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-B-ethylstyrene oxide, mznsstilbene 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$-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. Epoxidized compounds are an important group of xenobiotics which may either be present in our environment or form metabolically in vivo from olefinic or aromatic compounds. A number of these epoxides are potentially toxic, mutagenic and/or carcinogenic [l]. Such epoxides can undergo metabolic conjugation with glutathione (GSH), a reaction sometimes catalyzed by glutathione S-transferases (GSH transferases); be reduced enzymatitally to the corresponding olefin; or be hydrolyzed to diols by epoxide hydrolases [2-51. Of these metabolic routes, the greatest attention has been focused on epoxide hydrolases. The epoxide hydrolases are ubiquitous in nature, occurring in all vertebrate species tested [6] and in a variety of other organisms. These epoxide hydrolases have also been detected in almost all mammalian tissues tested [6,7]. Subcellular distribution studies initially indicated that styrene oxide hydrolase activity in such mammalian tissues occurred predominantly in the endoplasmic reticulum [8,9]. Subsequent studies, however, have shown that epox* Author to whom all correspondence should be addressed: Dr. Sarjeet S. Gill, Department of Entomology, University of California, Davis, CA 95616, U.S.A. t One becquerel (Bq) equals one disintegration per second (International System of Units). ide hydrolase activity is fairly widespread in the liver cells if different compounds are utilized as substrates. For instance, a higher level of epoxide hydrolase activity is observed in the 100,OOOg supernatant fraction than in any other subfraction if juvenile hormone mimics or epoxidized fatty acids are used as substrates [10-141, and epoxide hydrolase activity also occurs in the nuclear membrane of mammalian liver cells [15]. The nuclear epoxide hydrolase activity monitored with styrene oxide and benzo[a] pyrene 4,5-oxide appears to be due to the same enzyme as that present in the endoplasmic reticulum [16]. In routine subcellular distribution studies on epoxide hydrolase activity in mammalian liver, significant activity was encountered in the mitochondrial fraction [13,14]. This study verifies the occurrence of epoxide hydrolase activities in mammalian liver mitochondria using trans-fi-ethylstyrene oxide and other epoxides as substrates. MATERIALS AND METHODS Chemicals. 1-[4’-Ethyl-‘%-phenoxyl-3,7-dimethyl-6,7-epoxy-2E-octene (ethyl epoxide, 0.63 GBqt/mmole, > 96 per cent E) was obtained from the Stauffer Chemical Co., Mountain View, CA, U.S.A., and purified to >99 per cent as described [ll, 171. Methyl cis-9,10-epoxystarate was synthesized from [ 1-‘“Cl-oleic acid (1.1 GBqimmole,