Precocious development of uridine diphosphate glucuronosyltransferase activity during organ culture of foetal rat liver in the presence of glucocorticoids.
Ontology highlight
ABSTRACT:
SUBMITTER: Wishart GJ
PROVIDER: S-EPMC1165001 | biostudies-other | 1977 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The objective of this study was to determine the regiospecificity of the important uridine diphosphate glucuronosyltransferase (UGT) isoforms responsible for the glucuronidation of flavones and flavonols. We systematically studied the glucuronidation of 13 flavonoids (7 flavones and 6 flavonols, with hydroxyl groups at C-3, C-4', C-5, and/or C-7 positions in flavonoid structure) at a substrate concentration of 10 ?M by 8 recombinant human UGT isoforms mainly responsible for the metabolism of flavonoids, UGTs 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, and 2B7. At 10 ?M substrate concentration, different UGT isoforms gave different regiospecific glucuronidation patterns. UGT 1A1 equally glucuronidated 3-O (glucuronic acid substituted at C-3 hydroxyl group), 7-O, and 4'-O, whereas UGTs 1A8 and 1A9 preferably glucuronidated only 3-O and 7-O positions. UGT 1A1 usually showed no regiospecificity for glucuronidating any position, whereas UGT 1A8 and UGT 1A9 showed dominant, moderate, or weak regiospecificity for 3-O or 7-O position, depending on the structure of the compound. UGT 1A3 showed dominant regiospecificity for the 7-O position, whereas UGT 1A7 showed dominant regiospecificity for the 3-O position. We also showed that the glucuronidation rates of 3-O and 7-O positions in flavones and flavonols were affected by the addition of multiple hydroxyl groups at different positions as well as by the substrate concentrations (2.5, 10, and 35 ?M). In conclusion, regiospecific glucuronidation of flavonols was isoform- and concentration-dependent, whereas flavones were dominantly glucuronidated at the 7-O position by most UGT isoforms. We also concluded that UGTs 1A3 and 1A7 showed dominant regiospecificity for only the 7-O and 3-O positions, respectively. UGTs 1A8 and 1A9 showed moderate or weak preference on glucuronidating position 3-O over the 7-O position, whereas other UGT isoforms did not prefer glucuronidating any particular positions.
| S-EPMC3391414 | biostudies-literature
Project description:ObjectiveTo evaluate the role of bilirubin UDP-glucuronosyltransferase family 1, polypeptide A1 (UGT1A1) gene variations on prolonged unconjugated hyperbilirubinemia associated with breast milk feeding (breast milk jaundice [BMJ]).Study designUGT1A1 gene allelic variation was analyzed in 170 Japanese infants with BMJ with polymerase chain reaction-direct sequencing, and their genotypes compared with serum bilirubin concentrations. In 62 of 170 infants, serum bilirubin concentration was followed after 4 months of life. Genotypes were examined in 55 infants without BMJ.ResultsOf 170 infants with BMJ, 88 (51.8%) were homozygous UGT1A1*6. Serum bilirubin concentrations (21.8 ± 3.65 mg/dL) were significantly greater than in infants with other genotypes (P < .0001). The Gilbert UGT1A1*28 allele was not detected in infants with BMJ, except in an infant who was compound heterozygous with UGT1A1*6. At 4 months of age, serum bilirubin concentration improved to >1 mg/dL, except in 2 infants who were homozygous UGT1A1*7. Homozygous UGT1A1*6 was not detected in the control group.ConclusionOne-half of the infants with BMJ were homozygous UGT1A1*6 and exhibited a serum bilirubin concentration significantly greater than other genotypes. This finding indicates that UGT1A1*6 is a major cause of BMJ in infants in East Asia. Previous finding have demonstrated that 5β-pregnane-3α,20β-diol present in breast milk inhibits p.G71R-UGT1A1 bilirubin glucuronidation activity. Thus, prolonged unconjugated hyperbilirubinemia may develop in infants with UGT1A1*6 who are fed breast milk.
| S-EPMC4123958 | biostudies-literature
Project description:AimTo obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.MethodsThe full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with beta-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.ResultsThe gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418. The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.ConclusionCorrect sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.
| S-EPMC4205369 | biostudies-literature
Project description:UDP glucuronosyltransferases (UGTs) are an important class of Phase II enzymes involved in the metabolism and detoxification of numerous xenobiotics including therapeutic drugs and endogenous compounds (e.g. bilirubin). To date, there are 21 human UGT genes identified, and most of them contain single-nucleotide polymorphisms (SNPs). Non-synonymous SNPs (nsSNPs) of the human UGT genes may cause absent or reduced enzyme activity and polymorphisms of UGT have been found to be closely related to altered drug clearance and/or drug response, hyperbilirubinemia, Gilbert's syndrome, and Crigler-Najjar syndrome. However, it is unlikely to study the functional impact of all identified nsSNPs in humans using laboratory approach due to its giant number. We have investigated the potential for bioinformatics approach for the prediction of phenotype based on known nsSNPs. We have identified a total of 248 nsSNPs from human UGT genes. The two algorithms tools, sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen), were used to predict the impact of these nsSNPs on protein function. SIFT classified 35.5% of the UGT nsSNPs as "deleterious"; while PolyPhen identified 46.0% of the UGT nsSNPs as "potentially damaging" and "damaging". The results from the two algorithms were highly associated. Among 63 functionally characterized nsSNPs in the UGTs, 24 showed altered enzyme expression/activities and 45 were associated with disease susceptibility. SIFT and Polyphen had a correct prediction rate of 57.1% and 66.7%, respectively. These findings demonstrate the potential use of bioinformatics techniques to predict genotype-phenotype relationships which may constitute the basis for future functional studies.
| S-EPMC2758119 | biostudies-literature
Project description:Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.
| S-EPMC4837415 | biostudies-literature
Project description:Uridine diphosphate-glucuronosyltransferase 2B (UGT2B) enzymes conjugate testosterone metabolites to enable their excretion in humans. The functional significance of the UGT2B genetic variants has never been described in humans. We evaluated UGT2B variants in relation to plasma androstane-3?,17?-diol-glucuronide (AAG) levels and the prostate cancer risk.AAG levels were measured in sera from 150 controls and compared to the polymorphisms of UGT2B17, UGT2B15, and UGT2B7. Genomic DNA from controls (301) and cases (148) was genotyped for the polymorphisms, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using unconditional logistic regression analyses. Having two copies of UGT2B17 was associated with higher AAG levels in controls among Whites (p=0.02), but not Blacks (p=0.82). Logistic regression models adjusting for age and race revealed that homozygosity for the G allele of the UGT2B15(D85Y) polymorphism was directly associated with the prostate cancer risk (OR=2.70, 95% CI=1.28, 5.55).While the small sample size limits inference, our findings suggest that an association between the UGT2B17 copy number variant (CNV) and serum AAG levels in Whites, but unexpectedly not in Blacks. This novel observation suggests that genetic determinants of AAG levels in Blacks are unrelated to the UGT2B17 CNV. This study replicates the results that show an association of UGT215(D85Y) with an increased prostate cancer risk.
| S-EPMC3525886 | biostudies-literature
Project description:AimTo investigate the association of variations in the cyclooxygenase-2 (COX2) and uridine diphosphate glucuronosyltransferase 1A6 (UGT1A6) genes and non-steroidal anti-inflammatory drugs (NSAIDs) use with risk of colon cancer.MethodsNSAIDs, which are known to reduce the risk of colon cancer, act directly on COX2 and reduce its activity. Epidemiological studies have associated variations in the COX2 gene with colon cancer risk, but others were unable to replicate this finding. Similarly, enzymes in the UGT1A6 gene have been demonstrated to modify the therapeutic effect of NSAIDs on colon adenomas. Polymorphisms in the UGT1A6 gene have been statistically shown to interact with NSAID intake to influence risk of developing colon adenomas, but not colon cancer. Here we examined the association of tagging single nucleotide polymorphisms (SNPs) in the COX2 and UGT1A6 genes, and their interaction with NSAID consumption, on risk of colon cancer in a population of 422 colon cancer cases and 481 population controls.ResultsNo SNP in either gene was individually statistically significantly associated with colon cancer, nor did they statistically significantly change the protective effect of NSAID consumption in our sample. Like others, we were unable to replicate the association of variants in the COX2 gene with colon cancer risk (P > 0.05), and we did not observe that these variants modify the protective effect of NSAIDs (P > 0.05). We were able to confirm the lack of association of variants in UGT1A6 with colon cancer risk, although further studies will have to be conducted to confirm the association of these variants with colon adenomas.ConclusionOur study does not support a role of COX2 and UGT1A6 genetic variations in the development of colon cancer.
| S-EPMC2682239 | biostudies-literature
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:The elimination of numerous endogenous compounds and xenobiotics via glucuronidation by uridine-5'-diphosphate glycosyltransferase enzymes (UGTs) is an essential process of the body's chemical defense system. UGTs have distinct but overlapping substrate preferences, but the molecular basis for their substrate specificity remains poorly understood. Three-dimensional protein structures can greatly enhance our understanding of the interactions between enzymes and their substrates, but because of the inherent difficulties in purifying and crystallizing integral endoplasmic reticulum membrane proteins, no complete mammalian UGT structure has yet been produced. To address this problem, we have created a homology model of UGT1A6 using I-TASSER to explore, in detail, the interactions of human UGT1A6 with its substrates. Ligands were docked into our model in the presence of the cosubstrate uridine-5'-diphosphate-glucuronic acid, interacting residues were examined, and poses were compared to those cocrystallized with various plant and bacterial glycosyltransferases (GTs). Our model structurally resembles other GTs, and docking experiments replicated many of the expected UGT-substrate interactions. Some bias toward the template structures' protein-substrate interactions and binding preferences was evident.
| S-EPMC7114752 | biostudies-literature
Project description:Flavone glycosides, their aglycones, and metabolites are the major phytochemicals in dietary intake. However, there are still many unknowns about the cellular utilization and active sites of these natural products. Uridine diphosphate glucuronosyltransferases (UGTs) in the endoplasmic reticulum have gene polymorphism distribution in the population and widely mediate the absorption and metabolism of endogenous and exogenous compounds by catalyzing the covalent addition of glucuronic acid and various lipophilic chemicals. Firstly, we found that rutin, a typical flavone O-glycoside, has a stronger UGT2B7 binding effect than its metabolites. After testing a larger number of flavonoids with different aglycones, their aglycones, and metabolites, we demonstrated that typical dietary flavone O-glycosides generally have high binding affinities towards UGT2B7 protein, but the flavone C-glycosides and the phenolic acid metabolites of flavones had no significant effect on this. With the disposition of 4-methylumbelliferone examined by HPLC assay, we determined that 10 μM rutin and nicotifiorin could significantly inhibit the activity of recombinant UGT2B7 protein, which is stronger than isovitexin, vitexin, 3-hydroxyphenylacetic acid and 3,4-dihydroxyphenylacetic acid. In addition, in vitro experiments showed that in normal and doxorubicin-induced lipid composition, both flavone O-glycosides rutin and flavone C-glycosides isovitexin at 10 μM had no significant effect on the expression of UGT1A1, UGT2B4, UGT2B7, and UGT2B15 genes for 24 h exposure. The obtained results enrich the regulatory properties of dietary flavone glycosides, aglycones, and metabolites towards the catalysis of UGTs and will contribute to the establishment of a precise nutritional intervention system based on lipid bilayers and theories of nutrients on endoplasmic reticulum and mitochondria communication.
| S-EPMC10708323 | biostudies-literature