ABSTRACT
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.
Subject(s)
Glucuronosyltransferase/biosynthesis , Amino Acid Sequence , Animals , Cell Line , Female , Gene Expression Regulation, Enzymologic/physiology , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Humans , Male , Mice , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Substrate Specificity , Tissue DistributionABSTRACT
UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major UGT contributing to the glucuronidation of small phenolic compounds. The gene for rat 1A6 is expressed using two promoters, a distal promoter P1 and a proximal promoter P2. Transcripts from P2 are high in liver, gastrointestinal tract, and kidney, whereas P1 transcripts predominate in other tissues. Here we report evidence for primary control of the P2 promoter by hepatic nuclear factor 1 (HNF1). Transient transfection of a P2 reporter plasmid, p(-1354/+65) 1A6P2-luc, resulted in enhanced luciferase activity in HepG2 but not Hepa1 cells compared to cells transfected with pGL3-Basic control vector. A truncated reporter under the control of -224 to +65 exhibited comparable activity. Footprint analysis of the -224/+65 fragment revealed specific binding by rat liver nuclear protein to a region between bases -60 and -37. The binding activity was also observed with HepG2 cell but not Hepa1 cell extract. Electrophoretic mobility shift assays were consistent with the presence of HNF1 in the binding complexes. The functionality of an HNF1-binding site at -51/-37 is also supported by (1) marked decreases in the activity of P2 reporter plasmids containing a three-base substitution in the proposed HNF1 binding site and (2) the enhancement of P2 reporter activity following cotransfection of an HNF1alpha expression plasmid. The UGT1A6 P1 promoter lacks an HNF1 binding site in the analogous position and showed little response to HNF1 overexpression. Although these data do not strictly rule out an interaction between the P1 promoter and HNF1 bound to -51/-37 of P2, the results suggest a mechanism for the more abundant expression of P2-derived UGT1A6 transcripts in liver and other HNF1-enriched tissues.
Subject(s)
DNA-Binding Proteins , Gene Expression Regulation, Enzymologic , Glucuronosyltransferase/genetics , Monosaccharide Transport Proteins , Nuclear Proteins , Promoter Regions, Genetic , Transcription Factors/physiology , Animals , Binding Sites , Carcinoma, Hepatocellular/pathology , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Hepatocytes/enzymology , Hepatocytes/metabolism , Humans , Mice , Transcription Factors/biosynthesis , Transfection , Tumor Cells, CulturedABSTRACT
The utility of oltipraz as a cancer chemopreventive agent is thought to depend on the induction of enzymes involved in phase 2 xenobiotic detoxification. Although studies of some enzymes induced by oltipraz implicate a novel transcriptional activating pathway involving Nrf2 and antioxidant-response elements (AREs), the mechanism of phenol UGT induction has remained unclear. Previous work showed that UGT1A6 is transcribed from two promoters, P1 and P2, that are both induced by oltipraz in rat liver. The effect also occurs in rat hepatocytes treated with oltipraz (concentrations >3 microM). To investigate the mechanism, luciferase reporter plasmids under the control of P1 [p(-1078/+27)1A6P1-luc] or P2 [p(-1354/+65)1A6P2-luc] were transfected into rat hepatocytes and tested for inducibility. P1, but not P2, showed responsiveness to oltipraz (2- to 5-fold increase) and 3-methylcholanthrene (10- to 30-fold increase). Because P1 contained no visible AREs, the role of a xenobiotic response element (XRE) centered between bases -134 and -129 was evaluated. Mutation of the XRE core reduced the effects of both oltipraz and 3-methylcholanthrene on the P1 reporter. The 1A6 XRE conferred oltipraz responsiveness on the simian virus 40 promoter of pGL3-Promoter. Comparative effects of oltipraz and 3-methylcholanthrene on transfected cytochrome P4501A1 reporters support the general but relatively weak XRE-stimulating activity of oltipraz. The involvement of the aryl hydrocarbon receptor (AHR) and aryl hydrocarbon nuclear translocator (ARNT) in mediating the effects of oltipraz on the XRE is supported by electrophoretic mobility supershift data and AHR/ARNT overexpression studies. These data raise questions about the contribution of AHR and other secondary induction pathways in the mechanism of oltipraz.
Subject(s)
DNA-Binding Proteins , Glucuronosyltransferase/biosynthesis , Hepatocytes/drug effects , Monosaccharide Transport Proteins , Pyrazines/pharmacology , Receptors, Aryl Hydrocarbon/metabolism , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator , Binding Sites , Cells, Cultured , Hepatocytes/metabolism , Humans , Nuclear Proteins/metabolism , Rats , Thiones , Thiophenes , Transcription Factors/metabolism , Transcription, Genetic/drug effects , XenobioticsABSTRACT
Gunn rats glucuronidate acetaminophen (APAP) at reduced rates and show increased susceptibility to APAP-induced hepatotoxicity. This defect is presumed to involve UDP-glucuronosyltransferase (UGT) 1A6, which is nonfunctional in Gunn rats, but it is currently unclear whether other 1A family members are also involved. In humans, two 1A isoforms are known to be active (1A6 and 1A9) but 1A6 form has a 25-fold lower apparent K(m) (2 mM). Rat liver microsomal APAP UGT activity is induced by in vivo treatment with beta-naphthoflavone or oltipraz, an effect correlating with induction of 1A6 and 1A7. To address a possible role of 1A7 in APAP glucuronidation relative to other 1A forms, cDNAs encoding UGTs 1A1, 1A5, 1A6, 1A7, and 1A8 were expressed in human embryonic kidney cells and the contents of expressed enzyme in prepared membrane fractions determined by quantitative immunoblotting. At 2.5 mM APAP, 1A7 showed the highest specific activity (2.8 nmol/min/nmol 1A7 protein), followed by 1A6 (1.1 nmol/min/nmol), and 1A8 (0.27 nmol/min/nmol). 1A1 and 1A5 were essentially inactive. Kinetic comparisons indicated 1A7 had a similar apparent K(m) as 1A6 (4.7 versus 3.9 mM, respectively) but a 2.4-fold higher catalytic activity. These data suggest that in rats, 1A7 plays a major role in APAP glucuronidation and contributes to protection against APAP-induced hepatotoxicity. The involvement of other UGTs besides 1A6 is further underscored by the presence of significant residual APAP-glucuronidating activity by Gunn rat hepatocytes, indicating the activity of an unknown UGT2 family member.