Human liver epoxide hydrolases were characterized by several criteria and a cytosolic cis-stilbene oxide hydrolase (cEHCSO) was purified to apparent homogeneity. Styrene oxide and five phenylmethyloxiranes were tested as substrates for human liver epoxide hydrolases. With microsomes activity was highest with trans-2-methylstyrene oxide, followed by styrene 7,8-oxide, cis-2-methylstyrene oxide, cis-1,2-dimethylstyrene oxide, trans-1,2-dimethylstyrene oxide and 2,2-dimethylstyrene oxide. With cytosol the same order was obtained for the first three substrates, whereas activity with 2,2-dimethylstyrene oxide was higher than with cis-1,2-dimethylstyrene oxide and no hydrolysis occurred with trans-1,2-dimethylstyrene oxide. Generally, activities were lower with cytosol than with microsomes. The isoelectric point for both microsomal styrene 7,8-oxide and cis-stilbene oxide hydrolyzing activity was 7.0, whereas cEHCSO had an isoelectric point of 9.2 and cytosolic trans-stilbene oxide hydrolase (cEHTSO) of 5.7. The cytosolic epoxide hydrolases could be separated by anion-exchange chromatography and gel filtration. The latter technique revealed a higher molecular mass for cEHCSO than for cEHTSO. Both cytosolic epoxide hydrolases showed higher activities at pH 7.4 than at pH 9.0, whereas the opposite was true for microsomal epoxide hydrolase. The effects of ethanol, methanol, tetrahydrofuran, acetonitrile, acetone and dimethylsulfoxide on microsomal epoxide hydrolase depended on the substrate tested, whereas both cytosolic enzymes were not at all, or only slightly, affected by these solvents. Effects of different enzyme modulators on microsomal epoxide hydrolase also depended on the substrates used. Trichloropropene oxide and styrene 7,8-oxide strongly inhibited cEHCSO whereas cEHTSO was moderately affected by these compounds. Immunochemical investigations revealed a close relationship between cEHCSO and rat liver microsomal, but not cytosolic, epoxide hydrolase. Interestingly, cEHTSO has no immunological relationship to rat microsomal, nor to rat cytosolic epoxide hydrolase. cEHTSO from human liver differed also from its counterpart in the rat in that it was only moderately affected by tetrahydrofuran, acetonitrile and trichloropropene oxide. Five steps were necessary to purify cEHCSO. The enzyme has a molecular mass (49 kDa) identical to that of rat liver microsomal epoxide hydrolase.
        
Title: Inhibition of mono-oxygenase activities by 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrase, in rat liver microsomes Shimada T, Sato R Ref: Biochemical Pharmacology, 28:1777, 1979 : PubMed
Title: Effects of the epoxide hydrase inhibitor, 1,1,1-trichloropropane-2,3-oxide on the genetic activity of aflatoxin B1 metabolites in in vitro activation test systems Callen DF, Ong TM Ref: Mutat Res, 49:371, 1978 : PubMed
The epoxide hydrase inhibitor 1,1,1-trichloroprophane-2,3-oxide (TCPO) was genetically active to cells of S. cerevisiae and conidia of N. crassa. This genetic activity could be eliminated or reduced to near spontaneous levels in the presence of the S-9 fraction of hamster liver homogenate. The addition of TCPO to an in vitro activation system containing aflatoxin B1 resulted in an increase in the genetic activity of aflatoxin B1, and this increase was dependent on the dose of TCPO. These results are discussed in relation to the possible metabolism of the promutagen aflatoxin B1.
        
9 lessTitle: Oviductal microsomal epoxide hydrolase (EPHX1) reduces reactive oxygen species (ROS) level and enhances preimplantation mouse embryo development Cheong AW, Lee YL, Liu WM, Yeung WS, Lee KF Ref: Biol Reprod, 81:126, 2009 : PubMed
Somatic cell-embryo coculture enhances embryo development in vitro by producing embryotrophic factor(s) and/or removing harmful substances from the culture environment. Yet, the underlying molecular mechanisms on how somatic cells remove the toxicants from the culture medium remain largely unknown. By using suppression subtractive hybridization, we identified a number of mouse oviductal genes that were up-regulated when developing preimplantation embryos were present in the oviduct. Epoxide hydrolase 1, microsomal (Ephx1 previously known as mEH) was one of these genes. EPHX1 detoxifies genotoxic compounds and participates in the removal of reactive oxygen species (ROS). The transcript of Ephx1 increases in the oviductal epithelium at the estrus stage and in Day 3 of pregnancy as well as in the uterus of ovariectomized mice injected with estrogen or progesterone. Human oviductal epithelial cells OE-E6/E7 express EPHX1 and improve mouse embryo development in vitro. Addition of an EPHX1 inhibitor, cyclohexene oxide (CHO) or 1,1,1-trichloropropene 2,3-oxide (TCPO), to the culture medium increased intracellular and extracellular ROS levels of OE-E6/E7 cells and suppressed the beneficial effect of the cells on embryo development; CHO and TCPO at these concentrations had no adverse effect on OE-E6/E7 growth and embryo development in vitro. Taken together, EPHX1 in oviductal cells may enhance the development of cocultured embryos by protecting them from oxidative stress. Our result supports the notion that somatic cell coculture may enhance embryo development via removal of deleterious substances in the culture medium.
        
Title: Phenytoin embryopathy: effect of epoxide hydrolase inhibitor on phenytoin exposure in utero in C57BL/6J mice Hartsfield JK, Jr., Holmes LB, Morel JG Ref: Biochemical & Molecular Medicine, 56:131, 1995 : PubMed
Previous animal research has suggested that the phenytoin arene oxide metabolite is teratogenic in acute studies and that the fetal effects were increased after injecting an inhibitor of microsomal epoxide hydrolase (mEH) (Martz et al., Pharmacol Exp Ther 203:231-239, 1977, Barcellona et al., Teratog Carcinog Mutagen 7:159-168, 1987). We have studied the effects of chronic oral phenytoin exposure in utero and the mEH inhibitor trichloropropene oxide (TCPO) on the prenatal growth and development of an inbred mouse strain with a low incidence of spontaneous oral clefting (C57BL/6J). Chronic daily gastric gavage of phenytoin produced a plasma level (mean 10.7 micrograms/ml on gestation Day 8) within the range recommended to prevent epilepsy in humans; this did not produce an increase in oral clefting or ventricular septal defects in the exposed C57BL/6J pups. It did produce a significant delay in prenatal growth and development, including phalangeal ossification. However, except for percentage resorptions/implantation, there was no synergism between phenytoin and TCPO in contrast to the finding reported by Martz et al. in Swiss mice. This issue was also assessed in a test of the fetal effect of phenytoin injected with TCPO, as had been done by Martz et al. There were no oral clefts or ventricular septal defects or a difference (P > 0.05) in prenatal growth and development in these C57BL/6J pups compared to the chronic gastric phenytoin plus TCPO group. This suggests either that differences in the genotypes of Swiss and C57BL/6J mice may be a contributing factor or that other teratogenic mechanisms were involved.
Chemically reactive epoxide metabolites have been implicated in various forms of drug and chemical toxicity. Naphthalene, which is metabolized to a 1,2-epoxide, has been used as a model compound in this study in order to investigate the effects of perturbation of detoxication mechanisms on the in vitro toxicity of epoxides in the presence of human liver microsomes. Naphthalene (100 microM) was metabolized to cytotoxic, protein-reactive and stable, but not genotoxic, metabolites by human liver microsomes. The metabolism-dependent cytotoxicity and covalent binding to protein of naphthalene were significantly higher in the presence of phenobarbitone-induced mouse liver microsomes than with human liver microsomes. The ratio of trans-1,2-dihydrodiol to 1-naphthol was 8.6 and 0.4 with the human and the induced mouse microsomes, respectively. The metabolism-dependent toxicity of naphthalene toward human peripheral mononuclear leucocytes was not affected by the glutathione transferase mu status of the co-incubated cells. Trichloropropene oxide (TCPO; 30 microM), an epoxide hydrolase inhibitor, increased the human liver microsomal-dependent cytotoxicity (19.6 +/- 0.9% vs 28.7 +/- 1.0%; P = 0.02) and covalent binding to protein (1.4 +/- 0.3% vs 2.8 +/- 0.2%; P = 0.03) of naphthalene (100 microM), and reversed the 1,2-dihydrodiol to 1-naphthol ratio from 6.6 (without TCPO) to 2.6, 0.6 and 0.1 at TCPO concentrations of 30, 100 and 500 microM, respectively. Increasing the human liver microsomal protein concentration reduced the cytotoxicity of naphthalene, while increasing its covalent binding to protein and the formation of the 1,2-dihydrodiol metabolite. Co-incubation with glutathione (5 mM) reduced the cytotoxicity and covalent binding to protein of naphthalene by 68 and 64%, respectively. Covalent binding to protein was also inhibited by gestodene, while stable metabolite formation was reduced by gestodene (250 microM) and enoxacin (250 microM). The study demonstrates that human liver cytochrome P450 enzymes metabolize naphthalene to a cytotoxic and protein-reactive, but not genotoxic, metabolite which is probably an epoxide. This is rapidly detoxified by microsomal epoxide hydrolase, the efficiency of which can be readily determined by measurement of the ratio of the stable metabolites, naphthalene 1,2-dihydrodiol and 1-naphthol.
Human liver epoxide hydrolases were characterized by several criteria and a cytosolic cis-stilbene oxide hydrolase (cEHCSO) was purified to apparent homogeneity. Styrene oxide and five phenylmethyloxiranes were tested as substrates for human liver epoxide hydrolases. With microsomes activity was highest with trans-2-methylstyrene oxide, followed by styrene 7,8-oxide, cis-2-methylstyrene oxide, cis-1,2-dimethylstyrene oxide, trans-1,2-dimethylstyrene oxide and 2,2-dimethylstyrene oxide. With cytosol the same order was obtained for the first three substrates, whereas activity with 2,2-dimethylstyrene oxide was higher than with cis-1,2-dimethylstyrene oxide and no hydrolysis occurred with trans-1,2-dimethylstyrene oxide. Generally, activities were lower with cytosol than with microsomes. The isoelectric point for both microsomal styrene 7,8-oxide and cis-stilbene oxide hydrolyzing activity was 7.0, whereas cEHCSO had an isoelectric point of 9.2 and cytosolic trans-stilbene oxide hydrolase (cEHTSO) of 5.7. The cytosolic epoxide hydrolases could be separated by anion-exchange chromatography and gel filtration. The latter technique revealed a higher molecular mass for cEHCSO than for cEHTSO. Both cytosolic epoxide hydrolases showed higher activities at pH 7.4 than at pH 9.0, whereas the opposite was true for microsomal epoxide hydrolase. The effects of ethanol, methanol, tetrahydrofuran, acetonitrile, acetone and dimethylsulfoxide on microsomal epoxide hydrolase depended on the substrate tested, whereas both cytosolic enzymes were not at all, or only slightly, affected by these solvents. Effects of different enzyme modulators on microsomal epoxide hydrolase also depended on the substrates used. Trichloropropene oxide and styrene 7,8-oxide strongly inhibited cEHCSO whereas cEHTSO was moderately affected by these compounds. Immunochemical investigations revealed a close relationship between cEHCSO and rat liver microsomal, but not cytosolic, epoxide hydrolase. Interestingly, cEHTSO has no immunological relationship to rat microsomal, nor to rat cytosolic epoxide hydrolase. cEHTSO from human liver differed also from its counterpart in the rat in that it was only moderately affected by tetrahydrofuran, acetonitrile and trichloropropene oxide. Five steps were necessary to purify cEHCSO. The enzyme has a molecular mass (49 kDa) identical to that of rat liver microsomal epoxide hydrolase.
        
Title: Purification of microsomal epoxide hydrolase from liver of rhesus monkey: partial separation of cis- and trans-stilbene oxide hydrolase Moody DE, Hammock BD Ref: Archives of Biochemistry & Biophysics, 258:156, 1987 : PubMed
Solubilized rhesus monkey liver microsomes were used as the starting material for the purification of epoxide (cis-stilbene oxide) hydrolase. Successive chromatography over DEAE-Sephacel followed by CM-cellulose resulted in two peaks of activity, CM A and CM B. Passage of these two eluates over separate hydroxyapatite columns resulted in two peaks of activity from CM A, HA A1, and HA A2, and one peak from CM B and HA B, with respective recoveries of 1, 7, and 0.2% of cis-stilbene oxide hydrolase activities. A similar recovery was found for benzo[a]pyrene-4,5-oxide hydrolase, while trans-stilbene oxide hydrolase activity coeluted only in HA A2. Fraction HA A1 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblots of the three eluates and solubilized microsomes incubated with anti-HA A1 demonstrated a single band at 49 kDa in each fraction. The three eluates were differentially affected by the inhibitors of epoxide hydrolase, trichloropropene oxide and 4-phenylchalcone oxide, and addition of Lubrol PX and phospholipid. Immunoprecipitation of HA A2 resulted in coprecipitation of cis- and trans-stilbene oxide hydrolase activity. Upon immunoprecipitation of solubilized microsomes, all the cis-stilbene oxide and benzo[a]pyrene-4,5-oxide, but only 50-60% of trans-stilbene oxide hydrolase activity was precipitated. These studies support findings with other species that (i) an immunochemically distinct cytosolic-like epoxide hydrolase exists in microsomes, and (ii) microsomal epoxide hydrolase activity can be separated during ion-exchange chromatography giving proteins with similar molecular weights and immunochemical cross-reactivity. The precipitation of cis- and trans-stilbene oxide hydrolase activity in eluate HA A2 provides convincing evidence that these isozymes are not structurally identical.
        
Title: Selective inhibition and selective induction of multiple microsomal epoxide hydrolases Guenthner TM Ref: Biochemical Pharmacology, 35:839, 1986 : PubMed
The inhibition in vitro and induction in vivo of microsomal trans-stilbene oxide hydrolase have been studied. This microsomal epoxide hydrolase activity is distinguishable from the previously well-defined microsomal arene oxide hydrolase by a number of catalytic criteria. Two substituted chalcone oxides, 4-phenylchalcone oxide and 4'-phenylchalcone oxide, are potent inhibitors of microsomal trans-stilbene oxide hydrolase, but have no apparent activity against benzo[a]pyrene 4,5-oxide hydrolase. Conversely, compounds that are potent inhibitors of benzo[a]pyrene 4,5-oxide hydrolase, including styrene oxide, cyclohexene oxide, and trichloropropene oxide, inhibit microsomal trans-stilbene oxide hydrolase only at very high (millimolar) concentrations. The chalcone oxides inhibit microsomal trans-stilbene oxide hydrolase noncompetitively, and have micromolar or nanomolar affinity constants for the enzyme. Attempts were made to induce microsomal trans-stilbene oxide hydrolase in vivo. Compounds that induced microsomal benzo[a]pyrene 4,5-oxide hydrolase levels in mice did not simultaneously induce trans-stilbene oxide hydrolase levels. Clofibrate was an exception; it induced levels of both enzymes to a small but statistically significant degree. The two microsomal hydrolase activities have, therefore, very different catalytic sites and appear to be under separate genetic control. 4-Phenylchalcone oxide and 4'-phenylchalcone oxide are selective inhibitors of microsomal trans-stilbene oxide hydrolase and may prove to be very useful in assessing the involvement of this enzyme in the metabolism of endogenous or xenobiotic epoxides.
        
Title: Nuclear metabolism. II. Further studies on epoxide hydrolase activity Gazzotti G, Garattini E, Salmona M Ref: Chemico-Biological Interactions, 35:311, 1981 : PubMed
Apparent Km- and Vmax-values of nuclear styrene 7,8-oxide hydrolase were determined at different protein concentrations. In the protein concentrations range used no significant differences in the apparent Km-values were observed. The influence of the incubation with different modifiers (i.e. SKF-525A, metyrapone, 1,2-epoxy-3,3,3 trichloropropane, cyclohexene oxide) at two different concentrations on this enzyme activity was also determined. Cyclohexene oxide and 1,2-epoxy-3,3,3-trichloropropane, two well known inhibitors of the microsomal epoxide hydrolase(s) caused a marked inhibition, metyrapone had a strong activating effect whereas SKF-525A had no effect. In vivo pretreatment with phenobarbital significantly induced the nuclear epoxide hydrolase whereas beta-naphthoflavone caused a lower degree of induction. This pattern is quantitatively different but qualitatively very similar to the microsomal one. Moreover a toxifying to detoxifying enzymatic activity balance is attempted for the metabolization of the alkenic double bond of styrene, taking into account the ratio between the styrene monooxygenase (toxifying enzyme) and the styrene, 7,8-oxide hydrolase (detoxifying enzyme) after the above mentioned pretreatments, both in the microsomal and nuclear fractions.
        
Title: Inhibition of mono-oxygenase activities by 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrase, in rat liver microsomes Shimada T, Sato R Ref: Biochemical Pharmacology, 28:1777, 1979 : PubMed
Title: Effects of the epoxide hydrase inhibitor, 1,1,1-trichloropropane-2,3-oxide on the genetic activity of aflatoxin B1 metabolites in in vitro activation test systems Callen DF, Ong TM Ref: Mutat Res, 49:371, 1978 : PubMed
The epoxide hydrase inhibitor 1,1,1-trichloroprophane-2,3-oxide (TCPO) was genetically active to cells of S. cerevisiae and conidia of N. crassa. This genetic activity could be eliminated or reduced to near spontaneous levels in the presence of the S-9 fraction of hamster liver homogenate. The addition of TCPO to an in vitro activation system containing aflatoxin B1 resulted in an increase in the genetic activity of aflatoxin B1, and this increase was dependent on the dose of TCPO. These results are discussed in relation to the possible metabolism of the promutagen aflatoxin B1.
        
Title: Benzo(a)pyrene metabolism in mouse epidermis. Analysis by high pressure liquid chromatography and DNA binding Berry DL, Bracken WR, Slaga TJ, Wilson NM, Butty SG, Juchau MR Ref: Chemico-Biological Interactions, 18:129, 1977 : PubMed
Mouse epidermal homogenates contain an inducible aryl hydrocarbon hydroxylase (AHH) complex that catalyzes the formation of benzo(a)pyrene-7,8-dihydrodiol from benzo(a)pyrene (BP) as assessed by high pressure liquid chromatography (HPLC). 5,6-Benzoflavone (5,6-BF), 7,8-benzoflavone (7,8-BF) and 17-beta-estradiol decreased and butylated hydroxytoluene (BHT) enhanced oxidative metabolism of BP when added in vitro. Epoxide hydrase activity (hydration of benzo(a)pyrene-4,5-epoxide) (BP-4,5-epoxide) was enhanced by 17-beta-estradiol, 5,6-BF, and 7,8-BF. BHT exhibited no significant effect and 1,2-epoxy-3,3,3-trichloropropane (TCPO) inhibited hydrase activity. The capacity of epidermal homogenates to catalyze the covalent binding of BP to DNA indicated that addition of both 5,6-BF and 7,8-BF decreased binding. BHT and TCPO did not significantly affect DNA-binding.
        
Title: Phenytoin teratogenesis: correlation between embryopathic effect and covalent binding of putative arene oxide metabolite in gestational tissue Martz F, Failinger C, 3rd, Blake DA Ref: Journal of Pharmacology & Experimental Therapeutics, 203:231, 1977 : PubMed
The possibility that phenytoin (DPH) teratogenesis is due to an arene oxide (epoxide) metabolite was examined. On gestational day 11, Swiss mice were given teratogenic doses of DPH (50, 75 and 100 mg/kg) with and without a nonteratogenic dose of 1,2-epoxy-3,3,3-trichloropropane (TCPO; 100 mg/kg), an epoxide hydratase inhibitor. TCPO significantly increased the incidence of DPH-induced cleft lip and palate and enhanced the embryolethality 2-fold over DPH alone. Four hours after treatment with 14C-DPH (75 mg/kg, 80-90 muCi) the covalent binding of DPH radioactivity in fetuses and placentae was enhanced 2-fold in groups cotreated with TCPO (100 mg/kg). Enhancement was still evident in placentae 24 hours after treatment. There was no effect of TCPO on maternal plasma DPH level, which was comparable to that found in clinical therapeutics (20-30 microgram/ml). Likewise, fetal and placental DPH uptake was not increased by TCPO. DPH teratogenesis is postulated to result from DPH-epoxide formation and covalent binding of epoxide, the ultimate teratogen, to constituents of gestational tissue.
The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is aobut the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg), did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.