Genetic disruption of soluble epoxide hydrolase is protective against streptozotocin-induced diabetic nephropathy

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important roles in regulating cardiovascular functions. The anti-inflammatory, antiapoptotic, proangiogenic, and antihypertensive properties of EETs suggest a beneficial role for EETs in diabetic nephropathy. Endogenous EET levels are maintained by a balance between synthesis by CYP epoxygenases and hydrolysis by epoxide hydrolases into physiologically less active dihydroxyeicosatrienoic acids. Genetic disruption of soluble epoxide hydrolase (sEH/EPHX2) results in increased EET levels through decreased hydrolysis. This study investigated the effects of sEH gene disruption on diabetic nephropathy in streptozotocin-induced diabetic mice. Streptozotocin-induced diabetic manifestations were attenuated in sEH-deficient mice relative to wild-type controls, with significantly decreased levels of Hb A(1c), creatinine, and blood urea nitrogen and urinary microalbumin excretion. The sEH-deficient diabetic mice also had decreased renal tubular apoptosis that coincided with increased levels of antiapoptotic Bcl-2 and Bcl-xl, and decreased levels of the proapoptotic Bax. These effects were associated with activation of the PI3K-Akt-NOS3 and AMPK signaling cascades. sEH gene inhibition and exogenous EETs significantly protected HK-2 cells from TNFα-induced apoptosis in vitro. These findings highlight the beneficial role of the CYP epoxygenase-EETs-sEH system in the pathogenesis of diabetic nephropathy and suggest that the sEH inhibitors available may be potential therapeutic agents for this condition.     

Opposite regulation of cholesterol levels by the phosphatase and hydrolase domains of soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme with two catalytic domains: a C-terminal epoxide hydrolase domain and an N-terminal phosphatase domain. Epidemiology and animal studies have attributed a variety of cardiovascular and anti-inflammatory effects to the C-terminal epoxide hydrolase domain. The recent association of sEH with cholesterol-related disorders, peroxisome proliferator-activated receptor activity, and the isoprenoid/cholesterol biosynthesis pathway additionally suggest a role of sEH in regulating cholesterol metabolism. Here we used sEH knock-out (sEH-KO) mice and transfected HepG2 cells to evaluate the phosphatase and hydrolase domains in regulating cholesterol levels. In sEH-KO male mice we found a approximately 25% decrease in plasma total cholesterol as compared with wild type (sEH-WT) male mice. Consistent with plasma cholesterol levels, liver expression of HMG-CoA reductase was found to be approximately 2-fold lower in sEH-KO male mice. Additionally, HepG2 cells stably expressing human sEH with phosphatase only or hydrolase only activity demonstrate independent and opposite roles of the two sEH domains. Whereas the phosphatase domain elevated cholesterol levels, the hydrolase domain lowered cholesterol levels. Hydrolase inhibitor treatment in sEH-WT male and female mice as well as HepG2 cells expressing human sEH resulted in higher cholesterol levels, thus mimicking the effect of expressing the phosphatase domain in HepG2 cells. In conclusion, we show that sEH regulates cholesterol levels in vivo and in vitro, and we propose the phosphatase domain as a potential therapeutic target in hypercholesterolemia-related disorders.     

Targeted disruption of the microsomal epoxide hydrolase gene. Microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12-dimethylbenz[a]anthracene.

Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.     

Adamantyl thioureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing one or two thiourea groups has been developed. Inhibition potency of the described compounds ranges from 50?μM to 7.2?nM. 1,7-(Heptamethylene)bis[(adamant-1-yl)thiourea] (6f) was found to be the most potent sEH inhibitor, among the thioureas tested. The inhibitory activity of the thioureas against the human sEH is closer to the value of activity against rat sEH rather than murine sEH. While being less active, thioureas are up to 7-fold more soluble than ureas, which makes them more bioavailable and thus promising as sEH inhibitors.     

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing two urea groups has been developed. Inhibition potency of the described compounds ranges from 2.0 μM to 0.4 nM. 1,6-(Hexamethylene)bis[(adamant-1-yl)urea] (3b) was found to be a potent slow tight binding inhibitor (IC₅₀ = 0.5 nM) with a strong binding to sEH (K_i = 3.1 nM) and a moderately long residence time on the enzyme (k_off = 1.05 × 10⁻³ s⁻¹; t_1/2 = 11 min).     

Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase

Hepoxilins are lipid signaling molecules derived from arachidonic acid through the 12-lipoxygenase pathway. These trans-epoxy hydroxy eicosanoids play a role in a variety of physiological processes, including inflammation, neurotransmission, and formation of skin barrier function. Mammalian hepoxilin hydrolase, partly purified from rat liver, has earlier been reported to degrade hepoxilins to trioxilins. Here, we report that hepoxilin hydrolysis in liver is mainly catalyzed by soluble epoxide hydrolase (sEH): i) purified mammalian sEH hydrolyses hepoxilin A₃ and B₃ with a V_max of 0.4–2.5 μmol/mg/min; ii) the highly selective sEH inhibitors N-adamantyl-N’-cyclohexyl urea and 12-(3-adamantan-1-yl-ureido) dodecanoic acid greatly reduced hepoxilin hydrolysis in mouse liver preparations; iii) hepoxilin hydrolase activity was abolished in liver preparations from sEH^−/− mice; and iv) liver homogenates of sEH^−/− mice show elevated basal levels of hepoxilins but lowered levels of trioxilins compared with wild-type animals. We conclude that sEH is identical to previously reported hepoxilin hydrolase. This is of particular physiological relevance because sEH is emerging as a novel drug target due to its major role in the hydrolysis of important lipid signaling molecules such as epoxyeicosatrienoic acids. sEH inhibitors might have undesired side effects on hepoxilin signaling.     

Soluble epoxide hydrolase expression in a porcine model of arteriovenous graft stenosis and anti-inflammatory effects of a soluble epoxide hydrolase inhibitor

Synthetic arteriovenous (AV) grafts, placed between an artery and vein, are used for hemodialysis but often fail due to stenosis, typically at the vein-graft anastomosis. This study recorded T lymphocyte and macrophage accumulation at the vein-graft anastomosis, suggesting a role for inflammation in stenosis development. Epoxyeicosatrienoic acids (EETs), products of cytochrome P-450 epoxidation of arachidonic acid, have vasculoprotective and anti-inflammatory effects including inhibition of platelet activation, cell migration, and adhesion. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to less active diols. The effects of a specific inhibitor of sEH (sEHI) on cytokine release from human monocytes and mouse bone marrow-derived macrophages (BMMΦ) from wild-type (WT) and sEH knockout (KO) animals were investigated. Expression of sEH protein increased over time at the anastomosis as evaluated by immunohistochemistry. Pre-exposure of adherent human monocytes to sEHI (5 μM) significantly inhibited lipopolysaccharide-induced release of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α and enhanced the EET-to-diol ratio. Release of MCP-1 from WT BMMΦ was significantly inhibited but release from sEH KO BMMΦ was not attenuated indicating the specificity of the sEHI. In contrast, sEHI did not inhibit the release of macrophage inflammatory protein-1 or interleukin-6. Nuclear translocation of NF-κB, as assessed by immunocytochemical staining, was not decreased with sEHI in monocytes, but the phosphorylation of JNK was completely abrogated, suggesting this pathway is the target of sEHI effects in monocytes. These results suggest that sEHI may be useful for inhibition of inflammation and subsequently stenosis in AV grafts.     

Effect of soluble epoxide hydrolase inhibition on epoxyeicosatrienoic acid metabolism in human blood vessels

We investigated the effects of soluble epoxide hydrolase (sEH) inhibition on epoxyeicosatrienoic acid (EET) metabolism in intact human blood vessels, including the human saphenous vein (HSV), coronary artery (HCA), and aorta (HA). When HSV segments were perfused with 2 micromol/l 14,15-[3H]EET for 4 h, >60% of radioactivity in the perfusion medium was converted to 14,15-dihydroxyeicosatrienoic acid (DHET). Similar results were obtained with endothelium-denuded vessels. 14,15-DHET was released from both the luminal and adventitial surfaces of the HSV. When HSVs were incubated with 14,15-[3H]EET under static (no flow) conditions, formation of 14,15-DHET was detected within 15 min and was inhibited by the selective sEH inhibitors N,N'-dicyclohexyl urea and N-cyclohexyl-N'-dodecanoic acid urea (CUDA). Similarly, CUDA inhibited the conversion of 11,12-[3H]EET to 11,12-DHET by the HSV. sEH inhibition enhanced the uptake of 14,15-[3H]EET and facilitated the formation of 10,11-epoxy-16:2, a beta-oxidation product. The HCA and HA converted 14,15-[3H]EET to DHET, and this also was inhibited by CUDA. These findings in intact human blood vessels indicate that conversion to DHET is the predominant pathway for 11,12- and 14,15-EET metabolism and that sEH inhibition can modulate EET metabolism in vascular tissue.     

The regulation of cytosolic epoxide hydrolase in mice

The concentration of cytosolic epoxide hydrolase in untreated and clofibrate-treated mouse liver extracts was estimated by immunoblotting. Clofibrate treatment of mice was found to increase liver cytosolic epoxide hydrolase concentration by two fold, showing that the increase in cytosolic epoxide hydrolase in mouse liver after clofibrate treatment is primarily due to induction. The induced and uninduced cytosolic epoxide hydrolase, and epoxide hydrolase in the cytosolic and mitochondrial fractions were compared and found to be identical or very similar. Cytosolic epoxide hydrolases in kidney and liver were similar in molecular weight and antigenic properties.     

Optimization of piperidyl-ureas as inhibitors of soluble epoxide hydrolase

Inhibition of sEH is hypothesized to lead to an increase in epoxyeicosatrienoic acids resulting in the potentiation of their anti-inflammatory and vasodilatory effects. In an effort to explore sEH inhibition as an avenue for the development of vasodilatory and cardio- or renal-protective agents, a lead identified through high-throughput screening was optimized, guided by the determination of a solid state co-structure with sEH. Replacement of potential toxicophores was followed by optimization of cell-based potency and ADME properties to provide a new class of functionally potent sEH inhibitors with attractive in vitro metabolic profiles and high and sustained plasma exposures after oral administration in the rat.     

Discovery of potent non-urea inhibitors of soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is a novel target for the treatment of hypertension and vascular inflammation. A new class of potent non-urea sEH inhibitors was identified via high throughput screening (HTS) and chemical modification. IC₅₀s of the most potent compounds range from micromolar to low nanomolar.     

Cloning and expression of soluble epoxide hydrolase from potato

Five cDNAs encoding a putative soluble epoxide hydrolase (sEH) from potato were isolated and characterized. The cDNAs contained open reading frames encoding 36 kDa polypeptides which were highly homologous to the carboxy terminal region of mammalian sEH. When one of the cDNAs was expressed in a baculovirus system a soluble 38 kDa protein with epoxide hydrolase activity was produced. The recombinant enzyme hydrolyzed a commonly used diagnostic substrate for the soluble form of mammalian EH. Inhibitor profiles of the recombinant potato and mammalian sEH were also similar. The expression of sEH in potato was found to be regulated by both developmental and environmental signals. Levels of mRNA for sEH were higher in meristematic tissue than in mature leaves. This mRNA was also observed to accumulate on wounding and application of exogenous methyl jasmonate.     

Inhibition of soluble epoxide hydrolase by fulvestrant and sulfoxides

The soluble epoxide hydrolase (sEH) is a key enzyme in the metabolism of epoxy-fatty acids, signaling molecules involved in numerous biologies. Toward finding novel inhibitors of sEH, a library of known drugs was tested for inhibition of sEH. We found that fulvestrant, an anticancer agent, is a potent (K_I = 26 nM) competitive inhibitor of sEH. From this observation, we found that alkyl-sulfoxides represent a new kind of pharmacophore for the inhibition of sEH.     

Brassica napus soluble epoxide hydrolase (BNSEH1).

Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the metabolism of epoxy fatty acids and in mediating defence responses. We report the cloning of a full-length epoxide hydrolase cDNA (BNSEH1) from oilseed rape (Brassica napus) obtained by screening of a cDNA library prepared from methyl jasmonate induced leaf tissue, and the 5'-RACE technique. The cDNA encodes a soluble protein containing 318 amino acid residues. The identity on the protein level is 85% to an Arabidopsis soluble epoxide hydrolase (sEH) and 50-60% to sEHs cloned from other plants. A 5 x His tag was added to the N-terminus of the BNSEH1 and the construct was over-expressed in the yeast Pichia pastoris. The recombinant protein was recovered at high levels after Ni-agarose chromatography of lysed cell extracts, had a molecular mass of 37 kDa on SDS/PAGE and cross-reacted on Western blots with antibodies raised to a sEH from Arabidopsis thaliana. BNSEH1 was shown to be a monomer by gel filtration analysis. The activity was low towards cis-stilbene oxide but much higher using trans-stilbene oxide as substrate with Vmax of 0.47 micro mol.min.mg-1, Km of 11 micro m and kcat of 0.3 s-1. The optimum temperature of the recombinant enzyme was 55 degrees C and the optimum pH 6-7 for trans-stilbene oxide hydrolysis. The isolation of BNSEH1 will facilitate metabolic engineering of epoxy fatty acid metabolism for functional studies of resistance and seed oil modification in this important oilcrop.     

Identifying simultaneous matrix metalloproteinases/soluble epoxide hydrolase inhibitors

Molecular and Cellular Biochemistry - Matrix metalloproteinase (MMP) and soluble epoxide hydrolase (sEH) have completely unrelated biological functions; however, their dysregulation produce similar...     

Attenuation of cisplatin nephrotoxicity by inhibition of soluble epoxide hydrolase

Cisplatin is a highly effective chemotherapeutic agent against many tumors; however, it is also a potent nephrotoxicant. Given that there have been no significant advances in our ability to clinically manage acute renal failure since the advent of dialysis, the development of novel strategies to ablate nephrotoxicity would represent a significant development. In this study, we investigated the ability of an inhibitor of soluble epoxide hydrolase (sEH), n-butyl ester of 12-(3-adamantan-1-yl-ureiido)-dodecanoic acid (nbAUDA), to attenuate cisplatin-induced nephrotoxicity. nbAUDA is quickly converted to AUDA and results in maintenance of high AUDA levels in vivo. Subcutaneous administration of 40 mg/kg of nbAUDA to C3H mice every 24 h resulted in elevated blood levels of AUDA; this protocol was also associated with attenuation of nephrotoxicity induced by cisplatin (intraperitoneal injection) as assessed by BUN levels and histological evaluation of kidneys. This is the first report of the use of sEH inhibitors to protect against acute nephrotoxicity and suggests a therapeutic potential of these compounds.     

Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues.

Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using mouse and rat liver suggested that sEH may be cytosolic and/or peroxisomal. In this study we applied immunofluorescence and confocal microscopy using markers for different subcellular compartments to evaluate sEH colocalization in an array of human tissues. Results showed that sEH is both cytosolic and peroxisomal in human hepatocytes and renal proximal tubules and exclusively cytosolic in other sEH-containing tissues such as pancreatic islet cells, intestinal epithelium, anterior pituitary cells, adrenal gland, endometrium, lymphoid follicles, prostate ductal epithelium, alveolar wall, and blood vessels. sEH was not exclusively peroxisomal in any of the tissues evaluated. Our data suggest that human sEH subcellular localization is tissue dependent, and that sEH may have tissue- or cell-type-specific functionality. To our knowledge, this is the first report showing the subcellular localization of sEH in a wide array of human tissues.