Nicole Freiberg

Interaction between Oxidative Stress Sensor Nrf2 and Xenobiotic-activated Aryl Hydrocarbon Receptor in the Regulation of the Human Phase II Detoxifying UDP-glucuronosyltransferase 1A10

The defense against oxidative stress is a critical feature that prevents cellular and DNA damage. UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of xenobiotics, mutagens, and reactive metabolites and thus act as indirect antioxidants. Aim of this study was to elucidate the regulation of UGTs expressed in the mucosa of the gastrointestinal tract by xenobiotics and the main mediator of antioxidant defense, Nrf2 (nuclear factor erythroid 2-related factor 2). Xenobiotic (XRE) and antioxidant (ARE) response elements were detected in the promoters of UGT1A8, UGT1A9, and UGT1A10. Reporter gene experiments demonstrated XRE-mediated induction by dioxin in addition to tert-butylhydroquinone (ARE)-mediated induction of UGT1A8 and UGT1A10, which are expressed in extrahepatic tissue in humans in vivo. The responsible XRE and ARE motifs were identified by mutagenesis. Small interfering RNA knockdown, electrophoretic mobility shifts, and supershifts identified a functional interaction of Nrf2 and the aryl hydrocarbon receptor (AhR). Induction of UGT1A8 and UGT1A10 requires Nrf2 and AhR. It proceeds by utilizing XRE- as well as ARE-binding motifs. In summary, we demonstrate the coordinated AhR- and Nrf2-dependent transcriptional regulation of human UGT1As. Cellular protection by glucuronidation is thus inducible by xenobiotics via AhR and by oxidative metabolites via Nrf2 linking glucuronidation to cellular protection and defense against oxidative stress.

Coffee Induces Expression of Glucuronosyltransferases by the Aryl Hydrocarbon Receptor and Nrf2 in Liver and Stomach

BACKGROUND & AIMS Coffee is one of the most widely consumed beverages worldwide. Epidemiologic data indicate that coffee consumption protects against the progression of chronic liver disease and development of hepatocellular carcinoma and diabetes, but the mechanisms are not clear. UDP glucuronosyltransferases (UGT1A) are proteins with indirect antioxidant, cytoprotective, and genoprotective capabilities; we examined UGT1A regulation in response to coffee in cultured cells and mice. METHODS HepG2 and CaCo2 cells were incubated with regular, metal- or paper-filtered, decaffeinated, or instant coffee; green or black tea; cocoa; or metabolic products of caffeine. The effects of UGT1A regulation were investigated with reporter gene assays, immunoblot, TaqMan polymerase chain reaction, mutagenesis, and short interfering (si)RNA analyses. We also studied the effects of coffee in humanized transgenic mice that express human UGT1A. RESULTS Incubation of cells with coffee induced transcription of UGT1A1 (5.4-fold), UGT1A3 (5.2-fold), UGT1A4 (4.8-fold), UGT1A7 (6.2-fold), UGT1A8 (5.2-fold), UGT1A9 (3.5-fold), and UGT1A10 (6.1-fold). Induction was independent of caffeine, methylxanthines, or the diterpenes cafestol and kahweol. Mutagenesis and short interfering RNA knockdown studies showed that UGT1A is regulated by the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid-related factor 2 (Nrf2) by cis-acting antioxidant and xenobiotic response elements (ARE/XRE). In transgenic UGT1A mice, administration of coffee resulted in a 10- and 14-fold induction of UGT1A transcription in liver and stomach, respectively. CONCLUSIONS UGT1A genes are induced in vitro and in vivo by coffee, independent of caffeine content, cafestol, or kahweol. Coffee up-regulates glucuronidation by AhR signaling and Nrf2 binding to the ARE/XRE. Glucuronidation could mediate the protective and antioxidant effects of coffee.

Aryl hydrocarbon receptor-mediated regulation of the human estrogen and bile acid UDP-glucuronosyltransferase 1A3 gene

UDP-glucuronosyltransferases contribute to the detoxification of drugs by forming water soluble β-d-glucopyranosiduronic acids. The human UGT1A3 protein catalyzes the glucuronidation of estrogens, bile acids and xenobiotics including non-steroidal anti-inflammatory drugs and lipid lowering drugs. Regulation of UGT1A3 by xenobiotic response elements is likely, but the responsible elements are yet uncharacterized. In addition, genetic promoter variants may affect UGT1A3 regulation and potential induction by xenobiotics. The UGT1A3 promoter was analyzed by mutagenesis, reporter gene, and mobility shift analyses. Three hundred and eighty-nine blood donors were genotyped for promoter single nucleotide polymorphisms (SNPs) showing an allelic frequency of 42% of variants at −66 (T to C) and −204 (A to G). A xenobiotic response element regulating aryl hydrocarbon receptor (AhR)-mediated UGT1A3 transcription was identified and characterized. UGT1A3 transcription was reduced in the presence of promoter SNPs. These data demonstrate xenobiotic induced regulation of the UGT1A3 gene by the AhR, which shows genetic variability.

Gilbert syndrome redefined: A complex genetic haplotype influences the regulation of glucuronidation

Gilbert syndrome (GS) is characterized by intermittent unconjugated hyperbilirubinemia without structural liver damage, affecting about 10% of the white population. In GS the UGT1A1*28 variant reduces bilirubin conjugation by 70% and is associated with irinotecan and protease inhibitor side effects. The aim of this study was to characterize potential in vivo consequences of UGT1A gene variability in GS. Three hundred GS patients (UGT1A1*28 homozygous) and 249 healthy blood donors (HBD) were genotyped for UGT1A (UGT1A1*28, UGT1A3‐66 T>C, UGT1A6*3a, UGT1A7*3) and transporter single nucleotide polymorphisms (SNPs) (SCLO1B1 p.V174A, SCLO1B1 p.N130D, ABCC2 p.I1324I, ABCC2‐24 UTR) using TaqMan‐5′‐nuclease‐assays. A humanized transgenic UGT1A‐SNP and corresponding wildtype mouse model were established carrying the GS‐associated UGT1A variant haplotype. UGT1A transcript and protein expression, and transcriptional activation were studied in vivo. Homozygous UGT1A1*28 GS individuals were simultaneously homozygous for UGT1A3‐66 T>C (91%), UGT1A6*2a (77%), and UGT1A7*3 (77%). Seventy‐six percent of GS and only 9% of HBD were homozygous for the variant haplotype spanning four UGT1A genes. SCLO1B1 and ABCC2 SNPs showed no differences. In transgenic humanized UGT1A SNP and wildtype mice this UGT1A haplotype led to lower UGT1A messenger RNA (mRNA) expression and UGT1A protein synthesis. UGT1A transcriptional activation by dioxin, phenobarbital, and endotoxin was significantly reduced in SNP mice. Conclusion: Our data redefine the genetic basis behind GS. In vivo data studying the genotype present in 76% of GS individuals suggest that transcription and transcriptional activation of glucuronidation genes responsible for conjugation and detoxification is directly affected, leading to lower responsiveness. This study suggests that GS should be considered a potential risk factor for drug toxicity. (HEPATOLOGY 2012;55:1912–1921)

Gender matters: Estrogen receptor alpha (ERα) and histone deacetylase (HDAC) 1 and 2 control the gender-specific transcriptional regulation of human uridine diphosphate glucuronosyltransferases genes (UGT1A)

BACKGROUND & AIMS Gender influences incidence, progression, and therapy of hepatogastrointestinal diseases. The aim of this study was to elucidate the molecular mechanism of gender-specific UDP-glucuronosyltransferases (UGT1A) regulation, representing important hepatogastrointestinal detoxification enzymes for xenobiotics, drugs, and endobiotics. METHODS UGT1A-gene activation was studied by reporter gene experiments and estrogen receptor alpha (ESR1/ERα) co-transfection using KYSE70- and HepG2 cells (male origin), and SW403 cells (female origin). Cell lines, and humanized transgenic UGT1A (htgUGT1A) mice (female/male) were treated with the ERα inhibitor tamoxifen. UGT1A mRNA expression was analyzed by TaqMan PCR, the recruitment of ERα, histone deacetylases (HDAC), and the aryl hydrocarbon receptor (AhR) by chromatin immunoprecipitation (ChIP), and ERα expression in gastrointestinal mouse tissues by Western blot and immunofluorescence. RESULTS In KYSE70 cells (male), UGT1A gene expression was induced 5-10 fold, and inhibited in the presence of ERα by 55-77%. In SW403 (female) cells, absent inducibility was restored after tamoxifen treatment. In the jejunum and colon of tgUGT1A mice, UGT1A induction that was exclusively detected in male mice could be restored in female mice after tamoxifen pre-treatment. ChIP assays demonstrated the recruitment of ERα and HDACs to the xenobiotic response elements of UGT1A promoters during gene repression. Western blot showed higher ERα expression in the female jejunum and colon. CONCLUSIONS We show gender-specific transcriptional control of UGT1A genes in jejunum and colon, which is repressed by ERα and the recruitment of HDCAs to the UGT1A promoter sequence in females. A molecular mechanism controlling gender-specific drug metabolism and its therapeutic reversal is demonstrated.

Shared Regulation of UGT1A7 by Hepatocyte Nuclear Factor (HNF) 1α and HNF4α

Substrates for glucuronidation include endogenous and xenobiotic compounds such as environmental carcinogens and drugs, as well as the chemotherapeutic agent irinotecan. The UDP-glucuronosyltransferase (UGT) 1A7 gene is expressed in the upper gastrointestinal tract and the lung but is not expressed in the liver. The transcriptional regulation of UGT1A7 and the putative influence of single nucleotide polymorphisms (SNPs) are incompletely characterized. UGT1A8, UGT1A9, and UGT1A10, which are highly homologous to UGT1A7, have been reported to be transcriptionally regulated by hepatocyte nuclear factors (HNFs). In this study, we show the activation of UGT1A7 by the aforementioned transcription factors. Sequence analyses, mutagenesis, reporter gene experiments, small interfering RNA silencing, chromatin immunoprecipitation, and electromobility shift assays identified five HNF binding sites in the proximal promoter region of UGT1A7 that were regulated by HNF1α and HNF4α. Activation by HNF1α was lower in the presence of the UGT1A7 −57G SNP. In contrast to liver-expressed UGT1A9, transcriptional activation of UGT1A7 by HNF4α was lower and dependent on higher HNF4α concentrations, which may contribute to the observed differences in tissue expression patterns. Therefore, a specific role of HNF in the transcriptional control of UGT1A7 is shown and characterized, which may contribute to its tissue specificity and function.

960 THE FXR AGONIST GW4064 INCREASES LIVER INJURY LEADING TO AN INHIBITION OF THE TRANSCRIPTIONAL ACTIVATION OF UGT1A GENES IN BILE DUCT LIGATED tgUGT1A WT MICE

958 EXTRAHEPATIC MALIGNANCIES AFTER LIVER TRANSPLANTATION FOR PRIMARY SCLEROSING CHOLANGITIS – A LONG-TERM FOLLOW-UP OBSERVATIONAL STUDY OF A LARGE, MULTICENTER COHORT T. Horn, N. Pannicke, A. Dechene, D. Gotthardt, G. Kirchner, K. Herzer, H. Lenzen, H. Barg-Hock, M. Sterneck, U. Spengler, M.P. Manns, C.P. Strassburg, C. Schramm, T.J. Weismuller, German PSC Study Group. Gastroenterology, Hepatology und Endokrinology, Hannover Medical School, Hannover, 1st Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Department of Gastroenterology and Hepatology, University Hospital Essen, Essen, Department of Medicine, University Hospital of Heidelberg, Heidelberg, Department of Internal Medicine I, University Hospital of Regensburg, Regensburg, General, Visceral and Transplantation Surgery, University Hospital of Essen, Essen, General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Department of Internal Medicine 1, University of Bonn, Bonn, Germany E-mail: tobias.weismueller@gmx.de