The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Chantal Guillemette - One of the best experts on this subject based on the ideXlab platform.
-
in vitro glucuronidation of fenofibric acid by human udp glucuronosyltransferases and liver microsomes
Drug Metabolism and Disposition, 2009Co-Authors: Jelena Tojcic, Robert J. Straka, Marieodile Enoitbiancamano, Michael H Cou, Patrick Caro, Chantal GuillemetteAbstract:Fenofibric acid (FA), the active moiety of fenofibrate, is an agonist of the peroxisome proliferator-activated nuclear receptor α that modulates triglyceride and cholesterol profiles. Lipid response to fenofibrate and FA serum concentrations is highly variable. Although FA is reported to be almost exclusively inactivated by UDP-glucuronosyltransferases (UGTs) into FA-glucuronide (FA-G), the contribution of UGT isoenzymes has never been systematically assessed. Heterologously expressed human UGT1A and UGT2B and their coding variants were tested for FA glucuronidation using liquid chromatography/mass spectrometry. Recombinant UGT2B7 presented the highest Vmax/Km value (2.10 μl/min/mg), 16-fold higher than the activity of other reactive UGTs, namely, UGT1A3, UGT1A6, and UGT1A9 (0.13, 0.09, and 0.02 μl/min/mg, respectively). UGT2B7.1 (His268) and UGT2B7.2 (Tyr268) enzyme activity was similar, whereas UGT1A3.2 (R11A47), UGT1A3.3 (Trp11), and UGT1A9.3 (Thr33) showed 61 to 96% reduced Vmax/Km values compared with the respective (1) reference proteins. FA-G formation by a human liver bank (n = 48) varied by 10-fold, but the rate of formation was not associated with common genetic variations in UGT1A3, UGT1A6, UGT1A9, and UGT2B7. Correlation with activities for the probe substrates zidovudine (UGT2B7; r2 = 0.75), mycophenolic acid (UGT1A9; r2 = 0.42), fulvestrant (UGT1A3; r2 = 0.36), but not serotonin (UGT1A6; r2 = 0.06) indicated a primary role for UGT2B7 and lesser roles of UGT1A9 and UGT1A3 in hepatic FA glucuronidation. This was confirmed by a strong correlation of FA-G formation with UGT2B7 protein content and inhibition by fluconazole, a known UGT2B7 selective inhibitor. Additional studies are required to identify genetic factors contributing to the observed FA glucuronidation variability.
-
ugt1a8 and ugt1a9 as molecular determinants of mycophenolate mofetil mmf pharmacokinetics
Blood, 2006Co-Authors: Eric Levesque, Marieodile Benoit Biancamano, Felix Couture, Robert Delage, Chantal GuillemetteAbstract:Background: Mycophenolic acid (MPA), the active metabolite of the prodrug mycophenolate mofetil (MMF), is a standard immunosuppressive drug used after haematopoietic stem cell and solid organ transplantation. The pharmacokinetic profile of the drug and its phenolic (MPAG) and acyl (AcMPAG) glucuronides is characterized by unexplained interindividual variation. Despite the remarkable variability, a unique dosage is still currently used in clinics. A better knowledge of the factors influencing MMF disposition in patients is essential in order to minimize risk for the development of acute rejection and prevent toxicity. In this work, we were interested in the variability in MMF pharmacokinetics, particularly as a function of genetic background in the main metabolic pathway of MPA involving UDP-glucuronosyltransferase (UGT) enzymes. Our previous work demonstrated that the formation of the main metabolite (MPAG) is catalyzed by UGT1A9, present in both hepatic and extrahepatic tissues, and UGT1A8, expressed in the gastrointestinal tract and kidney. UGT2B7 forms the minor metabolite AcMPAG, in hepatic and extrahepatic tissues, while UGT1A8 is also involved to a minor extent in its formation. Methods : To evaluate the contribution of genetic variation in UGT1A8 (MPAG>AcMPAG) and UGT1A9 (MPAG) to the variability of MPA pharmacokinetics, 52 healthy volunteers were given a single 1.5g oral dose of MMF. These individuals were selected among 307 for the absence (n=17; controls) or the presence of UGT1A8 * 2 (A 173 G) (n=9), UGT1A8 * 3 (C 277 Y) (n=4), UGT1A9 * 3 (M 33 T) (n=5) and UGT1A9 −275/−2152 (n=17). Pharmacokinetics was measured in plasma and urine by high performance liquid chromatography coupled with tandem mass spectrometry over 12 h after drug intake. The mean age of pharmacokinetics participants was 29.2 ± 9.6 (range 20–54) and this cohort was composed of 31 and 21 healthy males and females, respectively. Results : Compared to controls, MPA exposure is lower in subjects with the low activity UGT1A8 * 3 but elevated in those with the high activity UGT1A9 * 3 (p UGT1A9 * 3 (p=0.021). As a result, the metabolic ratio of AcMPAG/MPAG is higher in UGT1A9 * 3 carriers but lower in carriers of the high activity UGT1A9 −275/−2152 and those carrying UGT1A8 * 3 . MPAG is similar in all groups except in subjects with UGT1A9 −275/−2152. In this group, a trend toward higher C max and AUC of MPAG were observed, consistent with the expected higher glucuronidation capacity for the formation of MPAG associated with this genotype in vitro . Conclusions : Overall, findings indicate that the UGT1A8 and UGT1A9 genotypes significantly alter the pharmacokinetic profile of MPA and its primary glucuronide metabolites, and this influence is detectable after a single dose of MMF. The UGT1A9 −275/−2152 genotype is likely associated with an increase in MPAG formation. In contrast, it is speculated that lower UGT1A9 activity due to UGT1A9 * 3 , may lead to a reduced amount of MPAG by this enzyme in the liver. This metabolic state would provide an increased availability of MPA for the UGT1A8- and UGT2B7-mediated pathways, therefore increasing the overall AcMPAG formation. In support of this hypothesis, the reverse situation was observed with carriers of a deficient UGT1A8 * 3 allele.
-
the novel ugt1a9 intronic i399 polymorphism appears as a predictor of 7 ethyl 10 hydroxycamptothecin glucuronidation levels in the liver
Drug Metabolism and Disposition, 2006Co-Authors: Hugo Girard, Patrick Caron, Louis-charles Fortier, Lisa L Von Moltke, Michael H Court, Lyne Villeneuve, David J Greenblatt, Chantal GuillemetteAbstract: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 r 2 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 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 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.
-
the novel ugt1a9 intronic i399 polymorphism appears as a predictor of 7 ethyl 10 hydroxycamptothecin glucuronidation levels in the liver
Drug Metabolism and Disposition, 2006Co-Authors: Hugo Girard, Lyne Villeneuve, Michael H Cou, Louischarles Fortie, Qi Hao, Lisa L Von Moltke, David J Greenbla, Patrick Caro, Chantal GuillemetteAbstract: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 –118T9>10), three novel UGT1A9 variants (–5366G>T, –4549T>C, and I399C>T), and UGT1A1 (–53TA6>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
-
hepatic expression of the ugt1a9 gene is governed by hepatocyte nuclear factor 4α
Molecular Pharmacology, 2005Co-Authors: Olivier Barbier, Franck J Gonzalez, Helene Duez, Yusuke Inoue, Akihide Kamiya, Jean Charles Fruchart, Chantal Guillemette, Lyne Villeneuve, Hugo Girard, Bart StaelsAbstract:UDP-glucuronosyltransferase (UGT) enzymes catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT enzyme family members, the UGT1A7, UGT1A8, UGT1A9, and UGT1A10 isoforms are issued from a single gene through differential splicing. However, these enzymes display distinct tissue-specific expression patterns. Indeed, UGT1A7, UGT1A8, and UGT1A10 are exclusively expressed in extrahepatic tissues, whereas UGT1A9 transcripts are found at high concentrations in liver. In the present study, we report that the liver-enriched hepatocyte nuclear factor 4 (HNF4)-α controls the hepatic expression of the UGT1A9 enzyme. Liver-specific disruption of the HNF4 α gene in mice drastically decreases liver UGT1A9 mRNA levels. Furthermore, an HNF4α response element (HNF4α RE) was identified in the promoter of human UGT1A9 at position -372 to -360 base pairs by transient transfection, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments. It is interesting that this response element is absent in the proximal UGT1A7, UGT1A8, and UGT1A10 gene promoters. In conclusion, the present study identifies HNF4α as a major factor for the control of UGT1A9 hepatic expression and suggests that the absence of UGT1A7, UGT1A8, and UGT1A10 expression in the liver is caused by, at least in part, a few base pair changes in their promoter sequences in the region corresponding to the HNF4α RE of the UGT1A9 gene.
Curtis D Klaassen - One of the best experts on this subject based on the ideXlab platform.
-
induction of mouse udp glucuronosyltransferase mrna expression in liver and intestine by activators of aryl hydrocarbon receptor constitutive androstane receptor pregnane x receptor peroxisome proliferator activated receptor α and nuclear factor eryt
Drug Metabolism and Disposition, 2009Co-Authors: David B Buckley, Curtis D KlaassenAbstract:UDP-glucuronosyltransferases (UGTs) catalyze the addition of UDP-glucuronic acid to endo- and xenobiotics, enhancing their water solubility and elimination. Many exogenous compounds, such as microsomal enzyme inducers (MEIs), alter gene expression through xenobiotic-responsive transcription factors, namely, the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor α (PPARα), and nuclear factor erythroid 2-related factor 2 (Nrf2). These transcription factors regulate xenobiotic-inducible expression of hepatic and intestinal biotransformation enzymes and transporters. The purpose of this study was to determine hepatic and intestinal inducibility of mouse Ugt mRNA by MEIs. Male C57BL/6 mice were treated for four consecutive days with activators of AhR [2,3,7,8-tetrachlorodibenzodioxin (TCDD), polychlorinated biphenyl 126, and β-naphthoflavone], CAR [1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), phenobarbital, and diallyl sulfide], PXR [pregnenolone-16α-carbonitrile (PCN), spironolactone, and dexamethasone], PPARα (clofibrate, ciprofibrate, and diethylhexylphthalate), and Nrf2 (oltipraz, ethoxyquin, and butylated hydroxyanisole), respectively. Ugt1a1 mRNA expression in liver was induced by activators of all five transcription factor pathways, Ugt1a5 by Nrf2 activators, Ugt1a6 by all the pathways except CAR, and Ugt1a9 by all the pathways except Nrf2. Ugt2b35 mRNA in liver was induced by AhR activators and Ugt2b36 by CAR and PPARα activators. Throughout the small and large intestine, the AhR ligand TCDD increased Ugt1a6 and Ugt1a7 mRNA. In small intestine, the PXR activator PCN increased Ugt1a1, Ugt1a6, Ugt1a7, Ugt2b34, and Ugt2b35 mRNA in the duodenum. In conclusion, chemical activation of AhR, CAR, PXR, PPARα, and Nrf2 in mouse results in induction of distinct Ugt gene sets in liver and intestine, predominantly the Ugt1a isoforms.
-
induction of mouse udp glucuronosyltransferase mrna expression in liver and intestine by activators of aryl hydrocarbon receptor constitutive androstane receptor pregnane x receptor peroxisome proliferator activated receptor α and nuclear factor eryt
Drug Metabolism and Disposition, 2009Co-Authors: David B Buckley, Curtis D KlaassenAbstract:UDP-glucuronosyltransferases (UGTs) catalyze the addition of UDP-glucuronic acid to endo- and xenobiotics, enhancing their water solubility and elimination. Many exogenous compounds, such as microsomal enzyme inducers (MEIs), alter gene expression through xenobiotic-responsive transcription factors, namely, the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor α (PPARα), and nuclear factor erythroid 2-related factor 2 (Nrf2). These transcription factors regulate xenobiotic-inducible expression of hepatic and intestinal biotransformation enzymes and transporters. The purpose of this study was to determine hepatic and intestinal inducibility of mouse Ugt mRNA by MEIs. Male C57BL/6 mice were treated for four consecutive days with activators of AhR [2,3,7,8-tetrachlorodibenzodioxin (TCDD), polychlorinated biphenyl 126, and β-naphthoflavone], CAR [1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), phenobarbital, and diallyl sulfide], PXR [pregnenolone-16α-carbonitrile (PCN), spironolactone, and dexamethasone], PPARα (clofibrate, ciprofibrate, and diethylhexylphthalate), and Nrf2 (oltipraz, ethoxyquin, and butylated hydroxyanisole), respectively. Ugt1a1 mRNA expression in liver was induced by activators of all five transcription factor pathways, Ugt1a5 by Nrf2 activators, Ugt1a6 by all the pathways except CAR, and Ugt1a9 by all the pathways except Nrf2. Ugt2b35 mRNA in liver was induced by AhR activators and Ugt2b36 by CAR and PPARα activators. Throughout the small and large intestine, the AhR ligand TCDD increased Ugt1a6 and Ugt1a7 mRNA. In small intestine, the PXR activator PCN increased Ugt1a1, Ugt1a6, Ugt1a7, Ugt2b34, and Ugt2b35 mRNA in the duodenum. In conclusion, chemical activation of AhR, CAR, PXR, PPARα, and Nrf2 in mouse results in induction of distinct Ugt gene sets in liver and intestine, predominantly the Ugt1a isoforms.
-
induction of rat udp glucuronosyltransferases in liver and duodenum by microsomal enzyme inducers that activate various transcriptional pathways
Drug Metabolism and Disposition, 2006Co-Authors: Melinda K Shelby, Curtis D KlaassenAbstract:Microsomal enzyme inducers (MEIs) up-regulate phase I biotransformation enzymes, most notably cytochromes P450. Transcriptional up-regulation by MEIs occurs through at least three nuclear receptor mechanisms: constitutive androstane receptor (CAR; CYP2B inducers), pregnane X receptor (PXR; CYP3A inducers), and peroxisome proliferator-activated receptor alpha (PPARalpha; CYP4A inducers). Other mechanisms include transcription factors aryl hydrocarbon receptor (AhR; CYP1A inducers), and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2; NADPH-quinone oxidoreductase inducers). UDP-glucuronosyltransferases (UGTs) are phase II biotransformation enzymes that are predominantly expressed in liver and intestine. MEIs increase UGT activity; however, transcriptional regulation of individual UGT isoforms is not completely understood. The purpose of this study was to examine inducibility of individual UGT isoforms and potential mechanisms of transcriptional regulation in rat liver and duodenum. UGT mRNA levels were assessed in liver and duodenum of rats treated with MEIs that activate various transcriptional pathways. All four CAR activators induced UGT2B1 in liver, but not duodenum. UGT1A1, 1A5, 1A6, and 2B12 were induced by at least two CAR activators in liver only. Two PXR ligands induced UGT1A2, but only in duodenum. Two PPARalpha ligands induced UGT1A1 and 1A3 in liver only. AhR ligands induced UGT1A6 and 1A7 in liver, but not duodenum. Nrf2 activators increased UGT2B3 and 2B12 in both liver and duodenum, and UGT1A6, 1A7, and 2B1 in liver only. In summary, only UGT1A2 and 1A8 were not inducible in liver by MEIs. MEIs differentially regulate hepatic expression of individual UGT isoforms, although no one transcriptional pathway dominated. In duodenum, MEIs had minimal effects on UGT expression.
Hugo Girard - One of the best experts on this subject based on the ideXlab platform.
-
the novel ugt1a9 intronic i399 polymorphism appears as a predictor of 7 ethyl 10 hydroxycamptothecin glucuronidation levels in the liver
Drug Metabolism and Disposition, 2006Co-Authors: Hugo Girard, Patrick Caron, Louis-charles Fortier, Lisa L Von Moltke, Michael H Court, Lyne Villeneuve, David J Greenblatt, Chantal GuillemetteAbstract: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 r 2 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 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 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.
-
the novel ugt1a9 intronic i399 polymorphism appears as a predictor of 7 ethyl 10 hydroxycamptothecin glucuronidation levels in the liver
Drug Metabolism and Disposition, 2006Co-Authors: Hugo Girard, Lyne Villeneuve, Michael H Cou, Louischarles Fortie, Qi Hao, Lisa L Von Moltke, David J Greenbla, Patrick Caro, Chantal GuillemetteAbstract: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 –118T9>10), three novel UGT1A9 variants (–5366G>T, –4549T>C, and I399C>T), and UGT1A1 (–53TA6>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
-
hepatic expression of the ugt1a9 gene is governed by hepatocyte nuclear factor 4α
Molecular Pharmacology, 2005Co-Authors: Olivier Barbier, Franck J Gonzalez, Helene Duez, Yusuke Inoue, Akihide Kamiya, Jean Charles Fruchart, Chantal Guillemette, Lyne Villeneuve, Hugo Girard, Bart StaelsAbstract:UDP-glucuronosyltransferase (UGT) enzymes catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT enzyme family members, the UGT1A7, UGT1A8, UGT1A9, and UGT1A10 isoforms are issued from a single gene through differential splicing. However, these enzymes display distinct tissue-specific expression patterns. Indeed, UGT1A7, UGT1A8, and UGT1A10 are exclusively expressed in extrahepatic tissues, whereas UGT1A9 transcripts are found at high concentrations in liver. In the present study, we report that the liver-enriched hepatocyte nuclear factor 4 (HNF4)-α controls the hepatic expression of the UGT1A9 enzyme. Liver-specific disruption of the HNF4 α gene in mice drastically decreases liver UGT1A9 mRNA levels. Furthermore, an HNF4α response element (HNF4α RE) was identified in the promoter of human UGT1A9 at position -372 to -360 base pairs by transient transfection, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments. It is interesting that this response element is absent in the proximal UGT1A7, UGT1A8, and UGT1A10 gene promoters. In conclusion, the present study identifies HNF4α as a major factor for the control of UGT1A9 hepatic expression and suggests that the absence of UGT1A7, UGT1A8, and UGT1A10 expression in the liver is caused by, at least in part, a few base pair changes in their promoter sequences in the region corresponding to the HNF4α RE of the UGT1A9 gene.
-
novel functional polymorphisms in the ugt1a7 and ugt1a9 glucuronidating enzymes in caucasian and african american subjects and their impact on the metabolism of 7 ethyl 10 hydroxycamptothecin and flavopiridol anticancer drugs
Journal of Pharmacology and Experimental Therapeutics, 2003Co-Authors: Lyne Villeneuve, Jeanfrancois Gagne, Louis-charles Fortier, Hugo Girard, Chantal GuillemetteAbstract:In vitro metabolic studies revealed that along with UDP-glucuronosyltransferase (UGT) 1A1, the hepatic UGT1A9 and the extrahepatic UGT1A7 are involved in the biotransformation of the active and toxic metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38). Variant UGT1A1 and UGT1A7 alleles have been reported but the polymorphic nature of the UGT1A9 gene has not been revealed yet. To further clarify the molecular determinants of irinotecan-induced toxicity, we have identified and characterized the functionality of novel UGT1A9 polymorphisms and determined whether additional missense polymorphisms exist in UGT1A7. Using direct DNA sequencing, four single nucleotide polymorphisms (SNPs) were identified in the first exons of UGT1A7 and UGT1A9. One of the two amino acid substitutions found in the UGT1A9 gene, UGT1A9*3 (M33T), results in a dramatic decrease in SN-38 glucuronide formation, with 3.8% of the activity of the UGT1A9*1 allele. In turn, the glucuronidation of flavopiridol, an anticancer drug biotransformed predominantly by UGT1A9, remains unaffected, indicating a substrate-dependent impact of this variant. UGT1A9*3 is detected only in Caucasians and 4.4% of the population tested was found heterozygous (*1/*3). Two additional UGT1A7 SNPs were found exclusively in African-American subjects and generate five alleles (UGT1A7*5 to *9) when combined to the four known SNPs present in UGT1A7*2, *3, and *4. Upon functional analysis with SN-38, five out of nine UGT1A7 allozymes exhibited much lower SN-38 glucuronidation activities compared with UGT1A7*1, all having in common the mutational changes at codons 115 or 208. Results suggest that these low SN-38 glucuronidating alleles may represent additional molecular determinants of irinotecan-induced toxicity and warrant further investigations.
Lyne Villeneuve - One of the best experts on this subject based on the ideXlab platform.
-
the novel ugt1a9 intronic i399 polymorphism appears as a predictor of 7 ethyl 10 hydroxycamptothecin glucuronidation levels in the liver
Drug Metabolism and Disposition, 2006Co-Authors: Hugo Girard, Patrick Caron, Louis-charles Fortier, Lisa L Von Moltke, Michael H Court, Lyne Villeneuve, David J Greenblatt, Chantal GuillemetteAbstract: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 r 2 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 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 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.
-
the novel ugt1a9 intronic i399 polymorphism appears as a predictor of 7 ethyl 10 hydroxycamptothecin glucuronidation levels in the liver
Drug Metabolism and Disposition, 2006Co-Authors: Hugo Girard, Lyne Villeneuve, Michael H Cou, Louischarles Fortie, Qi Hao, Lisa L Von Moltke, David J Greenbla, Patrick Caro, Chantal GuillemetteAbstract: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 –118T9>10), three novel UGT1A9 variants (–5366G>T, –4549T>C, and I399C>T), and UGT1A1 (–53TA6>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
-
hepatic expression of the ugt1a9 gene is governed by hepatocyte nuclear factor 4α
Molecular Pharmacology, 2005Co-Authors: Olivier Barbier, Franck J Gonzalez, Helene Duez, Yusuke Inoue, Akihide Kamiya, Jean Charles Fruchart, Chantal Guillemette, Lyne Villeneuve, Hugo Girard, Bart StaelsAbstract:UDP-glucuronosyltransferase (UGT) enzymes catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT enzyme family members, the UGT1A7, UGT1A8, UGT1A9, and UGT1A10 isoforms are issued from a single gene through differential splicing. However, these enzymes display distinct tissue-specific expression patterns. Indeed, UGT1A7, UGT1A8, and UGT1A10 are exclusively expressed in extrahepatic tissues, whereas UGT1A9 transcripts are found at high concentrations in liver. In the present study, we report that the liver-enriched hepatocyte nuclear factor 4 (HNF4)-α controls the hepatic expression of the UGT1A9 enzyme. Liver-specific disruption of the HNF4 α gene in mice drastically decreases liver UGT1A9 mRNA levels. Furthermore, an HNF4α response element (HNF4α RE) was identified in the promoter of human UGT1A9 at position -372 to -360 base pairs by transient transfection, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments. It is interesting that this response element is absent in the proximal UGT1A7, UGT1A8, and UGT1A10 gene promoters. In conclusion, the present study identifies HNF4α as a major factor for the control of UGT1A9 hepatic expression and suggests that the absence of UGT1A7, UGT1A8, and UGT1A10 expression in the liver is caused by, at least in part, a few base pair changes in their promoter sequences in the region corresponding to the HNF4α RE of the UGT1A9 gene.
-
novel functional polymorphisms in the ugt1a7 and ugt1a9 glucuronidating enzymes in caucasian and african american subjects and their impact on the metabolism of 7 ethyl 10 hydroxycamptothecin and flavopiridol anticancer drugs
Journal of Pharmacology and Experimental Therapeutics, 2003Co-Authors: Lyne Villeneuve, Jeanfrancois Gagne, Louis-charles Fortier, Hugo Girard, Chantal GuillemetteAbstract:In vitro metabolic studies revealed that along with UDP-glucuronosyltransferase (UGT) 1A1, the hepatic UGT1A9 and the extrahepatic UGT1A7 are involved in the biotransformation of the active and toxic metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38). Variant UGT1A1 and UGT1A7 alleles have been reported but the polymorphic nature of the UGT1A9 gene has not been revealed yet. To further clarify the molecular determinants of irinotecan-induced toxicity, we have identified and characterized the functionality of novel UGT1A9 polymorphisms and determined whether additional missense polymorphisms exist in UGT1A7. Using direct DNA sequencing, four single nucleotide polymorphisms (SNPs) were identified in the first exons of UGT1A7 and UGT1A9. One of the two amino acid substitutions found in the UGT1A9 gene, UGT1A9*3 (M33T), results in a dramatic decrease in SN-38 glucuronide formation, with 3.8% of the activity of the UGT1A9*1 allele. In turn, the glucuronidation of flavopiridol, an anticancer drug biotransformed predominantly by UGT1A9, remains unaffected, indicating a substrate-dependent impact of this variant. UGT1A9*3 is detected only in Caucasians and 4.4% of the population tested was found heterozygous (*1/*3). Two additional UGT1A7 SNPs were found exclusively in African-American subjects and generate five alleles (UGT1A7*5 to *9) when combined to the four known SNPs present in UGT1A7*2, *3, and *4. Upon functional analysis with SN-38, five out of nine UGT1A7 allozymes exhibited much lower SN-38 glucuronidation activities compared with UGT1A7*1, all having in common the mutational changes at codons 115 or 208. Results suggest that these low SN-38 glucuronidating alleles may represent additional molecular determinants of irinotecan-induced toxicity and warrant further investigations.
Tsuyoshi Yokoi - One of the best experts on this subject based on the ideXlab platform.
-
preparation of a specific monoclonal antibody against human udp glucuronosyltransferase ugt 1a9 and evaluation of ugt1a9 protein levels in human tissues
Drug Metabolism and Disposition, 2012Co-Authors: Miki Nakajima, Masahiko Hatakeyama, Tatsuki Fukami, Tsuyoshi YokoiAbstract:Glucuronidation is a major detoxification pathway of drugs and xenobiotics that are catalyzed by the UDP-glucuronosyltransferase (UGT) superfamily. Determination of the protein levels of the individual UGT isoforms in human tissues is required for the successful extrapolation of in vitro metabolic data to in vivo clearance. Most previous studies evaluating UGT isoform expression were limited to the mRNA level because of the high degree of amino acid sequence homology between UGT isoforms that has hampered the availability of isoform-specific antibodies. In this study, we generated a peptide-specific monoclonal antibody against human UGT1A9. We demonstrated that this antibody does not cross-react with the other UGT1A isoforms including UGT1A7, UGT1A8, and UGT1A10 and shows a high degree of amino acid sequence similarity with UGT1A9. Using this antibody, we found that UGT1A9 protein is expressed in the kidney and the liver but not in the jejunum or the ileum, consistent with previous reports of mRNA expression. In a panel of 20 individual human livers, the UGT1A9 protein levels exhibited 9-fold variability. It is noteworthy that the relative UGT1A9 protein levels were not correlated with the UGT1A9 mRNA level ( r = −0.13), like other UGT isoforms reported previously, suggesting the importance of evaluating UGT isoform expression at protein levels. In conclusion, we generated a specific monoclonal antibody against UGT1A9 and evaluated the distribution and relative expression levels of the UGT1A9 protein in human tissues. This antibody may serve as a useful tool for further studies of UGT1A9 to evaluate its physiological, pharmacological, and toxicological roles in human tissues.
-
preparation of a specific monoclonal antibody against human ugt1a9 and evaluation of ugt1a9 protein levels in human tissues
Drug Metabolism and Disposition, 2012Co-Authors: Miki Nakajima, Masahiko Hatakeyama, Tatsuki Fukami, Tsuyoshi YokoiAbstract:Glucuronidation is a major detoxification pathway of drugs and xenobiotics that is catalyzed by the UDP-glucuronosyltransferase (UGT) superfamily. Determination of the protein levels of the individual UGT isoforms in human tissues is required for the successful extrapolation of in vitro metabolic data to in vivo clearance. Most previous studies evaluating UGT isoform expression were limited to the mRNA level because of the high degree of amino acid sequence homology between UGT isoforms that has hampered the availability of isoform-specific antibodies. In this study, we generated a peptide-specific monoclonal antibody against human UGT1A9. We demonstrated that this antibody does not cross-react with the other UGT1A isoforms including UGT1A7, UGT1A8, and UGT1A10, showing a high degree of amino acid sequence similarity with UGT1A9. Using this antibody, we found that UGT1A9 protein is expressed in the kidney and the liver, but not in the jejunum or the ileum, consistent with previous reports of mRNA expression. In a panel of 20 individual human livers, the UGT1A9 protein levels exhibited 9-fold variability. Interestingly, the relative UGT1A9 protein levels were not correlated with the UGT1A9 mRNA level (r = 0.21), likely other UGT isoforms reported previously, suggesting the importance of evaluating UGT isoform expression at protein levels. In conclusion, we generated a specific monoclonal antibody against UGT1A9 and evaluated the distribution and relative expression levels of the UGT1A9 protein in human tissues. This antibody may serve as a useful tool for further studies of UGT1A9 to evaluate its physiological, pharmacological, and toxicological roles in human tissues.
-
interactions between human ugt1a1 UGT1A4 and ugt1a6 affect their enzymatic activities
Drug Metabolism and Disposition, 2007Co-Authors: Ryoichi Fujiwara, Miki Katoh, Miki Nakajima, Hiroyuki Yamanaka, Tsuyoshi YokoiAbstract:Protein-protein interactions between human UDP-glucuronosyltransferase (UGT) 1A1, UGT1A4, and UGT1A6 were investigated using double expression systems in HEK293 cells (UGT1A1/UGT1A4, UGT1A1/UGT1A6, and UGT1A4/UGT1A6). The substrates specific for UGT1A1 (estradiol and bilirubin), UGT1A4 (imipramine and trifluoperazine), and UGT1A6 (serotonin and diclofenac) were used to determine the effects of the coexpression of the other UGT1A isoforms on the enzymatic activity. The coexpression of UGT1A4 and UGT1A6 decreased the S(50) and V(max) values of UGT1A1-catalyzed estradiol 3-O-glucuronide formation and increased the V(max) value of UGT1A1-catalyzed bilirubin O-glucuronide formation. The coexpression of UGT1A1 decreased the V(max) value of UGT1A4-catalyzed imipramine N-glucuronide formation but had no effect on UGT1A4-catalyzed trifluoperazine N-glucuronide formation. The coexpression of UGT1A6 had no effect on UGT1A4-catalyzed imipramine N-glucuronide formation but increased the K(m) and V(max) of UGT1A4-catalyzed trifluoperazine N-glucuronide formation. The coexpression of both UGT1A1 and UGT1A4 increased the V(max) values of UGT1A6-catalyzed serotonin and diclofenac O-glucuronide formation. Thus, the effects of the coexpression of other UGT1A isoforms on the kinetics of specific activities were different depending on the UGT1A isoforms and substrates. Native polyacrylamide gel electrophoresis analysis of the double expression systems showed multiple bands at approximately 110 kDa, indicating the existence of heterodimers as well as homodimers of UGTs. In conclusion, we found that human UGT1A1, UGT1A4, and UGT1A6 interact with each other, possibly by heterodimerization, and that their effects on the enzymatic activities are complex depending on the isoforms and substrates.
-
stereoselective glucuronidation of 5 4 hydroxyphenyl 5 phenylhydantoin by human udp glucuronosyltransferase ugt 1a1 ugt1a9 and ugt2b15 effects of ugt ugt interactions
Drug Metabolism and Disposition, 2007Co-Authors: Miki Nakajima, Miki Katoh, H Yamanaka, Ryoichi Fujiwara, Tsuyoshi YokoiAbstract:5-(4′-Hydroxyphenyl)-5-phenylhydantoin (4′-HPPH), a major metabolite of phenytoin in human, is exclusively metabolized to a glucuronide. 4′-HPPH has a chiral center. ( S )-4′-HPPH is a predominant form produced from phenytoin in humans, and ( R )-4′-HPPH is an extremely toxic form with respect to gingival hyperplasia. In the present study, we investigated stereoselective 4′-HPPH O -glucuronide formation in human liver microsomes. Human liver microsomes predominantly formed ( S )-4′-HPPH O -glucuronide rather than ( R )-4′-HPPH O -glucuronide from racemic 4′-HPPH. Among human UDP-glucuronosyltransferase (UGT) enzymes, UGT1A1, UGT1A9, and UGT2B15 showed 4′-HPPH O -glucuronide formation. Interestingly, UGT1A1 stereoselectively formed ( R )-4′-HPPH O -glucuronide, whereas UGT1A9 and UGT2B15 stereoselectively formed ( S )-4′-HPPH O -glucuronide from racemic 4′-HPPH. By using UGT1A double-expression systems in HEK293 cells that we previously established, the effects of UGT-UGT interactions on 4′-HPPH O -glucuronide formation were investigated. It was demonstrated that coexpression of UGT1A4 increased the V max values of ( S )- and ( R )-4′-HPPH O -glucuronide formation catalyzed by UGT1A1 but decreased the V max values of ( S )- and ( R )-4′-HPPH O -glucuronide formation catalyzed by UGT1A9. Coexpression of UGT1A6 increased the S 50 values and decreased the V max values of ( S )- and ( R )-4′-HPPH glucuronide formation catalyzed by UGT1A1 and UGT1A9. However, the interaction did not alter the stereoselectivity. In conclusion, we found that 4′-HPPH O -glucuronide formation in human liver microsomes is catalyzed by UGT1A1, UGT1A9, and UGT2B15 in a stereoselective manner, being modulated by interaction with other UGT1A isoforms.
-
effects of coexpression of ugt1a9 on enzymatic activities of human ugt1a isoforms
Drug Metabolism and Disposition, 2007Co-Authors: Ryoichi Fujiwara, Akiko Nakamura, Miki Katoh, H Yamanaka, Miki Nakajima, Toshiyuki Sakaki, Shin Ichi Ikushiro, Tsuyoshi YokoiAbstract:We established stable HEK293 cell lines expressing double isoforms, UGT1A1 and UGT1A9, UGT1A4 and UGT1A9, or UGT1A6 and UGT1A9, as well as stable cell lines expressing each single isoform. To analyze the protein-protein interaction between the UGT1As, we investigated the thermal stability and resistance to detergent. UGT1A9 uniquely demonstrated thermal stability, which was enhanced in the presence of UDP-glucuronic acid (>90% of control), and resistance to detergent. Interestingly, UGT1A1, UGT1A4, and UGT1A6 acquired thermal stability and resistance to detergent by the coexpression of UGT1A9. An immunoprecipitation assay revealed that UGT1A6 and UGT1A9 interact in the double expression system. Using the single expression systems, it was confirmed that estradiol 3-O-glucuronide, imipramine N-glucuronide, serotonin O-glucuronide, and propofol O-glucuronide formations are specific for UGT1A1, UGT1A4, UGT1A6, and UGT1A9, respectively. By kinetic analyses, we found that the coexpressed UGT1A9 significantly affected the kinetics of estradiol 3-O-glucuronide formation (decreased Vmax), imipramine N-glucuronide formation (increased Km and Vmax), and serotonin O-glucuronide formation (decreased Vmax) catalyzed by UGT1A1, UGT1A4, and UGT1A6, respectively. On the other hand, the coexpressed UGT1A1 increased Km and decreased the Vmax of the propofol O-glucuronide formation catalyzed by UGT1A9. The coexpressed UGT1A4 and UGT1A6 also increased the Vmax of the propofol Oglucuronide formation by UGT1A9. This is the first study showing that human UGT1A isoforms interact with other isoforms to change the enzymatic characteristics.
