Distribution and properties of a mammalian soluble epoxide hydrase.

Two substrates, 1-(4’-ethylphenoxy)-3,7-dimethyl-6,7-epoxy-trans-2-octene and cis-epoxymethyl stearate were used to determine the distribution of epoxide hydrase activity in mammals. The highest epoxide hydrase activity in liver subcellular fractions was found in the 100,000 g supernatant and the mitochondrial fraction, while activity in washed microsomes is lower. The 100,000 g supernatant epoxide hydrase activity is present in all organs studied. This soluble epoxide hydrase activity which is highest in the liver and kidney of mice and rabbits is also present in the duodenum, muscle, colon, lung and spleen in decreasing order of activity. The level of soluble epoxide hydrase activity, although present in all mammalian species studied, is highest in female rabbits and male mice and in comparison, significantly lower in male rats. The level of epoxide hydrase activity varies with the strain of mice used and is higher in male mice than female mice. Epoxide hydrase activity in both male and female mice increases with age particularly after mice are 5 weeks old. The soluble epoxide hydrase activity requires no cofactor and has a molecular weight of approximately 130,000 as estimated by gel filtration on Sephacryl S-200. This molecular weight is about 2.5 times that reported for the solubihzed microsomal epoxide hydrase. The soluble epoxide hydrase is inhibited by inorganic ions, particularly Cu*+ and stabilized by the addition of diisopropyl fluorophosphate to the incubation mixture. Based on this data the presence of at least two epoxide hydrases in most mammalian tissues, one in the soluble fraction and the other in the microsomal fraction, is evident. Aromatic and olefinic compounds can be metabolized by mammalian oxidases to give epoxides [l, 21. In addition, epoxide containing compounds are known to occur naturally [3, 41. Some epoxides, whether naturally occurring or metabolically formed, are potentially toxic, mutagenic and/or carcinogenic. These epoxides can be metabolically transformed in mammals by conjugation with glutathione, a reaction often catalyzed by glutathione S-epoxide transferases, or hydrated by epoxide hydrases to 1,Zdiols [2,5-71. These investigative efforts, which have been directed to the role of membrane bound microsomal epoxide hydrases of mammalian liver, are based on studies first reported by Oesch and coworkers [S-lo] in which epoxide hydrase activity was found to occur predominantly in the microsomal fraction of mammalian liver using styrene oxide as a substrate. Subsequent studies utilizing other epoxides have rarely investigated the subcellular distribution of epoxide hydrase activity even when using a variety of different epoxide containing substrates [9-121. In an investigation of the metabolism of a potentially useful insect juvenile hormone mimic, significant epoxide hydrase activity was observed in the 100,000 g soluble fraction of mammalian liver [13, 141. A subsequent study [15] confirmed these initial findings that in contrast to all previous reports, significant epoxide hydrase activity is in the 100,000 g soluble fraction. The present study designates some properties and the distribution of the soluble epoxide hydrase in various species of mammals, tissues and subcellular fractions. MATERIALS AND METHODS Chemicals, l-(4’-Ethyl-‘4C-phenoxy)-3,7-dimethyl-6,7-epoxy-trans-2-octene (ethyl epoxide, 0.63 GBqimmol, >96 per cent tram) was obtained from Stauffer Chemical Company, Mountain View, CA, U.S.A. and was purified to >99 per cent as described earlier [14, 161. Unlabeled ethyl epoxide and the corresponding ethyl diol were synthesized as reported earlier [13, 161. Oleic acid (ICN, Irvine CA, l-i4C, 1.11 GBq/mmol) was esterified with diazomethane and oxidized with m-chloroperoxybenzoic acid to yield cis-9,10-epoxymethyl stearate (epoxymethyl stearate). The radioactive product showed one radioactive spot on thin-layer chromatography (tic) (Silica gel GF) in several solvent systems including hexane-ethyl acetate-acetic acid (30:20:1) and it cochromatographed with authentic epoxymethyl stearate. All other chemicals used were either of analytical grade or nanograde. Animals. For routine studies, male Swiss-Webster mice (3@-40 g, 9-10 weeks old, Hilltop Laboratories, Chatsworth, CA) were used. Alternatively, SwissWebster mice used for studies on the level of soluble epoxide hydrase activity in males and females with respect to age and male Sprague-Dawley rats (150200 g) were obtained from Simonsen Laboratories, Gilroy, CA. Swiss-Webster, AK& C57Bl and BALB mice used for studies of soluble epoxide hydrase activity in different strains were obtained from Jackson Laboratories, Bar Harbor, ME. Female New Zealand white rabbits (2-4 kg) were obtained from Vista, CA.