UGT1A1 mRNA Research Articles

Studies on the expression of liver detoxifying enzymes in rats fed seaweed ( Monostroma nitidum)

The expression level of phase I (CYP1A1 and CYP1A2) and phase II (GST, and UGT) enzyme-coded genes were measured in liver microsomes of 30 Sprague–Dawley rats fed sea weed ( Monostroma nitidum). Quantitative and qualitative analysis of the detoxifying enzymes were investigated using reverse transcription polymerase reaction (RT-PCR) and real time polymerase reaction (Real-time PCR) techniques. The antioxidative properties of seaweed were screened and investigated for its hepatoprotective activity in rat. There was no significant induction of GSTYa1, GSTYa2, and CYP1A2. However, an M. nitidum diet was found to significantly increase UGT1A1 and UGT1A6 mRNA levels and to decrease CYP1A1 mRNA levels in rat liver. Structural studies confirmed the presence of sulfated polysaccharides in the seaweed samples. The results demonstrate the potential of seaweed as a natural source of sulfated polysaccharide substances with potential use in chemoprevention medicine.

Hepatocyte nuclear factor-1 alpha is associated with UGT1A1, UGT1A9 and UGT2B7 mRNA expression in human liver

Experimental evidence suggests HNF1alpha regulates UGT expression. This study investigates (1) whether the variability in HNF1alpha expression is associated with the variability in UGT1A1, UGT1A9 and UGT2B7 expression in human livers and (2) the functionality of 12 HNF1alpha variants using mRNA expression as phenotype. Controlling for known UGT variation in cis-acting elements known to affect UGT expression, we demonstrate that a combination of HNF1alpha mRNA levels and UGT genotype predicts variance in UGT expression to a higher extent than UGT genotype alone. None of the HNF1alpha polymorphisms studied, however, seem to have an effect on HNF1alpha, UGT1A1, UGT1A9 and UGT2B7 expression, ruling out their functional role. Our data provide evidence for HNF1alpha being a determinant of UGT1A1, UGT1A9 and UGT2B7 mRNA expression. However, the amount of UGT intergenotype variability explained by HNF1alpha expression appears to be modest, and further studies should investigate the role of multiple transcription factors.

Hepatocyte Nuclear Factor 1 Alpha and 4 Alpha are Factors Involved in Interindividual Variability in the Expression of UGT1A6 and UGT1A9 but not UGT1A1, UGT1A3 and UGT1A4 mRNA in Human Livers

UDP-glucuronosyltransferases (UGTs) catalyze phase-II biotransformation reaction of a variety of substances. Among the UGT1A isoforms, UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9 are predominantly expressed in the liver. Interindividual variability in expression of these isoforms would cause interindividual differences in drug response, toxicity and cancer susceptibility. In the present study, we investigated the interindividual variability in UGT1A mRNA expression and whether hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF4alpha were factors responsible for their variability in human livers. The amounts of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, HNF1alpha and HNF4alpha mRNA in 18 human livers were measured by quantitative real-time polymerase chain reaction. The largest and smallest interindividual differences in expression levels were observed in UGT1A1 (8.6-fold) and UGT1A4 (2.5-fold) mRNA, respectively. The amounts of HNF1alpha and HNF4alpha mRNA were strongly correlated with the amount of UGT1A9 mRNA and moderately correlated with that of UGT1A6 mRNA, whereas no significant correlation was found with the amounts of UGT1A1, UGT1A3 and UGT1A4 mRNA. Our results suggest that HNF1alpha and HNF4alpha are the factors involved in the interindividual variability of UGT1A6 and UGT1A9 mRNA expression. Further studies of other transcription factors are needed to clarify the factor(s) determining the interindividual variations in UGT1A1, UGT1A3 and UGT1A4 mRNA expression.

Human UGT1A8 and UGT1A10 mRNA Are Expressed In Primary Human Hepatocytes

It is widely believed that the UGT1A isoforms, UGT1A8 and -1A10, are expressed exclusively in extrahepatic tissues. In this work, human primary hepatocytes from six donors were analyzed for UGT1A8 and -1A10 mRNA expression by semi-quantitative RT-PCR. New primers to amplify UGT1A8 mRNA were designed and found to differ from those previously published. We demonstrated that UGT1A8 and -1A10 mRNA are expressed in hepatocytes. Although basal UGT mRNA levels were detected in untreated hepatocytes, significant up-regulation of the levels of mRNA for these isoforms were seen after treatment with 3-methylcholanthrene (3-MC) and rifampicin (Rif). RT-PCR products for all UGTs were sequenced and unambiguously identified as matching the corresponding cDNA. The discovery of these isoforms in hepatocytes is a novel discovery and will stimulate studies on the potential role for these isoforms in hepatic detoxification.

Dietary Sesame Seed and Its Lignan Increase Both Ascorbic Acid Concentration in Some Tissues and Urinary Excretion by Stimulating Biosynthesis in Rats

We previously showed that the intake of sesamin, a major lignan in sesame seed, decreased lipid peroxidation and elevated tocopherol concentration in rat tissues. In this study, we examined the effect of dietary sesame seed and sesamin on the ascorbic acid concentration in rat tissues. Rats (4-wk-old) were fed either a vitamin E-free diet, or a diet containing 50 mg gamma-tocopherol/kg, one containing 2 g sesamin/kg, one containing 50 mg gamma-tocopherol/kg and 2 g sesamin/kg, or one containing 200 g sesame seed/kg for 28 d. The dietary sesamin and sesame seed elevated ascorbic acid concentrations in the liver and kidney, and increased urinary excretion in those Wistar rats. The dietary sesamin also elevated the hepatic mRNA levels of cytochrome P450 (CYP) 2B, and UDP-glucuronosyltransferase (UGT) 1A and 2B. In contrast, neither the sesamin nor the sesame seed affected the liver concentration of ascorbic acid in ODS rats with a hereditary defect in ascorbic acid synthesis, though the dietary sesame seed elevated the UGT1A and 2B mRNA levels in the liver. In addition, the sesame seed elevated the gamma-tocopherol concentration in the various ODS rat tissues and the ascorbic acid concentrations in the kidney, heart and lung, while reducing the thiobarbituric acid reactive substance concentration in the heart and kidney. These results suggest that dietary sesame seed and its lignan stimulate ascorbic acid synthesis as a result of the induction of UGT1A and the 2B-mediated metabolism of sesame lignan in rats. The data of ODS rat studies also suggest that dietary sesame seed enhances antioxidative activity in the tissues by elevating the levels of two antioxidative vitamins, vitamin C and E.

Stereoselective Metabolism of Carvedilol by the .BETA.-Naphthoflavone-Inducible Enzyme in Human Intestinal Epithelial Caco-2 Cells

Treatment of Caco-2 cells with beta-naphthoflavone (beta-NF) and 1alpha,25-dihydroxyvitamin D(3) (VD(3)) induces UDP-glucuronosyltransferases (UGTs) and cytochrome P450 (CYP) 3A4, respectively. In the present study, we evaluated the metabolism of carvedilol in beta-NF- and VD(3)-treated Caco-2 cells. The metabolism of R-carvedilol was not significant in non-treated Caco-2 cells, whereas S-carvedilol was significantly metabolized in the cells. The metabolism of R- and S-carvedilol was significantly increased by the treatment of Caco-2 cells with 50 microM beta-NF for 3 d. In contrast, the treatment of Caco-2 cells with 250 nM VD(3) for 2 weeks did not induce a significant change in the metabolism of R- and S-carvedilol. The metabolism of carvedilol in beta-NF-treated Caco-2 cells was markedly inhibited by a substrate of UGTs, baicalein. In addition, the expression of UGT1A1, 1A6, and 1A9 mRNA was increased in beta-NF-treated Caco-2 cells as compared with non-treated cells. These findings indicated that carvedilol was metabolized stereoselectively by the beta-NF-inducible enzyme in Caco-2 cells. The UGT1A subfamily in intestinal epithelial cells may be partly responsible for first-pass (presystemic) metabolism of the drug.

Effects of Green Tea Compounds on Irinotecan Metabolism

The effects of green tea compounds on the metabolism of irinotecan have never been investigated. We aimed to study whether catechins [(-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epicatechin] affect the inactivation metabolism of irinotecan into 7-ethyl-10-[4-N-(1-piperidino)-1-amino]carbonyloxycamptothecin (NPC) (by CYP3A4) and 7-ethyl-10-hydroxycamptothecin (SN-38) into 7-ethyl-10-hydroxycamptothecin glucuronide (SN-38G) (by UGT1A1). Human liver microsomes, hepatocytes and Hep G2 cells were incubated with catechins and treated with irinotecan and/or SN-38. NPC and SN-38G formation was measured by high-performance liquid chromatography. UGT1A1 mRNA levels were measured by real-time polymerase chain reaction. In human liver microsomes, a concentration-dependent decrease in the formation of NPC and SN-38G was observed. In human hepatocytes, a significant increase in SN-38G production was observed in 33% (EGCG), 44% (ECG), and 44% (EGC) of the hepatocyte preparations. Phenobarbital increased the formation of SN-38G in 100% of the same hepatocyte preparations. In Hep G2 cells, no increase in SN-38G formation was observed. With the exception of ECG in one liver, catechins did not increase UGT1A1 mRNA levels. NPC production was also significantly increased in 40% of the hepatocyte preparations for each catechin. However, the production of 6beta-hydroxytestosterone remained unaffected in other hepatocyte preparations. At pharmacologically relevant concentrations, catechins are unlikely to inhibit the formation of irinotecan inactive metabolites when administered concomitantly. The induction effect of catechins on UGT1A1 seems to be modest and highly variable. Catechins do not induce CYP3A4 activity. The effect of acute and prolonged use of green tea on the pharmacokinetics of irinotecan in patients remains to be evaluated.

Murine hepatoma (Hepa1c1c7) cells: A responsive in vitro system for chemoprotective enzyme induction by organoselenium compounds

Murine (Hepa1c1c7) hepatoma cells are a suitable in vitro system for investigating the regulation of chemoprotective enzymes by selenazolidines, novel l-selenocysteine prodrugs developed as potential chemopreventive agents. They are less sensitive to the cytotoxic effects of both selenite and the less toxic selenazolidines than rat hepatoma (H4IIE) cells. All four selenazolidine 4-carboxylic acid (SCA) derivatives examined elevated thioredoxin reductase (Txnrd1), alpha-class glutathione transferases (Gsta), and UDP-glucuronosyltransferase (Ugt)1a6 mRNAs. NAD(P)H-quinone oxidoreductase (Nqo1) was induced by the three 2-alkyl derivatives (2-cyclohexylSCA, 2-butylSCA, and 2-methylSCA) but not SCA itself. Transcripts of mu- and pi-class glutathione transferases were induced only by 2-cyclohexylSCA and 2-butylSCA. Only Gsta and Txnrd1 transcripts were elevated by l-selenomethionine, l-selenocystine, or Se-methyl- l-selenocysteine. Txnrd1, Gsta, Nqo1, and Gstp responses to selenazolidines were all abolished by actinomycin D while Ugt1a6 responses were not. Induction responses to the selenazolidines were also eliminated (most) or reduced (Txnrd1 by 2-methylSCA) by cycloheximide, with the exception of Ugt1a6. The Ugt1a6 mRNA levels in the presence of SCAs and cycloheximide were similar to those with cycloheximide alone, and were almost double those of vehicle-treated cells. Thus, Hepa1c1c7 cells appear to provide a viable platform for the study of protective enzyme regulation by selenocompounds, and with the exception of Ugt1a6, the mRNA elevations from selenazolidines are transcriptionally dependent.

Irinotecan Inactivation Is Modulated by Epigenetic Silencing ofUGT1A1in Colon Cancer

Irinotecan is used in the first-line treatment of metastatic colorectal cancer. The UGT1A1-metabolizing enzyme, expressed in liver and colon, is primarily involved in the inactivation of its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). Herein, we explored the role of DNA methylation in the silencing of UGT1A1 gene expression in colon cancer and its influence on cellular SN-38 detoxification. UGT1A1 mRNA was repressed in most primary tumors (41 of 50; 82%) and in three colon cancer cell lines (HCT-116, HCT-15, and COLO-320DM). Bisulfite sequencing of the UGT1A1 gene revealed the aberrant methylation of specific CpG islands in UGT1A1-negative cells. Conversely, hypomethylation was observed in HT-29, HT-115, and LOVO cells that overexpress UGT1A1. Direct methylation of the UGT1A1 promoter resulted in the complete repression of transcriptional activity. Treatment with demethylating and histone deacetylase inhibitor agents had the capacity to reverse aberrant hypermethylation and to restore UGT1A1 expression in hypermethylated UGT1A1-negative cells but not in hypomethylated cells. Loss of UGT1A1 methylation was further associated with an increase in UGT1A1 protein content and with an enhanced inactivation of SN-38 by 300% in HCT-116 cells. We conclude that DNA methylation represses UGT1A1 expression in colon cancer and that this process may contribute to the level of tumoral inactivation of the anticancer agent SN-38 and potentially influence clinical response.

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-α (PPARα), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARα did not exhibit significant ontogenetic changes. RARβ mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARα levels. Levels of RXRα and RXRβ mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.

Differential UGT1A1 Induction by Chrysin in Primary Human Hepatocytes and HepG2 Cells

Chrysin, a dietary flavonoid, has been shown to markedly induce UGT1A1 expression and activity in HepG2 and Caco-2 cell lines; thus, it has been suggested to have clinical utility in the treatment of UGT1A1-mediated deficiencies, such as unconjugated hyperbilirubinemia or the prevention of 7-ethyl-10-hydroxycamptothecin (SN-38) toxicity. However, little is known about its induction potential in a more physiologically relevant model system, such as primary hepatocyte culture. In this study, induction of UGT1A1 expression (mRNA, protein, and activity) was investigated in primary human hepatocyte cultures after treatment with chrysin and other prototypical inducers. Endogenous nuclear receptor-mediated UGT1A1 induction was studied using transient transfection reporter assays in primary human hepatocytes and HepG2 cells. Results indicated that induction of UGT1A1 expression was minimal in human hepatocytes treated with chrysin compared with that in HepG2 cells (1.2-versus 11-fold, respectively). Subsequent experiments to determine whether the differential response was due to its metabolic stability revealed strikingly different elimination rate constants between the two cell systems (half-life of 13 min in human hepatocytes versus 122 min in HepG2 cell suspensions). Further study demonstrated that UGT1A1 mRNA expression could be induced in human hepatocyte cultures by either increasing the chrysin dosing frequency or by modulating chrysin metabolism, suggesting that the differential induction observed in hepatocytes and HepG2 cells was due to differences in the metabolic clearance of chrysin. In conclusion, this study suggests that the metabolic stability of chrysin likely would limit its ability to induce UGT1A1 in vivo.

ISOFORM-SPECIFIC EXPRESSION AND INDUCTION OF UDP-GLUCURONOSYLTRANSFERASE IN IMMUNOACTIVATED PERITONEAL MACROPHAGES OF THE RAT

Phase I drug-metabolizing enzymes such as cytochrome P450 in immunocytes are known to play a role in metabolic activation of toxic and immunosuppressive compounds such as polycyclic aromatic hydrocarbon (PAH). UDP-glucuronosyltransferase (UGT), a drug-metabolizing phase II enzyme, accelerates elimination of these compounds; however, there is little information on the expression and function of UGT in immunocytes. In this study, we investigated the expressions of UGT isoforms in rat peritoneal macrophages and the role of UGT in macrophage functions. Expressions of UGT1A1, 1A6, and 1A7 were observed in macrophages by immunohistochemical staining and reverse transcriptase-polymerase chain reaction. When macrophage cells cultured in plates were exposed to 1-naphthol and 3-hydroxybenzo-[a]pyrene (3-OH-B[a]P), these glucuronides increased in the medium, indicating that macrophages glucuronidated the chemicals. The production of the glucuronides of 1-naphthol and 3-OH-B[a]P was induced by lipopolysaccharide (LPS) treatment of the cultured macrophage cells. Northern blot analysis revealed that UGT1A7 mRNA was induced by LPS treatment. This result is the first evidence that a drug-metabolizing enzyme is induced by immunoactivation. The results indicated that macrophages can detoxify various toxic and immunosuppressive compounds with UGT, and that ability is enhanced by immunoactivation. We propose that macrophages contribute to protection against not only macromolecules as immunocytes but also small molecules such as the immunosuppressive agents PAHs in peripheral blood and interstitial tissues.

Modulation of UDP‐glucuronosyltransferase 1A1 in primary human hepatocytes by prototypical inducers

The primary objective of this study was to evaluate the modulation of UGT1A1 expression in human hepatocytes using prototypical CYP450 inducers. A bank of 16 human livers was utilized to obtain an estimate of the range of UGT1A1 protein expression and catalytic activity. Concentration-dependent changes in UGT1A1 response were evaluated in hepatocyte cultures after treatment with 3-methylchloranthrene, beta-napthoflavone, rifampicin, or phenobarbital. Pharmacodynamic analyses of UGT1A1 expression were conducted and compared to those of CYP450 after treatment with inducers in 2-3 different hepatocyte preparations. Additionally, expression of UGT1A1 mRNA and protein was evaluated in human hepatocytes treated with 14 different compounds known to activate differentially the human pregnane-X-receptor or constitutive androstane receptor. Pharmacodynamic modeling revealed EC50 values statistically significant between UGT1A1 and CYP2B6 after treatment with PB, but not statistically distinguishable between UGT1A1 and CYP's 1A2 or 3A4 after treatment with 3-methylchloranthrene or rifampicin, respectively. UGT1A1 was most responsive to the pregnane-X-receptor-agonists rifampicin, ritonavir, and clotrimazole at the mRNA level and, to a lesser extent, the constitutive androstane receptor-activators, phenobarbital and phenytoin. Pharmacodynamic analyses support a mechanism of coordinate regulation between UGT1A1 and a number of CYP450 enzymes by multiple nuclear receptors.

Induction of uridine 5'-diphosphate-glucuronosyltransferase gene expression by sulforaphane and its mechanism: experimental study in human colon cancel cells

To study the induction of expression of uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) 1A in colon cancer cells by sulforaphane (SFN) and its possible mechanism. Human colon cancer cells of the line Caco-2 were cultured and added with SFN of different terminal concentrations, all below the concentration of IC(50). RT-PCR was used to examine the expression of UGT1A mRNA induced by SFN. Western blotting was used to detect the expression of UGT1A protein. The glucuronidation rate of N-hydroxy-PhIP was measured by high performance liquid chromatography (HPLC). The nuclear localization of transcription factor Nrf2 was observed by confocal laser microscopy. (1) Expression of UGT1A mRNA was observed in the Cac0-2 cells induced by SFN of the concentrations of 10 micromol/L approximately 35 micromol/L in a dose-independent manner (P < 0.05). Sulforaphane of the concentration of 25 micromol/L induced the UGT1A mRNA expression time-dependently. The levels of UGT1A1, UGT1A8, and UGT1A10 mRNA expression were significantly increased in the cells treated with 25 micromol/L sulforaphane compared to that in the controls (P = 0.006, P = 0.017, and P = 0.008 respectively). (2) The UGT1A protein band intensity increased significantly in the Coco-2 cells treated with sulforaphane of the concentrations 10 micromol/L approximately 30 micromol/L for 24 h in comparison with the control cells. (3) When the microsomes from the untreated Caco-2 cells were incubated with N-hydroxy-PhIP there was a minor HPLC peak at the expected retention time for N-hydroxy-PhIP-N2-glucuronide. This peak was dramatically increased in the sulforaphane-treated cells, suggesting higher activities of glucuronidation of N-hydroxy-PhIP. (4) Cytoplasmic labeling of NF-E2-related factor 2 (Nrf2), a transcription factor, with no nuclear staining was observed in the non-stimulated cells, whereas an intense nuclear labeling was observed in the sulforaphane-treated cells, indicating the induction of nuclear translocation of Nrf2 by sulforaphane. (1) Low dose sulforaphane induces the expression of UGT1A, UGT1A1, UGT1A A8, and UGT1A A10 mRNA significantly. These changes are accompanied by an increase in UGT1A1 protein and increase in heterocyclic aromatic amine glucuronidation. (2) The induction of the phase II enzyme activity by SFN occurs at the transcriptional level and is regulated by Nrf2.

Serotonin glucuronidation by Ah receptor- and oxidative stress-inducible human UDP-glucuronosyltransferase (UGT) 1A6 in Caco-2 cells

Caco-2 cells are a widely used model in drug development to study intestinal drug transport and metabolism. Recently, serotonin (5-hydroxytryptamine, 5-HT) has been characterized as a highly selective substrate of human UDP-glucuronosyltransferase UGT1A6 [Krishnaswamy S, Duan SX, von Moltke LL, Greenblatt DJ, Court MH. Validation of serotonin (5-hydroxytryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase (UGT) 1A6. Drug Metab Disp 2003; 31:133–9], an isoform which conjugates planar phenols and is inducible by Ah receptor agonists and by oxidative/electrophile stress. To gain more insight into intestinal 5-HT disposition, uptake and metabolism of this neurotransmitter was studied in Caco-2 cell monolayers. It was found that 5-HT was taken up from the basolateral and to a lesser extent from the apical surface. It was mainly excreted basolaterally as 5-HT glucuronide. 5-HT UGT activity and UGT1A6 mRNA were induced by Ah receptor agonists and by oxidative stress generated by tert-butylhydroquinone and by isomeric thymoquinone, a potential antitumor agent and constituent of Nigella sativa seeds, commonly used as a condiment in the Middle East. While UGT1A6 induction was clearly detectable in NAD(P)H:quinone oxidoreductase 1 (NQO1)-deficient Caco-2 cells, it was not induced in NQO1-efficient HT-29 colon adenocarcinoma cells. The results suggest that – in addition to its detoxification function – intestinal UGT1A6 contributes to intestinal homeostasis of 5-HT from dietary sources and from release by enterochromaffin cells.

UDP Glucuronosyltransferase (UGT) 1A6 Pharmacogenetics: I. Identification of Polymorphisms in the 5′-Regulatory and Exon 1 Regions, and Association with Human Liver UGT1A6 Gene Expression and Glucuronidation

UDP glucuronosyltransferase (UGT) 1A6 is a major isoform in human liver that glucuronidates numerous drugs, toxins, and endogenous substrates with high interindividual variability. The molecular basis for this variability remains unknown, although it likely involves genetic and environmental factors. Phenotype-genotype studies were conducted using a well characterized human liver bank (n = 54) and serotonin glucuronidation as a UGT1A6-specific phenotype marker. A positive moderate-to-heavy alcohol use history (>14 drinks per week) was the only demographic factor examined that correlated with phenotype and was associated with 2-fold higher serotonin glucuronidation (p < 0.001), UGT1A6 protein content (p = 0.004), and UGT1A6 mRNA content (p = 0.025). UGT1A6 gene resequencing identified three nonsynonymous polymorphisms (S7A, T181A, and R184S) in exon 1 and eight novel polymorphisms in the 5'-regulatory region (to -2052 base pairs). S7A was in complete linkage disequilibrium with three 5'-regulatory region polymorphisms (-1710c-->g, -1310del5, and -652g-->a). Initial univariate analyses did not identify any significant phenotype-genotype associations. However, in livers without substantial alcohol exposure, 50% lower UGT1A6 mRNA levels (p = 0.026) were found in carriers of the linked S7A-enhancer polymorphisms compared with noncarriers but without significant effect on UGT1A6 protein content or glucuronidation activities. Three major haplotypes, including (*)1A (reference), (*)1B (-1535g-->a and -427g-->c), and (*)2 (-1710c-->g, -1310del5, -652g-->a, S7A, T181A, and R184S), were identified, accounting for 90% of alleles. No association of haplotype with any of the phenotype measures could be discerned. In conclusion, although the identified UGT1A6 polymorphisms did not explain the observed glucuronidation variability, there does seem to be a significant role for environmental factors associated with alcohol consumption.

Role of HNF-1? in the regulation of UGT1A1, UGT1A9 and UGT2B7 MRNA expression

Background The hepatocyte nuclear factor 1α (HNF-1α) is potentially involved in the regulation of the expression of UGT1A1, UGT1A9 and UGT2B7. A variant (79A>C) in HNF-1α has been associated with insulin resistance and glucose intolerance among nondiabetic individuals; however, its effect on HNF1-α function is not known. We aim to investigate the role of HNF-1α in the regulation of the above UGTs. Methods Liver samples (n=83) were genotyped for HNF-1α 79A>C and for UGT variants affecting their expression. HNF-1α and UGT mRNA levels were measured by real time PCR. Results No correlation was observed between HNF-1α expression and that of UGT1A1, UGT1A9 and UGT2B7 (r2=0.0002, p>0.05; r2=0.09, p=0.02; r2=0.04, p>0.05, respectively; log transformed). HNF-1α genotypes were not associated with UGT mRNA levels (p>0.05). Significant correlations were found between UGT1A1 expression and the *28 polymorphism (6/6>6/7>7/7, p=0.03, nonparametric trend), UGT1A9 expression and the *22 T indel polymorphism (9/9+9/10 vs. 10/10, p=0.02, Mann-Whitney), and UGT2B7 expression and an intronic gene variant (A/A vs. A/G+G/G; p=0.01, Mann-Whitney). HNF-1α genotypes seemed not to predict UGT mRNA levels in multivariate analysis. Conclusion Our data do not provide evidence of a significant role of HNF-1α in the prediction of interindividual variability in UGT1A1, UGT1A9 and UGT2B7 expression. Variation in cis-acting regulatory UGT elements might have a stronger phenotypic effect. Clinical Pharmacology & Therapeutics (2005) 77, P61–P61; doi: 10.1016/j.clpt.2004.12.125

ANALYSIS OF SUBSTRATE SPECIFICITIES AND TISSUE EXPRESSION OF RAT UDP-GLUCURONOSYLTRANSFERASES UGT1A7 AND UGT1A8

The UGT1 complex codes for a subfamily of homologous "1A7-like" UDP-glucuronosyltransferases (UGTs), including UGT1A7 and UGT1A8. Little information is available regarding either the substrate specificities or regulation of the UGT1A7-like forms from rats. We compared the activities and tissue expression of UGT1A7 and UGT1A8, which exhibit 77% identity in their amino terminal sequence. UGT1A7 shows broad specificity, catalyzing the glucuronidation of 31 of 40 randomly selected substrates (100 muM) at rates >0.1 nmol/mg/min. UGT1A7 substrates included both planar and nonplanar compounds, mono- and polycyclic aromatics, and compounds with bulky side chain ring substitutions. UGT1A8 exhibited a narrower substrate specificity that completely overlapped with UGT1A7. UGT1A8 was most active toward the 1-OH, 4-OH, 5-OH, 6-OH, 7-OH, 10-OH, 11-OH, and 12-OH derivatives of benzo[a]pyrene. Other effective UGT1A8 substrates (>0.1 nmol/mg/min) included 9-OH-benzo[a]pyrene, 1-naphthol, 4-methylumbelliferone, 7-hydroxycoumarin, chrysin, quercetin, 4-nitrophenol, and estriol. In general, substrates preferred by UGT1A8 were polyaromatic planar structures with nonbulky substituents and a superimposable 1-naphtho ring structure. Studies of the tissue expression of the UGT1A7 and 1A8 mRNAs using RNase protection analysis suggested that each is expressed in liver and kidney of control rats. A major difference is the higher expression of UGT1A7 mRNA in intestine. These studies suggest complementary functions of the UGT1A7 and UGT1A8 forms in xenobiotic metabolism. Further studies are necessary to determine whether their relative contributions change as a function of development, hormonal status, or exposure to inducing agents.

Induction of gene expression of xenobiotic metabolism enzymes and ABC-transport proteins by PAH and a reconstituted PAH mixture in human Caco-2 cells

It was shown recently that in epithelial Caco-2 cells the food contaminant benzo[ a]pyrene (B[ a]P) is metabolized and B[ a]P-sulfate metabolites were transported out of the cells. The aim of this study was to investigate whether B[ a]P and other polycyclic aromatic hydrocarbons (PAH) such as chrysene, phenanthrene, benzo[ k]fluoranthene (B[ k]F), dibenzo[ a,l]pyrene (DB[ a,l]P), and pyrene alone or in a mixture in a ratio as they occur in tobacco smoke have effects on gene expression of intestinal cytochrome P450 enzymes (CYP), Phase II enzymes and ATP-binding cassette (ABC)-transport proteins in the human Caco-2 cells. B[ a]P induced its own metabolism. Treatment of the Caco-2 cells with B[ a]P, chrysene, B[ k]F, or DB[ a,l]P induced mRNA expression of CYP1A1 and CYP1B1 specifically as measured by RT-PCR. In contrast, the mRNA expression of the microsomal epoxide hydrolase (mEH) was not affected by PAH. The gene expression of the Phase II enzymes UDP-glucuronosyltransferase 1A6 (UGT1A6) and UGT1A7 was also induced by these PAH but treatment with them had no effect on gene expression of sulfotransferases (SULT) at all. Of the ABC-transport proteins, MDR1 mRNA expression was induced by treatment with carcinogenic PAH, whereas MRP2 mRNA expression was not changed. The mixture of PAH also induced CYP1A1, CYP1B1, UGT1A6, and UGT1A7 mRNA expression. We conclude that B[ a]P, chrysene, B[ k]F, and DB[ a,l]P have specific effects on intestinal CYP1A1, CYP1B1, UGT1A6, and UDP1A7 mRNA expression but no effects on the expression of SULT.

Hepatic expression of the UGT1A9 gene is governed by hepatocyte nuclear factor 4alpha.

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)-alpha controls the hepatic expression of the UGT1A9 enzyme. Liver-specific disruption of the HNF4alpha gene in mice drastically decreases liver UGT1A9 mRNA levels. Furthermore, an HNF4alpha response element (HNF4alpha 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 HNF4alpha 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 HNF4alpha RE of the UGT1A9 gene.