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.     

Altered activation/detoxication enzymology following neonatal diethylstilbestrol treatment

The effects of neonatal exposure to diethylstilbestrol (DES) on hepatic activation/detoxication enzyme levels in the adult rat were investigated. Neonatal exposure of male rats to DES (DES males) decreased the endogenous levels of UDP-glucuronyltransferase as compared to control males. Female rats exposed neonatally to DES (DES females) had higher endogenous epoxide hydrolase and glutathione transferase activity levels than control females. Adult animals treated neonatally with DES also had altered metabolic potential following exposure to 3-methylcholanthrene and phenobarbital. The DES males treated in adulthood with 3-methylcholanthrene had higher benzo(a)pyrene hydroxylase activities and lower UDP-glucuronyltransferase activity levels than did control males treated in adulthood with 3-methylcholanthrene. The DES males exposed in adulthood to phenobarbital had reduced cytochrome P-450 and glutathione transferase activity levels as compared with respective controls. The DES females treated in adulthood with 3-methylcholanthrene had lower benzo(a)pyrene hydroxylase and epoxide hydrolase activity levels than control females receiving 3-methylcholanthrene. The DES females challenged in adulthood with phenobarbital also had decreased benzo(a)pyrene hydroxylase, epoxide hydrolase, UDP- glucuronyltransferase, and glutathione transferase activity levels as compared with respective controls. Our results demonstrated that neonatal exposure to DES changed the endogenous levels of specific hepatic enzymes and altered the metabolic response of these adult animals to a carcinogen and a drug.     

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.     

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.     

Induction of DT-diaphorase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (ICDD).

The compounds 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin (TCDD) are both inducers of the enzyme system aryl hydrocarbon hydroxylase. It has recently been reported that 3-MC is also an inducer of DT-diaphorase activity in rat liver. In this report the ability of TCDD to induce hepatic DT-diaphorase activity was examined. The results indicate that TCDD is approximately 17,000 times more potent as an inducer of DT-diaphorase activity than 3-MC.     

Dependency of the in vitro stabilization of differentiated functions in liver parenchymal cells on the type of cell line used for co-culture

Summary The differentiation status in cultures of primary rat liver parenchymal cells was determined by measuring the activities of various xenobiotic metabolizing enzymes. Most enzyme activities dropped rather rapidly in monocultures of parenchymal cells. The protein content and the activities of cytosolic epoxide hydrolase, glutathione S-transferase, andα-naphthol UDP-glucuronosyl transferase were, however, well stabilized in 7-day-old co-cultures of parenchymal cells with two different lines of rat liver nonparenchymal epithelial cells (NEC1 and NEC2). Phenol sulfotransferase and microsomal epoxide hydrolase activity were reduced in this coculture system after 7 days to about 30 and 20% of the initial activity. Generally, higher enzyme activities were measured in co-cultures with one specific epithelial cell line (NEC2) as compared to those with the other line (NEC1). C3H 10T1/2 mouse embryo fibroblasts supported the parenchymal cells even better than the two epithelial lines, because the activity of microsomal epoxide hydrolase was also stabilized. Glutathione transferase activity was increased over time in this co-culture system. Our results show that the differentiation status of liver parenchymal cells was much better stabilized in co-cultures than in monocultures but that, depending on the type of cells used for co-culture, great quantitative differences existed. The entire pattern of xenobiotic metabolizing enzyme activities could not be stabilized at the kind of levels found in freshly isolated parenchymal cells.     

Effect of various chemicals on the aldehyde dehydrogenase activity of the rat liver cytosol

The cytosolic activity of aldehyde dehydrogenase (ALDH) was studied in the rat liver, after acute administration of various carcinogenic and chemically related compounds. Male Wistar rats were treated with 27 different chemicals, including polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, azo dyes, as well as with some known direct-acting carcinogens. The cytosolic ALDH activity of the liver was determined either with propionaldehyde and NAD (P/NAD), or with benzaldehyde and NADP (B/NADP). The activity of ALDH remained unaffected after treatment with 1-naphthylamine, nitrosamines and also with the direct-acting chemical carcinogens tested. On the contrary, polycyclic aromatic hydrocarbons, polychlorinated biphenyls (Arochlor 1254) and 2-naphthylamine produced a remarkable increase of ALDH. In general, the response to the effectors was disproportionate between the two types of enzyme activity, being much in favour for the B/NADP activity. This fact resulted to an inversion of the ratio B/NADP vs. P/NAD, which under constitutive conditions is lower than 1. In this respect, the most potent compounds were found to be polychlorinated biphenyls, 3-methylcholanthrene, benzo(a)pyrene and 1,2,5,6-dibenzoanthracene. Our results suggest that the B/NADP activity of the soluble ALDH is greatly induced after treatment with compounds possessing aromatic ring(s) in their molecule. It is not known, if this response of the hepatocytes is related with the process of chemical carcinogenesis.     

Reduction of Brain Antioxidant Defense Upon Treatment with Butylated Hydroxyanisole (BHA) and Sudan III in Syrian Golden Hamster

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1. 4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.Deceased 4th November 1998     

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)     

Effect of inducers of DT-diaphorase on the toxicity of 2-methyl- and 2-hydroxy-1,4-naphthoquinone to rats

It has previously been shown that rats pre-treated with butylated hydroxyanisole (BHA), a well-known inducer of the enzyme DT-diaphorase, are protected against the toxic effects of 2-methyl-1,4-naphthoquinone but are made more susceptible to the harmful action of 2-hydroxy-1,4-naphthoquinone. In the present experiments, the effects of BHA have been compared with those of other inducers of DT-diaphorase. Rats were dosed with BHA, butylated hydroxytoluene (BHT), ethoxyquin (EQ), dimethyl fumarate (DMF) or disulfiram (DIS) and then challenged with a toxic dose of the naphthoquinones. All the inducers protected against the haemolytic anaemia induced by 2-methyl-1,4-naphthoquinone in rats, with BHA, BHT and EQ being somewhat more effective than DMF and DIS. A similar order of activity was recorded in the relative ability of these substances to increase hepatic activities of DT-diaphorase, consistent with a role for this enzyme in facilitating conjugation and excretion of this naphthoquinone. In contrast, all the compounds increased the haemolytic activity of 2-hydroxy-1,4-naphthoquinone. DMF and DIS were significantly more effective in this regard than BHA, BHT and EQ. DMF and DIS also caused a much greater increase in levels of DT-diaphorase in the intestine, suggesting that 2-hydroxy-1,4-naphthoquinone is activated by this enzyme in the gut. BHA, BHT and EQ had no effect on the nephrotoxicity of 2-hydroxy-1,4-naphthoquinone, but the severity of the renal lesions was decreased in rats pre-treated with DMF and DIS. The results of the present experiments show that modulation of tissue levels of DT-diaphorase may not only alter the severity of naphthoquinone toxicity in vivo, but may also change the relative toxicity of these substances to different target organs.     

Differential induction of class alpha glutathione S-transferases in mouse liver by the anticarcinogenic antioxidant butylated hydroxyanisole. Purification and characterization of glutathione S-transferase Ya1Ya1.

A novel cytosolic Alpha class glutathione S-transferase (GST) that is not normally expressed in mouse liver was found to be markedly induced (at least 20-fold) by the anti-carcinogenic compound butylated hydroxyanisole. This enzyme (designated GST Ya1 Ya1) did not bind to either the S-hexylglutathione-Sepharose or the glutathione-Sepharose affinity matrices, and purification was achieved by using bromosulphophthalein-glutathione-Sepharose. The purified isoenzyme, which comprises subunits of Mr 25,600, was characterized, and its catalytic, electrophoretic, immunochemical and structural properties are documented. GST Ya1 Ya1 was shown to be distinct from the Alpha class GST that is expressed in normal mouse liver and is composed of 25,800-Mr subunits; the Alpha class isoenzyme that is constitutively expressed in the liver is now designated GST Ya3 Ya3. Hepatic concentrations of GST Ya3 Ya3 were not significantly affected when mice were treated with butylated hydroxyanisole. Both Pi class GST (subunit Mr 24,800) and Mu class GST (subunit Mr 26,400) from female mouse liver were induced by dietary butylated hydroxyanisole. By contrast, hepatic concentrations of microsomal GST (subunit Mr 17,300) were unaffected.     

Multiplicity of induction patterns of rat liver microsomal mono-oxygenases and other polypeptides produced by administration of various xenobiotics.

Induction of hepatic microsomal mono-oxygenase species after administration of various xenobiotics is a well-documented phenomenon. To examine the number and specific species of rat liver microsomal membrane polypeptides involved in such responses, we have used sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to analyse microsomal fractions from animals treated with a number of important xenobiotics. The following are the principal points to have emerged from this study. 1. A minimum of twelve electrophoretically distinct patterns of induction of haemopolypeptides and other polypeptides could be distinguished after administration, either singly or in certain combinations, of phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls, 2-acetylaminofluorene, safrole (or isosafrole), pregnenolone-16 alpha-carbonitrile and ethanol. The patterns consisted of various permutations of the amounts of eight polypeptides of 47000-56000 mol.wt., of which at least three were haemopolypeptides. The possible identities of these polypeptides, which included species of cytochrome P-450, cytochrome P-448 and epoxide hydratase, are discussed. 2. Agents (3-methylcholanthrene, benzo[a]-pyrene, polychlorinated biphenyls, 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone) that result in the induction of cytochrome P-448 caused a marked increase in two polypeptides of 54000 and 56000 mol.wt., whereas safrole and isosafrole induced only the former polypeptide. 3. Administration of 2-acetylaminofluorene resulted in the induction of two polypeptides; evidence is presented that suggests that one of these is a species of epoxide hydratase [cf. Levin, Lu, Thomas, Ryan, Kizer & Griffin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3240-3243] ANd that the other may be a novel haemopolypeptide. 4. The overall results emphasize the complexity of the responses exhibited by rat liver microsomal fractions to the administration of xenobiotics.     

Species differences in cytochromes P-450 and epoxide hydrolase: comparisons of xenobiotic-induced hepatic microsomal polypeptides in hamsters and rats

Cytochromes P-450 and epoxide hydrolase in hamsters were studied by using two-dimensional gel electrophoresis of hepatic microsomes from untreated animals and those treated with phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, trans-stilbene oxide, and pregnenolone-16 alpha-carbonitrile. Coelectrophoresis with corresponding microsomes from rats and in situ peptide mapping were used to identify resolved microsomal polypeptides as cytochromes P-450 or epoxide hydrolase. Two forms of hepatic microsomal epoxide hydrolase were shown to exist in hamsters; these evidenced extensive structural homology with the corresponding enzyme in rats and were induced by the same xenobiotics. At least eight inducible polypeptides in microsomes from hamsters were tentatively identified as cytochromes P-450. Two of these were electrophoretically identical and structurally related with previously characterized forms of the enzyme in rats. Homologues of several major cytochromes P-450 induced by pregnenolone-16 alpha-carbonitrile and/or phenobarbital in the rat were apparently not present in the hamster. In most cases, putative forms of inducible cytochrome P-450 in the hamster existed at significant levels in microsomes from untreated animals whereas in rats the levels of most inducible forms of the enzyme were low in control microsomes, being more strictly dependent on xenobiotic pretreatment. In contrast with epoxide hydrolase, the molecular complexity of hepatic cytochrome P-450 seems to be comparable for rats and hamsters, but the structure and control of these hemoproteins appear to have markedly diverged.     

Topically applied nitropyrenes are potent inducers of cutaneous and hepatic monooxygenases

The inducibility of skin and liver microsomal cytochrome P-450 dependent aryl hydrocarbon hydroxylase and other monooxygenases by a mixture of nitropyrenes was assessed and compared with the parent non-nitrated compound, pyrene. A single topical application of nitropyrenes to neonatal rats resulted in highly significant induction of aryl hydrocarbon hydroxylase, ethoxycoumarin O-de-ethylase, and ethoxyresorufin O-de-ethylase activities in skin and liver after 24 hours. Inducibility of the skin and liver enzymes was 3.9-5.7 fold and 1.8-10.3 fold respectively. On the other hand, aminopyrine N-demethylase, benzphetamine N-demethylase and epoxide hydrolase activities in the liver were unaffected by topically applied nitropyrenes. Furthermore, treatment with nitropyrenes produced a 1 nm shift to the blue region in the wavelength maximum of hepatic microsomal cytochrome P-450. Topically applied pyrene produced only marginal or no effects on cutaneous and hepatic enzyme activities. Our results suggest that nitration of pyrene, a relatively ineffective enzyme inducer, produces nitropyrenes which are potent inducers of hepatic and cutaneous monooxygenases and they resemble 3-methylcholanthrene in this inducing effect.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

The effect of perfluordecaline on the activity of phase III xenobiotic transformation enzymes

Injection of perfluorodecaline to rats caused an increase of the phase II xenobiotic biotransformation enzyme activities followed by cytochrome P-450 induction. The activities of liver microsomal UDP-glucuronosyl transferase and glutathione transferase increased by 130 and 40%, respectively, against the control level. The increase of the cytosolic glutathione transferase activity was insignificant In contrast, the activity of sulfotransferase decreased about 2-fold. The role of modification of xenobiotic biotransformation enzymes in the biological effect of perfluorodecaline is discussed.     

Chalcone oxides—potent selective inhibitors of cytosolic epoxide hydrolase

The systematic screening of over 150 compounds for inhibitory activity on mammalian cytosolic epoxide hydrolase led to identification of chalcone oxide (trans-1-benzoyl-2-phenyloxirane) as an optimal inhibitory structure. Important structural features for inhibition include two hydrophobic moieties preferably orientating in a trans manner from an electrophilic center such as an activated olefin or epoxide, with the epoxide giving maximal activity. Synthesis of chalcone oxide derivatives bearing a single p-substituent on either phenyl ring has led to very potent inhibitors of the enzyme, the best being 4-phenylchalcone oxide (50% inhibition at 6.4 × 10^?8m). Multiple factorial analysis on the inhibition data for the two series of chalcone oxides prepared (phenyl or benzoyl substituted) revealed both the essentialness of hydrophobic interactions and the apparent nonequivalence of the two hydrophobic sites involved in the inhibitory process. Steric factors were considerably less crucial while electronic effects were unimportant in the compounds examined. The chalcone oxides were either inactive or only weak inhibitors of the other major epoxide-metabolizing enzymes in mouse liver, cytosolic glutathione S-transferase, and microsomal epoxide hydrolase. The nature of the inhibition of cytosolic epoxide hydrolase by chalcone oxides was further investigated through steady-state kinetic analysis and the use of amino acid modifiers. Chalcone oxides give a slowly reversible mixed-noncompetitive inhibition. They may interact covalently with a cysteine residue possibly essential to the catalytic action of cytosolic epoxide hydrolase, and may indeed be alternative substrates with very low turnover. The cytosolic and microsomal epoxide hydrolases can be clearly distinguished by these inhibitors, further indicating different catalytic mechanisms.     

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.