Soluble Epoxide Hydrolase Regulation of Lipid Mediators Limits Pain.

The role of lipids in pain signaling is well established and built on decades of knowledge about the pain and inflammation produced by prostaglandin and leukotriene metabolites of cyclooxygenase and lipoxygenase metabolism, respectively. The analgesic properties of other lipid metabolites are more recently coming to light. Lipid metabolites have been observed to act directly at ion channels and G protein-coupled receptors on nociceptive neurons as well as act indirectly at cellular membranes. Cytochrome P450 metabolism of specifically long-chain fatty acids forms epoxide metabolites, the epoxy-fatty acids (EpFA). The biological role of these metabolites has been found to mediate analgesia in several types of pain pathology. EpFA act through a variety of direct and indirect mechanisms to limit pain and inflammation including nuclear receptor agonism, limiting endoplasmic reticulum stress and blocking mitochondrial dysfunction. Small molecule inhibitors of the soluble epoxide hydrolase can stabilize the EpFA in vivo, and this approach has demonstrated relief in preclinical modeled pain pathology. Moreover, the ability to block neuroinflammation extends the potential benefit of targeting soluble epoxide hydrolase to maintain EpFA for neuroprotection in neurodegenerative disease.

Expression of human microsomal epoxide hydrolase in Saccharomyces cerevisiae reveals a functional role in aflatoxin B1 detoxification.

The metabolism and genotoxicity of the carcinogenic mycotoxin, aflatoxin B1 (AFB), was studied in the lower eukaryotic yeast Saccharomyces cerevisiae. Recombinant strains of yeast were engineered to express human cDNAs for CYP1A1, CYP1A2, and microsomal epoxide hydrolase (mEH). Coexpression of mEH with CYP1A1 or CYP1A2 resulted in significant decreases in measurements of AFB genotoxicity. In cells expressing CYP1A2 and mEH, the level of AFB-DNA adducts was decreased by 50% relative to cells expressing CYP1A2 alone. Mitotic recombination, as assayed by gene conversion at the trp5 locus, was diminished by 50% or greater in cells coexpressing mEH and CYP1A2 compared to CYP1A2 alone. The mutagenicity of AFB in the Ames assay was also decreased by approximately 50% when AFB was incubated with microsomes containing CYP1A1 or CYP1A2 and mEH versus CYP1A1 or CYP1A2 alone. The biotransformation of AFB by CYPs is known to involve the generation of a reactive epoxide intermediate, AFB-8,9-epoxide, but previous direct biochemical and kinetic studies have failed to demonstrate any functional role for mEH in AFB detoxification. By reconstructing a metabolic pathway in intact yeast, we have shown, for the first time, that mEH may play a role in mitigating the carcinogenic effects of AFB.

Mouse endometrium stromal cells express a polycyclic aromatic hydrocarbon-inducible cytochrome P450 that closely resembles the novel P450 in mouse embryo fibroblasts (P450EF).

Stromal cells from mouse endometrium, E041 cells, at passages 21, 23 and 26 were metabolically active towards 7,12-dimethylbenz[a]anthracene (DMBA). The total DMBA-metabolizing activity of E041 cells was preferentially increased by benz[a]anthracene (BA) relative to 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) treatment (7-fold by BA and 4-fold by TCDD), but the relative proportions of DMBA metabolites formed remained unchanged. Profiles of DMBA metabolites generated from E041 cell microsomes were very different from that of mouse P4501A1 but exhibited similarities to that of P450EF, the polycyclic aromatic hydrocarbon (PAH)-inducible cytochrome P450 in the mouse embryo fibroblast cell line C3H10T1/2 (10T1/2). Notably, both induced and uninduced E041 cell microsomes were very effective in the formation of the proximate carcinogen DMBA-3,4-dihydrodiol (15-20% of total) and DMBA-10,11-dihydrodiol (13-18% of total) but ineffective in forming the 7-hydroxymethyl derivative of DMBA (< 1% of total). Antibodies to P450EF completely inhibited the DMBA-metabolizing activities of both induced and uninduced E041 cell microsomes, with an effectiveness similar to that in microsomes prepared from identically treated 10T1/2 cells. Anti-P4501A1 had no inhibitory effect on DMBA metabolism by either induced or uninduced E041 cell microsomes. Total DMBA-metabolizing activities in BA- and TCDD-induced E041 cells were consistently 2-fold lower compared to those in similarly treated 10T1/2 cells. In addition, both induced and uninduced E041 cell microsomes formed a lower proportion of DMBA dihydrodiols relative to phenols in comparison to identically treated 10T1/2 cell microsomes (0.5 versus 1). Addition of exogenous epoxide hydrolase to E041 cell microsomes resulted in a product distribution indistinguishable from that in 10T1/2 cells. Immunoblots demonstrated 5-fold lower levels of epoxide hydrolase in E041 cell microsomes compared to 10T1/2 cell microsomes. Anti-P450EF immunoblotted a 55 kd protein in E041 cell microsomes that was induced 14-fold by BA and 6-fold by TCDD, thus paralleling the increases in the respective DMBA metabolism. It is therefore concluded that following PAH exposure endometrial stromal cells express the novel PAH-inducible mouse embryo fibroblast P450 and fail to express P4501A1.