Differential induction of cytosolic epoxide hydrolase, microsomal epoxide hydrolase, and glutathione S-transferase activities

Three major enzyme systems have been shown to metabolize epoxidized xenobiotics in vertebrate tissues, and this study demonstrates that these enzyme systems can be differentially induced. The cytosolic epoxide hydrolase activity was routinely monitored with trans-beta-ethylstyrene oxide, the microsomal epoxide hydrolase activity with benzo(a)pyrene, 4,5-oxide, and the glutathione S-transferase activity with 2,4-dichloro-4-nitrobenzene. Commonly used inducers of microsomal mixed-function oxidase, microsomal epoxide hydrolase, and cytosolic glutathione S-transferase activities failed to cause significant induction of the cytosolic epoxide hydrolase while leading to the expected induction of the other epoxide metabolizing enzymes. The compounds tested by ip injection into male Swiss-Webster mice included phenobarbital, 3-methylcholanthrene, Aroclor 1254, trans- and cis-stilbene oxides, pregnenolone-16 alpha-carbonitrile, chalcone, and 4-bromochalcone. To determine if there were strain, sex, or species differences, the enzymes were monitored in male C57BL/6 mice, female Swiss-Webster mice, and male Sprague-Dawley rats following ip injection of phenobarbital, 3-methylcholanthrene, and/or pregnenolone-16 alpha-carbonitrile. The time dependence of enzyme induction was followed in Sprague-Dawley rats following trans-stilbene oxide administration. Male Swiss-Webster mice were additionally exposed to dietary alpha-naphthoflavone and 2(3)-tert-butyl-4-hydroxyanisole while male Sprague-Dawley rats were fed 2,6-di-tert-butyl-4-methylphenol. In no case was significant induction of cytosolic epoxide hydrolase activity observed. Dietary di-(2-ethylhexyl)phthalate, 2-ethyl-l-hexanol, and clofibrate proved to be potent inducers of the cytosolic epoxide hydrolase in male Swiss-Webster mice while probucol (a nonperoxisome proliferating hypolipidemic drug) failed to cause significant induction. Data from isoelectric focusing experiments and other data are consistent with the epoxide hydrolase activities induced by 2-ethyl-l-hexanol and clofibrate being due to the same protein that is present in control animals. The lack of induction of the cytosolic epoxide hydrolase by a variety of compounds which were selected to demonstrate induction of other xenobiotic metabolizing enzymes, may indicate that the cytosolic epoxide hydrolase has a constitutive role whereas its induction by clofibrate could be related to some of the pharmacological and/or carcinogenic actions of this drug.     

Induction of epoxide hydrolase in cultured rat hepatocytes and hepatoma cell lines

The expression of epoxide hydrolases was studied in cultured rat hepatocytes and hepatoma cell lines. Styrene 7,8-oxide and benzo[a]pyrene 4,5-oxide were used as substrates for microsomal epoxide hydrolase and trans-stilbene oxide for the cytosolic form of this enzyme. In freshly isolated hepatocytes from control rats, microsomal epoxide hydrolase activity was 7.7 and 10.8 nmoles/mg cellular protein/min with benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide as substrates respectively. This enzyme activity increased by more than 2-fold in hepatocytes after 24 hr in culture and remained elevated throughout 96 hr using both substrates. In cultured hepatocytes from rats pretreated in vivo with phenobarbital, trans-stilbene oxide, 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene, both benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide hydrolase activities were increased greater than 1.8 relative to controls. Hepatocytes from 2-acetylaminofluorene-pretreated animals at 24 hr in culture had approximately 9-fold higher activities than control hepatocytes. In marked contrast to microsomal epoxide hydrolase activity, the cytosolic enzyme showed an initial activity of 191 pmoles/mg cellular protein/min in freshly isolated hepatocytes, decreased by 75% after 24 hr in culture, and was barely detectable at 96 hr. A similar trend was apparent in hepatocytes from the pretreated animals. In vitro treatment of hepatocytes with trans-stilbene oxide and phenobarbital increased microsomal epoxide hydrolase, while this activity was refractory to 2-acetylaminofluorene treatment. Styrene 7,8-oxide hydrolase activity was increased in the McA-RH-7777 rat hepatoma cell line by phenobarbital, trans-stilbene oxide and 2-acetylaminofluorene treatment. Similarly, benzo[a]pyrene 4,5-oxide hydrolase activity was also increased in this cell line by treatment with phenobarbital and trans-stilbene oxide but not by 2-acetylaminofluorene. Microsomal epoxide hydrolase activity in rat H4-II-E hepatoma cells was refractory to induction, except by trans-stilbene oxide treatment, which caused a 70% increase in benzo[a]pyrene 4,5-oxide hydrolase activity.     

Effect of dietary clofibrate on epoxide hydrolase activity in tissues of mice

The effects of dietary clofibrate on the epoxide-metabolizing enzymes of mouse liver, kidney, lung and testis were evaluated using trans-stilbene oxide as a selective substrate for the cytosolic epoxide hydrolase, cis-stilbene oxide and benzo[a]pyrene 4,5-oxide as substrates for the microsomal form, and cis-stilbene oxide as a substrate for glutathione S-transferase activity. The hydration of trans-stilbene oxide was greatest in liver followed by kidney greater than lung greater than testis. Its hydrolysis was increased significantly in the cytosolic fraction of liver and kidney of clofibrate-treated mice and in the microsomes from the liver. Isoelectric focusing indicates that the same enzyme is responsible for hydrolysis of trans-stilbene oxide in normal and induced liver and kidney. Clofibrate induced glutathione S-transferase activity on cis-stilbene oxide only in the liver. Hydrolysis of both cis-stilbene oxide and benzo[a]pyrene 4,5-oxide was highest in testis followed by liver greater than lung greater than kidney. Hydration of cis-stilbene oxide was induced significantly in both liver and kidney by clofibrate but that of benzo[a]pyrene 4,5-oxide was induced only in the liver. These and other data based on ratios of hydration of benzo[a]pyrene 4,5-oxide to cis-stilbene oxide in tissues of normal and induced animals indicate that there are one or more novel epoxide hydrolase activities which cannot be accounted for by either the classical cytosolic or microsomal hydrolases. These effects are notable in the microsomes of kidney and especially in the cytosol of testis.     

Microsomal and cytosolic epoxide hydrolase in Drosophila melanogaster

Subcellular fractions from Drosophila melanogaster and rat liver were investigated on their epoxide hydrolase activity. Both microsomes and the post-microsomal supernatant of Drosophila appeared to contain epoxide hydrolase activity using styrene-7,8-oxide as the substrate. Based on body weight, these activities were in the same order of magnitude. Rat liver cytosol was able to catalyze the hydrolysis of styrene oxide only if the glutathione S-transferase activity was blocked.     

Stereoselectivity and regioselectivity of purified human glutathione transferases pi, alpha-epsilon, and mu with alkene and polycyclic arene oxide substrates

The stereoselectivities of three biochemically distinct human glutathione transferases, the acidic isoenzyme (pi) purified from placenta and the basic (alpha-epsilon) and the near-neutral (mu) isoenzymes purified from liver, were determined with (+/-)-benzo(a)pyrene-4,5-oxide, pyrene-4,5-oxide, and (+/-)-styrene-7,8-oxide as substrates. Transferase mu was highly selective (greater than 95%) for reaction of glutathione with R-configured oxirane carbon atoms of (+/-)-benzo(a)pyrene-4,5-oxide and pyrene-4,5-oxide, whereas transferase pi was highly stereoselective (greater than 95%) for S-configured epoxide carbon atoms of (+/-)-benzo(a)pyrene-4,5-oxide and pyrene-4,5-oxide. The basic transferases (alpha-epsilon) showed relatively low stereoselectivity with these polycyclic arene oxide substrates; glutathione reaction at R-configured oxirane carbons was preferred, but only by about 2-fold. With (+/-)-benzo(a)pyrene-4,5-oxide as substrate, transferases mu and alpha-epsilon were enantioselective for (4R,5S)-benzo(a)pyrene-4,5-oxide (about 6-fold), whereas transferase pi showed little enantioselectivity. With (+/-)-styrene-7,8-oxide as substrate, transferases mu and pi were selective for (7S)-styrene-7,8-oxide, but this enantioselectivity was not great (1.3- to 1.8-fold); enantioselectivity could not be accurately determined with alpha-epsilon due to the low enzymatic turnover. Transferase pi selectively catalyzed the reaction of glutathione with the benzylic oxirane carbon (C-7) of (+/-)-styrene-7,8-oxide whereas alpha-epsilon preferentially catalyzed reaction with the terminal epoxide carbon (C-8) atom.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferation of peroxisomes and induction of cytosolic and microsomal epoxide hydrolases in different strains of mice and rats after dietary treatment with clofibrate

1. The effects of dietary clofibrate (0.5%, w/w, for 10 days) on seven inbred strains of mice--C57BL/6, C57BL/B10A(5R), ATL/OLA, C3H/HE/OLA, BALB/C, CBA/CA and A/J/OLA--and three strains of rats--Sprague-Dawley, Wistar and LOU/OLA--have been investigated. Liver weight, peroxisome proliferation, catalase activity, cytosolic, microsomal and mitochondrial epoxide hydrolase activities, cytochrome oxidase activity, microsomal cytochrome P-450 content and cytosolic glutathione transferase activity in liver were determined, together with cytosolic and microsomal epoxide hydrolase and cytosolic glutathione transferase activities in the kidneys. 2. In all cases peroxisome proliferation and induction of cytosolic epoxide hydrolase were observed in livers of rodents exposed to clofibrate. Thus, no non-responsive strains were found and further evidence for a coupling between these two phenomena was provided. In many cases significant increases in the liver microsomal cytochrome P-450 content and decreases in the hepatic cytosolic glutathione transferase activity were also seen. 3. High levels of cytosolic epoxide hydrolase were found in the rat kidney. In several strains of mice and rats renal cytosolic epoxide hydrolase activity was increased by clofibrate. 4. There were often considerable strain differences. However, in general mice had higher cytosolic epoxide hydrolase and glutathione transferase activities, whereas rats had higher microsomal epoxide hydrolase activities.     

Proliferation of peroxisomes and induction of cytosolic and microsomal epoxide hydrolases in different strains of mice and rats after dietary treatment with clofibrate

1. The effects of dietary clofibrate (0.5%, w/w, for 10 days) on seven inbred strains of mice—C57BL/6, C57BL/B10A(5R), ATL/OLA, C3H/HE/OLA, BALB/C, CBA/CA and A/J/OLA—and three strains of rats—Sprague-Dawley, Wistar and LOU/OLA—have been investigated. Liver weight, peroxisome proliferation, catalase activity, cytosolic, microsomal and mitochondrial epoxide hydrolase activities, cytochrome oxidase activity, microsomal cytochrome P-450 content and cytosolic glutathione transferase activity in liver were determined, together with cytosolic and microsomal epoxide hydrolase and cytosolic glutathione transferase activities in the kidneys.

2. In all cases peroxisome proliferation and induction of cytosolic epoxide hydrolase were observed in livers of rodents exposed to clofibrate. Thus, no non-responsive strains were found and further evidence for a coupling between these two phenomena was provided. In many cases significant increases in the liver microsomal cytochrome P-450 content and decreases in the hepatic cytosolic glutathione transferase activity were also seen.

3. High levels of cytosolic epoxide hydrolase were found in the rat kidney. In several strains of mice and rats renal cytosolic epoxide hydrolase activity was increased by clofibrate.

4. There were often considerable strain differences. However, in general mice had higher cytosolic epoxide hydrolase and glutathione transferase activities, whereas rats had higher microsomal epoxide hydrolase activities.     

Concomitant induction of microsomal epoxide hydrolase and UDP-glucuronosyltransferase activities by dipyridine compounds.

The coordinated response of the major rat hepatic phase II xenobiotic-metabolizing enzymes following 3-day exposure to diaryl compounds was investigated. Four diaryl compounds containing heterocyclic nitrogen atoms elevated microsomal epoxide hydrolase activity from 2- to 4-fold. Equivalent compounds lacking the heteroatom, when given in the same dosing regimen (75 mg/kg, ig, daily for 3 days), did not induce this or any other drug-metabolizing enzyme activity. Epoxide hydrolase activity closely paralleled UDP-glucuronosyltransferase activity toward three aglycones: 4-nitrophenol (r = 0.87), morphine (r = 0.84), and 1-naphthol (r = 0.78). There was less correlation (r = 0.60) between epoxide hydrolase activity and both UDP-glucuronosyltransferase activity toward testosterone and cytosolic glutathione S-transferase activity. There was no correlation between microsomal epoxide hydrolase activity and cytochrome P-450 or the monooxygenase reaction (4-nitrophenol hydroxylase) preferentially induced by pyridine-containing compounds. Induction of rat hepatic microsomal epoxide hydrolase activity by some pyridine-containing compounds appears coordinately regulated with glucuronidation rather than oxidation enzymes.     

Epoxide hydrolase activity in the mitochondrial and submitochondrial fractions of mouse liver

Distribution of epoxide hydrolase activity in subcellular fractions of livers from male Swiss-Webster mice and Sprague-Dawley rats was monitored with trans-β-ethylstyrene oxide, trans-stilbene oxide and benzo[a]pyrene 4,5-oxide following differential centrifugation. With the former two substrates the highest activity was encountered in the cytosolic fraction; however, significant activity was found in the mitochondrial fraction. These fractions hydrated benzo[a]pyrene 4,5-oxide very slowly, and the major benzo[a]pyrene 4,5-oxide hydrolyzing activity was recovered in the microsomal fraction. Using Triton WR-1339-treated mice, it was shown that trans-β-ethylstyrene oxide hydrolyzing activity was predominantly localized in the mitochondria rather than in lysosomes and peroxisomes. Subsequent separation of the mitochondrial fraction into submitochondrial components by swelling, shrinking, and sonication, followed by sucrose density gradient centrifugation, showed that most of the epoxide hydrolyzing activity was present in the matrix and intermembrane space fraction. Significant activity was also present in the outer and inner membrane fractions. However, epoxide hydrolyzing activity in these fractions was reduced if either increased sonication times were used or the fractions were washed, indicating possible contamination of these fractions by the matrix and intermembrane space enzyme(s). The epoxide hydrolase activity in the mitochondrial and cytosolic fractions in mice appeared similar with regard to inhibition, molecular weight, and substrate selectivity.     

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.     

Effect of hypolipidemic compounds on lauric acid hydroxylation and phase II enzymes

Treatment of male Fischer 344 rats with various hypolipidemic drugs of different peroxisome proliferating potency (1-benzylimidazole, acetylsalicylic acid, clofibrate, tiadenol) led to an induction of liver lauric acid hydroxylase, whereas probucol, which is not a peroxisome proliferator, did not induce this enzyme. Activity of bilirubin UDP-glucuronosyltransferase was increased by all the compounds tested. The highest increase was observed after treatment with acetylsalicylic acid (2.3-fold). High correlation (r = 0.953) was observed between the activities of lauric acid hydroxylase and the corresponding activities of cytosolic epoxide hydrolase reported previously. The amount of microsomal epoxide hydrolase was not changed by any of the compounds. Whereas clofibrate and tiadenol decreased glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate, 1-benzylimidazole and probucol increased this activity. With 4-hydroxynonenal as a substrate qualitatively the same results were obtained with the exception that probucol did not affect the enzyme activity. When glutathione S-transferase activity was measured with cis-stilbene oxide as substrate only the more than five-fold increase after treatment with 1-benzylimidazole was significantly different from control values. Activity of dihydrodiol dehydrogenase was increased after treatment of rats with 1-benzylimidazole (1.5-fold), whereas application of tiadenol led to a decrease of enzyme activity. Feeding of male guinea pigs with clofibrate did not change the activity of peroxisomal beta-oxidation, cytosolic epoxide hydrolase or lauric acid hydroxylase. However, treatment with tiadenol caused an increase of these activities.     

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

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. 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. 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. These cells also glucuronidated 1-naphthol efficiently (6 nmol/min per mg) and, at a lower extent, bilirubin (12 pmol/min per mg). 5. Addition of fenofibrate (70 microM) 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.     

Influence of modulators of epoxide metabolism on the cytotoxicity of trans-anethole in freshly isolated rat hepatocytes.

The effect of modulating epoxide metabolism by inhibiting microsomal and cytosolic epoxide hydrolases and depleting glutathione, on the cytotoxicity of trans-anethole has been examined in freshly isolated rat hepatocytes in suspension. Hepatocytes derived from female Sprague-Dawley CD rats by collagenase perfusion were incubated in suspension and sampled at intervals over a 6-hr period. Cytotoxicity was assessed by the leakage of lactate dehydrogenase into the culture medium and in the cells after lysis. Glutathione was determined by fluorimetry. Anethole showed a dose-dependent cytotoxicity at concentrations ranging from 5 x 10(-4) to 5 x 10(-3) M, with concentrations of 10(-3) M and above causing greater than 63% leakage of lactate dehydrogenase in 6 hr. Microsomal epoxide hydrolase was inhibited by trichloropropene oxide (10(-4) M) and cyclohexene oxide (10(-3) M), and cytosolic epoxide hydrolase by 4-fluorochalcone oxide (5 x 10(-4) M). Cellular glutathione was depleted by diethyl maleate (5 x 10(-4) M), and its synthesis inhibited by 2.5 x 10(-3) M-L-buthionine (S,R)-sulphoximine. Suspensions treated with a sub-cytotoxic concentration of anethole (5 x 10(-4) M) showed a rapid increase in cytotoxicity when 4-fluorochalcone oxide was present (complete loss of viability within 2 hr), while pretreatment of hepatocytes with diethyl maleate in combination with buthionine sulphoximine, to deplete glutathione, slowly increased the cytotoxic response at later times (after 4 hr of incubation). The association of the effects of 4-fluorochalcone oxide with the inhibition of cytosolic epoxide hydrolase is strengthened by the inability of chalcone oxide, a close structural analogue of 4-fluorochalcone oxide, which has no effect on epoxide hydrolase or glutathione conjugation, to influence the effects of anethole on hepatocytes. These data are discussed in terms of the role of anethole epoxide in the cytotoxicity of trans-anethole.     

Characterization of multiple epoxide hydrolase activities in mouse liver nuclear envelope

A nuclear envelope-associated epoxide hydrolase in mouse liver that hydrates trans-stilbene oxide has been identified and characterized. This epoxide hydrolase is distinct from the enzyme in nuclear envelopes that hydrates benzo[a]pyrene 4,5-oxide and other arene oxides. This distinction was demonstrated by the criteria of pH optima, response to specific inhibitors in vitro, and precipitation by specific antibodies. The new epoxide hydrolase had a pH optimum of 6.8, was poorly inhibited by trichloropropene oxide, was potently inhibited by 4-phenylchalcone oxide, and did not bind to antiserum against benzo[a]pyrene 4,5-oxide hydrolase. This nuclear enzyme is similar in many of its properties to cytosolic and microsomal trans-stilbene oxide hydrolases and may be nuclear envelope-bound form of these other epoxide hydrolases. It differed from these other trans-stilbene oxide hydrolases in that its affinities for both trans-stilbene oxide (measured as apparent Km) and 4-phenylchalcone oxide (measured as I50) were 4- to 20-fold lower than those of either the cytosolic or microsomal forms.     

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-alpha-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.     

Valpromide inhibits human epoxide hydrolase.

The effect of antipileptic drug valpromide (VPM) on the activity of epoxide hydrolase was studied in human adult and foetal liver, kidneys, lungs, intestine and in placenta. The activity of the epoxide hydrolase was measured with both styrene oxide and benzo(a)pyrene-4,5-oxide as substrates. VPM inhibited the epoxide hydrolase obtained from all organs studied. The degree of inhibition was independent of the substrate used. A lowering of the epoxide hydrolase activity by 50% was observed when the concentration of VPM was similar to that of the substrates. VPM competitively inhibited the activity of adult liver epoxide hydrolase with styrene oxide as substrate.     

Xenobiotic metabolizing enzymes are not restricted to parenchymal cells in rat liver

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

Characterization of distinct forms of cytochromes P-450, epoxide metabolizing enzymes and UDP-glucuronosyltransferases in rat skin

Study of drug metabolizing enzyme activity was undertaken in skin microsomal and cytosolic fractions of male and female rats. The presence of several isoforms was revealed from their activities towards selected substrates and from their cross immunoreactivity using antibodies raised against purified hepatic or renal cytochromes P-450, epoxide hydrolase and UDP-glucuronosyltransferases. Cytochrome P-450 content was precisely quantified by second derivative spectrophotometry, 23.1 and 16.5 pmol/mg protein in males and females, respectively. The monooxygenase activity associated to cytochromes P-450IIB1 and P-450IA1 was determined through O-dealkylation of ethoxy-; pentoxy- and benzoxyresorufin. The activity ranged between 4 and 2 nmol/min/mg protein for male and female rats, respectively. These results and Western blot analysis indicated that rat skin microsomes contain both monooxygenase systems associated with cytochromes P-450IIB1 and P-450IA1. By contrast lauric acid hydroxylation, supported by cytochrome P-450IVA1, was not detectable. Activities of epoxide metabolizing enzymes (microsomal and cytosolic epoxide hydrolases; glutathione S-transferase) were also characterized in skin. Microsomes catalysed the hydratation of benzo(a)pyrene-4,5-oxide and cis-stilbene oxide at the same extent, whatever the sex, although the specific activity was 10 times lower than in liver. The hydratation of trans-stilbene oxide by soluble epoxide hydrolase was four times lower than in the liver. Conjugation of cis-stilbene oxide with glutathione in skin and liver proceeded at essentially similar rates, as the specific activity of glutathione S-transferase in skin was only two times less than that measured in hepatic cytosol. Glucuronidation of 1-naphthol, bilirubin but not of testosterone could be followed in the microsomal fraction. Revelation by Western blot indicated that both the isoforms involved in conjugation of phenols and bilirubin were present in skin microsomes. By contrast, the isoform catalysing the conjugation of testosterone was apparently missing. When immunoblotting was carried out using specific antibodies raised against the renal isoforms, the same result was obtained. In addition, an intense staining corresponding to a 57 kD-protein was observed.     

Induction of liver microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic glutathione transferase in different rodent species by 2-acetylaminofluorene or 3-methylcholanthrene

Control activities vary 12-fold for microsomal epoxide hydrolase, two-fold for UDP-glucuronyl transferase and five-fold for cytosolic glutathione (GSH) transferase among the different rodents (rat, hamster, guinea-pig, mouse) examined. For all three enzymes the activities in rat liver are towards the lower values. In these rodents, except for a 100% increase in microsomal epoxide hydrolase in guinea-pig liver, 2-acetylaminofluorene induces the three phase 2 enzymes only in rat. Treatment with 3-methylcholanthrene also produces the largest effects on these three enzyme activities in rat liver; exceptions are its failure to induce microsomal epoxide hydrolase in female rat and the large induction of cytosolic GSH transferase in hamster liver. Quantitatively, hepatic microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic GSH transferase activities, and their inducibility by 2-acetylaminofluorene or 3-methylcholanthrene, in male Sprague-Dawley rats are not representative for other rodent species or even, in all cases, for female rat.     

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.