Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We describe a novel transcriptional suppressor element found in the control region of the gene that encodes rat microsomal epoxide hydrolase (mEH), an inducible xenobiotic metabolizing enzyme. This element consists of the juxtaposition of two distinct factor-binding regions. The first region is composed of a series of five tandemly repeated factor-binding sequences, and the second region is an unique AT-rich factor-binding sequence. Although each region binds its cognate factor(s) in vitro, a single region does not function as a suppressor independently of the other. Transcriptional suppression was observed only when the two regions were combined. Thus, we propose that this regulatory element is a bipartite suppressor, requiring two distinct factor-binding regions for its function. The element displayed position-independent but orientation-dependent suppressor activity. The level of suppressor activity was proportional to the number of repetitive sites in region 1. We speculate that this region could mediate the dose-response behavior of mEH gene expression induced by chemical carcinogens in vivo. A qualitative difference in the region 2 binding factor(s) was observed between normal liver cells and a hepatoma cell line or carcinogen-treated liver cells. The possible relationship between this observation and the deregulation of mEH gene expression during the course of hepatocarcinogenesis is discussed.
Mol Cell Biol 1992 Oct
PMID:A bipartite suppressor: conjunction of two distinct factor-binding sites is essential for down-regulation in rat epoxide hydrolase gene expression. 140 38

Microsomes prepared from COS-1 cells transiently expressing rabbit cytochromes P450 2C1 and 2C2 catalyzed the metabolism of arachidonic acid to predominantly 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) when microsomal epoxide hydrolase activity was inhibited by 0.2 mM 1,2-epoxy-3,3,3-trichloropropane. P450 2C2 catalyzed the formation of 11,12-EET and 14,15-EET at a ratio of 3.0 and also produced 19-hydroxyeicosatetraenoic acid (19-HETE). The 11,12-EET, 14,15-EET, and 19-HETE represented 48.3, 15.9, and 12.8%, respectively, of the total metabolites formed. P450 2C1 produced a similar but distinct ratio of 11,12-EET to 14,15-EET (2.0) and did not produce any detectable 19-HETE. The 11,12-EET and 14,15-EET represented 63.0 and 31.1%, respectively, of the total metabolites formed. The 8,9- and 5,6-EETs were not detected with either enzyme. The ratio of the 11,12-EET to 14,15-EET was 1.5 with P450 2CAA, a P450 arachidonic acid epoxygenase (P450 2CAA) that had an amino-terminal sequence identical to that of P450 2C2 [J. Biol. Chem. 267:5552-5559 (1992)]. P450 2C1, 2C2, and 2CAA metabolized lauric acid. The ratio of omega-1- to omega-hydroxylated laurate was 3.6, 3.4, and 2.4 for P450 2CAA, P450 2C2, and P450 2C1, respectively. Purified P450 2CAA had a slightly greater apparent molecular weight than expressed P450 2C2 on sodium dodecyl sulfate-polyacrylamide gels. The results clearly establish that rabbit P450 2C1 and 2C2 are arachidonic acid epoxygenases, and they suggest that P450 2CAA and 2C2 are very similar but may not be identical isoforms.
Mol Pharmacol 1992 Dec
PMID:Identification of rabbit cytochromes P450 2C1 and 2C2 as arachidonic acid epoxygenases. 148 Jan 36

The effects of imidazole antifungal agents, including ketoconazole, clotrimazole, miconazole, and econazole, on the expression and regulation of microsomal epoxide hydrolase (mEH) were examined in rat hepatic tissue (doses of agents, 150 mg/kg of body weight/day, orally). Immunoblot analyses revealed that administration of either ketoconazole or clotrimazole caused a approximately 4-5-fold increase in mEH levels, whereas either miconazole or econazole resulted in a approximately 7-fold increase in mEH at day 3 after treatment. RNA hybridization analyses, probed with a 1.3-kilobase mEH cDNA, revealed that administration of these imidazole antifungal agents caused substantial elevation of hepatic mEH mRNA in total RNA. Hepatic mEH mRNA levels in total RNA were elevated approximately 11-, 15-, and 18-fold at 12, 24, and 72 hr, respectively, after ketoconazole treatment, whereas mEH mRNA levels were increased approximately 14-, 19-, and 22-fold, respectively, relative to control, at the same time points after clotrimazole treatment. The rate of increase of mEH mRNA caused by miconazole was more rapid than the rates observed for the other agents examined, with a maximal increase in mRNA being noted at 12 hr after treatment. The degree of mEH mRNA increase after 3 consecutive days of miconazole treatment was appreciably less than that observed at 12 hr after a single treatment. Econazole caused a maximal increase at 24 hr and subsequent decline in mEH mRNA levels after 3 consecutive days of treatment. Elevation of mEH mRNA levels by these antimycotic agents was confirmed in poly(A)+ RNA, as assessed by both Northern and slot blot hybridization analyses. Nuclear run-on analyses revealed that administration of ketoconazole, clotrimazole, or miconazole stimulated the rate of mEH gene transcription at 12 hr after treatment by 11-, 8.5-, and 9-fold, respectively, compared with control, whereas econazole resulted in a 4-fold increase in the rate of mEH gene transcription at the same time point. The transcription rates of mEH mRNA at 24 hr were significantly less than those observed at 12 hr after a single treatment with either ketoconazole, miconazole, or econazole, resulting in 6.5-, 2.5-, and 2-fold increases, respectively, relative to control. Clotrimazole, however, maintained the activated mEH transcription rate at 24 hr after treatment, exhibiting a 11-fold increase, compared with control. These results provide evidence that the imidazole antimycotic agents induce mEH and that the mEH induction involves large increases in mRNA, with transcriptional activation.
Mol Pharmacol 1992 Aug
PMID:Transcriptional regulation of rat microsomal epoxide hydrolase gene by imidazole antimycotic agents. 151 25

Expression of microsomal epoxide hydrolase (mEH), levels of mEH mRNA, and the rate of mEH mRNA transcription were examined in hepatic and renal tissues at 4, 24, 44, and 56 weeks of age in male and 4, 14, 24, 34, and 44 weeks of age in female Sprague-Dawley rats. Immunoblot analyses revealed that hepatic mEH levels in males increased in an age-dependent manner, with a maximal increase (approximately 3-fold) being noted at 44 weeks of age, whereas the expression of hepatic mEH in females decreased significantly at 14 weeks of age or older, by approximately 70%s, compared with that of 4-week-old rats. Microsomes from kidney tissue failed to exhibit an age-dependent change in either sex. mEH mRNA levels were measured in total and poly(A)+ RNA isolated from hepatic and renal tissues. RNA blot hybridization analyses, probed with a 1.3-kilobase mEH cDNA, revealed that the levels of hepatic mEH mRNA in total RNA isolated from males were elevated approximately 1.5-, 2.8-, and 2.3-fold at 24, 44, and 56 weeks of age, respectively, relative to those at 4 weeks of age, whereas the levels of hepatic mEH mRNA in poly(A)+ RNA from males failed to change in an age-dependent manner. In contrast, the levels of hepatic mEH mRNA in either total or poly(A)+ RNA from female animals were dramatically decreased from 4 to 14 weeks of age, by approximately 90%. The suppressed levels of mEH mRNA in females were maintained at 24, 34, and 44 weeks of age (approximately 80%). However, the levels of renal mEH mRNA failed to change in an age-dependent manner in either sex, which was consistent with there being no change in the levels of mEH protein in kidney. In order to examine whether the gender-related maturational changes in hepatic mEH mRNA levels could result from transcriptional regulation, nuclear run-on assays were performed. The rate of hepatic mEH gene transcription failed to change in either males or females at the ages that exhibited significant changes in both mRNA levels and protein expression, suggesting that transcriptional regulation is not associated with the gender-dependent modulation of mEH mRNA levels during maturation.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1992 Jul
PMID:Gender-related expression of rat microsomal epoxide hydrolase during maturation: post-transcriptional regulation. 163 54

Infection of mice with Leishmania donovani resulted in decreased activities of several liver enzymes involved in the metabolism of xenobiotics. Microsomal membranes from infected livers contained reduced amounts of cytochromes P450 and b5 and NADPH-cytochrome P450 reductase. Several cytochrome P450 isoenzymes (P450-PB1, P450-PB3, P450-PCN and P450-UT1) and P450-mediated reactions (aminopyrine demethylase, aniline hydroxylase, benzphentamine demethylase and ethoxycoumarin deethylase) were affected similarly. The metabolism of two carcinogens (nitrosodimethylamine and 7,12-dimethylbenz[a]anthracene) by liver microsomal membrane preparations was also reduced. Leishmania infection caused an increase of cytosolic epoxide hydrolase and microsomal epoxide hydrolase and NADH-cytochrome b5 reductase were unaffected. The results suggest that Leishmania-infected animals are likely to have altered responses to exogenous toxins compared to uninfected animals.
Mol Biochem Parasitol 1990 Jun
PMID:Changes in hepatic xenobiotic-metabolising enzymes in mouse liver following infection with Leishmania donovani. 211 55

The expression of a number of enzymes involved in drug metabolism, membrane function etc. was compared in hyperplastic and neoplastic lesions of the rat bladder and in human bladder tumours. Transitional cell carcinomas (TCC) in both rat and Man were characterized by decreased alkaline phosphatase (ALP) and increased gamma-glutamyl transpeptidase (GGT), beta-glucuronidase (beta-G1), succinate dehydrogenase (SD) and glucose-6-phosphate dehydrogenase (G6PD) activities. In addition, binding for antibodies specific for different cytochrome P-450 species (UT50, PB3a, MC1, MC2) and microsomal epoxide hydrolase (mEHb) was elevated in both murine and human tumours. Comparison of the enzyme phenotype in hyperplastic lesions induced by freeze ulceration or uracil administration with that in preneoplastic papillary or nodular hyperplasia (PNH) and TCC suggested, however, that most of the alteration in enzyme content or activity was non-specific and related to requirements for epithelial cell proliferation. On the other hand, the decreased ALP, and increased GGT and beta-G1 activity appeared more directly related to neoplastic transformation. The results suggested that qualitative differences exist between reactive hyperplasia and preneoplastic or neoplastic lesions in the urinary bladder. The finding of increased cytochrome P-450, in clear contrast to the reduction characteristic of preneoplastic hepatic lesions, may be important with regard to the observed difference in neoplastic transformation between the bladder and liver in response to drug metabolising enzyme inducers.
Virchows Arch B Cell Pathol Incl Mol Pathol 1989
PMID:Comparison of enzyme phenotypes in human bladder tumours and experimentally induced hyperplastic and neoplastic lesions of the rat urinary bladder. A combined histochemical and immunohistochemical approach. 256 27

The K-region 5,6-epoxide and non-K-region 1,2- and 3,4-epoxides of chrysene were isolated by normal phase high performance liquid chromatography (HPLC) from a mixture of products formed in the metabolism of chrysene by liver microsomes from untreated (control), phenobarbital-treated, or 3-methylcholanthrene-treated rats in the presence of an epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide. Epoxides were characterized by ultraviolet, mass, and circular dichroism spectral and chiral stationary phase HPLC analyses. Each of the metabolically formed epoxides was hydrated by rat liver microsomal epoxide hydrolase to a trans-dihydrodiol. The metabolically formed chrysene 5,6-epoxides were determined by chiral stationary phase HPLC and were found to contain (5S,6R):(5R,6S) enantiomer ratios of 68:32 (control), 71:29 (phenobarbital), and 5:95 (3-methylcholanthrene), respectively. The enantiomers of chrysene 1,2-epoxide and 3,4-epoxide were also resolved by chiral stationary phase HPLC. However, the enantiomeric compositions of the metabolically formed chrysene 1,2- and 3,4-epoxides, which racemized rapidly at room temperature, could not be directly determined. By using molecular oxygen-18 in the in vitro incubation of chrysene and by mass spectral analyses of the resulting oxygen-18-containing dihydrodiol metabolites and their acid-catalyzed dehydration (phenolic) products, both 1,2-epoxide and 3,4-epoxide were found to be converted by microsomal epoxide hydrolase-catalyzed water attack at predominantly (greater than or equal to 97%) the allylic carbons.
Mol Pharmacol 1987 Jul
PMID:Stereoselective formations of K-region and non-K-region epoxides in the metabolism of chrysene by rat liver microsomal cytochrome P-450 isozymes. 303 4

To characterize the distribution and inducibility of drug metabolizing enzymes within different hepatic cell populations, the activities of aminopyrine N-demethylase, ethoxyresorufin O-deethylase, microsomal epoxide hydrolase and cytosolic glutathione transferase were measured in liver parenchymal, Kupffer, and endothelial cells isolated from untreated rats or rats pretreated with phenobarbital, 3-methylcholanthrene, or Aroclor 1254. Enzyme activities, measurable in all cases, were 2.3- to 5.7-fold higher in parenchymal cells than in Kupffer and endothelial cells. Phenobarbital increased aminopyrine N-demethylase, microsomal epoxide hydrolase, and cytosolic glutathione transferase activities, whereas 3-methylcholanthrene enhanced ethoxyresorufin O-deethylase, epoxide hydrolase, and glutathione transferase activities in the three cell populations. Aroclor 1254 consistently induced each of the enzyme activities in parenchymal, Kupffer, and endothelial cells. Western blot analyses revealed clear differences in the expression of proteins immunologically related to cytochrome P-450 PB-1, and glutathione transferases B and X in parenchymal cells compared with the corresponding Kupffer and endothelial cells. In contrast, only minor differences between the cell types were apparent in the expression of cytochromes P-450 PB-4, P-450 MC1a, P-450 MC1b and microsomal epoxide hydrolase. These studies establish that oxidative and postoxidative drug metabolizing enzymes are not restricted to parenchymal cells: similar but distinguishable complements of these enzymes are also found in Kupffer and endothelial cells.
Mol Pharmacol 1987 Oct
PMID:Xenobiotic metabolizing enzymes are not restricted to parenchymal cells in rat liver. 367 Feb 81

Human liver microsomal epoxide hydrolase was purified to apparent homogeneity as judged by a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sheep antibody to human liver epoxide hydrolase reacted with the enzyme in detergent-solubilized human liver microsomes, giving a single immunoprecipitin band which formed a line of identity with the pure human enzyme. Antibody against the human enzyme reacted well with epoxide hydrolase in detergent-solubilized monkey liver microsomes by the Ouchterlony test but less strongly cross-reacted with the enzyme from rat liver and several other species. The antibody produced against purified human liver epoxide hydrolase precipitated the enzyme but did not inhibit catalytic activity, reminiscent of the relationship of rat liver epoxide hydrolase to its antibody. The absolute level of epoxide hydrolase in liver microsomal samples from 11 human subjects, as measured by a radial immunodiffusion assay, varied 3.4-fold whereas the rate of hydration of octene oxide varied 2.9-fold. The excellent correlation of the amounts of epoxide hydrolase determined catalytically or immunochemically (r = 0.99) indicated that interindividual variation in octene oxide hydration rates by human liver microsomes is a consequence of differences in amount of epoxide hydrolase protein which is present, and not the result of differences in levels of endogenous modulators of catalytic activity.
Mol Pharmacol 1982 Jul
PMID:Human liver microsomal epoxide hydrolase. Correlation of immunochemical quantitation with catalytic activity. 681 57

Human microsomal epoxide hydrolase (mEH) is a biotransformation enzyme that metabolizes reactive epoxide intermediates to more water-soluble trans-dihydrodiol derivatives. We compared protein-coding sequences from six full-length human mEH DNA clones and assessed potential amino acid variation at seven positions. The prevalence of these variants was assessed in at least 37 unrelated individuals using polymerase chain reaction experiments. Only Tyr/His 113 (exon 3) and His/Arg 139 (exon 4) variants were observed. The genotype frequencies determined for residue 113 alleles indicate that this locus may not be in Hardy-Weinberg equilibrium, whereas frequencies observed for residue 139 alleles were similar to expected values. Nucleotide sequences coding for the variant amino acids were constructed in an mEH cDNA using site-directed mutagenesis, and each was expressed in vitro by transient transfection of COS-1 cells. Epoxide hydrolase mRNA level, catalytic activity, and immunoreactive protein were evaluated for each construct. The results of these analyses demonstrated relatively uniform levels of mEH RNA expression between the constructs. mEH enzymatic activity and immunoreactive protein were strongly correlated, indicating that mEH specific activity was similar for each variant. However, marked differences were noted in the relative amounts of immunoreactive protein and enzymatic activity resulting from the amino acid substitutions. These data suggest that common human mEH amino acid polymorphisms may alter enzymatic function, possibly by modifying protein stability.
Hum Mol Genet 1994 Mar
PMID:Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants. 751 76


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