Transcriptional Activation of the UDP-Glucuronosyltransferase1A7 Gene in Rat Liver by Aryl Hydrocarbon Receptor Ligands and Oltipraz

Abstract

UDP-glucuronosyltransferase UGT1A7 catalyzes the glucuronidation of benzo(a)pyrene metabolites and other bulky aromatic compounds. Both UGT1A7 mRNA and an associated enzyme activity (benzo(a)pyrene-7,8-dihydrodioltransferase activity) are markedly increased in livers of rats treated with ␤-naphthoflavone or 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz). Nuclear runoff assays show that the effects of both inducers are primarily due to transcriptional activation. A 27-kilobase region that included the UGT1A7/ UGT1A6 promoter regions was cloned. Primer extension and RNase protection studies indicated >30 transcription start sites in five clusters between bases ؊85 and ؊40 respective to the translation start codon. There was no recognizable TATA box, but the promoter region is TA-rich. Sequence analysis revealed potential binding sites for CCAAT enhancer-binding protein, activator protein 1, and hepatic nuclear factors 1, 3, and 4, but no xenobiotic response elements or antioxidant response elements, implicated in the regulation of other genes by ␤-naphthoflavone or oltipraz, were found. A UGT1A7 gene reporter plasmid directed strong constitutive expression in transient transfection assays using primary rat hepatocytes. Treatment with 3-methylcholanthrene or oltipraz had no effect compared with similarly treated pGL3-Basic-transfected cells. These results suggest that the regulatory elements controlling xenobiotic inducibility of UGT1A7 transcription are located either 5 or 3 of bases ؊1600 to ؉54. One possibility is that the polycyclic aromatic-mediated regulation of UGT1A7 occurs via the xenobiotic response element flanking the UGT1A6 locus 7 kilobase pairs downstream.UDP-Glucuronosyltransferases (UGTs) 1 (EC 2.4.1.17) catalyze the reaction of UDP-glucuronic acid with substrates bearing a variety of different functional groups, including OH, NH 2 , SH 2 , and COOH. Because the glucuronides are usually less biologically active, more polar, and readily excreted from organisms, the UGTs are considered important for the general detoxification and elimination of a host of endogenous and exogenous substances, such as bilirubin, steroids, environmental pollutants, carcinogens, and dietary substances. Molecular cloning evidence indicates the existence of two large families of UGTs, UGT1 and UGT2 (1). UGT1 family members are encoded by a unique gene complex, UGT1, which utilizes exon sharing to generate Ն12 isozyme forms with homologous but unique amino-terminal (Ϸ287 amino acids) and identical carboxyl-terminal sequences (245 amino acids) (2, 3). UGT2 family members, in contrast, appear to be encoded by independent genes. Each UGT exhibits its own unique profile of substrate and tissue specificity and regulation by exposure to endogenous or xenobiotic compounds (1).A recent focus of our studies has been the UGT isozymes responsible for the glucuronidating activity of rat liver microsomes toward benzo(a)pyrene metabolites. Benzo(a)pyrene is a prototype of the polycyclic aromatic hydrocarbons (PAHs), a class of c...

Haplotype structures of the UGT1A gene complex in a Japanese population

“…Next, pairwise LD analysis was performed for the UGT1A genes using the polymorphisms detected in this study (1A8, 1A10, 1A9, 1A7, and 1A3) and previous studies (1A6, 1A4, 1A1, and common exons [2][3][4][5] 19-21 from the same 196 subjects, and the values for rho square (r 2 ), chi square (w 2 ), and |D 0 | were obtained. Variations found in only one subject were excluded from the analysis.…”

“…2,3 The UGT1A N-terminal domains (encoded by the exon-1's) determine the substrate-binding specificity and the C-terminal domain (encoded by exons 2-5) is important for binding to UDP-glucuronic acid. 1 Thus, the exon 1 segments confer the substrate specificity of UGT1A isoforms, 4 and the 5 0 -flanking region (and possibly the 3 0 -flanking region) of each exon 1 is acknowledged to independently regulate the expression of each isoform. 3,4 A number of genetic polymorphisms including single nucleotide polymorphisms (SNPs) in UGT1As have been identified and published on the UDP glucuronosyltransferase home page (http://som.flinders.edu.au/FUSA/ClinPharm/UGT/allele_table.html).…”

“…1 Thus, the exon 1 segments confer the substrate specificity of UGT1A isoforms, 4 and the 5 0 -flanking region (and possibly the 3 0 -flanking region) of each exon 1 is acknowledged to independently regulate the expression of each isoform. 3,4 A number of genetic polymorphisms including single nucleotide polymorphisms (SNPs) in UGT1As have been identified and published on the UDP glucuronosyltransferase home page (http://som.flinders.edu.au/FUSA/ClinPharm/UGT/allele_table.html). Some of these polymorphisms are known to affect glucuronidation rates.…”

Increase in Rat Liver UDP-Glucuronosyltransferase mRNA by Microsomal Enzyme Inducers that Enhance Thyroid Hormone Glucuronidation

“…Expression of liver UGT1A7 mRNA is increased following treatment with 3-MC and oltipraz, similar to UGT1A6 mRNA in response to these chemicals. However, no analogous XRE or transcriptional elements mediating the regulation of UGT1A7 by 3-MC or oltipraz have been identified in the upstream promoter region (Metz and Ritter, 1998). It is possible that UGT1A7 is under the regulation of the XRE in the UGT1A6 promoter.…”

Cloning and Characterization of the Human UDP-glucuronosyltransferase 1A8, 1A9, and 1A10 Gene Promoters

“…Analysis of the promoters of the human UGT1A1 and 1A6 genes showed that they were functional in liver and colon cells, respectively (8). Studies on the rat UGT1A6 and UGT1A7 genes have also shown that their promoter regions are capable of stimulating transcription in liver cells (9,10). In addition, UGT1A genes have recently been demonstrated to be induced by the nuclear receptors constitutive active receptor (CAR), aryl hydrocarbon receptor (AHR), pregnane X receptor (PXR) and peroxisome proliferator-activated receptor (PPAR) through binding to their promoter regions (9,(11)(12)(13)(14).…”