UDP-glucuronosyltransferase activity and bilirubin conjugation in the bullfrog.
Ontology highlight
ABSTRACT:
SUBMITTER: Cole KD
PROVIDER: S-EPMC1152043 | biostudies-other | 1983 May
REPOSITORIES: biostudies-other
Similar Datasets
Project description:BackgroundOxidative stress is a major mediator of adverse outcome after kidney transplantation. Bilirubin is produced by heme oxygenase-1 (HO-1), catalyzed by UDP-glucuronosyltransferase (UGT1A1), and has potential as an antioxidant. In this study, we investigated the effects of HO-1 and UGT1A1 sequence variations on kidney allograft outcomes.MethodsClinical data were collected from 429 Korean recipients who underwent kidney transplantation from 1990-2008. Genotyping for UGT1A1*28 and HO-1 (A-413T) was performed. Acute rejection and graft survival were monitored as end-points.ResultsSerum levels of total bilirubin were significantly increased after transplantation (0.41 ± 0.19 mg/dL to 0.80 ± 0.33 mg/dL, P<0.001). Post-transplant 1-year bilirubin level was higher in 6/7 or 7/7 carriers compared with 6/6 homozygotes in terms of the UGT1A1*28 polymorphism (6/6 vs. 6/7 vs. 7/7: 0.71 ± 0.27 vs. 1.06 ± 0.36 vs. 1.10 ± 0.45 mg/dL, P<0.001). According to an additive model of genotype analysis, the 7-allele genotype had a protective effect on the development of acute rejection compared with the 6-allele (odds ratio 0.43, 95% CI 0.25-0.73, P for trend = 0.006). Multivariate Cox regression analysis revealed that individuals carrying the 7-allele had a decreased risk of graft loss, by a factor of 0.36 (95% CI 0.15-0.85, P = 0.019). The HO-1 (A-413T) polymorphism had no effect on serum bilirubin levels or graft outcomes.ConclusionsThe UGT1A1*28 polymorphism is associated with changes in serum bilirubin and with graft outcome after kidney transplantation.
| S-EPMC3972238 | biostudies-literature
Project description:Crigler-Najjar (CN) disease is an inherited disorder of bilirubin metabolism. The disease is caused by a deficiency of the hepatic enzyme bilirubin UDP-glucuronosyltransferase (B-UGT). Patients with CN disease have high serum levels of the toxic compound, unconjugated bilirubin. The only defect in bilirubin metabolism of CN patients is the absence of B-UGT activity. The transplantation of cells able to glucuronidate bilirubin should therefore lower serum bilirubin levels. The Gunn rat is the animal model of CN disease. Primary Gunn rat fibroblasts (GURF) were transduced with a recombinant retrovirus, capable of transferring B-UGT cDNA. A cell line was obtained expressing B-UGT at a level comparable to hepatocytes. Bilirubin added to the culture medium of these cells was glucuronidated and excreted. The B-UGT activities of transduced GURF and freshly isolated Wistar hepatocytes were compared at different bilirubin concentrations. The specific B-UGT activities of these two cell types were comparable when physiological bilirubin concentrations (5-10 microM) were present in the culture media. At higher bilirubin concentrations (20-80 microM) the hepatocytes were more active than the transduced GURF. We conclude that with the addition of only one enzyme (B-UGT) fibroblasts can perform the complete set of reactions necessary for bilirubin glucuronidation. The difference in B-UGT activity between transduced GURF and hepatocytes at 20-80 microM bilirubin can be explained by lower UDP-glucuronic acid and glutathione S-transferase levels in GURF. Our findings also indicate that these cells could be used to develop extrahepatic gene therapy for CN disease.
| S-EPMC1217075 | biostudies-other
Project description:We aimed to investigate the effect of the genetic mutant G71R (c. 211G > A) in uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) on the glucuronidation of unconjugated bilirubin. The UGT1A1 wild-type and mutant G71R gene sequences were inserted into the lentiviral vector GV358 plasmid and then transfected into COS-7 cells. Real-time polymerase chain reaction and western blot analyses were used to determine mRNA and protein expression levels of UGT1A1, respectively. High-performance liquid chromatography was used to quantitate the levels of conjugated bilirubin. The results showed no significant difference in the mRNA and protein expression levels between the UGT1A1 wild-type and G71R homozygous and heterozygous mutants. The level of conjugated bilirubin reached a maximum in wild-type UGT1A1-transfected COS-7 cells. However, relative to the UGT1A1 wild-type, conjugated bilirubin concentrations were 71 and 22% with G71R heterozygous- and G71R homozygous-transfected COS-7 cells, respectively. In conclusion, we successfully established in vitro cell models of the UGT1A1 wild-type and the G71R homozygous and heterozygous mutants using a lentiviral vector. Furthermore, the catalytic activity for unconjugated bilirubin was lower in the mutant G71R than the UGT1A1 wild-type enzyme, and a weaker effect was observed in the homozygote.
| S-EPMC8934855 | biostudies-literature
Project description:Herb-drug interaction (HDI) limits clinical application of herbs and drugs, and inhibition of herbs towards uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) has gained attention as one of the important reasons to cause HDIs. Sauchinone, an active lignan isolated from aerial parts of Saururus chinensis (Saururacease), possesses anti-oxidant, anti-inflammatory, and anti-viral activities. In pharmacokinetics of sauchinone, sauchinone is highly distributed to the liver, forming extensive metabolites of sauchinone via UGTs in the liver. Thus, we investigated whether sauchinone inhibited UGTs to explore potential of sauchinone-drug interactions. In human liver microsomes (HLMs), sauchinone inhibited activities of UGT1A1, 1A3, 1A6, and 2B7 with IC50 values of 8.83, 43.9, 0.758, and 0.279 ?M, respectively. Sauchinone also noncompetitively inhibited UGT1A6 and 2B7 with Ki values of 1.08 and 0.524 ?M, respectively. In in vivo interaction study using mice, sauchinone inhibited UGT2B7-mediated zidovudine metabolism, resulting in increased systemic exposure of zidovudine when sauchinone and zidovudine were co-administered together. Our results indicated that there is potential HDI between sauchinone and drugs undergoing UGT2B7-mediated metabolism, possibly contributing to the safe use of sauchinone and drug combinations.
| S-EPMC6017115 | biostudies-literature
Project description:The UDP-glucuronosyltransferase (UGT) 1A enzymes are involved in the phase II metabolism of many important endogenous and exogenous compounds. The nine UGT1A isoforms exhibit high interindividual differences in expression, but their epigenetic regulation is not well understood. The purpose of the present study was to examine microRNA (miRNA) regulation of hepatic UGT1A enzymes and determine whether or not that regulation impacts enzymatic activity. In silico analysis identified miRNA 491-3p (miR-491-3p) as a potential regulator of the UGT1A gene family via binding to the shared UGT1A 3'-untranslated region common to all UGT1A enzymes. Transfection of miR-491-3p mimic into HuH-7 cells significantly repressed UGT1A1 (P < 0.001), UGT1A3 (P < 0.05), and UGT1A6 (P < 0.05) mRNA levels. For UGT1A1, this repression correlated with significantly reduced metabolism of raloxifene into raloxifene-6-glucuronide (ral-6-gluc; P < 0.01) and raloxifene-4'-glucuronide (ral-4'-gluc; P < 0.01). In HuH-7 cells with repressed miR-491-3p expression, there was a significant increase (~80%; P < 0.01) in UGT1A1 mRNA and a corresponding increase in glucuronidation of raloxifene into ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (22%; P < 0.01). Knockdown of endogenous miR-491-3p in HepG2 cells did not significantly alter UGT1A1 mRNA levels but did increase the formation of ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (34%; P < 0.001). A significant inverse correlation between miR-491-3p expression and both UGT1A3 (P < 0.05) and UGT1A6 (P < 0.01) mRNA levels was observed in a panel of normal human liver specimens, with a significant (P < 0.05) increase in UGT1A3 and UGT1A6 mRNA levels observed in miR-491-3p nonexpressing versus expressing liver specimens. These results suggest that miR-491-3p is an important factor in regulating the expression of UGT1A enzymes in vivo.
| S-EPMC3935146 | biostudies-literature
Project description:UDP-glucuronosyltransferase (UGT) isozymes catalyze detoxification of numerous chemical toxins present in our daily diet and environment by conjugation to glucuronic acid. The special properties and enzymatic mechanism(s) that enable endoplasmic reticulum-bound UGT isozymes to convert innumerable structurally diverse lipophiles to excretable glucuronides are unknown. Inhibition of cellular UGT1A7 and UGT1A10 activities and of [33P]orthophosphate incorporation into immunoprecipitable proteins after exposure to curcumin or calphostin-C indicated that the isozymes are phosphorylated. Furthermore, inhibition of UGT phosphorylation and activity by treatment with PKCepsilon-specific inhibitor peptide supported PKC involvement. Co-immunoprecipitation, colocalization by means of immunofluorescence, and cross-linking studies of PKCepsilon and UGT1A7His revealed that the proteins reside within 11.4 angstroms of each other. Moreover, mutation of three PKC sites in each UGT isozyme demonstrated that T73A/G and T202A/G caused null activity, whereas S432G-UGT1A7 caused a major shift of its pH-8.5 optimum to 6.4 with new substrate selections, including 17beta-estradiol. S432G-UGT1A10 exhibited a minor pH shift without substrate alterations. PKCepsilon involvement was confirmed by the demonstration that PKCepsilon overexpression enhanced activity of UGT1A7 but not of its S432 mutant and the conversion of 17beta-[14C]estradiol by S432G-UGT1A7 but not by UGT1A7. Consistent with these observations, treatment of UGT1A7-transfected cells with PKCepsilon-specific inhibitor peptide or general PKC inhibitors increased 17beta-estradiol catalysis between 5- and 11-fold, with parallel decreases in phosphoserine-432. Here, we report a mechanism involving PKC-mediated phosphorylation of UGT such that phosphoserine/threonine regulates substrate specificity in response to chemical exposures, which possibly confers survival benefit.
| S-EPMC1088353 | biostudies-literature
Project description:Bilirubin UDP-glucuronosyltransferase (UDPGT) activity in sealed hepatic microsomes from clofibrate-treated rats was highly latent and was fully expressed by disruption of vesicles with detergents. Antibodies raised against purified bilirubin UDPGT were used to study the transmembrane orientation of the protein to provide a molecular understanding of the UDPGT latency. Immunoblot analysis of sealed microsomes, and microsomes after treatment with proteinases, showed that only a small portion of the protein resides on the cytoplasmic side of the microsomal vesicles. Treatment of microsomes with sodium deoxycholate allowed subtilisin and proteinase K to cleave the transferase, causing loss of activity and the release of smaller immunodetectable peptides. Treatment of the purified bilirubin UDPGT with peptide N-glycosidase F indicated that the enzyme was a glycoprotein. A working model of the transmembrane topology of bilirubin UDPGT is described.
| S-EPMC1138556 | biostudies-other
Project description:Background and purposeCytochrome P450 (CYP, P450) 3A4 is involved in the metabolism of 50% of drugs and its catalytic activity in vivo is not explained only by hepatic expression levels. We previously demonstrated that UDP-glucuronosyltransferase (UGT) 2B7 suppressed CYP3A4 activity through an interaction. In the present study, we target UGT1A9 as another candidate modulator of CYP3A4.Experimental approachWe prepared co-expressed enzymes using the baculovirus-insect cell expression system and compared CYP3A4 activity in the presence and absence of UGT1A9. Wistar rats were treated with dexamethasone and liver microsomes were used to elucidate the role of CYP3A-UGT1A interactions.Key resultsUGT1A9 and UGT2B7 interacted with and suppressed CYP3A4. Kinetic analyses showed that both of the UGTs significantly reduced Vmax of CYP3A4 activity. In addition, C-terminal truncated mutants of UGT1A9 and UGT2B7 still retained the suppressive capacity. Dexamethasone treatment induced hepatic CYP3As and UGT1As at different magnitudes. Turnover of CYP3A was enhanced about twofold by this treatment.Conclusion and implicationsThe changes of kinetic parameters suggested that UGT1A9 suppressed CYP3A4 activity with almost the same mechanism as UGT2B7. The luminal domain of UGTs contains the suppressive interaction site(s), whereas the C-terminal domain may contribute to modulating suppression in a UGT isoform-specific manner. CYP3A-UGT1A interaction seemed to be disturbed by dexamethasone treatment and the suppression was partially cancelled. CYP3A4-UGT interactions would help to better understand the causes of inter/intra-individual differences in CYP3A4 activity.
| S-EPMC7042106 | biostudies-literature
Project description:Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17β-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70-100 and 80-90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130-140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D(3) enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-(33)P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55-58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions.
| S-EPMC3397865 | biostudies-literature
Project description:Glucuronidation by UDP-glucuronyltransferase 2B enzymes (UGT2Bs) is a major pathway for the elimination of endobiotics and xenobiotics, including therapeutic drugs. Morphine, a probe drug for UGT2B7, is metabolized to morphine-3-beta-glucuronide (M3G) and morphine-6-beta-glucuronide (M6G) in humans. Morphine has been used in a series of experiments in the baboon to characterize developmental changes in fetal glucuronidation. This study identifies the baboon UGT2B family of enzymes, compares them with that of the human and the monkey (Macaca fascicularis), and measures the activity of the individual baboon UGT2Bs toward morphine. UGT2B cDNAs were cloned from the liver of adult and newborn baboons and expressed in human embryonic kidney 293 cells. The UGT activity toward morphine was assessed by the rate of formation of M3G and M6G by high-performance liquid chromatography. Eight baboon UGT2Bs were cloned and identified: UGT2B41 and UGT2B42, which are 90% homologous to human UGT2B4; UGT2B43, which is 93% homologous to human UGT2B15; and UGT2B39, UGT2B40, UGT2B44, UGT2B45, and UGT2B46, which are 89 to 91% homologous to human UGT2B7. Homology between baboon and monkey UGT2B ranged from 92.6 to 99.1%, with the primary protein structure of UGT2B43 being 99.1% identical to monkey UGT2B20, including a unique R96I substitution. Gene conversion interfered with the phylogenetic signal in the baboon UGT2B7-like and the monkey UGT2B4-like groups and led to concerted evolution of these enzymes. All of the baboon UGT2Bs metabolized morphine to both M3G and M6G. This study lays the foundation for investigating the regulation of UGT2B enzymes during fetal and neonatal development in the baboon.
| S-EPMC2845934 | biostudies-literature