Comparison of the sex and subcellular distributions, catalytic and immunochemical reactivities of hepatic epoxide hydrolases in seven mammalian species

Sex and species differences in hepatic epoxide hydrolase activities towards cis- and trans-stilbene oxide were examined in common laboratory animals, as well as in monkey and man. In general trans-stilbene oxide was found to be a good substrate for epoxide hydrolase activity in cytosolic fractions, whereas the cis isomer was selectively hydrated by the microsomal fraction (with the exception of man, where the cytosol also hydrated this isomer efficiently). The specific cytosolic epoxide hydrolase activity was highest in mouse, followed by hamster and rabbit. Epoxide hydrolase activity in the crude 'mitochondrial' fraction towards trans-stilbene oxide was also highest in mouse and low in all other species examined. Microsomal epoxide hydrolase activity was highest in monkey, followed by guinea pig, human and rabbit, which all had similar activities. Sex differences were generally small, but where significant, male animals had higher catalytic activities than females of the same species in most cases. Antibodies raised against microsomal epoxide hydrolase purified from rat liver reacted with microsomes from all species investigated, indicating structural conservation of this protein. Antibodies directed towards cytosolic epoxide hydrolase purified from mouse liver reacted only with liver cytosol from mouse and hamster and with the 'mitochondrial' fraction from mouse in immunodiffusion experiments. Immunoblotting also revealed reaction with rat liver cytosol. The cytosolic and 'mitochondrial' epoxide hydrolases in all three mouse strains and in both sexes for each strain were immunochemically identical. The anomalies in human liver epoxide hydrolase activities observed here indicate that no single common laboratory animal is a good model for man with regard to these activities.     

Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse

Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.     

Hepatic levels of cytosolic, microsomal and 'mitochondrial' epoxide hydrolases and other drug-metabolizing enzymes after treatment of mice with various xenobiotics and endogenous compounds

This study was performed in order to study the response of epoxide hydrolases in different subcellular compartments of mouse liver to treatment with various compounds. Male C57BL/6 mice were treated with 31 different compounds--including traditional inducers of xenobiotic-metabolizing systems, liver carcinogens, stilbene derivatives, endogenous compounds and various other drugs and xenobiotics. The effects on liver somatic index; protein contents in 'mitochondria', microsomes and cytosol prepared from the liver; epoxide hydrolase activity towards trans- or cis-stilbene oxide in these three fractions; microsomal cytochrome P-450 content; cytosolic and 'mitochondrial' glutathione transferase activity and cytosolic DT-diaphorase activity were then determined. Cytosolic epoxide hydrolase activity was induced by chlorinated paraffins, di(2-ethylhexyl)phthalate and clofibrate and depressed by alpha-naphthylisothiocyanate, 3-methylcholanthrene, benzil and quercitin. Radial immunodiffusion revealed similar changes in the amount of enzyme protein present, except for two cases, where the increase in amount was larger; and the enzyme seems to be inhibited by benzil. Microsomal epoxide hydrolase activity was induced by these same compounds and several others as well, including dibenzoylmethane, butylated hydroxyanisole and polychlorinated biphenyls. 'Mitochondrial' epoxide hydrolase activity towards trans-stilbene oxide was not affected by those compounds which induced the cytosolic enzyme, but increased about two-fold after treatment with 2-acetylaminofluorene, DL-ethionine, aflatoxin B1 and phenobarbital. There does not seem to be any co-regulation of different forms of epoxide hydrolase in mouse liver. In general small effects were observed on liver weight and protein contents in the different subcellular fractions. Polychlorinated biphenyls were the most potent of the 8 compounds which induced cytochrome P-450, while butylated hydroxyanisole induced cytosolic glutathione transferase activity to the highest extent. 'Mitochondrial' glutathione transferase activity was most induced by certain of the stilbene derivatives. The most potent inducers of DT-diaphorase activity were 3-methylcholanthrene, polychlorinated biphenyls and dinitrotoluene.     

Continuous spectrophotometric assays for cytosolic epoxide hydrolase

Two convenient and sensitive continuous spectrophotometric assays for cytosolic epoxide hydrolase are described. The assays are based on the differences in the ultraviolet spectra of the epoxide substrates and their diol products. The hydrolysis of 1,2-epoxy-1-(p-nitrophenyl)pentane (ENP5) is accompanied by a decrease in absorbance at 302 nm, while the hydration of 1,2-epoxy-1-(2-quinolyl)pentane (EQU5) produces an increase in absorbance at 315.5 nm. The Km, Vmax values for ENP5 and EQU5 with purified mouse liver cytosolic epoxide hydrolase were 1.7 microM, 11,700 nmol/min/mg and 25 microM, 8300 nmol/min/mg, respectively. Both substrates are hydrolyzed significantly faster than trans-stilbene oxide, which is currently the most commonly used substrate for measuring cytosolic epoxide hydrolase activity. No spontaneous hydrolysis of the substrates is detectable under normal assay conditions. The assays are applicable to whole tissue homogenates as well as purified enzyme preparations. p-Nitrostyrene oxide and p-nitrophenyl glycidyl ether were also examined and found to be very poor substrates for cytosolic epoxide hydrolase from mouse liver.     

Immunocytochemical analysis of soluble epoxide hydrolase and catalase in mouse and rat hepatocytes demonstrates a peroxisomal localization before and after clofibrate treatment

The intracellular localization of soluble epoxide hydrolase and catalase was investigated in hepatocytes from untreated and clofibrate-treated male C57B1/6 mice and from untreated male Sprague-Dawley rats. Polyclonal rabbit antibodies directed against purified mouse liver cytosolic epoxide hydrolase and rat liver catalase were used and their specificity ascertained by Ouchterlony immunodiffusion and immunoblotting. The IgG fraction was purified and incubated with cryosections of isolated hepatocytes or liver tissue, priorly fixed in 4% paraformaldehyde, and protein-A gold conjugates were used to visualize the antigen-antibody reaction. The soluble form(s) of epoxide hydrolase was found to be localized in the matrix of peroxisomes in hepatocytes from normal and clofibrate-treated mice and normal rats. No significant reactivity was found against plasma membrane, nuclei, mitochondria, the Golgi apparatus, endoplasmic reticulum, lysosomes, or cytosol. Catalase was also localized to peroxisomes in all samples investigated. Accordingly, both the catalase and the epoxide hydrolase activities routinely recovered in the high-speed supernatant after subfractionation of rat and mouse liver tissue mostly seemed to be due to extensive matrix leakage from peroxisomes, and this phenomenon may also be found in other species. Rat hepatocytes contained less epoxide hydrolase than mouse hepatocytes, as judged by both immunocytochemical labeling and biochemical data. Clofibrate treatment of mice decreased the labeling density of epoxide hydrolase and catalase in hepatocytes peroxisomes, as expected, and more unlabeled peroxisomes were observed.     

Biochemical characterization of a variant form of cytosolic epoxide hydrolase induced by parental exposure to N-ethyl-N-nitrosourea

1. ENU4 mice express a protein variant originally detected in a CBF1 mouse sired by a C57BL/6 mouse exposed to N-ethyl-N-nitrosourea. It appears to be an isolelectric point variant of cytosolic epoxide hydrolase. Affinity purified cytosolic epoxide hydrolase from ENU4 mice has a pI of approximately 5.1 compared to 5.6 in other mouse strains.2. Clofibrate induced cytosolic epoxide hydrolase to similar levels in five strains of mice. However, CBF1 and ENU4 mice were more sensitive to the induction of palmitoyl CoA oxidase activity.3. Except for isoelectric point, the physico- and immunochemical properties of cytosolic epoxide hydrolase from ENU4 mice were similar to those of the other mouse strains. Substrate specificities for five of six substrates tested were also similar.     

Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels

Improved affinity chromatography procedures for the purification of cytosolic epoxide hydrolase are described. An earlier affinity purification method using immobilized 7-methoxycitronellyl thiol (MCT) sporadically produced final enzyme preparations containing major impurities. To eliminate these impurities, we tested alternate ligands, spacer arms, and ligand concentrations. A series of alkyl and aryl thiols coupled to epoxy-activated Sepharose were found to exhibit markedly different binding characteristics as compared with commercially available alkyl- and aryl-Sepharose gels. Using one of these new matrices, benzylthio-Sepharose, cytosolic epoxide hydrolase from mouse liver was purified over 100-fold, appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was obtained with 60-90% recovery of enzyme activity. The impurities previously observed with the MCT-Sepharose procedure were reduced or eliminated by using an MCT ligand concentration of 5 microequivalents per gram or less. MCT-Sepharose and benzylthio-Sepharose provide rapid and convenient one-step procedures for obtaining purified cytosolic epoxide hydrolase from numerous species and tissues.     

In vitro metabolism of juvenile hormone III and juvenile hormone III bisepoxide by Drosophila melanogaster and mammalian cytosolic epoxide hydrolase

In vitro metabolism of juvenile hormone III (JH III) and juvenile hormone III bisepoxide was investigated using purified mouse liver cytosolic epoxide hydrolase (cEH) and cell fractions from Drosophila melanogaster. JH III was metabolized faster than JH III bisepoxide by epoxide hydrolase activity in D. melanogaster cell fractions and by cEH. After incubation with JH III bisepoxide, all cell fractions and cEH produced epoxy-diol, cis- and trans-tetrahydrofuran-diols, and tetraol as metabolites. An increase in the concentration of cEH resulted in an increase in the proportion of tetraol as a JH III bisepoxide metabolite but this trend was not observed in the D. melanogaster cell fractions. Differences between cell fractions in the metabolism of JH III and JH III bisepoxide suggests the presence of juvenile hormone epoxide hydrolase isozymes.     

Differential subcellular localization of endogenous and transfected soluble epoxide hydrolase in mammalian cells: evidence for isozyme variants

Endogenous, constitutive soluble epoxide hydrolase in mice 3T3 cells was localized via immunofluorescence microscopy exclusively in peroxisomes, whereas transiently expressed mouse soluble epoxide hydrolase (from clofibrate-treated liver) accumulated only in the cytosol of 3T3 and HeLa cells. When the C-terminal lie of mouse soluble epoxide hydrolase was mutated to generate a prototypic putative type 1 PTS (-SKI to -SKL), the enzyme targeted to peroxisomes. The possibility that soluble epoxide hydrolase-SKI was sorted slowly to peroxiosmes from the cytosol was examined by stably expressing rat soluble epoxide hydrolase-SKI appended to the green fluorescent protein. Green fluorescent protein soluble epoxide hydrolase-SKI was strictly cytosolic, indicating that -SKI was not a temporally inefficient putative type 1 PTS. Import of soluble epoxide hydrolase-SKI into peroxisomes in plant cells revealed that the context of -SKI on soluble epoxide hydrolase was targeting permissible. These results show that the C-terminal -SKI is a non-functional putative type 1 PTS on soluble epoxide hydrolase and suggest the existence of distinct cytosolic and peroxisomal targeting variants of soluble epoxide hydrolase in mouse and rat.     

Inhibition of epoxide hydrolases and glutathione S-transferases by 2-, 3-, and 4-substituted derivatives of 4'-phenylchalcone and its oxide

4'-Phenylchalcones, chalcone oxides, and related compounds were synthesized and tested as inhibitors of cytosolic epoxide hydrolase, microsomal epoxide hydrolase, and glutathione S-transferases from mouse and rat liver. Several compounds were more potent inhibitors of the cytosolic epoxide hydrolase than the parent 4'-phenylchalcone oxide while large substituents in the 4- and especially the 2-position caused a reduction in inhibition. The chalcone oxides showed selectivity as inhibitors of the cytosolic epoxide hydrolase acting on trans-stilbene oxide, while chalcones were inhibitors of cytosolic glutathione S-transferase acting on cis-stilbene oxide. Data are consistent with the hypothesis that much of the inhibition of the glutathione S-transferase is caused by the glutathione conjugate of the chalcone.     

The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates

The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5-oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg-yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in situ in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5-oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30-50% and towards androstene oxide 60-90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg-yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were found with different effectors. Activation of epoxide hydrolase activity towards styrene oxide by clotrimazole was found to be uncompetitive with the substrate and highly structure specific. On the other hand, inhibition of epoxide hydrolase activity towards androstene oxide by clotrimazole was found to be competitive in microsomes. It is concluded that the marked effects of these four modulators on microsomal epoxide hydrolase activity are due to an interaction with the enzyme protein itself, but that the presence of total microsomal phospholipids allows the maximal expression leading to similar degrees of modulation as those observed in intact microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. Purification procedure and physiochemical characterization of the pure enzymes

Cytosolic epoxide hydrolase was purified from the liver of untreated and clofibrate-treated male C57Bl/6 mice. The purification procedure involves chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxyapatite, takes two days to perform and results in a 120-fold purification and approximately 35% yield of the enzyme from untreated mice. The purified enzyme is a dimer with a molecular mass of 120 kDa, a Stokes' radius of 4.2 nm, a frictional ratio of 1.0 and an isoelectric point of 5.5. The subunits behave identically upon isoelectric focusing in 8 M urea and only one band with a molecular mass of 60 kDa is seen after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The form purified from clofibrate-treated mice had very similar properties and was apparently identical to the control form as judged by amino acid analysis and peptide mapping as well. These analyses also demonstrated that the cytosolic enzyme is clearly different from microsomal epoxide hydrolase isolated from rat liver. Furthermore, Ouchterlony immunodiffusion using antibodies raised in rabbits towards the control form of cytosolic epoxide hydrolase revealed identity between the two forms of cytosolic epoxide hydrolase, but no reaction with the microsomal epoxide hydrolase was observed. These findings indicate large structural differences between the cytosolic and microsomal forms of epoxide hydrolase in the liver.     

Purification and characterization of rat-liver cytosolic epoxide hydrolase

Rat liver cytosolic epoxide hydrolase has been purified and characterized. The enzyme was purified from tiadenol-induced rat liver 540-fold with respect to trans-stilbene oxide as a substrate. Similar purification was obtained with the substrates trans-beta-ethyl styrene oxide and styrene 7,8-oxide, the specific activities decreasing in the order trans-beta-ethyl styrene oxide greater than styrene 7,8-oxide greater than trans-stilbene oxide. The enzyme exerts highest activity at pH 7.4 Km and Vmax of the pure enzyme for trans-stilbene oxide were 1.7 microM and 205 nmol x min-1 x mg protein-1 respectively. With trans-stilbene oxide as a substrate, the inhibition by organic solvents (2.5% by vol.) increased in the order ethanol less than methanol less than acetone less than isopropanol = N,N-dimethyl formamide less than acetonitrile less than tetrahydrofuran. The native enzyme, with a molecular mass of 120 kDa, consists of two 61-kDa subunits. Digestion of rat liver cytosolic and microsomal epoxide hydrolase by three proteases resulted in markedly different peptide maps. Western-blot analysis with antiserum against rat liver cytosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofibrate-induced rats. No cross-reactivity was observed with purified rat microsomal epoxide hydrolase or microsomes. A positive reaction at the same molecular mass was obtained with liver cytosol of mouse, guinea pig, Syrian hamster and New Zealand white rabbit but not with that of green monkey.     

A new enzyme immunoassay of microsomal rat liver epoxide hydrolase

Antiserum against purified rat liver microsomal epoxide hydrolase was produced in the rabbit. We developed an enzyme-linked immunosorbent assay which is reliable with regard to its analytical criteria. The concentration of epoxide hydrolase was measured in liver microsomes of control rats and animals treated with F 1379 (250 mg/kg/day) for 5, 7, 14, and 21 days. This hypolipidemic drug was able to induce strong epoxide hydrolase activity and enhance protein concentration. The gradual increase in epoxide hydrolase concentration paralleled the increase of epoxide hydrolase activity, with stabilization occurring after the 14th until the 21st day of treatment.     

In vitro translation of cytosolic and peroxisomal epoxide hydrolase and catalase on liver polyribosomes from untreated and clofibrate-treated C57B1/6 mice

Free and membrane-bound polyribosomes were isolated from the livers of untreated and clofibrate-treated male C57B1/6 mice. The in vitro translation products were investigated in a rabbit reticulocyte cell-free system by immunoprecipitation of cytosolic epoxide hydrolase, catalase and albumin. The soluble forms of epoxide hydrolase present in cytosol and in peroxisomes were found to be synthesized on free polyribosomes and could not be distinguished from each other, since only one primary translation product was found with the methods used. Clofibrate treatment was found to increase total protein synthesis, synthesis of soluble epoxide hydrolase and translational efficiency of the isolated polyribosomes.     

Cytosolic epoxide hydrolase

Epoxide hydrolase activity is recovered in the high-speed supernatant fraction from the liver of all mammals so far examined, including man. For some as yet unexplained reason, the rat has a very low level of this activity, so that cytosolic epoxide hydrolase is generally studied in mice. This enzyme selectively hydrolyzes trans epoxides, thereby complementing the activity of microsomal epoxide hydrolase, for which cis epoxides are better substrates. Cytosolic epoxide hydrolase has been purified to homogeneity from the livers of mice, rabbits and humans. Certain of the physicochemical and enzymatic properties of the mouse enzyme have been thoroughly characterized. Neither the primary amino acid, cDNA nor gene sequences for this protein are yet known, but such characterization is presently in progress. Unlike microsomal epoxide hydrolase and most other enzymes involved in xenobiotic metabolism, cytosolic epoxide hydrolase is not induced by treatment of rodents with substances such as phenobarbital, 2-acetylaminofluorene, trans-stilbene oxide, or butylated hydroxyanisole. The only xenobiotics presently known to induce cytosolic epoxide hydrolase are substances which also cause peroxisome proliferation, e.g., clofibrate, nafenopin and phthalate esters. These and other observations indicate that this enzyme may actually be localized in peroxisomes in vivo and is recovered in the high-speed supernatant because of fragmentation of these fragile organelles during homogenization, i.e., recovery of this enzyme in the cytosolic fraction is an artefact. The functional significance of cytosolic epoxide hydrolase is still largely unknown. In addition to deactivating xenobiotic epoxides to which the organism is exposed directly or which are produced during xenobiotic metabolism, primarily by the cytochrome P-450 system, this enzyme may be involved in cellular defenses against oxidative stress.     

Human liver cytosolic epoxide hydrolases

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.     

Immunological similarity of epoxide hydrolase activity in the mitochondrial and cytosolic fractions of mouse liver

The light and heavy mitochondrial fractions of mouse liver have relatively high levels of epoxide hydrolase (EH) activity when monitored with trans-stilbene oxide as substrate. Using double diffusion analysis and immunoprecipitation experiments it was shown that EH activity in the mitochondrial fractions is immunologically similar to cytosolic EH, but immunologically dissimilar from microsomal EH. The EHs in the mitochondrial and cytosolic fractions also have a similar pI.     

Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.     

Properties of enzymes hydrating epoxides in human epidermis and liver

• 1.1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different.
• 2.2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity Chromatographic properties.
• 3.3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities.
• 4.4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes.
• 5.5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts.
• 6.6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.