This article is available online at http://dmd.aspetjournals.org ABSTRACT:This article is an updated report of a symposium held at the June 2000 annual meeting of the American Society for Pharmacology and Experimental Therapeutics in Boston. The symposium was sponsored by the ASPET Divisions for Drug Metabolism and Molecular Pharmacology. The report covers research from the authors' laboratories on the structure and regulation of UDP-glucuronosyltransferase (UGT) genes, glucuronidation of xenobiotics and endobiotics, the toxicological relevance of UGTs, the role of UGT polymorphisms in cancer susceptibility, and gene therapy for UGT deficiencies.
The nucleic acid and derived amino acid sequences of a chicken phenobarbital-inducible cytochrome P450 cDNA clone, pCHP7, are presented. The amino acid sequence shares 92% identity with that of a previously characterized chicken cytochrome P450 cDNA clone, pCHP3. The two clones, pCHP7 and pCHP3, represent two distinct mRNAs of 2.2 and 3.5 kb and appear to be transcribed from separate cytochrome P450 genes. Sequence comparisons with P450s from mammalian species indicate that the chicken P450s are most closely related to the IIC subfamily. According to the cytochrome P450 nomenclature (Nebert et al., 1989), the chicken P450 gene encoding the 3.5-kb mRNA is termed P450IIF1 and the P450 gene encoding the 2.2-kb mRNA is designated P450IIF2. When chick embryos were treated with 2-allyl-2-isopropylacetamide, the levels of the 3.5- and 2.2-kb P450 mRNAs in liver were elevated maximally by about 100-fold, while phenobarbital treatment resulted in a maximal increase of about 50-fold. These increases in mRNA levels were accompanied by a less than sixfold increase in the corresponding gene transcription rates. The results indicate that the increase in the amount of these two mRNAs is due to both transcriptional activation of the P450 genes and to a marked post-transcriptional mechanism. By contrast, the drug-induced hepatic mRNA levels for 5-aminolevulinate synthase, the rate-controlling enzyme of the heme biosynthetic pathway, could be accounted for predominantly by activation of gene transcription. A close correlation was observed between the time courses of hepatic mRNA accumulation for cytochrome P450 and 5-aminolevulilnate synthase following drug treatment of chick embryos. In adult hens it was demonstrated that a tissue-specific drug induction of cytochrome P450 mRNAs occurred with levels being substantially elevated in the liver, kidney, and small intestine. The mRNA for 5-aminolevulinate synthase was also drug-induced in the same tissue-specific fashion. The results are compatible with the gene for 5-aminolevulinate synthase being activated in response to an increased cytochrome P450 heme requirement.
The human UDP glucuronosyltransferase UGT2B17, glucuronidates androgens and is expressed in the liver and the prostate. Although evidence suggests that variations in UGT2B17 expression between tissues may be a critical determinant of androgen response, the factors that regulate UGT2B17 expression in the liver and prostate are unknown. In this study, we have isolated a 596 bp promoter of the UGT2B17 gene and studied its regulation in the liver cell line, HepG2 and the prostate cell line, LNCaP. The transcription start site of UGT2B17 was mapped and proteins that bound to the proximal promoter were detected by DNase1 footprint analysis. A region (-40 to -52 bp) which resembled a hepatocyte nuclear factor 1 (HNF1) binding site bound proteins in nuclear extracts from HepG2 cells, but did not bind proteins from LNCaP nuclear extracts. In HepG2 cells, HNF1alpha bound to this region and activated the UGT2B17 promoter, as assessed by functional and gel shift assays. HNF1alpha activation of the promoter was prevented by mutation or deletion of the putative HNF1 site. The related transcription factor HNF1beta, which is present in HepG2 cells, did not activate the promoter. The UGT2B17 promoter could also be activated by exogenous HNF1alpha in LNCaP cells. However, because these cells do not contain HNF1alpha, other transcription factors must regulate the UGT2B17 promoter. Cotransfection experiments showed that HNF1beta, elevates promoter activity in LNCaP cells. This activation did not involve the putative HNF1 region (-40 to -52 bp) since mutation of this region did not affect promoter activation by HNF1beta. These results suggest that the UGT2B17 promoter is regulated by different factors in liver-derived HepG2 and prostate-derived LNCaP cells.
It has been proposed that transcription of cytochrome-P450 genes is positively regulated by heme, the prosthetic group of cytochrome-P450 proteins. We have investigated this proposal in rats treated with succinylacetone, a known specific inhibitor of the heme biosynthetic pathway. While 2-allyl-2-isopropylacetamide, phenobarbitone, dexamethasone, p-naphthoflavone and clofibrate induced specific cytochrome-P450-mRNA species in rat liver, the levels of these induced mRNAs were not affected by succinylacetone administration. Synthesis of the first enzyme of the heme biosynthetic pathway, 5-aminolevulinate synthase, is known to be regulated by the end-product heme, with heme inhibiting 5-aminolevulinate-synthase-gene transcription. Hepatic 5-aminolevulinate-synthase mRNA was induced by drugs and the level increased further by succinylacetone. Furthermore, treatment of rats with succinylacetone alone resulted in elevated levels of 5-aminolevulinate-synthase mRNA but did not affect cytochrome-P450-mRNA levels.The results show that while lowered heme levels lead to an increase in 5-aminolevulinate-synthase-mRNA synthesis there is no effect on cytochrome-P450-mRNA levels, implying that heme is not required for the cytochrome-P450-gene transcription.Cytochrome P45Os (P450s) are heme-containing proteins which constitute a super-family of enzymes involved in the oxidative metabolism of either endogenous compounds or a vast range of foreign chemicals [l, 21. Considerable progress has been made in understanding the mechanism by which polycyclic aromatic hydrocarbons induce hepatic P450I genes and evidence indicates that a drug-receptor complex mediates induction by interaction with regulatory elements in the 5'-
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