Gene_locus Report for: human-EPHX1

KZR-616 is an irreversible tripeptide epoxyketone-based selective inhibitor of the human immunoproteasome. Inhibition of the immunoproteasome results in anti-inflammatory activity in vitro and, based on promising therapeutic activity in animal models of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), KZR-616 is being developed for potential treatment of multiple autoimmune and inflammatory diseases. The presence of a ketoepoxide pharmacophore presents unique challenges in the study of drug metabolism during lead optimization and clinical candidate profiling. This study presents a thorough and systematic in vitro and cell-based enzymatic metabolism and kinetic investigation to identify the major enzymes involved in the metabolism and elimination of KZR-616. Upon exposure to liver microsomes in the absence of NADPH, KZR-616 and its analogs were converted to their inactive diol derivatives with varying degrees of stability. Diol formation was also shown to be the major metabolite in pharmacokinetic studies in monkeys and correlated with in vitro stability results for individual compounds. Further study in intact hepatocytes and a hepatocellular carcinoma cell line revealed that KZR-616 metabolism was sensitive to an inhibitor of microsomal epoxide hydrolase (mEH) but not inhibitors of cytochrome P450 (CYP) or soluble epoxide hydrolase (sEH). Primary human hepatocytes were determined to be the most robust source of mEH activity for study in vitro These findings also suggest that the exposure of KZR-616 in vivo is unlikely to be affected by co-administration of inhibitors or inducers of CYP and sEH. Significance Statement This work presents a thorough and systematic investigation of metabolism and kinetic of KZR-616 and other peptide epoxyketones in in vitro and cell-based enzymatic systems. Gained information could be useful in assessing novel covalent proteasome inhibitors during lead compound optimization. The study also demonstrates a robust source of in vitro metabolism identification that correlated very well with in vivo PK metabolism for peptide epoxyketones.

Microsomal epoxide hydrolase (mEH) hydrolyzes a wide range of epoxide containing molecules. Although involved in the metabolism of xenobiotics, recent studies associate mEH with the onset and development of certain disease conditions. This phenomenon is partially attributed to the significant role mEH plays in hydrolyzing endogenous lipid mediators, suggesting more complex and extensive physiological functions. In order to obtain pharmacological tools to further study the biology and therapeutic potential of this enzyme target, we describe the development of highly potent 2-alkylthio acetamide inhibitors of the human mEH with IC50 values in the low nanomolar range. These are around 2 orders of magnitude more potent than previously obtained primary amine, amide and urea-based mEH inhibitors. Experimental assay results and rationalization of binding through docking calculations of inhibitors to a mEH homology model indicate that an amide connected to an alkyl side chain and a benzyl-thio function as key pharmacophore units.

Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxy phenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of (18)O from H2 (18)O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979.

OBJECTIVE: The aim: The aim of the present study was to establish a link between polymorphic variants of the microsomal epoxide hydrolase gene and the severity of COPD in patients with COPD and coronary heart disease. PATIENTS AND METHODS: Materials and methods: The study included 128 patients with COPD and IHD, who were divided into two groups: group 1 included 72 patients with infrequent exacerbations of COPD (0-1 per year) and group 2 included 56 patients with frequent exacerbations of COPD (exacerbation of COPD <=2 per year). The control groups consisted of 15 smokers without COPD and IHD, 11 practically healthy non-smokers and 11 patients with IHD who do not smoke. All patients underwent DNA isolation and purification, followed by determination of the Tyr113His polymorphism of the EPHX1 microsomal epoxide hydrolase gene (rs1051740). RESULTS: Results: There was a significant association of the carriage of the CC genotype of the EPHX1 gene in patients with COPD and IHD (RO = 21.326 [95.0% CI 4.217-107.846], p <0.001) with a more severe course of COPD compared with the TT genotype of the EPHX1 gene. CONCLUSION: Conclusions: Patients with COPD and coronary heart disease who were carriers of a homozygous variant of the EPHX1 gene have a reliable association with a more severe course of COPD with frequent exacerbations (higher class according to GOLD classification and more severe symptoms of COPD according to the AT questionnaire).

KZR-616 is an irreversible tripeptide epoxyketone-based selective inhibitor of the human immunoproteasome. Inhibition of the immunoproteasome results in anti-inflammatory activity in vitro and, based on promising therapeutic activity in animal models of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), KZR-616 is being developed for potential treatment of multiple autoimmune and inflammatory diseases. The presence of a ketoepoxide pharmacophore presents unique challenges in the study of drug metabolism during lead optimization and clinical candidate profiling. This study presents a thorough and systematic in vitro and cell-based enzymatic metabolism and kinetic investigation to identify the major enzymes involved in the metabolism and elimination of KZR-616. Upon exposure to liver microsomes in the absence of NADPH, KZR-616 and its analogs were converted to their inactive diol derivatives with varying degrees of stability. Diol formation was also shown to be the major metabolite in pharmacokinetic studies in monkeys and correlated with in vitro stability results for individual compounds. Further study in intact hepatocytes and a hepatocellular carcinoma cell line revealed that KZR-616 metabolism was sensitive to an inhibitor of microsomal epoxide hydrolase (mEH) but not inhibitors of cytochrome P450 (CYP) or soluble epoxide hydrolase (sEH). Primary human hepatocytes were determined to be the most robust source of mEH activity for study in vitro These findings also suggest that the exposure of KZR-616 in vivo is unlikely to be affected by co-administration of inhibitors or inducers of CYP and sEH. Significance Statement This work presents a thorough and systematic investigation of metabolism and kinetic of KZR-616 and other peptide epoxyketones in in vitro and cell-based enzymatic systems. Gained information could be useful in assessing novel covalent proteasome inhibitors during lead compound optimization. The study also demonstrates a robust source of in vitro metabolism identification that correlated very well with in vivo PK metabolism for peptide epoxyketones.

Epoxide hydrolases (EHs) regulate cellular homeostasis through hydrolysis of epoxides to less-reactive diols. The first discovered EH was EPHX1, also known as mEH. EH functions remain partly unknown, and no pathogenic variants have been reported in humans. We identified two de novo variants located in EPHX1 catalytic site in patients with a lipoatrophic diabetes characterized by loss of adipose tissue, insulin resistance, and multiple organ dysfunction. Functional analyses revealed that these variants led to the protein aggregation within the endoplasmic reticulum and to a loss of its hydrolysis activity. CRISPR-Cas9-mediated EPHX1 knockout (KO) abolished adipocyte differentiation and decreased insulin response. This KO also promoted oxidative stress and cellular senescence, an observation confirmed in patient-derived fibroblasts. Metreleptin therapy had a beneficial effect in one patient. This translational study highlights the importance of epoxide regulation for adipocyte function and provides new insights into the physiological roles of EHs in humans.

Microsomal epoxide hydrolase (mEH) hydrolyzes a wide range of epoxide containing molecules. Although involved in the metabolism of xenobiotics, recent studies associate mEH with the onset and development of certain disease conditions. This phenomenon is partially attributed to the significant role mEH plays in hydrolyzing endogenous lipid mediators, suggesting more complex and extensive physiological functions. In order to obtain pharmacological tools to further study the biology and therapeutic potential of this enzyme target, we describe the development of highly potent 2-alkylthio acetamide inhibitors of the human mEH with IC50 values in the low nanomolar range. These are around 2 orders of magnitude more potent than previously obtained primary amine, amide and urea-based mEH inhibitors. Experimental assay results and rationalization of binding through docking calculations of inhibitors to a mEH homology model indicate that an amide connected to an alkyl side chain and a benzyl-thio function as key pharmacophore units.

PURPOSE: The extensive drug resistance of hepatocellular carcinoma (HCC) has become a major cause of chemotherapy failure. A deeper understanding of the drug resistance mechanism of tumor cells is very significant for improving the clinical prognosis of patients with HCC. EXPERIMENTAL DESIGN: In this study, proteomic studies on the composition of 5-fluorouracil (5-Fu) resistant Bel/5Fu cell line and its parent Bel7402 cell line by using an ionic liquid assisted proteins extraction method with the advantage of extracting plasma membrane proteins to a wider extent are performed. Then the expression level and function of differentially expressed plasma membrane proteins are verified. RESULTS: In total, 25 plasma membrane proteins are shown differentially expressed in Bel/5Fu compared with Bel7402. Western blot analysis results further confirmed that the EPHX1 PLIN2 RAB27B SLC4A2 are upregulated in Bel/5Fu cells in accordance with the proteomics data. Moreover, cell viability assay and clonogenic survival assay results demonstrated that EPHX1 is closely related to the chemoresistance of Bel/5Fu to 5-Fu. CONCLUSIONS AND CLINICAL RELEVANCE: Plasma membrane protein EPHX1 is closely related to the chemotherapy resistance of Bel/5Fu cells and can be used as a new drug target to improve the clinical prognosis of patients with HCC.

BACKGROUND: Environmental insults and microsomal epoxide hydrolase 1 (EPHX1) single nucleotide polymorphisms (SNPs), Tyr113His T/C rs1051740 and His139Arg A/G rs2234922, aberrantly alters microRNA (miR) expression and are linked to low birthweights (LBW). OBJECTIVES: To investigate the interplay between pollution, EPHX1 SNPs and miRs during pregnancy and associated LBW outcomes.
METHODS: South African pregnant women (n=241) were recruited in the MACE birth cohort study in Durban, a city with high levels of industry and traffic related pollutants. EPHX1 SNPs were genotyped using PCR-RFLP and grouped into their respective phenotypes, i.e. normal (N), slow (S), very slow (VS) and fast (F). EPHX1, miR-26b-5p, miR-193b-3p and miR-1207-5p expression were determined using quantitative PCR.
RESULTS: Mothers with the Tyr113His SNP had low iron levels [TT vs. TC+CC: mean difference (MD)=0.67g/dl; p=0.0167], LBW [TT vs. TC+CC: MD=189.30g; p=0.0067], and low EPHX1 expression; p<0.0001. miR-26b-5p and miR-1207-5p expression were significantly higher in the CC genotypes compared to TT+TC groups; p<0.0001. The opposite trend occurred for miR-193b-3p; p=0.0045. Mothers with the VS phenotype had low iron levels [N vs. VS and VS vs. F: MD=2.03 and -1.96g/dl; p=0.0021, respectively], decreased gestational age [VS vs. F: MD=-2.14weeks; p=0.0051, respectively], and LBW [N vs. VS, VS vs. F and S vs. VS: MD=1000, -940.30 and 968.80g; p<0.0001, respectively]; F phenotype had the highest EPHX1 expression [N vs. F, VS vs. F and S vs. F: MD=-1.067, -1.854 and -1.379; p=0.0002, respectively]; and N phenotype had low miR-26b-5p [N vs. VS: MD=-0.6100; p=0.0159] and miR-1207-5p [N vs. VS and VS vs. F: MD=-0.834 and 1.103; p=0.0007, respectively] expression. miR-193b-3p expression between phenotypes remained unchanged. CONCLUSION: The Tyr113His T/C variant of rs1051740 and VS phenotype alters EPHX1, miR-26b-5p and miR-1207-5p expression, and contributes towards low blood iron levels and LBW.

Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxy phenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of (18)O from H2 (18)O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. We recently demonstrated that fatty amides, such as elaidamide, represent a new class of potent inhibitors of mEH. While these compounds are very active on recombinant mEH in vitro, they are quickly inactivated in liver extracts reducing their value in vivo. We investigated the effect of structural changes on mEH inhibition potency and microsomal stability. Results obtained indicate that the presence of a small alkyl group alpha to the terminal amide function and a thio-ether beta to this function increased mEH inhibition by an order of magnitude while significantly reducing microsomal inactivation. The addition of a hydroxyl group 9-10 carbons from the terminal amide function resulted in better inhibition potency without improving microsomal stability. The best compound obtained, 2-nonylsulfanyl-propionamide, is a competitive inhibitor of mEH with a K I of 72 nM. Furthermore, this new inhibitor significantly reduces mEH diol production in ex vivo lungs exposed to naphthalene, underlying the usefulness of the inhibitors described herein. These novel inhibitors could be valuable tools to investigate the physiological and biological roles of mEH.

Human microsomal epoxide hydrolase (EPHX1) is active in the metabolism of many potentially carcinogenic or otherwise genotoxic epoxides, such as those derived from the oxidation of polyaromatic hydrocarbons. EPHX1 is polymorphic and encodes allelic variation at least two amino acid positions, Y113H and H139R. In a number of recent molecular epidemiological investigations, EPHX1 polymorphism has been suggested as a susceptibility factor for several human diseases. To better evaluate the functional contribution of EPHX1 genetic polymorphism, we characterized the enzymatic properties associated with each of the respective variant proteins. Enzymatic profiles were evaluated with cis-stilbene oxide (cSO) and benzo[a]pyrene-4,5-epoxide (BaPO), two prototypical substrates for the hydrolase. In one series of experiments, activities of recombinant EPHX1 proteins were analyzed subsequent to their expression using the pFastbac baculovirus vector in Spodoptera frugiperda-9 (Sf9) insect cells, and purification by column chromatography. In parallel studies, EPHX1 activities were evaluated with human liver microsomes derived from individuals of known EPHX1 genotype. Using the purified protein preparations, rates of cSO and BaPO hydrolysis for the reference protein, Y113/H139, were approximately 2-fold greater than those measured with the other EPHX1 allelic variants. However, when activities were analyzed using human liver microsomal fractions, no major differences were evident in the reaction rates generated among preparations representing the different EPHX1 alleles. Collectively, these results suggest that the structural differences encoded by the Y113H and H139R variant alleles exert only modest impact on EPHX1-specific enzymatic activities in vivo.

Five novel single nucleotide polymorphisms (SNPs) were found in the EPHX1 gene from 96 Japanese epileptic patients. The detected SNPs were as follows: 1) SNP, MPJ6_EX1009; GENE NAME, EPHX1 ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-CCTCACTTCAGTG/ACTGGGCTTTGCC-3'. 2) SNP, MPJ6_EX1013; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-TCCGCAGCCAGGG/CAGGACGACAGCA-3'. 3) SNP, MPJ6_EX1026; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-GTTCTCCCTGGAC/TGACCTGCTGACC-3'. 4) SNP, MPJ6_EX1028; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-AGGCAGGGGGACG/AGCCAGTCTTGGG-3'. 5) SNP, MPJ6_EX1030; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-TGAAAAGTGGGTG/AAGGTTCAAGTAC-3'. The frequencies were 0.016 for MPJ6_EX1028 (IVS8+54G>A) and 0.005 for the other SNPs. The SNP MPJ6_EX1013 (130G>C) results in an amino acid alteration (E44Q). The other three SNPs in the coding region, MPJ6_EX1009 (30G>A), MPJ6_EX1026 (1056C>T), and MPJ6_EX1030 (1239G>A) result in synonymous changes (V10V, D352D, and V413V, respectively).

Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in carcinogen metabolism and is also able to mediate the sodium-dependent uptake of bile acids into hepatocytes. Studies have identified a subject (S-1) with extremely elevated serum bile salt levels in the absence of observable hepatocellular injury, suggesting a defect in bile acid uptake. In this individual, mEH protein and mEH mRNA levels were reduced by approximately 95% and 85%, respectively, whereas the expression and amino acid sequence of another bile acid transport protein (NTCP) was unaffected. Sequence analysis of the mEH gene (EPHX1) revealed a point mutation at an upstream HNF-3 site (allele I) and in intron 1 (allele II), which resulted in a significant decrease in EPHX1 promoter activity in transient transfection assays. Gel shift assays using a radiolabeled oligonucleotide from each region resulted in specific transcription factor binding patterns, which were altered in the presence of the mutation. These studies demonstrate that the expression of mEH is greatly reduced in a patient with hypercholanemia, suggesting that mEH participates in sodium-dependent bile acid uptake in human liver where its absence may contribute to the etiology of this disease.

This study determined whether genetic variability in exons 3 and 4 of the microsomal epoxide hydrolase gene jointly modifies individual preeclampsia risk. The study also determined whether genetic variability in the gene encoding for microsomal epoxide hydrolase (EPHX) contributes to individual differences in susceptibility to the development of preeclampsia. The study involved 133 preeclamptic and 115 healthy control pregnant women who were genotyped for two single nucleotide polymorphisms (SNPs), T-->C (Tyr113His) in exon 3 and A-->G (His139Arg) in exon 4, in the EPHX gene. Chi-square analysis was used to assess genotype and allele frequency differences between the preeclamptic and control groups. In addition, single-point analysis was expanded to pair of loci haplotype analysis to examine the estimated haplotype frequencies of the two SNPs, of unknown phase, among the preeclamptic and control groups. Estimated haplotype frequencies were assessed using the maximum-likelihood method, employing an expectation-maximization (EM) algorithm. Single-point allele and genotype distributions in exons 3 and 4 of the EPHX gene were not statistically different between the groups. However, according to the haplotype estimation analysis, we observed a significantly elevated frequency of haplotype T-A (Tyr113-His139) among the preeclampsia group vs the control group (P=0.01). The odds ratio for preeclampsia associated with the high-activity haplotype T-A (Tyr113-His139) was 1.61 (95% CI: 1.12-2.32). The use of two intragenic SNPs jointly in haplotype analysis of association demonstrated that the genetically determined high-activity haplotype T-A (Tyr113-His139) was significantly associated with preeclampsia.

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.

Human microsomal epoxide hydrolase (mEH) is a xenobiotic-metabolizing enzyme that detoxifies reactive epoxides to more water soluble dihydrodiol compounds. We have isolated and sequenced clones that encode the entire human mEH gene (EPHX1). The primary nuclear transcript, extending from the start of transcription to the site of poly(A) addition, is 20,271 nucleotides in length. The human mEH gene contains 9 exons, separated by 8 introns; canonical intron/exon boundary sites are observed at each junction. The introns vary in size from 335 to 6696 bp and contain numerous repetitive DNA elements, including 18 Alu sequences (each > 100 nucleotides in length) within 4 introns. Alu sequences were classified with respect to subfamily assignment. Two thousand eighteen nucleotides 5' of the transcription start and 2501 nucleotides 3' of the poly(A) addition sites were also sequenced. To evaluate the human mEH promoter, chimeric constructs were prepared linking portions of the 5' mEH flanking sequence (up to -693 bp) to a CAT reporter gene, followed by transient transfection in both COS-1. and HepG2 cells. Results from these expression experiments suggest that the human mEH gene contains a weak core promoter and that inclusion of DNA sequences 5' of the minimal promoter region negatively regulates constitutive transcription.

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).

Epoxide hydrolase (EC 3.3.2.3) was purified to electrophoretic homogeneity from human liver cytosol by using hydrolytic activity toward trans-8-ethylstyrene 7,8-oxide (TESO) as an assay. The overall purification was 400-fold. The purified enzyme has an apparent monomeric molecular weight of 58 000, significantly greater than the 50 000 found for human (or rat) liver microsomal epoxide hydrolase or for another TESO-hydrolyzing enzyme also isolated from human liver cytosol. Purified cytosolic TESO hydrolase catalyzes the hydrolysis of cis-8-ethylstyrene 7,8-oxide 10 times more rapidly than does the microsomal enzyme, catalyzes the hydrolysis of TESO and trans-stilbene oxide as rapidly as the microsomal enzyme, but catalyzes the hydrolysis of styrene 7,8-oxide, p-nitrostyrene 7,8-oxide, and naphthalene 1,2-oxide much less effectively than does the microsomal enzyme. Purified cytosolic TESO hydrolase does not hydrolyze benzo[a]pyrene 4,5-oxide, a substrate for the microsomal enzyme. The activities of the purified enzymes can explain the specific activities observed with subcellular fractions. Anti-human liver microsomal epoxide hydrolase did not recognize cytosolic TESO hydrolase in purified form or in cytosol, as judged by double-diffusion immunoprecipitin analysis, precipitation of enzymatic activity, and immunoelectrophoretic techniques. Cytosolic TESO hydrolase and microsomal epoxide hydrolase were also distinguished by peptide mapping. The results provide evidence that physically different forms of epoxide hydrolase exist in different subcellular fractions and can have markedly different substrate specificities.