Hepatic cytochrome P-448 and epoxide hydrase: enzymes of nuclear origin are immunochemically identical with those of microsomal origin.

Nuclear and microsomal sources of hepatic cytochrome P-448 and epoxide hydrase were compared using antibodies made against the pure antigens isolated from rat liver microsomes. Both antigens were easily detected in detergent-solubilized nuclei and microsomes from rats using the Ouchterlony double-diffusion technique. Epoxide hydrase from either whole nuclei or nuclear envelope was immunochemically identical with the enzyme isolated from microsomes. Similarly, in rats pretreated with 3-methylcholanthrene, the cytochrome P-448 of nuclear origin was immunochemically indistinguishable from the enzyme derived from microsomes. These results establish the immunochemical identity of these hepatic nuclear and microsomal enzymes and provide a firm basis for applying the knowledge gained with the microsomal system of metabolism to the nuclear system.     

Comparison of nuclear and microsomal epoxide hydrase from rat liver

The specific activities of hydration of nine arene and alkene oxides by purified nuclei prepared from the livers of 3-methylcholanthrene-pretreated rats were found to fall within the range of 2.2 to 9.1% of the corresponding microsomal values. Pretreatment with phenobarbital enhanced both the nuclear and microsomal hydration of phenanthrene-9,10-oxide, benzo(a)pyrene-11,12-oxide, and octene-1,2-oxide. 3-Methylcholanthrene pretreatment enhanced the nuclear hydration of these three substrates by 30–60% but had no significant effect on microsomal hydration. An epoxide hydrase modifier, metyrapone, stimulated the hydration of octene-1,2-oxide by the two organelles to quantitatively similar extents, but affected the nuclear and microsomal hydration of benzo(a)pyrene-4,5-oxide differentially. Cyclohexene oxide also exerted differential effects on nuclear and microsomal epoxide hydrase which were dependent both on the substrate and on the organelle. The inhibition by this agent of nuclear and microsomal epoxide hydrase was quantitatively similar only for a single substrate, benzo(a)anthracene-5,6-oxide. When purified by immunoaffinity chromatography, nuclear and microsomal epoxide hydrases from 3-methylcholanthrene-pretreated rats were shown to have identical minimum molecular weights (? 49,000) on polyacrylamide gels in the presence of sodium dodecyl sulfate. These findings support the assertion that microsomal metabolism can no longer be considered an exclusive index of the cellular activation of polycyclic aromatic hydrocarbons.     

The induction of hepatic cytochrome P-450 in C57 BL/10 and DBA/2 mice by isosafrole and piperonyl butoxide. A comparative study with other inducing agents

Styrene monooxygenase activity was measured in intact nuclear preparations from rat liver by means of a gas chromatographic method. Styrene epoxide formation is NADPH-dependent although it is enhanced when NADH is added with NADPH. This activity is inhibited by microsomal monooxygenase inhibitors SKF 525A and metyrapone and by microsomal epoxide hydrase inhibitors 1,2-epoxy-3,3,3-trichloropropene oxide and cyclohexene oxide. The percentage of inhibition is quantitatively dffferent for the four compounds. Known inducers of liver microsomal monooxygenase show different patterns of induction on nuclear preparations. Phenobarbital induces nuclear monooxygenase activity more than the respective microsomal activity, whereas the contrary holds true for β-naphthoflavone.     

Halogenated epoxides and related compounds as inhibitors of epoxide hydrase

A variety of chlorinated and fluorinated epoxides and related compounds were synthesized and evaluated as inhibitors of epoxide hydrase. The compounds were tested using chicken liver microsomes and a radiometric assay based on [³H]styrene oxide, and using partially purified chicken liver microsomal epoxide hydrase and a continuous photometric assay based on p-nitrostyrene oxide, whose hydration could be monitored at 310 nm. For the 16 compounds studied both assays gave similar patterns of inhibitory activity. As expected from the relative K_m values of the two substrates, all inhibitors were considerably more active against styrene oxide (K_m =1.0 mM) than against p-nitrostyrene oxide (K_m = 4.2 μM), and styrene oxide was a weak alternate-substrate inhibitor against p-nitrostyrene oxide. 1,1,1-Trichloropropene oxide, however, was a potent alternate-substrate inhibitor against p-nitrostyrene oxide. Addition of various substituents to the α-carbon of styrene oxide generated a series of compounds whose inhibitory potency toward p-nitrostyrene oxide increased in the order H ≈ CF₃ < CH₃ < CH₂Cl < CHCl₂ < CCl₃ ≈ 1,1,1-trichloropropene oxide. In contrast, addition of a CH₃ or CCl₃ group to the β-carbon of styrene oxide resulted in only a modest increase in inhibitory potency. 2-Phenyl- and 3-phenyloxetane showed no pronounced inhibitory activity toward either styrene oxide or p-nitrostyrene oxide, but pentafluorophenyl ethylene oxide and 1,1, 1-trichlorobutane-3,4-oxide were moderately active inhibitors, although significantly less potent than 1,1,1-trichloroproene oxide. These results show that electronegativity, steric effects, and hydrophobic effects are each important in governing the interaction of epoxide hydrase substrates with the enzyme, although it is not yet possible to analyze separately the effects of each of these parameters on K_m, V, and the catalytic mechanism.     

Induction, activation and inhibition of epoxide hydrase: an anomalous prevention of chlorobenzene-induced hepatotoxicity by an inhibitor of epoxide hydrase

Epoxide hydrase activity, measured with [³H]styrene oxide as substrate, is present in mammalian liver, kidney, lung, intestine and skin. The hepatic level of the enzyme, measured in vitro with [³H]styrene oxide, benzene oxide or naphthalene-1,2-oxide, is elevated substantially by pretreatment of rats with phenobarbital and to a lesser extent by pretreatment with 3-methylcholanthrene. Metyrapone and 1-(2-isopropylphenyl)-imidazole, two monooxygenase inhibitors, activate epoxide hydrase in vitro, but have no demonstrable effect on the enzyme in vivo. 3,3,3-Trichloropropene oxide, a potent in vitro inhibitor of epoxide hydrase, has no effect on monooxygenase activity measured in vitro with [³H]benzenesulfonanilide. Trichloropropene oxide is extremely toxic. In sub-lethal dosages, it does not significantly inhibit epoxide hydrase activity in vivo, although it and several other epoxides do react with and thereby reduce hepatic levels of glutathione. Cyclohexane oxide, another potent in vitro inhibitor of epoxide hydrase, reduces hepatic glutathione levels to 10% of control values. This relatively non-toxic substance should potentiate the hepatotoxicity of chlorobenzene by inhibiting further metabolism of the toxic chlorobenzene oxide intermediate through either hydration or conjugation with glutathione. Instead, co-administration of cyclohexene oxide and chlorobenzene significantly reduces the rate of metabolism of [¹⁴C]chlorobenzene and prevents the hepatic centrilobular necrosis caused by chlorobenzene in rats. Arene oxide-mediated hepatotoxicity apparently is dependent upon a variety of factors including both rates of formation and degradation of arene oxides in tissue. The presently known hydrase inhibitors are not sufficiently selective in their effects on liver cells to permit a quantitative assessment of the relative importance of these factors.     

The metabolism of α-naphthoflavone (7,8-benzoflavone) by hepatic microsomes from the marine fish Stenotomus versicolor

Incubation of α-naphthoflavone with fish (scup; Stenotomus versicolor) liver microsomes and NADPH resulted in the production of a major component and several minor components analyzed by high pressure liquid chromatography. The appearance of these components was dependent on time, native protein, NADPH, and O₂ and was strongly inhibited by carbon monoxide. The appearance of the major component was abolished by addition of the epoxide hydrase inhibitor trichloropropene oxide. Mass spectral analysis of the major component yielded a molecular weight of 306. The results strongly indicate that α-naphthoflavone is metabolized by scup hepatic microsomal mixed-function oxygenases and epoxide hydrase and that the major product is a dihydrodiol.     

Interrelationships of dietary protein level,aflatoxin B1 metabolism,and hepatic microsomal epoxide hydrase activity

In a continuation of studies on protein intake and aflatoxin B₁ (AFB₁) metabolism, weanling rats were fed semipurified diets containing either 20% casein or 5% casein for two weeks to determine the effect of dietary protein level on hepatic microsomal epoxide hydrase activity and AFB₁ metabolism in an effort to evaluate the role of protein intake on the formation and degradation of the reactive metabolite of AFB₁. Styrene oxide was used as substrate for epoxide hydrase since the hypothetical AFB₁ 2,3-epoxide (AFB-epox) cannot be synthesized because of its lability. Two groups of animals were fed 20% casein diets; one was fed ad libitum and the second was pair fed to the 5% casein group in order to control the effects of total feed intake. The depression of epoxide hydrase activities caused by the 5% casein diets was approximately equivalent to that previously seen with hepatic microsomal mixed function oxidase (MFO) activities with the identical protocol. Similarly, the metabolism of AFB₁ to AFQ₁ and AFM₁ was depressed by the 5% casein diets, with an increase in the production of chromatographically more polar material. The relationship of the MFO and epoxide hydrase activities to AFB₁ metabolism and formation of macromolecular adducts is discussed.     

Nuclear metabolism. II. Further studies on epoxide hydrolase activity

Apparent K_m- and V_max-values of nuclear styrene 7,8-oxide hydrolase were determined at different protein concentrations. In the protein concentrations range used no significant differences in the apparent K_m-values were observed. The influence of the incubation with different modifiers (i.e. SKF-525A, metyrapone, 1,2-epoxy-3,3,3 trichloropropane, cyclohexene oxide) at two different concentrations on this enzyme activity was also determined. Cyclohexene oxide and 1,2-epoxy-3,3,3-trichloropropane, two well known inhibitors of the microsomal epoxide hydrolase(s) caused a marked inhibition, metyrapone had a strong activating effect whereas SKF-525A had no effect. In vivo pretreatment with phenobarbital significantly induced the nuclear epoxide hydrolase whereas β-naphthoflavone caused a lower degree of induction. This pattern is quantitatively different but qualitatively very similar to the microsomal one. Moreover a toxifying to detoxifying enzymatic activity balance is attempted for the metabolization of the alkenic double bond of styrene, taking into account the ratio between the styrene monooxygenase (toxifying enzyme) and the styrene 7,8-oxide hydrolase (detoxifying enzyme) after the above mentioned pretreatments, both in the microsomal and nuclear fractions.     

NUCLEAR MEMBRANES FROM MAMMALIAN LIVER : I. Isolation Procedure and General Characterization

Nuclear membranes were isolated from rat and pig liver by sonication of highly purified nuclear fractions and subsequent removal of adhering nucleoproteins in a high salt medium. The fractions were examined in the electron microscope by both negative staining and thin sectioning techniques and were found to consist of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents still could frequently be recognized. The chemical composition of the nuclear membrane fractions was determined and compared with those of microsomal fractions prepared in parallel. For total nuclei as well as for nuclear membranes and microsomes, various enzyme activities were studied. The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids. Both membranous fractions had many proteins in common including some membrane-bound enzymes. Activities in Mg-ATPase and the two examined cytochrome reductases were of the same order of magnitude. The content of cytochrome b₅ as well as of P-450 was markedly lower in the nuclear membranes. The nuclear membranes were found to have a higher buoyant density and to be richer in protein. The glucose-6-phosphatase and Na-K-ATPase activities in the nuclear membrane fraction were very low. In the gel electrophoresis, in addition to many common protein bands, some characteristic ones for either microsomal or nuclear membranous material were detected. Significant small amounts of DNA and RNA were found to remain closely associated with the nuclear envelope fragments. Our findings indicate that nuclear and endoplasmic reticulum membranes which are known to be in morphological continuity have, besides a far-reaching similarity, some characteristic differences.     

Assay and partial purification of epoxide hydrase from rat liver microsomes

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [³H]styrene oxide, [³H]naphthalene oxide, [³H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [³H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [³H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.     

Subcellular and organ distribution of cholesterol epoxide hydrolase in the rat

The subcellular and organ distributions of microsomal epoxide hydrolases measured with cis-stilbene oxide and cholesterol 5,6 alpha-epoxide as substrates have been investigated. These two enzyme activities were found to have essentially the same subcellular distribution, with the highest total and specific activities localized in rough and smooth endoplasmic reticulum. Among the tissues studied (i.e., liver, kidney, lung, testis, spleen, brain and intestinal epithelium), the highest specific activities were recovered in liver microsomes, where the activities were at least 5-fold greater than in any of the other microsomal preparations.     

Properties of isolated frog liver and kidney nuclei

In order for a nuclear preparation to be used for analytical purposes, the method of isolation and composition of the suspension medium must be carefully examined. Accordingly, satisfactory techniques for the isolation of frog liver and kidney nuclei were developed. The medium for frog liver nuclei consisted of: 55% glycerol, 0.001 M magnesium chloride, 0.033 M sodium β-glycerophosphate and/or 0.002 M KH₂PO₄, K₂HPO₄ (pH 6.8), however, the addition of 0.15 M sucrose was essential for satisfactory isolation of kidney nuclei. Inclusion of sucrose (0.15 M ) in the isolation medium promoted nucleolar swelling and a decrease in nuclear volume in liver cell nuclei. Nucleolar migration and extrusion were noted in solutions with high cationic content. The morphological appearance of isolated nuclei was found to be extremely sensitive to the ionic strength of the isolation medium, as was the isolation procedure in toto. Effects were considered to be the result of precipitation and swelling of nucleoprotein. Dissociation of nucleoprotein was considered to be associated with temperature change. The uptake of supra-vital dyes aided in recognition of the morphological alterations and was also an indicator of nuclear viability. Trypsin readily altered the nuclear membrane and a rapid decrease in nuclear density occurred, but the nucleolus remained intact. The diverse response of liver and kidney nuclei as compared with the nucleated red blood cells (a contaminant) to treatment with trypsin was noted and its implications discussed.     

The metabolism of organochlorine compound by microsomal enzymes of the shag (Phalacrocorax aristotelis).

1. The activities of microsomal enzymes of adult male shags (Phalacrocorax aristotelis) towards the organochlorine substrates HHDN, HCE and Heom were compred with those of microsomal enzymes of the adult male Wistar rat. 2. Liver homogenates showed similar epoxide hydrase activity to kidney homogenates in the shag, but in the rat liver preparations was much more active than the kidney preparation. 3. Liver microsomes of the shag showed smaller than 8% of the epoxide hydrase activity and smaller than 14% of the hydroxylating capacity of liver microsomes from the rat. 4. The relatively low activity of these enzymes is probably the main reason why the shag has been found to contain relatively high levels of dieldrin in ecological studies.     

Fate of naturally occurring epoxy acids: a soluble epoxide hydrase, which catalyzes cis hydration, from Fusarium solani pisi.

A cell-free extract prepared from Fusarium solani pisi grown on cutin, catalyzed the hydration of 18-hydroxy-9,10-epoxyoctadecanoic acid to 9,10,18-trihydroxyoctadecanoic acid while extracts from glucose-grown cells contained <6% of this activity. The product was identified by Chromatographic techniques and by radio gas-liquid chromatography of its periodate oxidation products. This epoxide hydrase activity had a pH optimum at 9.0 and it was located mainly in the 100,000g supernatant fraction. Rate of hydration of the epoxy acid was linear up to 15 min and up to a protein concentration of 30 μg/ml. This fungal epoxide hydrase has a molecular weight of 35,000, as determined by Sephadex G-100 gel filtration. It was partially purified by ammonium sulfate fractionation and gel filtration. The apparent K_m and V of the enzyme was 2 × 10^?4m and 222 nmoles/min/mg, respectively. Parachloromercuribenzoate strongly inhibited the enzyme, while N-ethylmaleimide was a less potent inhibitor. 1,1,1,-Trichloropropylene-2,3-oxide at 10^?3m gave 50% inhibition of the hydration of 18-hydroxy-9,10-epoxyoctadecanoic acid. Kinetic analysis showed that trichloropropylene oxide was a competitive inhibitor. 18-Acetoxy-9,10-epox-yoctadecanoic acid, methyl 18-acetoxy-9,10-epoxyoctadecanoate, 9,10-epoxyoctadecanoic acid, and styrene oxide were not readily hydrated by this fungal epoxide hydrase showing that it has a stringent substrate specificity. Analysis of the enzymatic hydration product on boric acid-impregnated silica gel plates showed that the product obtained from the cis epoxide was exclusively erythro while acid hydrolysis of this epoxide gave rise to the expected threo product. This enzyme is novel in that it catalyzes cis hydration of epoxide while the other epoxide hydrases heretofore isolated catalyzed trans hydration of epoxides.     

UPD-glucuronosyltransferase,epoxide hydrolase and gultathione S-transferase

The activities of UDPglucuronosyltransferase, microsomal epoxide hydrolas and cytosolic glutathione S-transferase were measured in the liver of spontaneously (db/db and ob/ob) or streptozotocin-induced diabetic mice. An important (2–3-fold) increase of most phase II activities was observed instreptozotocin-treated animals, whereas sligher changes were detected in spontaneously diabetic animals. The latter exhibit physico-chemical modifications of liver microsomal membranes, as shown by the temperature-induced variations of epoxide hydrolase activity.     

Purification of human liver microsomal epoxide hydrase. Differences in the properties of the human and rat enzymes.

Human liver microsomal epoxide hydrase has been highly purified to a specific activity (570 to 620 nmol/min/mg of protein) comparable to that of the rat enzyme using styrene oxide as substrate. Like the purified rat liver microsomal epoxide hydrase, the human enzyme has a minimum molecular weight of 49,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and exhibits broad substrate specificity toward a variety of alkene and arene oxides. Despite these similarities, the human and rat enzymes are different proteins as judged by their immunochemical properties as well as their relative catalytic activities toward certain substrates.     

THE ISOLATION OF A CELL MEMBRANE FRACTION FROM RAT LIVER

A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO₃, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ~0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.     

Pentamidine activates T1 the hepatic microsomal glucose 6-phosphate transport protein of the glucose-6-phosphatase system

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) in vitro by pentamidine has been investigated in both intact and fully disrupted microsomes. The major effect of pentamidine is a 4.7-fold reduction in the K_m of glucose-6-phosphatase activity in intact diabetic rat liver microsomes. The site of action of pentamidine is T₁ the hepatic microsomal glucose 6-phosphate transport protein. The activation of T₁ by pentamidine may contribute to the disturbed blood glucose homeostasis see in many patients after administration of the drug pentamidine.     

PLA2 activity in rat liver nuclei

• 1.1. Subcellular fractions of rat liver were assayed for PLA₂ activity.
• 2.2. The PLA₂ assay measures the release of [³ H]oleic acid from phospholipids, using labeled E. coli as substrate.
• 3.3. Nuclear fractions contained PLA₂ activity, which was Ca²⁺ dependent and could not be explained from mitochondrial, microsomal or plasma membrane contamination.
• 4.4. The V_max value of nuclear PLA₂ is 0.30 ± 0.04 pmol oleic acid/min/mg protein; its K_m value is 0.86±0.12μM, similar to that of mitochondrial PLA₂.
• 5.5. We conclude that rat liver nuclei contain PLA₂ activity.