Induction, activation and inhibition of epoxide hydrase: an anomalous prevention of chlorobenzene-induced hepatotoxicity by an inhibitor of epoxide hydrase.
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Hydration of arene and alkene oxides by epoxide hydrase in human liver microsomes.
The comparative hydration of styrene 7,8-oxide, octene 1,2-oxide, naphthalene 1,2-oxide, phenanthrene 9,10-oxide, benzo[a]anthracene 5,6-oxide, 3-methylcholanthrene 11,12-oxide, dibenzo[a,h]anthracene 5,6-oxide, and benzo[a, 7,8-, 9,10-, and 11,12-oxides to their respective dihydrodiols was investigated in microsomes from nine human autopsy livers. The substrate specificity of the epoxide hydrase in human liver microsomes was very similar to that of the epoxide hydrase in rat liver microsomes. Phenanthrene 9,10-oxide was the best substrate for the human and rat epoxide hydrases and dibenzo[a,h]anthracene 5,6-oxide and benzo[a-a)pyrene 11, 12-oxide were the poorest substrates. Plotting epoxide hydrase activity obtained with one substrate against epoxide hydrase activity for another substrate for each of the nine human livers revealed excellent correlations for all combinations of the 11 substrates studied (r = 0.87 to 0.99). The data suggest the presence in human liver of a single epoxide hydrase with broad substrate specificity. However, the results do not exclude the possible presence in human liver of several epoxide hydrases that are under similar regulatory control. These results suggest the need for further investigation to determine whether there is a safe epoxide of a drug whose in vivo metabolism is predictive of the capacity of different individuals to metabolize a wide variety of epoxides of drugs and environmental chemicals.
Alkenes↗
Stimulation of mammalian epoxide hydrase activity by flavones.
1. A series of flavones have been examined for the ability to stimulate the activity in vitro of rat liver microsomal epoxide hydrase. Flavone and 7,8-benzoflavone both stimulate epoxide hydrase activity in vitro with styrene oxide as the substrate. The stimulation by flavone itself was several times that of 7,8-benzoflavone. 2. When tested in vitro under the same conditions, the stimulation by 7,8-benzoflavone was significantly greater than the stimulation by metyrapone, a recognized stimulator of the activity of epoxide hydrase in vitro. The stimulation by 6,7-benzoflavone was barely detectable; the 5,6-benzoflavone isomer did not stimulate epoxide hydrase activity. 3. Exposure of rate to either 7,8- or 5,6-benzoflavone for 4 days before killing did not increase the epoxide hydrase specific activity in the subsequently isolated microsomes. Exposure to flavone, however, did induce epoxide hydrase activity. 4. The possible relevance of these observations to the effects of benzoflavone upon arylhydrocarbon-induced tumours is discussed.
Animals↗
Rotation and interaction with epoxide hydrase of cytochrome P-450 in proteoliposomes.
Purified rat liver cytochrome P-450MC or P-450PB was co-reconstituted with epoxide hydrase in liposomal vesicles made of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a lipid to protein weight ratio of 5 by the cholate dialysis procedure. Rotational diffusion of the cytochromes was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane-normal" model. The measurements were used to investigate interactions of cytochrome P-450MC or P-450PB with epoxide hydrase. Different rotational mobilities of the two cytochromes were observed. The amount of mobile molecules was 78% for cytochrome P-450MC and 91% for P-450PB, and the rest was immobile within the experimental time range of 1 ms. In the presence of epoxide hydrase 85% of cytochrome P-450MC and 96% of P-450PB were mobile. Cross-linking of epoxide hydrase by anti-epoxide hydrase antibodies resulted in a drastic immobilization of the cytochromes, reducing the mobile population to 49% for P-450MC and to 60% for P-450PB. The rotational relaxation times phi of the mobile populations ranged from 210 to 283 microseconds. These results imply that both cytochromes P-450MC and P-450PB transiently associate with epoxide hydrase in liposomal membranes. Further analysis of the data showed that the angle between the heme plane of P-450MC and the membrane is 48 degrees or 62 degrees, different from the value of 55 degrees reported previously for P-450PB (Gut, J., Richter, C., Cherry, R. J., Winterhalter, K. H., and Kawato, S. (1983) J. Biol. Chem. 258, 8588-8594).
Animals↗
Characterization of rat lung epoxide (styrene oxide) hydrase with a modified radioactive assay of improved sensitivity.
The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is aobut the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg), did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.
Alcohols↗
Hepatic microsomal epoxide hydrase. Involvement of a histidine at the active site suggests a nucleophilic mechanism.
The effects of a wide variety of chemical modification reagents on the activity of purified rat liver microsomal epoxide hydrase have been investigated. Alkylating agents, such as the phenacyl bromides and benzyl bromide are potent inhibitors of epoxide hydrase. 2-Bromo-4'-nitroacetophenone (p-nitrophenacyl bromide) specifically and irreversibly inactivates epoxide hydrase. Pseudo-first order kinetics of inhibition is observed at higher inhibitor/enzyme ratios. The rate of inactivation is controlled by a group on the enzyme with an apparent pKa of 7.6. Inactivation of the enzyme with 14C-labeled 2-bromo-4'-nitroacetophenone leads to the incorporation of approximately 1 mol of radioactive inhibitor/mol of protein. Epoxide hydrase can be protected against this inactivation by the substrate phenanthrene-9,10-oxide. These results are consistent with the interpretation that 2-bromo-4'-nitroacetophenone acts as an active site-directed inhibitor. The site of alkylation by 2-bromo-4'-nitroacetophenone is a histidine residue of epoxide hydrase. The N-alkylated histidine derivative has been identified as 1-(p-nitrophenacyl)-4-histidine. A possible mechanism for the enzymatic hydration catalyzed by epoxide hydrase is discussed which involves a histidine residue of the enzyme serving as a general base catalyst for the nucleophilic addition of water.
Amino Acids↗
Epoxide hydrase and glutathione S-transferase activities with selected alkene and adrene oxides in several marine species.
Epoxide hydrase and glutathione (GSH) S-transferase activities were measured in subcellular fractions prepared from liver or hepatopancreas and some extrahepatic organs of a number of marine species common to Maine or Florida. These activities were easily detected in the species studied. In fish, hepatic GSH S-transferase activities were normally higher than hepatic epoxide hydrase activities for the alkene oxide (styrene oxide and octene oxide) and arene oxide (benzo[a]pyrene 4,5-oxide) substrates studied, whereas in crustacea, hepatopancreas epoxide hydrase activities were higher than hepatopancreas GSH S-transferase activities with the same substrates. Extrahepatic organs from fish and crustacea usually had higher GSH S-transferase activities than epoxide hydrase activities with the alkene and arene oxide substrates. GSH S-transferase activity was also found in liver or hepatopancreas of every aquatic species studied and in a number of extrahepatic organs, when 1,2-dichloro-4-nitrobenzene or 1-chloro-2,4-dinitrobenzene served as substrate.
Alkenes↗
Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.
Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.
Amino Acid Sequence↗
Effect of diastereoisomeric epoxyrotenones on hepatic epoxide hydrase activity.
The effect on epoxide hydrase activity of the two diastereomeric (6'R) and (6'S)-6',7'-epoxyrotenones formed during the oxidative metabolism of rotenone on the isopropenyl side chain is reported. The activity of microsomal epoxide hydrase was determined using styrene oxide as substrate. The results indicate that the two diastereomeric 6',7'-epoxyrotenones are good substrates for epoxide hydrase and are noncompetitive inhibitors for the hydration of styrene oxide. The more polar of the two isomers is more active as inhibitor of this enzyme.
Animals↗
Enhancement of microsomal monooxygenase, epoxide hydrase and UDPglucuronyltransferase by aldrin, dieldrin and isosafrole administrations in rat liver.
The effects of intraperitoneal administration of aldrin (10 mg/kg), dieldrin (10 mg/kg) and isosafrole (50 mg/kg) were investigated on the activities of drug biotransformation enzymes in rat liver. All the compounds studied were found to enhance the activities of microsomal monooxygenase (e.g. p-nitroanisole O-demethylase, aryl hydrocarbon hydroxylase), epoxide hydrase (styrene oxide as substrate) and UDPglucuronyltransferase (p-nitrophenol as aglycone). Dieldrin, the epoxidized derivative aldrin, the eposidized derivative of aldrin, was a more potent inducer than the parent compound itself. NADPH CYTOCHROME C REDUCTASE ACTIVITY WAS EHANCED 2.5-FOLD, P-NITROANISOLE O-demethylase 7-fold, benzpyrene hydroxylase 2-fold, and epoxide hydrase 5-fold after treating rats with dieldrin for 6 days. The increase in activity of the microsomal UDPglucuronyltransferase could be only detected after an in vitro digitonin treatment of microsomal membranes, the enhancement being about 1.5-fold after administering dieldrin for 6 days. The administration of isosafrole to rats increased especially p-nitroanisole O-demethylase activity in liver microsomes (10-fold in 3 days). NADPH cytochrome c reducatase activkty was increased 2-fold, cytochrome P-450 content 1.2-fold, benzpyrene hydrozylase activity 2.5-fold and epoxide hydrase activity 1.2-fold after treatment of rats for 3 days. UDPglucuronyltransferase activity increased 2.2-fold by treating rats for 6 days with isosafrole. This increase was, however, only to be seen in in vitro digitonin-activated microsomes due to the latency of UDPglucuronyltransferase.
Aldrin↗
Identification of epoxide hydrase as the preneoplastic antigen in rat liver hyperplastic nodules.
A liver microsomal protein, previously referred to as preneoplastic antigen, from hyperplastic nodules of rats fed a diet containing 2-acetylaminofluorene has been identified as the enzyme epoxide hydrase [glycol hydro-lyase (epoxideforming), EC 4.2.1.63]. Purified preneoplastic antigen from hyperplastic nodules and purified rat liver microsomal epoxide hydrase are immunochemically identical on the basis of Ouchterlony double-diffusion analysis. In addition, the purified proteins have identical minimum molecular weights in sodium dodecyl sulfate/polyacrylamide gels, and both proteins catalyze the hydration of arene oxides to dihydrodiols. Chronic feeding of 2-acetylaminofluorene to rats results in a 5- to 7-fold increase in epoxide hydrase activity in rat liver. The induced level of the enzyme is maintained in developing hyperplastic nodules and hepatomas but not in the nontumor tissue after removal of the carcinogen from the diet.
2-Acetylaminofluorene↗
Liver microsomal epoxide hydrase.
1. The substrate specificity of membrane-bound and purified epoxide hydrase from rat liver microsomes has been studied. Both enzyme preparations catalyzed the hydration of a variety of alkene oxidase as well as arene oxides of several polycyclic aromatic hydrocarbons. 2. Unlike the membrane-bound enzyme, the rate of hydration for most of the substrates catalyzed by the purified epoxide hydrase was constant for only 1 or 2 min. The addition of dilauroyl phosphatidylcholine or heated microsomes to the incubation mixture extended the linearity of the reaction. 3. When rat liver microsomes were used as the source of the enzyme, the apparent Km values for many of the substrates were dependent on the amount of microsomes used. When purified epoxide hydrase was used as the enzyme source and benzo(a)pyrene 11,12-oxide as substrate, the apparent Km for benzo(a)pyrene 11,12-oxide was independent of enzyme concentration but dependent on added lipid concentration. Thus, in the absence of added dilauroyl phosphatidylcholine or in the presence of this lipid at a concentration below its critical micelle concentration, the observed Km for benzo(a)pyrene 11,12-oxide remained constant. However, when the lipid concentration was greater than the critical micelle concentration, the apparent Km value increased linearly with lipid concentration. These results are consistent with a model based on the partition of lipid-soluble substrate between the lipid micelle and the aqueous medium.
Animals↗
Isolation and incorporation of rabbit liver epoxide hydrase into phospholipid vesicles.
Methods are described for the incorporation into phospholipid vesicles of epoxide hydrase isolated from liver microsomes of phenobarbital-treated rabbits. Chromatography on a Sephadex G-50 column of epoxide hydrase and egg yolk phosphatidylcholine treated with sodium cholate yielded homogeneous vesicles with a diameter of about 25 nm and containing 80 to 85% of the protein applied. At high substrate concentrations, the vesicles catalyzed the hydration of benzo(a)pyrene-4,5-oxide and styrene-7,8-epoxide at a rate similar to that obtained with the enzyme in a soluble form. However, the kinetics of styrene glycol formation catalyzed by the vesicular or microsomal preparations were complex. Convex Lineweaver-Burk plots and concave Hill plots were obtained, whereas normal Michaelis-Menten kinetics characterized the hydration catalyzed by the enzyme in a soluble form. The results could be explained if reconstitution of the enzyme into the vesicles gives rise to low affinity high capacity sites for the substrate on the enzyme, or alternatively facilitates the interaction of the substrate with such sites already present. It is suggested that reconstituted liposomes containing both the liver microsomal hydroxylase system and epoxide hydrase may prove to be a good model system for evaluating substrate specificity and factors of importance in the formation of toxic and carcinogenic metabolites by these enzymes.
Animals↗
The effect of alpha-1-acetylmethadol hydrochloride on rat hepatic aryl hydrocarbon hydroxylase and epoxide hydrase.
Male and female Wistar rats were administered alpha-1-acetylmethadol via their drinking water for one to two weeks. Hepatic aryl hydrocarbon hydroxylase, cytochrome P-450 and epoxide hydrase activities were measured after various doses of this drug. In female rats, a significant increase in aryl hydrocarbon hydroxylase and cytochrome P-450 occurred at doses as low as 4.7 mg/Kg/day. However, no change in epoxide hydrase activity occurred. In male rats, a significant increase in epoxide hydrase activity occurred at all doses tested. From these results, it is concluded that alpha-1-acetylmethadol induces a number of hepatic drug metabolizing enzyme systems.
Animals↗
Expression of mouse carbonic anhydrase VII in E. coli and demonstration of its CO2 hydrase activity.
The alpha-carbonic anhydrase (alpha-CA) gene family in mammals encodes 10 CA or CA-like proteins (CA I-CA X). Although the gene for human CA VII has been cloned and characterized, the corresponding protein has not previously been purified, and hence, the CO2 hydrase activity of its product has not as yet been demonstrated. In this study, we have cloned the mouse CA VII cDNA in an E. coli, glutathione-S-transferase (GST) expression vector. The CO2 hydrase activity of the expressed protein is about 4% that of the high-activity CAII isozyme, demonstrating that this evolutionarily highly conserved protein is a catalytically active member of this CA gene family.
Amino Acid Sequence↗
trans-Stilbene oxide: an inducer of rat hepatic microsomal and nuclear epoxide hydrase and mixed-function oxidase activities.
The administration of trans-stilbene oxide to rats resulted in increased hepatic microsomal and nuclear epoxide hydrase (with styrene oxide (SO), benzo[a]pyrene 4,5-oxide (4,5-BP) as substrates) and aryl hydrocarbon hydroxylase (AHH) activities. Hepatic microsomal aminopyrine N-demethylase, benzphetamine N-demethylase, and ethylmorphine N-demethylase activities were also increased. These increases in microsomal enzyme activity were dose- and time-dependent (about 100% at 200 mg/kg body weight, administered for 2 consecutive days). However, only marginal increases in hepatic microsomal NADPH-cytochrome c reductase activity and cytochrome P-450 content were observed. No apparent proliferation of hepatic endoplasmic reticulum occurred in trans-stilbene oxide pretreated rats. The administration of trans-stilbene oxide has no effect on hepatic glutathione S-transferase activities (with SO or 4,5-BPO as substrates). None of the parameters were affected in pulmonary microsomes from treated rats. The in vitro addition of trans-stilbene oxide (10(-6)--10(-2) M) did not affect hepatic epoxide hydrase or glutathione S-transferase activities.
Animals↗
Effects of inducers and epoxide hydrase on the metabolism of benzo(a)pyrene by liver microsomes and a reconstituted system: analysis by high pressure liquid chromatography.
The mobilities of 24 potential metabolites of benzo[a]pyrene were examined with high pressure liquid chromatography. Twelve phenols, five quinones, four dihydrodiols, and three oxides were studied. The chromatographic procedure employed allowed the separation and quantitation of benzopyrene metabolites into three major groups consisting of phenols, quinones, and dihydrodiols. Two of the benzopyrene oxides were unstable during chromatography, whereas the third oxide was more stable and chromatographed in the quinone fraction. Treatment of rats with phenobarbital or 3-methylcholanthrene enhanced the metabolism of benzopyrene by liver microsomes and altered the relative amounts of the various metabolites formed. In the absence of epoxide hydrase (EC 4.2.1.63), benzopyrene was metabolized primarily to phenols and quinones but was not appreciably metabolized to dihydrodiols by a solubilized, reconstituted cytochrome P-448 monooxygenase system. Addition of partially purified epoxide hydrase resulted in the formation of benzopyrene dihydrodiols with a concomitant decrease in the formation of phenolic metabolites, indicating that benzopyrene undergoes metabolism via arene oxides that are precursors for dihydrodiols and phenols.
Animals↗
Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2-acetylaminofluorene.
A knowledge of the biological characteristics of carcinogen-induced hyperplastic nodules of rat liver may be important in the understanding of cancer development. Although its biological role remains to be elucidated, the level of microsomal epoxide hydrase (epoxide hydrolase, EC 3.3.2.3) is 5- to 7-fold greater in hyperplastic nodules nodules induced by feeding the hepatocarcinogen 2-acetylaminofluorene than in liver of control rats. After removal of the carcinogen from the diet, the high level of the enzyme is maintained in those nodules that persist and in the hepatocellular carcinomas that subsequently develop. The availability of antibody to the epoxide hydrase made it possible to use electron microscopic immunocytochemistry to localize this enzyme in the cells of hyperplastic nodules. The immunocytochemical procedure provides direct visual evidence for the presence of this enzyme in smooth endoplasmic reticulum and also in rough endoplasmic reticulum (including the nuclear envelope) of the nodule's parenchymal cells.
2-Acetylaminofluorene↗