Soluble epoxide hydrolase (sEH) hydrolyzes a wide variety of endogenous and exogenous epoxides. Many of these epoxides are believed to be formed by cytochrome P450 epoxygenases. Here we report the distribution of sEH and cytochrome P450 epoxygenases 2C8, 2C9, and 2J2 by immunohistochemistry. A large number of different tissues from different organs were evaluated using high-throughput tissue microarrays. sEH was found in the liver, kidney, and in many other organs, including adrenals, pancreatic islets, pituitary gland, lymphoid tissues, muscles, certain vascular smooth muscles, and epithelial cells in the skin, prostatic ducts, and the gastrointestinal tract. Immunolabeling for sEH was highly specific for particular tissues and individual cell types. CYP2C9 was also found in almost all of these organs and tissues, suggesting that 2C9 and sEH are very similar in their tissue-specific patterns of expression. CYP2C8 and 2J2 were also widely distributed in human tissues but were less frequently associated with sEH. The results suggest potentially distinct pathways of endogenous fatty acid epoxide production and hydrolysis in a variety of human tissues.

Human soluble epoxide hydrolase (hsEH) metabolizes a variety of epoxides to the corresponding vicinal diols. Arachidonic and linoleic acid epoxides are thought to be endogenous substrates for hsEH. Enzyme activity in humans shows high interindividual variation (e.g., 500-fold in liver) suggesting the existence of regulatory and/or structural gene polymorphisms. We resequenced each of the 19 exons of the hsEH gene (EPHX2) from 72 persons representing black, Asian, and white populations. A variety of polymorphisms was found, six of which result in amino acid substitutions. Amino acid variants were localized on the crystal structure of the mouse sEH, resulting in the prediction that at least two of these (Arg287Gln and Arg103Cys) might significantly affect enzyme function. The six variants of the hsEH cDNA corresponding to each single polymorphism and one corresponding to a double polymorphism were then constructed by site-directed mutagenesis and expressed in insect cells. As predicted, Arg287Gln and the double mutant Arg287Gln/Arg103Cys showed decreased enzyme activity using trans-stilbene oxide, trans-diphenylpropene oxide, and 14,15-epoxyeicosatrienoic acid as substrates. Lys55Arg and Cys154Tyr mutants had elevated activity for all three substrates. Detailed kinetic studies revealed that the double mutant Arg287Gln/Arg103Cys showed significant differences in K m and V max . In addition, stability studies showed that the double mutant was less stable than wild-type protein when incubated at 37°C. These results suggest that at least six hsEH variants exist in the human population and that at least four of these may influence hsEH-mediated metabolism of exogenous and endogenous epoxide substrates in vivo.Epoxide hydrolases (EC 3.3.2.3) metabolize exogenous and endogenous epoxides by hydrolyzing them to vicinal diols, which are usually less reactive and less mutagenic because of their higher hydrophilicity. sEH is one of five epoxide hydrolases (the others are hepoxilin EH, leukotriene A 4 hydrolase, cholesterol EH, and microsomal EH), which differ in molecular weight, subcellular localization, pI and substrate specificity.Previous work suggests the existence of one hsEH gene localized to chromosomal region 8p21-p12 (Larsson et al., 1995). The human sEH gene (EPHX2) consists of 19 exons encoding 555 amino acids (Sandberg and Meijer, 1996). Because the human and mouse proteins are 73% identical (Beetham et al., 1995) with 100% identity in residues forming the catalytic triad, the crystal structure of murine sEH (Argiriadi et al., 1999) is a good model for predicting structurefunction correlations of the hsEH. Each monomer of the homodimeric mouse sEH has two domains: an N-terminal domain and a C-terminal catalytic domain connected by a proline rich linker (Argiriadi et al., 1999). The catalytic mechanism involves formation of a covalent alkylenzyme ester intermediate as a result of nucleophilic attack by Asp333. This is subsequently hydrolyzed with assistance of the general base His523 in a charge relay with...

R source code for the method is available upon request.

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