Cytosolic Epoxide Hydrolase Activity Research Articles

Age- and gender-related changes in the hepatic metabolism of 2-methylpropene and relationship to epoxide metabolizing enzymes

The effect of age and gender on the in vitro biotransformation of 2-methylpropene, an alkene metabolized to 2-methyl-1,2-epoxypropane, was studied. The epoxide concentration and the epoxide metabolizing enzymatic activities were investigated in male and female Brown Norway rats of different ages. Liver tissue of senescent rats was exposed to smaller 2-methyl-1,2-epoxypropane concentrations than that of young animals, although changes during ageing were rather modest. With advancing age a feminization of male glutathione S-transferase and cytosolic epoxide hydrolase activities was found, as well as a significant decline of the female microsomal epoxide hydrolase activity and an increase of the cytochrome P-450 content in the oldest female rats.

Perfluorooctane sulfonic acid is a potent inducer of peroxisomal fatty acid beta-oxidation and other activities known to be affected by peroxisome proliferators in mouse liver.

Perfluorooctane sulfonic acid is almost as potent as perfluorooctanoic acid in causing increases in peroxisomal fatty acid beta-oxidation, peroxisomal catalase activity, omega-hydroxylation of lauric acid, cytosolic epoxide hydrolase activity and cytosolic DT-diaphorase activity. Octane sulfonic acid was ineffective at doses used for perfluorooctane sulfonic acid and perfluorooctanoic acid. The results support the theory of co-regulation of these parameters and peroxisome proliferation. The fact that perfluorooctane sulfonic acid causes peroxisome proliferation challenges the hypothesis that the first step in this process is formation of a thioester between the proliferator (the carboxylic group) and coenzyme A.

Effects of perfluoro fatty acids on xenobiotic-metabolizing enzymes, enzymes which detoxify reactive forms of oxygen and lipid peroxidation in mouse liver

Male mice were exposed via their diet to perfluoro fatty acids of various chain-lengths (2–10 carbon atoms) at different doses (0.02 and 0.1% weight) and for different periods of time (2–10 days). Thereafter, we monitored effects on liver and body weights and a number of hepatic parameters, including mitochondrial protein content, microsomal contents of cytochromes P450 and b 5, NADPH-cytochrome P450 reductase activity [measured as NADPH-cytochrome c reductase (EC 1.6.2.3)], microsomal and cytosolic epoxide hydrolase (EC 3.3.2.3) activities, cytosolic DT-diaphorase (EC 1.6.99.2), glutathione transferase (EC 2.5.1.18), glutathione peroxidase (EC 1.11.1.9) and super-oxide dismutase (EC 1.15.1.1) activities, and levels of thiobarbituric acid-reactive material (as an indicator of lipid peroxidation) in the mitochondrial subtraction. The most dramatic changes observed were a 5–9-fold increase in mitochondrial protein, a 3–6-fold increase in the microsomal content of cytochrome P450, a 3–10-fold increase in cytosolic DT-diaphorase activity, an approximately 2-fold increase in cytosolic epoxide hydrolase activity and as much as a 60% decrease in the level of thiobarbituric acid-reactive compounds in the mitochondrial fraction. Smaller increases in microsomal epoxide hydrolase activity and decreases in cytosolic glutathione peroxidase activity were also observed. Of the perfluoro fatty acids tested, perfluorooctanoic acid caused the largest changes in the parameters examined here. Dietary exposure of mice to a 0.02% dose of this substance for 10 days results in a maximal or near-maximal effect in most cases.

Cigarette smoke inhibits cytosolic but not microsomal epoxide hydrolase of human lung.

The effect of cigarette smoke exposure on the activity of cytosolic and microsomal epoxide hydrolase (EH) has been investigated in human lung. Patients were classified as 'recent smokers' (n = 9) or 'non-recent smokers' (n = 10) according to whether they were or were not still smoking 1 month before surgery. Cytosolic EH was measured with [3H]trans-stilbene oxide as a substrate, whereas microsomal EH was measured with [7-3H]styrene oxide as a substrate. Microsomal EH activity did not differ between recent smokers (2.51 +/- 0.93 nmol min-1 mg-1) and non-recent smokers (2.74 +/- 1.10 nmol min-1 mg-1), whereas cytosolic EH activity was significantly lower in recent smokers (6.46 +/- 1.79 pmol min-1 mg-1) than in non-recent smokers (8.41 +/- 2.09 pmol min-1 mg-1, P less than 0.05). Cytosolic EH activity was correlated with the number of days that had passed since the cessation of smoking (r = 0.58, P less than 0.05) and the effect was dose-dependent, since the enzyme activity was inversely correlated with the number of cigarettes smoked per day (r = 0.85, P less than 0.01). This suggests that recent smoking exposure inhibits the activity of cytosolic EH but not microsomal EH, and that the inhibition increases with the number of cigarettes smoked per day. The contribution of cytosolic enzymes to xenobiotic metabolism may be remarkable in extrahepatic tissues. The inhibition of cytosolic EH by tobacco smoke may reduce the inactivation of carcinogenic epoxides in human lung tissues and so may increase a person's susceptibility to lung cancer.

The effect of structurally divergent herbicides on mouse liver xenobiotic-metabolizing enzymes (P-450-dependent mono-oxygenases, epoxide hydrolases and glutathione S-transferases) and carnitine acetyltransferase

Male mice were treated with structurally diverse herbicides to study their effect on liver xenobiotic-metabolizing enzymes. Chlorfiurecol, trifluralin, alachlor, propham, MCPP and 2,4-DP caused increases in phase I (cytochrome P-450, ethoxycoumarin O-deethylase, and/or aminopyrine N-demethylase) and phase II (microsomal epoxide hydrolase and cytosolic glutathione S-transferase) activities. MCPP and 2,4-DP also increased cytosolic epoxide hydrolase and carnitine acetyltransferase activities suggestive of peroxisome proliferation. Benthiocarb and molinate increased only some phase II enzyme activities. Dicamba, at the dose employed, caused mortality and decreases in some of the enzymes monitored. Most of the herbicides tested induced xenobiotic-metabolizing enzyme activities, the pattern of induction being dependent on herbicide structure.

Influence of the level of cytosolic epoxide hydrolase on the induction of sister chromatid exchanges by trans-β-ethylstyrene 7,8-oxide in human lymphocytes

trans-β-Ethylstyrene 7,8-oxide, a substrate of cytosolic epoxide hydrolase, and 4-fluorochalcone oxide, an inhibitor of this enzyme, were investigated on induction of sister chromatid exchanges (SCE) in human lymphocytes. Both epoxides enhanced the frequency of SCE. 4-Fluorochalcone oxide at low concentration (2.5μM) inhibited cytosolic epoxide hydrolase activity towards trans-β-ethylstyrene 7,8-oxide in lymphocytes by 74% and had no effect on glutathione transferase activity using this substrate. At this concentration it did not induce SCE itself, but it potentiated the effect of trans-β-ethylstyrene 7,8-oxide several fold. In lymphocytes from different subjects, the number of SCE induced by a low concentration of trans-β-ethylstyrene 7,8-oxide correlated negatively with the individual cytosolic epoxide hydrolase activity ( r = −0.72; −0.73 in two series of experiments). The number of SCE induced by a high concentration of trans-β-ethylstyrene 7,8-oxide did not correlate with cytosolic epoxide hydrolase activity ( r = 0.004; −0.24), but a negative correlation was found with glutathione transferase activity ( r = −0.50). This finding is consistent with the results of biochemical studies in lymphocytes in which we determined the relative contribution of cytosolic epoxide hydrolase and glutathione transferase to the metabolism of trans-β-ethylstyrene 7,8-oxide at varying substrate concentrations. The study demonstrates that the level of genotoxic effects induced in human lymphocytes is influenced by the individual level of detoxifying enzymes. At low concentrations, cytosolic epoxide hydrolase was more important than glutathione transferase activity.

Effects of environmentally encountered epoxides on mouse liver epoxide-metabolizing enzymes

Male mice were treated (i.p.) for 3 days with 15 different environmentally encountered epoxides, and the effects of these compounds on liver microsomal and cytosolic epoxide hydrolase (mEH and cEH), glutathione S-transferase (mGST and cGST) and carboxylesterase (mCE) activities were determined. The epoxides included the pesticides: heptachlor epoxide, dieldrin, tridiphane, and juvenoid R-20458; the natural products: disparlure, limonin, nomilin, and epoxymethyloleate; the endogenous steroids: lanosterol epoxide, cholesterol-α-epoxide, and progesterone epoxide; and the industrial or synthetic epoxides: epichlorohydrin, araldite, trans-stilbene oxide, and 4'-phenylchalcone oxide. The pesticide epoxides were the most effective inducers of liver weight, microsomal protein, and the enzyme activities measured, with mEH and cEH activities towards cis-stilbene oxide (mEHcso and cEHcso), cGST activities towards four of five substrates, and mCE towards clofibrate (mCEclof) and p-nitrophenylacetate (mCEpna) increased following treatment with most of the pesticides. The synthetic epoxides increased some of the same activities, while the natural products, except for increases in cGST activities, and endogenous steroid epoxides were generally not inductive. cEH activity towards trans-stilbene oxide (cEHtso) was increased only following treatment with the peroxisome proliferator, tridiphane, but decreased following treatment with several of the epoxides, while microsomal cholesterol epoxide hydrolase (mEHchol) was increased only moderately by disparlure. Microsomes could effectively conjugate glutathione to chlorodinitrobenzene (mGSTcdnb) and cis-stilbene oxide (mGSTcso). These two activities were differentially induced by a few of the epoxides, suggesting that they may be selective substrates for different isozymes of mGST. Correlation coefficients were determined for the relative response of liver weight, subfraction protein, and enzyme activities. A relatively high correlation was found between the response of liver weight and cytosolic hydrolysis of trans-stilbene oxide ( r = 0.73) and cis-stilbene oxide ( r = 0.62), and cytosolic glutathione conjugation of dichloronitrobenzene ( r = 0.66) and trans-stilbene oxide ( r = 0.75). In addition, relatively high correlations were found between the different cGST activities, in particular for dichloronitrobenzene with trans-stilbene oxide ( r = 0.89). These studies show that there exists a wide variation in the response of xenobiotic-metabolizing enzymes to environmentally encountered epoxides and that a fairly strong correlation exists between the increases in liver size and increases in certain cytosolic enzyme activities; they also suggest further studies concerning the possibility of an additional isozyme of mGST.

Expression of arylhydrocarbon hydroxylase, epoxide hydrolases, glutathione S-transferase and UDP-glucuronosyltransferases in H5-6 hepatoma cells

1. 1. The presence of arylhydrocarbon hydroxylase (cytochrome P-450 IA1 dependent), glutathione S-transferase, two distinct forms of epoxide hydrolases and UDP-glucuronosyltransferases was detected in H5-6 hepatoma cell homogenates using model substrates, selective inhibitors and specific antibodies. 2. 2. The activity of arylhydrocarbon hydroxylase decreased strongly at the first days after plating and remained at a minimal value (1.5 pmol/min per mg) after 5 days of culture. 3. 3. The hydratation of trans-stilbene oxide catalyzed by the soluble form of epoxide hydrolase was very low (11.0 pmol/min per mg), whereas the hepatoma cells contained appreciable amounts of the membrane-bound epoxide hydrolase and glutathione S-transferase measured with cis-stilbene oxide as substrate (maximal specific activity: 1.46 and 2.73 nmol/min per mg, respectively). 4. 4. These cells also glucuronidated 1-naphthol efficiently (6 nmol/min per mg) and, at a lower extent, bilirubin (12 pmol/min per mg). 5. 5. Addition of fenofibrate (70 μM) into the culture medium for 1–3 days failed to significantly stimulate the activity of cytosolic epoxide hydrolase. Only bilirubin glucuronidation increased 2-fold after 2 days of presence of the drug.

Inhibition of catalase and epoxide hydrolase by the renal cystogen 2-amino-4,5-diphenylthiazole and its metabolites

Subchronic feeding of 2-amino-4,5-diphenylthiazole (DPT) to rats results in the development of renal cysts and has been used as a model system to study polycystic kidney disease. Because previous studies revealed changes in renal enzymes following DPT administration, a possible direct effect of DPT and its phenolic metabolites on catalase and a related enzyme, epoxide hydrolase, was examined. Experiments with three in vitro systems (suspensions of rabbit renal tubules, rat kidney homogenates, and commercially obtained bovine liver catalase) revealed direct inhibition of catalase activity by the diphenolic metabolite (diOH- DPT: 2-amino-4,5di(4′-hydroxyphenyl)-thiazole), the known renal cystogen nordihydroquaiaretic acid (NDGA) 2-amino-4(4′- hydroxyphenyl),5-phenyl-thiazole (4OH-DPT), and the known catalase inhibitor 3-amino-1,2,4-triazole; DPT did not inhibit catalase activity. Following oral administration to rats of the DPT congeners, 4OH-DPT caused the greatest decrease in both renal catalase and cytosolic epoxide hydrolase activityes and the shortest time to onset of cystic lesions. In vitro, mouse liver cytosolic epoxide hydrolase activity was substantially inhibited by 4OH-DPT and dioH-DPT, and NDGA, but not by 2-amino-4-phenyl,5- (4′-hydroxyphenyl)-thiazole (5OH-DPT) or DPT itself. Microsomal epoxide hydrolase (mEH) activity was inhibited by 4OH-DPT, unaffected by DPT or dioH-DPT, and stimulated 2-fold by 5OH-DPT. Finally, mEH activity was substantially higher in samples of normal human kidney than in samples of kidney derived from a patient with autosomal recessive polycystic kidney disease; no differences were observed in cEH activity in these samples. Although the role of altered catalase and epoxide hydrolase activities in cystogenesis is unknown, DPT-induced cyst formation is associated with loss of these enzyme activities in kidney tissue. To our knowledge, this is the first report of an in vivo diminution of cytosolic epoxide hydrolase activity by xenobiotics.

Selective inhibition of cytosolic epoxide hydrolase activity in vitro by compounds that inhibit catalase.

The ability of a number of known inhibitors of catalase activity to affect cytosolic and microsomal epoxide hydrolase activities in vitro, measured as enzymatic trans-stilbene oxide hydrolysis and styrene oxide hydrolysis, respectively, was investigated. Catalase and cytosolic epoxide hydrolase activities are inhibited by hydroxylated metabolites of 2-amino-4,5-diphenylthiazole (DPT). The metabolite hydroxylated on the 4-phenyl ring (4OH-DPT) and the metabolite hydroxylated on both phenyl rings (4,5-DIOH-DPT) are potent inhibitors of both enzymes; the metabolite hydroxylated on the 5-phenyl ring (5OH-DPT) is less potent. Unmetabolized DPT has no effect on either enzyme. 4OH-DPT inhibits, but 5OH-DPT enhances, microsomal epoxide hydrolase activity. 4,5-DIOH-DPT and DPT have no effect on this enzyme. Other compounds that inhibit both catalase and cytosolic epoxide hydrolase activities, but do not inhibit microsomal epoxide hydrolase activity, are nordihydroguaiaretic acid and 2-aminothiazole. Microsomal epoxide hydrolase activity is enhanced by 2-aminothiazole and levamisole in vitro. Thus these inhibitors of catalase are selective epoxide hydrolase inhibitors in that they inhibit cytosolic epoxide hydrolase activity in vitro, but have either no effect on, or increase the activity of, microsomal epoxide hydrolase in vitro. Conversely, the selective cytosolic epoxide hydrolase inhibitors 4-phenylchalcone oxide and 4'-phenylchalcone oxide do not inhibit catalase activity, nor does trichloropropene oxide, a selective microsomal epoxide hydrolase inhibitor.

Effect of diabetes and starvation on the activity of rat liver epoxide hydrolases, glutathione S-transferases and peroxisomal β-oxidation

The activities of peroxisomal β-oxidation, cytosolic and microsomal epoxide hydrolase as well as soluble glutathione S-transferases have been determined in the livers of alloxan- and streptozotocin-diabetic male Fischer-344 rats. Five, seven and ten days after initiation of diabetes serum glucose levels were elevated 3.6-, 5.7- to 6.2- and 6-fold, while the activities of peroxisomal β-oxidation and cytosolic epoxide hydrolase were elevated 1.5- and 2.5-fold, 1.4- and 2.7-fold and 1.3- and 2.0-fold, respectively. The activities of microsomal epoxide hydrolase and glutathione S-transferases were reduced to about 71% and 80% of controls. Application of 10 I.U./kg depot insulin twice a day for 10 consecutive days to alloxan-diabetic individuals approximately restored the initial glucose levels and enzyme activities except for peroxisomal β-oxidation. Starvation of Fischer-344 rats for 48 hours and 5 days similarly resulted in a 1.3-fold to 2.1-fold and 1.2- to 1.6-fold increase in peroxisomal β-oxidation and cytosolic epoxide hydrolase activity, respectively. Microsomal epoxide hydrolase was significantly decreased to 57% and 61% of control activity whereas glutathione S-transferase was only marginally reduced to 91% and 92%. Except for glutathione S-transferases initial enzyme activities were restored upon refeeding within 10 days. These results are similar to those obtained upon feeding of hypolipidemic compounds with peroxisome proliferating activity, and may indicate that high levels of free fatty acids or their metabolites which are known to accumulate in liver in both metabolic states may act as endogenous peroxisome proliferators.

Microsomal and cytosolic epoxide hydrolase and glutathione S-transferase activities in the gill, liver, and kidney of the rainbow trout, Salmo gairdneri: Baseline levels and optimization of assay conditions

Microsomal and cytosolic epoxide hydrolase (mEH and cEH respectively) and glutathione S-transferase (GST) activities were measured in the liver, kidney, and gills of rainbow trout. Assays were optimized for time, pH, and temperature, using trans-stilbene oxide (TSO) and cis-stilbene oxide (CSO) as substrates for cEH and mEH, respectively. Optimal pH values for mEH, cEH, and GST were similar to mammalian values (i.e. 8.5, 7.5, and 9). Temperature optima differed between tissues and cell fractions. Specific activity of cEH-TSO was 3–14 times greater than mEH-CSO for all three tissues, and 8–60 times greater on a tissue weight basis. Liver and, to a lesser extent, kidney mEH were active against benzo[ a]pyrene 4,5-oxide, whereas gill mEH was not active against this substrate. Liver cytosolic GST was active against CSO and 1-chloro-2,4-dinitrobenzene (CDNB) but not TSO, whereas gill and kidney cytosolic GST were active only against CDNB. Liver and kidney microsomal GST were active against CDNB, but no activity was found in gill microsomes. The results are discussed in relation to possible endogenous substrates and uninduced xenobiotic metabolizing capacities of different trout tissues.

Similarities between catalase and cytosolic epoxide hydrolase.

Cytosolic epoxide hydrolase, measured as trans-stilbene oxide hydrolase activity, was isolated and purified from human and guinea pig liver cytosol. Antiserum to the guinea pig liver preparation reacted strongly with bovine liver catalase. We determined that this lack of selectivity of the antiserum was due to catalase contamination of the epoxide hydrolase preparation. We also determined that several commercial catalase preparations are contaminated with cytosolic epoxide hydrolase. Our human epoxide hydrolase preparation contained no detectable catalase contamination, yet antiserum to this protein also cross-reacted slightly with catalase, indicating some intrinsic similarity between the two enzymes. We conclude that catalase and cytosolic epoxide hydrolase contain some similar immunogenic epitopes, and we surmise that similarities between the subunits of these two enzymes may lead to their partial copurification. Functional similarities between the two enzymes are also demonstrated, as several compounds that inhibit catalase are also shown to inhibit cytosolic epoxide hydrolase activity in the same concentration range and rank order.

Cytosolic epoxide hydrolase in humans: development and tissue distribution.

Cytosolic epoxide hydrolase activity was measured towards trans-stilbene oxide in 41 human adult livers, in 40 fetal livers, in 17 placentas and in fetal and adult lungs, kidneys and gut. The cytosolic epoxide hydrolase activity was measurable in all specimens investigated. The rate of formation of trans-stilbene glycol (pmol/min per mg protein, mean +/- SD) was 55.2 +/- 89.6 (fetal liver). 303.2 +/- 73.2 (adult liver) and 18.8 +/- 13.1 (placenta) In the fetal extrahepatic tissues, the cytosolic epoxide hydrolase activity was 70.0 +/- 9.4 (adrenals), 47.6 +/- 7.2 (gut), 69.4 +/- 22.5 (kidneys) and 43.2 +/- 19.2 (lungs) pmol/min per mg protein, whereas in the adult tissues it was 131.2 +/- 63.1 (kidneys), 27.8 +/- 20.3 (intestine), 8.5 +/- 2.8 (lungs) and 7.2 +/- 4.2 (urinary bladder) pmol/min per mg protein.

Organ distribution of epoxide hydrolases in cytosolic and microsomal fractions of normal and nafenopin-treated male DBA/2 mice

Using trans-stilbene oxide and styrene oxide as substrates, epoxide hydrolase activities were measured in cytosolic and microsomal fractions from liver, kidney, heart, lung and testis of male DBA/ 2 mice. The activities towards these two substrates are remarkably organ specific: trans-stilbene oxide was most effectively hydrolyzed in subcellular fractions from liver, kidney and heart, whereas styrene oxide was predominantly hydrolyzed in those from liver, lung and testis. Immunoblotting experiments were performed with two polyclonal antibodies isolated from goat antisera. Using an anti-mouse liver cytosolic epoxide hydrolase antibody, the corresponding antigen protein was predominantly detected in both cytosolic and microsomal fractions from liver, kidney and heart. An anti-rat liver microsomal epoxide hydrolase antibody proved to be cross-reactive with the mouse enzyme and stained SDS-gels run with microsomal fractions from liver, lung and testis. The anti-mouse liver cytosolic epoxide hydrolase antibody precipitated cytosolic epoxide hydrolase activities from liver, kidney and heart cytosolic fractions. Dietary exposure to the hypolipidemic agent nafenopin (2000 ppm/10 days) caused an induction of trans-stilbene oxide hydrolase and styrene oxide hydrolase activities in cytosolic and microsomal liver fractions whereas, in the other organs, the same activities were unaffected by this treatment. This finding was in accordance with the increased amounts of antigen protein as detected with the antibodies in liver fractions from treated animals. The anti-mouse liver cytosolic epoxide hydrolase antibody was found to precipitate the whole trans-stilbene oxide hydrolase activity also from liver cytosol of nafenopin-treated mice, which indicates the presence of a single cytosolic epoxide hydrolase following induction.

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 μm, 11,700 nmol/min/mg and 25 μm, 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.

Microsomal and cytosolic epoxide hydrolases, the peroxisomal fatty acid beta-oxidation system and catalase. Activities, distribution and induction in rat liver parenchymal and non-parenchymal cells.

A number of structurally unrelated hypolipidaemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in rodent liver, but there is relatively limited data regarding the specific effects of these drugs on liver non-parenchymal cells. In the present study, liver parenchymal, Kupffer and endothelial cells from untreated and fenofibrate-fed rats were isolated and the activities of two enzymes associated with peroxisomes (catalase and the peroxisomal fatty acid beta-oxidation system) as well as cytosolic and microsomal epoxide hydrolase were measured. Microsomal epoxide hydrolase, cytosolic epoxide hydrolase and catalase activities were 7-12-fold higher in parenchymal cells than in Kupffer or endothelial cells from untreated rats; the peroxisomal fatty acid beta-oxidation activity was only detected in parenchymal cells. Fenofibrate increased catalase, cytosolic epoxide hydrolase and peroxisomal fatty acid beta-oxidation activities in parenchymal cells by about 1.5-, 3.5- and 20-fold, respectively. The induction of catalase (2-3-fold) and cytosolic epoxide hydrolase (3-5-fold) was also observed in Kupffer and endothelial cells; furthermore, a low peroxisomal fatty acid beta-oxidation activity was detected in endothelial cells. Morphological examination by electron microscopy showed that peroxisomes were confined to liver parenchymal cells in untreated animals, but could also be observed in endothelial cells after administration of fenofibrate.

Cytosolic epoxide hydrolase in human placenta.

It has been shown that cytosol from human term placenta contains cytosolic epoxide hydrolase activity. This cytosolic epoxide hydrolase was enriched more than 700-fold by affinity chromatography and appears similar to the enzyme from mouse and human liver in terms of molecular mass (Mr 59,000) and antigenic reactivity.

Induction of cytosolic and microsomal epoxide hydrolases in mouse liver by peroxisome proliferators, with special emphasis on structural analogues of 2-ethylhexanoic acid

Using dietary administration, mice were exposed to eight substances known to cause peroxisome proliferation (i.e. clofibrate clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, ICI-55.897, S-8527 and Wy-14.643) or the related substance p-chlorophenoxyacetic acid (group A). Other animals received di(2-ethylhexyl)phthalate, mono(2-ethylhexyl)phthalate, 2-ethylhexanoic acid, or one of 12 other metabolically and/or structurally related compounds (group B). The effects of these treatments on liver cytosolic and microsomal epoxide hydrolases, microsomal cytochrome P-450, cytosolic glutathione transferase activity, the liver-somatic index and the protein contents of the microsomal and cytosolic fractions prepared from liver were subsequently monitored. In general, peroxisome proliferation was accompanied by increases in cytosolic epoxide hydrolase activity. Many peroxisome proliferators also caused increases in microsomal epoxide hydrolase activity, although the correlation was poorer in this case. Immunochemical quantitation by radial immunodiffusion demonstrated that the increases observed in both of these enzyme activities reflected equivalent increases in enzyme protein, i.e. that induction truly occurred. Induction of total microsomal cytochrome P-450 was obtained after dietary exposure to clofibrate, clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, Wy-14.643, di(2-ethylhexyl)phthalate and di(2-ethylhexyl)phosphate. The most pronounced effects on cytosolic glutathione transferase activity were the decreases obtained after treatment with clofibrate, clofibric acid and Wy-14.643. Our results, together with those reported by others, suggest that the processes of peroxisome proliferation and induction of cytosolic epoxide hydrolase are intimately related. One possible explanation for this is presented.

Hepatic epoxide hydrolase activities and their induction by clofibrate and diethylhexylphthalate in various strains of mice

The presence of epoxide hydrolase activity in cytoplasm, microsomes and mitochondrial fraction in livers from twelve strains of mice (AKR/J, A/J, BALB/cByJ, CBA/J, C3H/HeJ, G57BL/6J, C57BL/10J, DBA/2J, NZB/B1NJ, PL/J, SEC/1ReJ and SW), and the influence of orally administered clofibrate and di(2-ethylhexyl)phthalate (DEHP) (0.5 and 2%, respectively, in diet) on epoxide hydrolase activities, were studied. Significant differences in basal cytosolic epoxide hydrolase activity, which ranged from 5.6 to 11.2 nmol diol · min −1 · (mg protein) −1 using trans-stilbene oxide (TSO) as substrate, were noted among the mice. The highest and lowest enzyme levels were observed in the A/J and DBA/ 2J strains respectively. Similarly, microsomal epoxide hydrolase activity, monitored with cis-stilbene oxide (CSO), varied with the mouse strain, with the highest and lowest microsomal epoxide hydrolase activity being observed in A/J and SW strains respectively. Variations were also noted in the epoxide hydrolase activity in the mitochondrial fraction (monitored with TSO) with the highest and lowest levels observed in C57BL/6J and SW strains respectively. Clofibrate or DEHP treatment induced both cytosolic and microsomal epoxide hydrolases in nearly all of the strains examined. In contrast, the hydrolysis of TSO by the mitochondrial fraction in these strains was either not affected or decreased by clofibrate or DEHP treatment. The induction of cytosolic epoxide hydrolase was found to range between 1.2- and 2.8-fold, with generally a higher level of induction in mouse strains with low basal levels of cytosolic epoxide hydrolase activity. This level of cytosolic epoxide hydrolase activity, monitored with TSO as substrate, closely reflected the level of cytosolic epoxide hydrolase protein detected by immunoblot. There were also no significant differences observed in the molecular weight, immunological characteristics, pH-dependence and heat stability of hepatic cytosolic epoxide hydrolase activities of control and clofibrate-treated mice from various strains. These results suggest that clofibrate and DEHP induce both cytosolic and microsomal epoxide hydrolases but not the epoxide hydrolase in the mitochondrial fraction.