UDP-glucuronosyltransferase 1A4 (UGT1A4) polymorphisms in a Jordanian population

Glucuronidation is one of the most important phase II metabolic pathways. It is catalyzed by a family of UDP-glucuronosyltransferase enzymes (UGTs). One of the subfamilies is UGT1A. Allele frequencies in UGT1A4 differ among ethnic groups. The aim of this study was to determine the allelic frequency of two most common defective alleles: UGT1A4*2 and UGT1A4*3 in a Jordanian population. A total of 216 healthy Jordanian Volunteers (165 males and 51 females) were included in this study. Genotyping for UGT1A4*1, UGT1A4*2 and UGT1A4*3 was done using a well established polymerase chain reaction-restriction fragment length polymorphism test. Among 216 random individuals studied for UGT1A4*2 mutation there were 26 individuals who were heterozygous, giving a prevalence of 12% and an allele frequency of 6.5%. Only one individual was homozygous for UGT1A4*2. The UGT1A4*3 mutation was detected as heterozygous in 9 of 216 individuals indicating a prevalence of 4.2% and allele frequency of 3.5%. Three individuals were homozygous for the UGT1A4*3 indicating a prevalence of 1.4%. The prevalence of UGT1A4*2 is similar to the Caucasians but different from other populations whilst the UGT1A4*3 prevalence in the Jordanian population is distinct from other populations. Our results provide useful information for the Jordanian population and for future genotyping of Arab populations in general.     

Genetic polymorphisms in the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene and prostate cancer risk in Caucasian men

Background: Catechol-estrogen metabolites can induce carcinogenesis by acting as endogenous tumor initiators. Glucuronidation, mediated by the UDP-glucuronosyltransferase 1A1 (UGT1A1) enzyme, is a main metabolic pathway of estrogen detoxification in steroid target tissues, such as the prostate. The aim of our study was to investigate the possible correlation between UGT1A1 promoter gene polymorphisms and prostate cancer risk. Patients and methods: 129 patients with prostate cancer and 260 healthy controls were included in our study. A(TA)TAA promoter polymorphism of UGT1A1 gene was studied using the Fragment Analysis Software of an automated DNA sequencer and three genotypes (homozygous 7/7, heterozygous 6/7 and normal homozygous 6/6) were identified. Results: No significant differences were observed between the cancer group and controls regarding the genotyping distribution of the three UGT1A1 promoter genotypes (P > 0.05). Also, no association was found between overall disease risk and the presence of the polymorphic homozygous genotype (TA(7)/TA(7) vs TA(6)/TA(7) + TA(6)/TA(6)) (P = 0.18). In addition, no association was revealed between UGT1A1 genotype distribution and Gleason score (P = 0.55). Conclusion: Our data suggest that the TA repeat polymorphism of UGT1A1 gene does not seem to alter prostate cancer risk susceptibility in Caucasian men.     

Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity

Our discovery of rapid down-regulation of human bilirubin UDP-glucuronosyltransferase (UGT) in colon cell lines that was transient and irreversible following curcumin- and calphostin-C-treatment, respectively, suggested phosphorylation event(s) were involved in activity. Likewise, bilirubin-UGT1A1 expressed in COS-1 cells was inhibited by curcumin and calphostin-C. Because calphostin-C is a highly specific protein kinase C (PKC) inhibitor, we examined and found 4 to 5 predicted PKC phosphorylation sites in 11 UGTs examined. UGT1A1 incorporated [33P]orthophosphate, which was inhibited by calphostin-C. Also triple mutant, T75A/T112A/S435G-UGT1A1, at predicted PKC sites failed to incorporate [33P]orthophosphate. Individual or double mutants exhibited dominant-negative, additive, or no effect, while the triple mutant retained 10-15% activity towards bilirubin and two xenobiotics. Compared to wild-type, S435G and T112A/S435G shifted pH-optimum for eugenol, but not for bilirubin or anthraflavic acid, toward alkaline and acid conditions, respectively. This represents the first evidence that a UGT isozyme requires phosphorylation for activity.     

Genotype frequencies of UDP-glucuronosyltransferase 1A1 promoter gene polymorphism in the population of healthy Croatian pre-scholars

Increased serum bilirubin levels in patients with Gilbert syndrome (GS) are caused by reduction of hepatic activity of bilirubin glucuronosyltranferase to about 30% of normal. UGT1A1 genetic polymorphism with absent or very low bilirubin UDP-glucuronosyltransferase (B-UGT) activity is associated with Gilbert's syndrome (GS) and other hyperbilirubinemias. The genetic basis of GS is the insertion of two additional TA nucleotides (resulting in seven repeats of TA) in the TATAA box, present in proximal promoter of UGT1A1 gene. This study included 323 Croatian pre-scholars, including 164 boys and 159 girls. Statistical analysis showed significant difference for total bilirubin concentration between different genotypes (p < 0.001). Also, statistically significant difference for total bilirubin concentration was emphasized between genotypes 6/6 and 7/7 (p < 0.001) as well as 6/7 and 7/7 (p < 0.001). Higher total plasma bilirubin concentrations are significantly correlated with 7/7 genotype which is present in 9.8% of population studied.     

UDP-glucuronosyltransferase 1A1 (UGT1A1) gene haplotypes and their effect on serum bilirubin concentration in healthy Indian adults

The aim of the present study was to investigate the allele and genotype frequencies and haplotype structures of the variants in the UGT1A1 gene and their association with serum bilirubin levels in healthy adults. Total serum bilirubin levels were measured in 300 healthy adults (normal hematology and liver function test) and genotyping of seven SNPs was performed by PCR-RFLP, Gene Scan analysis and direct sequencing on the ABI Prism 310 Genetic Analyzer. Of the seven SNPs, four were found to be polymorphic and the frequencies of minor alleles were 0.336, 0.431, 0.353 and 0.066 for − 53(TA)7, − 3279G, − 3156A and 211A respectively. Individuals who carried the − 53(TA)7, − 3279G and − 3156A mutant alleles in homozygous or heterozygous states had significantly higher mean serum bilirubin levels. Five major promoter haplotypes were observed: − 53(TA)6/− 3279T/− 3156G was the most common haplotype, followed by − 53(TA)7/− 3279G/− 3156A, − 53(TA)6/− 3279G/− 3156G, − 53(TA)6/− 3279G/− 3156A and − 53(TA)7/− 3279T/− 3156G with an estimated frequency of 0.445, 0.230, 0.083, 0.065 and 0.050 respectively. Furthermore, the mutant haplotype (− 53(TA)7/− 3279G/− 3156A) was found to have a significant effect on bilirubin concentrations. Promoter polymorphisms and a common haplotype of the UGT1A1 gene are associated with serum bilirubin concentrations and could be a genetic risk factor for hyperbilirubinemia in Indians.     

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A)

A major component of phase II drug metabolism is the covalent addition of glucuronic acid to metabolites and xenobiotics. This activity is carried out by UDP-glucuronosyltransferases (UGT) which bind the UDP-glucuronic acid donor and catalyze the covalent addition of glucuronic acid sugar moieties onto a wide variety of substrates. UGTs play important roles in drug detoxification and were recently shown to act in an inducible form of multi-drug resistance in cancer patients. Despite their biological importance, structural understanding of these enzymes is limited. The C-terminal domain is identical for all UGT1A family members and required for binding to UDP-glucuronic acid as well as involved in contacts with substrates. Here, we report the backbone assignments for the C-terminal domain of UGT1A. These assignments are a critical tool for the development of a deeper biochemical understanding of substrate specificity and enzymatic activity.     

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

The human UDP-glucuronosyltransferase 1A6 (UGT1A6) isoform is actively involved in the detoxication of phenolic compounds. In an effort to gain insight on active-site amino acids, we investigated the functional relevance of cysteinyl residues in the glucuronidation process. The enzyme was irreversibly inactivated upon exposure to thiol-specific reagents, especially N-phenylmaleimide. Site-directed mutagenesis of the conserved Cys126 into valine led to a fully inactive mutant, whereas conservative substitution with serine significantly restored the glucuronidation activity toward 4-methylumbelliferone used as a reference substrate. This mutant exhibited a reduced affinity toward the acceptor substrate, as evidenced by a 10-times increase in K(m) value, compared to the wild-type enzyme. The two mutations did not alter the stability of UGT1A6 nor change the subcellular localization of the protein in the endoplasmic reticulum of recombinant cells. These results support the conclusion that Cys126 is an essential residue for the integrity of the substrate binding site of UGT1A6.     

Influence of GSTM1, GSTT1, GSTP1, and EPHX gene polymorphisms on DNA adduct level and HPRT mutant frequency in coke-oven workers

To evaluate the influence of individual susceptibility factors on the level of polyaromatic (PAH) hydrocarbon DNA adducts and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants in peripheral lymphocytes, 70 coke-oven workers exposed to PAH were genotyped for four metabolic enzyme polymorphisms of potential importance in PAH metabolism. The examined genetic polymorphisms concerned glutathione S-transferases M1 (GSTM1; gene deletion; 96 workers), T1 (GSTT1; gene deletion), P1 (GSTP1; Ile→Val substitution at codon 104 or Ile→Val at codon 104 and Val→Ala at codon 113), and microsomal epoxide hydrolase (EPHX; Tyr→His substitution at codon 113 and His→Arg at codon 139). The workers were classified in a high- and low-exposure group on the basis of urinary concentration of 1-pyrenol. The GSTM1 null genotype increased the number of DNA adducts in smoking coke-oven workers with high PAH exposure. DNA adducts were affected by PAH-exposure in non-smokers and in GSTM1 null smokers and by smoking in GSTM1 null individuals. In a multiple linear regression analysis, the interaction of the GSTM1 genotype was statistically significant (p=0.04) with smoking (yes/no) and of borderline significance (p=0.06) with PAH-exposure (high/low). As smoking also increased urinary 1-pyrenol, the genotype modification seemed to concern DNA adducts due to smoking rather than occupational exposure. GSTT1 positive individuals showed an elevated level of DNA adducts in comparison with GSTT1 null subjects (p=0.04), and EPHX genotypes associated with slow hydroxylation reaction yielded a higher (p=0.05) HPRT mutant frequency than fast EPHX genotypes; these findings were, however, based on small numbers of subjects and need to be clarified in further studies. In conclusion, our findings indicate that homozygous deletion of GSTM1 results in an increased sensitivity to genotoxic PAHs in tobacco smoke, which is seen as an increase in aromatic DNA adducts in blood mononuclear cells.     

Accelerated degradation of mislocalized UDP-glucuronosyltransferase family 1 (UGT1) proteins in Gunn rat hepatocytes

Gunn rat is a hyperbilirubinemic rat strain that is inherently deficient in the activity of UDP-glucuronosyltransferase form 1A1 (UGT1A1). A premature termination codon is predicted to produce truncated UGT1 proteins that lack the COOH-terminal 116 amino acids in Gunn rat. Pulse-chase experiments using primary cell cultures showed that the truncated UGT1A1 protein in Gunn rat hepatocytes was synthesized similarly to wild-type UGT1A1 protein in normal Wistar rat hepatocytes. However, the truncated UGT1A1 protein was degraded rapidly with a half-life of about 50 min, whereas the wild-type UGT1A1 protein had a much longer half-life of about 10 h. The rapid degradation of truncated UGT1A1 protein was inhibited partially but not completely by treating Gunn rat hepatocytes with proteasome inhibitors such as carbobenzoxy-Leu-Leu-leucinal and lactacystin. By contrast, neither the lysosomal cysteine protease inhibitor nor the calpain inhibitor slowed the degradation. Our findings show that the absence of UGT1 protein from Gunn rat hepatocytes is due to rapid degradation of the truncated UGT1 protein by the proteasome and elucidate the molecular basis underlying the deficiency in bilirubin glucuronidation.     

Transcriptional enhancement of UDP-glucuronosyltransferase form 1A2 (UGT1A2) by nuclear factor I-A (NFI-A) in rat hepatocytes

In cultured primary hepatocytes UDP-glucuronosyltransferase form 1A2 (UGT1A2) mRNA level is 80 times higher than that found in rat liver. We previously identified an enhancer sequence in the UGT1A2 promoter, and designated it as culture-associated expression responsive enhancer module (CEREM). Affinity chromatography with DNA fragments containing CEREM allowed enrichment of nuclear factor I (NFI) proteins from cultured hepatocytes. The NFI family is encoded by four distinct genes, NFI-A, NFI-B, NFI-C, and NFI-X. Immunoblot analysis with isoform-specific antibodies showed that NFI-A1 existed as a major component in rat liver and cultured hepatocytes. By contrast, NFI-C1 was present in rat liver but disappeared immediately upon cultivation of hepatocytes. Only trace amounts of NFI-B and NFI-X were detectable in rat liver and cultured hepatocytes. NFI-A1 elevated expression of the reporter gene that is under the control of CEREM, while NFI-C1 had an inhibitory effect. Co-expression of a constant amount of NFI-A1 with an increasing amount of NFI-C1 led to a concentration-dependent decrease in the expression of the CEREM-controlled reporter gene mediated by NFI-A1. Activation of UGT1A2 expression by NFI-A1 is suppressed by the coexistence of NFI-C1 in the liver, and culture-associated expression of UGT1A2 is triggered by the rapid disappearance of NFI-C1 in cultured hepatocytes.     

Mutations in the hypoxanthine guanine phosphoribosyltransferase gene (HPRT1) in Asian HPRT deficient families

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified 34 mutations in 28 Japanese, 7 Korean, and 1 Indian families with the patients manifesting different clinical phenotypes, including two rare cases in female subjects, by the analysis of all nine exons of HPRT from the genomic DNA and reverse transcribed mRNA using PCR technique coupled with direct sequencing.     

Effects of deoxyribonucleosides and cell-stimulation on frequencies of ultraviolet-B-induced micronuclei in human peripheral blood lymphocytes

In the present study the involvement of deoxyribonucleotides (dNTPs) in the clastogenicity of ultraviolet-B (UVB) in unstimulated peripheral blood lymphocytes (G(0)-PBLs) was investigated. This was studied by analyzing the frequency of UVB-induced micronuclei (MN), either after adding a cocktail of the four deoxyribonucleosides to the PBLs immediately after exposure to UVB, or by stimulating the cells before exposure. In total, PBLs obtained from two different donors were investigated. For both donors, it could be demonstrated that addition of deoxyribonucleosides to UVB-irradiated G(0)-PBLs resulted in a significant reduction of the clastogenic effect of UVB. A gradual reduction of the clastogenic effect of UVB could also be realized by irradiating PBLs that were progressively more stimulated with the lectin PHA before exposure. The latter finding is explained by upregulation of intracellular pool sizes of dNTPs in stimulated PBLs.     

4-hydroxyretinoic acid, a novel substrate for human liver microsomal UDP-glucuronosyltransferase(s) and recombinant UGT2B7

It is suggested that formation of more polar metabolites of all-trans-retinoic acid (atRA) via oxidative pathways limits its biological activity. In this report, we investigated the biotransformation of oxidized products of atRA via glucuronidation. For this purpose, we synthesized 4-hydroxy-RA (4-OH-RA) in radioactive and nonradioactive form, 4-hydroxy-retinyl acetate (4-OH-RAc), and 5,6-epoxy-RA, all of which are major products of atRA oxidation. Glucuronidation of these retinoids by human liver microsomes and human recombinant UDP-glucuronosyltransferases (UGTs) was characterized and compared with the glucuronidation of atRA. The human liver microsomes glucuronidated 4-OH-RA and 4-OH-RAc with 6- and 3-fold higher activity than atRA, respectively. Analysis of the glucuronidation products showed that the hydroxyl-linked glucuronides of 4-OH-RA and 4-OH-RAc were the major products, as opposed to the formation of the carboxyl-linked glucuronide with atRA, 4-oxo-RA, and 5,6-epoxy-RA. We have also determined that human recombinant UGT2B7 can glucuronidate atRA, 4-OH-RA, and 4-OH-RAc with activities similar to those found in human liver microsomes. We therefore postulate that this human isoenzyme, which is expressed in human liver, kidney, and intestine, plays a key role in the biological fate of atRA. We also propose that atRA induces its own oxidative metabolism via a cytochrome P450 (CYP26) and is further biotransformed into glucuronides via UGT-mediated pathways.     

Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications

Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme.