enzyme activity) or zidovudine glucuronidation (a marker of UGT2B7 enzyme activity) and levels of immunoreactive protein for each enzyme. Glucuronidation rates for both enzymes were measured at K(m)/S(50) and 10 times K(m)/S(50) concentrations. RESULTS: UGT1A1 and UGT2B7 enzyme activities varied up to 16-fold and sixfold, respectively. Rates at K(m)/S(50) concentration closely correlated with rates at 10 times K(m)/S(50) concentration for both enzymes (but not at 1/10th K(m) for UGT2B7). Enzyme activities correlated with relative levels of immunoreactive protein for UGT1A1 and UGT2B7. Furthermore, rates of zidovudine glucuronidation correlated well with rates of glucuronidation of the UGT2B7 substrate gemcabene, but did not correlate with UGT1A1 enzyme activities. For the UGT1A1*28 polymorphism, consistent with levels of UGT1A1 immunoreactive protein, mean UGT1A1 activity was 2.5- and 3.2-fold lower for TA(6)/TA(7) (P < 0.05) and TA(7)/TA(7) (P < 0.001) genotypes in comparison with the TA(6)/TA(6) genotype. CONCLUSIONS: Relative to the observed 16-fold variability in UGT1A1 activity, these data indicate only a partial (approximately 40%) contribution of the UGT1A1*28 polymorphism to variability of interindividual differences in UGT1A1 enzyme activity. For the UGT2B7*2 polymorphism, genotype had no influence on immunoreactive UGT2B7 protein or the rate of 3'-azido-3'-deoxythymidine glucuronidation.     

Glucuronidation of acetaminophen is independent of UGT1A1 promotor genotype

The metabolism of acetaminophen (paracetamol) is thought to be altered in patients with Gilbert's syndrome (GS), a chronic unconjugated hyperbilirubinemia. The underlying cause of GS is a polymorphism in the promotor region of the uridine diphosphate glucuronosyltransferase isoform 1A1 gene (UGT1A1*28), its encoded enzyme being responsible for the glucuronidation of bilirubin and presumably acetaminophen. Decreased enzyme activity results in elevated bilirubin levels and may activate various metabolic pathways leading to higher amounts of potentially hepatotoxic acetaminophen metabolites. Patients with GS might be more susceptible to unexpected side effects while taking acetaminophen and other drugs which are substrates of UGT1A1. The possibility of a correlation between glucuronidation capacity with respect to acetaminophen, UGT1A1 promotor polymorphism and the bilirubin serum level were investigated in 23 healthy male volunteers selected for UGT1A1 genotype (6 wildtypes, 9 mutants and 8 heterozygotes). One gram acetaminophen was administered p.o. and urine was collected over 2 4-hour periods. Unchanged acetaminophen and its glucuronide metabolite were determined using HPLC. The metabolic ratios unchanged acetaminophen/acetaminophen glucuronide in UGT1A1-wildtypes, heterozygotes and mutants showed no statistically significant differences. An association between metabolic ratio and serum bilirubin level could not be detected in any of the urine collection periods. These data confirm that there is no correlation between the capacity to glucuronidate acetaminophen, the UGT1A1 genotype and the bilirubin serum level. Acetaminophen is likely to be substrate of a UGT isoform other than the UGT1A1.     

Population differences in major functional polymorphisms of pharmacokinetics/pharmacodynamics-related genes in Eastern Asians and Europeans: implications in the clinical trials for novel drug development

Drug lag, recently discussed extensively in Japan, can be divided into two phases: clinical development time and application review time. The former factor is still an important problem that might be improved by promoting multi-regional clinical trials and considering the results from other similar populations with Japanese, such as Koreans and Chinese. In this review, we compare the allelic or genotype frequencies of 30 relatively common functional alleles mainly between Eastern Asians and Europeans as well as among 3 major populations in Eastern Asian countries, Japan, Korea, and China, in 12 pharmacokinetics (PK)/pharmacodynamics (PD)-related genes; CYP2C9 (*2 and *3), CYP2C19 (*2, *3 and *17), 13 CYP2D6 haplotypes including *4, *5 and *10, CYP3A5 (*3), UGT1A1 (*28 and *6), NAT2 (*5, *6 and *7), GSTM1 and GSTT1 null genotypes, SLCO1B1 521T>C, ABCG2 421C>A, and HLA-A*31:01 and HLA-B*58:01. In this review, differences in allele frequencies (AFs) or genotype frequencies (GFs) less than 0.1 (in the cases of highest AF (GF) ≥0.1) or less than 0.05 (in the cases of lowest AF (GF) <0.1) were regarded as similar. Between Eastern Asians and Europeans, AFs (or GFs) are regarded as being different for many alleles such as CYP2C9 (*2), CYP2C19 (*2, *3 and *17), CYP2D6 (*4 and *10), CYP3A5 (*3), UGT1A1 (*28 and *6), NAT2 (*5*7), GSTT1 null and ABCG2 421C>A. Among the 3 Eastern Asian populations, however, only AFs of CYP2C19*3, CYP2D6*10, HLA-A*31:01 and HLA-B*58:01 are regarded as dissimilar. For CYP2C19*3, the total functional impact on CYP2C19 could be small if the frequencies of the two null alleles CYP2C19*2 and *3 are combined. Regarding CYP2D6*10, frequency difference over 0.1 is observed only between Japanese and Chinese (0.147). Although environmental factors should be considered for PK/PD differences, we could propose that among Japan, Korea, and China, genetic differences are very small for the analyzed common PK-related gene polymorphisms. On the other hand, AFs of the two HLA alleles important for cutaneous adverse drug reactions are diverse even among Eastern Asians and thus should be taken into account.     

Genetic variants of human UGT1A3: functional characterization and frequency distribution in a Chinese Han population.

UDP-glucuronosyltransferase 1A3 (UGT1A3) contributes to glucuronidation of many important endogenous compounds and xenobiotics, including some flavonoids. Recently, a total of six single nucleotide polymorphisms (SNPs) have been identified in the human UGT1A3 gene. Among them, four SNPs (A17G, Q6R; T31C, W11R; C133T, R45W; and T140C, V47A) cause amino acid substitutions. Variants caused by these SNPs showed an activity change in estrone metabolism, whereas their activities toward other substrates were not examined. In the present study, three common flavonoids, quercetin, luteolin, and kaempferol, were used as substrates for glucuronidation by wild-type and variant UGT1A3s. Our results demonstrated that the activities of three variants, UGT1A3.2, UGT1A3.3, and UGT1A3.5, were remarkably lower than that of UGT1A3.1. In contrast, UGT1A3.4 exhibited an increase in glucuronidation efficiency of approximately 4 times and a clear preference to quercetin 7- and 3-hydroxyl groups. The frequency distributions of UGT1A3 alleles and SNPs in UGT1A3 in a Chinese Han population were statistically different from the reported value in German-Caucasians (p < 0.05). UGT1A3 variants have an altered glucuronidation activity toward quercetin, luteolin, and kaempferol and may alter human susceptibility to flavonoid exposure.     

Epirubicin glucuronidation is catalyzed by human UDP-glucuronosyltransferase 2B7.

Epirubicin is one of the most active agents for breast cancer. The formation of epirubicin glucuronide by liver UDP-glucuronosyltransferase (UGT) is its main inactivating pathway. This study aimed to investigate epirubicin glucuronidation in human liver microsomes, to identify the specific UGT isoform for this reaction, and to correlate epirubicin glucuronidation with other UGT substrates. Microsomes from human livers were used. UGTs specifically expressed in cellular systems, as well as two UGT2B7 variants, were screened for epirubicin glucuronidation. Epirubicin, morphine, and SN-38 glucuronides were measured by high-pressure liquid chromatography. The mean +/- S.D. formation rate of epirubicin glucuronide in human liver microsomes (n = 47) was 138 +/- 37 pmol/min/mg (coefficient of variation, 24%). This phenotype was normally distributed. We screened commercially available UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 for epirubicin glucuronidation. Only UGT2B7 converted epirubicin to its glucuronide. No differences in epirubicin glucuronidation were found in HK293 cells expressing the two UGT2B7 variants at position 268. Catalytic efficiency (V(max)/K(m)) of epirubicin glucuronidation was 1.4 microl/min/mg, a value higher than that observed for morphine, a substrate of UGT2B7. Formation of epirubicin glucuronide was significantly related to that of morphine-3-glucuronide (r = 0.76, p < 0.001) and morphine-6-glucuronide (r = 0.73, p < 0.001). No correlation was found with SN-38, a substrate of UGT1A1 (r = 0.04). UGT2B7 is the major human UGT catalyzing epirubicin glucuronidation, and UGT2B7 is the candidate gene for this phenotype. The reported tyrosine to histidine polymorphism in UGT2B7 does not alter the formation rate of epirubicin glucuronide, and undiscovered genetic polymorphisms in UGT2B7 might change the metabolic fate of this important anticancer agent.     

Urinary excretion of phenytoin metabolites, 5-(4'-hydroxyphenyl)-5-phenylhydantoin and its O-glucuronide in humans and analysis of genetic polymorphisms of UDP-glucuronosyltransferases

The anticonvulsant agent phenytoin (5,5-diphenylhydantoin) is mainly excreted as 5-(4'-hydroxyphenyl)-5-phenylhydantoin (4'-HPPH) O-glucuronide in humans. Previously, we demonstrated that the glucuronidation of 4'-HPPH is catalyzed by multiple UDP-glucuronosyltransferases (UGTs) of UGT1A1, UGT1A4, UGT1A6, and UGT1A9. Since 4'-HPPH may be bioactivated to a reactive metabolite by peroxidase, the glucuronidation in considered to be a detoxification pathway. In the present study, we investigated the relationship between the extent of interindividual variability in the urinary excretion levels of 4'-HPPH and its O-glucuronide and genotyping of CYP2C9, CYP2C19, UGT1A1, UGT1A6, and UGT1A9. 4'-HPPH and its glucuronide in urine samples from 15 patients to whom phenytoin was administered were measured by liquid chromatography-tandem mass spectrometry. When the molar ratio of 4'-HPPH O-glucuronide/4'-HPPH was calculated as an index of glucuronidation, a large interindividual variability (11 fold) was observed in the 15 patients. Phenytoin is metabolized to 4'-HPPH by CYP2C9 and CYP2C19 in which there are genetic polymorphisms. Although 5 patients were genotyped as heterozygotes of mutated alleles of CYP2C9 or CYP2C19 genes, no relationship with the interindividual difference in the total excretion levels of 4'-HPPH and its O-glucuronide was observed. The UGT1A1*6, UGT1A1*28, UGT1A1*60 and UGT1A6*2 alleles were found in 1, 3, 6, and 8 patients, respectively. Although there was no relationship between the genetic polymorphisms of UGT1As and the interindividual difference in the 4'-HPPH glucuronidation, the large interindividual variability of 4'- HPPH glucuronidation may contribute to interindividual differences in toxic reactions to phenytoin.     

The novel UGT1A9 intronic I399 polymorphism appears as a predictor of 7-ethyl-10-hydroxycamptothecin glucuronidation levels in the liver.

Polymorphisms in UGT1A9 were associated with reduced toxicity and increased response to irinotecan in cancer patients. UDP-glucuronosyltransferase (UGT) protein expression, glucuronidation activities for 7-ethyl-10-hydroxycamptothecin (SN-38), and probe substrates of the UGT1A9 and UGT1A1 were measured in 48 human livers to clarify the role of UGT1A9 variants on the in vitro glucuronidation of SN-38. Genotypes were assessed for UGT1A9 (-2152C>T, -275T>A, and -118T(9>10)), three novel UGT1A9 variants (-5366G>T, -4549T>C, and I399C>T), and UGT1A1 (-53TA(6>7), -3156G>A, and -3279T>G). Of all the variants, the UGT1A9 I399C>T was associated with the most dramatic change in SN-38-glucuronide (SN-38G) (2.64-fold; p = 0.0007). Compared with UGT1A9 I399C/C, homozygous I399T/T presented elevated UGT1A1 and UGT1A9 proteins and higher glucuronidation of UGT1A9 and UGT1A1 substrates (p < 0.05). The very low linkage disequilibrium (r(2) < 0.19) between UGT1A9 I399 and all the other UGT1A1 and UGT1A9 variants suggests a direct effect or linkage to unknown functional variant(s) relevant to SN-38 glucuronidation. The UGT1A9 -118T(9/10) was also linked to alteration of SN-38 glucuronidation profiles in the liver (p < 0.05) and was associated with higher UGT1A1 protein (p = 0.03). However, UGT1A9 -118T(10) appears to have low functional impact as a result of the lack of correlation with UGT1A9 protein levels and a modest 1.4-fold higher reporter gene expression associated with the -118T(10) allele in HepG2 cells (p = 0.004). In contrast, the UGT1A9 -5366T, -4549C, -2152T, and -275A, associated with higher UGT1A9 protein (2-fold; p < 0.05), have no influence on SN-38G. Despite limitations resulting from sample size, results indicate that UGT1A9 I399 and -118T(9/10) may represent additional candidates in combination with UGT1A1 promoter haplotypes for the prediction of SN-38 glucuronidation profile in vivo.     

Inhibition and active sites of UDP-glucuronosyltransferases 2B7 and 1A1.

Two human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A1, catalyze the glucuronidation of many endo- and xenobiotics. Although UGT1A1 uniquely catalyzes the glucuronidation of the endobiotic, bilirubin, and UGT2B7 uniquely catalyzes the glucuronidation of morphine to both the 3-0 glucuronide and the 6-0 glucuronide, both catalyze the glucuronidation of the mixed opioid agonist/antagonist buprenorphine with high efficiency. Etonitazenyl, a mu opioid receptor antagonist, was found to inhibit competitively opioid, steroid, and other substrate glucuronidation reactions catalyzed by UGT2B7. Data showing several benzodiazepines and alternative substrates interacting competitively support previous work, which indicates a single binding domain within UGT2B7. Etonitazenyl also competitively inhibited the glucuronidation of buprenorphine catalyzed by UGT1A1. However, neither etonitazenyl nor buprenorphine inhibited bilirubin glucuronidation except at very high concentrations. Therefore, it is unlikely that buprenorphine therapy for opioid or other drug addiction would influence bilirubin glucuronidation and lead to hyperbilirubenmia. Anthraflavic acid and catechol estrogen glucuronidation, catalyzed by UGT1A1, was also not inhibited by etonitazenyl or buprenorphine. Reactions catalyzed by UGT1A6 were not affected by etonitazenyl. These studies indicate that UGT2B7 has one binding site and that UGT1A1 has two or more binding sites for xenobiotics and endobiotics.