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Cancer therapy reduces tumor burden via tumor cell death ("debris"), which can accelerate tumor progression via the failure of inflammation resolution. Thus, there is an urgent need to develop treatment modalities that stimulate the clearance or resolution of inflammation-associated debris. Here, we demonstrate that chemotherapy-generated debris stimulates metastasis by up-regulating soluble epoxide hydrolase (sEH) and the prostaglandin E 2 receptor 4 (EP4). Therapy-induced tumor cell debris triggers a storm of proinflammatory and proangiogenic eicosanoid-driven cytokines. Thus, targeting a single eicosanoid or cytokine is unlikely to prevent chemotherapy-induced metastasis. Pharmacological abrogation of both sEH and EP4 eicosanoid pathways prevents hepato-pancreatic tumor growth and liver metastasis by promoting macrophage phagocytosis of debris and counterregulating a protumorigenic eicosanoid and cytokine storm. Therefore, stimulating the clearance of tumor cell debris via combined sEH and EP4 inhibition is an approach to prevent debris-stimulated metastasis and tumor growth., Competing Interests: Competing interest statement: Y.S., K.-K.H., X.Y., and B.D.H. work with Ionova and EicOsis, respectively, on the development of EP4 and epoxide hydrolase inhibitors for clinical use. D.P., A.G., and B.D.H., and colleagues C. N. Serhan and P. Sime were coauthors on an earlier commentary suggesting modulating the eicosanoid and cytokine storms with a soluble epoxide hydrolase inhibitor to modulate severity of COVID infections, introducing the same mechanism as is suggested here for reduction of the cytokine storm following cancer therapy., (Copyright © 2021 the Author(s). Published by PNAS.)

The inhibition of the enzyme soluble epoxide hydrolase (sEH) has demonstrated clinical therapeutic effects in several peripheral inflammatory-related diseases, with 3 compounds in clinical trials. However, the role of this enzyme in the neuroinflammation process has been largely neglected. Herein, we disclose the pharmacological validation of sEH as a novel target for the treatment of Alzheimer's disease (AD). Evaluation of cognitive impairment and pathological hallmarks were used in 2 models of age-related cognitive decline and AD using 3 structurally different and potent sEH inhibitors as chemical probes. sEH is upregulated in brains from AD patients. Our findings supported the beneficial effects of central sEH inhibition, regarding reducing cognitive impairment, neuroinflammation, tau hyperphosphorylation pathology, and the number of amyloid plaques. This study suggests that inhibition of inflammation in the brain by targeting sEH is a relevant therapeutic strategy for AD.

Objective: To investigate the spatial and temporal changes of soluble epoxide hydrolase (sEH) in the human placenta throughout gestation and to study the effects of hypoxia-reoxygenation (HR) on the expression of sEH in villous explants in vitro., Materials and Methods: Placental samples were obtained from women of different gestation and grouped as early (8-12 weeks, n = 10), mid- (16-28 weeks, n = 6), and late gestation (38-39 weeks, n = 10) according to gestational age. Immunohistochemistry, western blot, and real-time quantitative PCR were used to assess the cellular distribution and temporal changes of sEH. Villous explant cultures were used to study the effect of HR (8 h at 2% oxygen, followed by 16 h at 8% oxygen, two cycles) on the expression of sEH., Results: Using a mouse monoclonal antibody against human sEH, immunoreactivity of sEH was observed mainly localized in the cytotrophoblasts and, to a lesser extent, the syncytiotrophoblast in the villous tissues throughout gestation. Compared to villous tissues of early gestation, the levels of sEH mRNA and protein were significantly increased in villous samples of mid- and late gestation. Furthermore, villous explants subjected to HR had significantly higher levels of sEH mRNA and protein compared to villous tissues kept at 8% oxygen throughout the experiment., Conclusion: Our results indicate that sEH is likely to play an essential role in the development of human placenta and HR is a possible factor regulating the expression of sEH in the placenta., (Copyright © 2019. Published by Elsevier B.V.)

Maternal infection during pregnancy increases risk of neurodevelopmental disorders such as schizophrenia and autism spectrum disorder (ASD) in offspring. In rodents, maternal immune activation (MIA) yields offspring with schizophrenia- and ASD-like behavioral abnormalities. Soluble epoxide hydrolase (sEH) plays a key role in inflammation associated with neurodevelopmental disorders. Here we found higher levels of sEH in the prefrontal cortex (PFC) of juvenile offspring after MIA. Oxylipin analysis showed decreased levels of epoxy fatty acids in the PFC of juvenile offspring after MIA, supporting increased activity of sEH in the PFC of juvenile offspring. Furthermore, expression of sEH (or EPHX2 ) mRNA in induced pluripotent stem cell-derived neurospheres from schizophrenia patients with the 22q11.2 deletion was higher than that of healthy controls. Moreover, the expression of EPHX2 mRNA in postmortem brain samples (Brodmann area 9 and 40) from ASD patients was higher than that of controls. Treatment with 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), a potent sEH inhibitor, in juvenile offspring from prenatal day (P) 28 to P56 could prevent cognitive deficits and loss of parvalbumin (PV) immunoreactivity in the medial PFC of adult offspring after MIA. In addition, dosing of TPPU to pregnant mothers from E5 to P21 could prevent cognitive deficits, and social interaction deficits and PV immunoreactivity in the medial prefrontal cortex of juvenile offspring after MIA. These findings suggest that increased activity of sEH in the PFC plays a key role in the etiology of neurodevelopmental disorders in offspring after MIA. Therefore, sEH represents a promising prophylactic or therapeutic target for neurodevelopmental disorders in offspring after MIA., Competing Interests: The authors declare no conflict of interest.

CYP epoxygenases metabolize arachidonic acid into four regioisomers of epoxyeicosatrienoic acids (EETs) which are hydrolysed into their corresponding diols by soluble epoxide hydrolase (sEH). EETs are very biologically active molecules. They promote proliferation and inhibit apoptosis as well as numerous other functions within organisms. Peroxisome proliferator-activated receptor α (PPARα) play role in regulation of CYP epoxygenases and sEH. PPARα is the ligand-dependent transcriptional factor which is activated by various compounds, including fibrates. The latter are widely used in clinical practice. This study investigates the changes in expression of CYP2C8, CYP2J2, and sEH in HEK293, HepG2, and HT-29 cell lines after fibrate treatment using two different incubation times. The results demonstrate that the effect of fibrates on arachidonic acid-metabolizing enzymes expression is concentration-dependent. Although CYP2C8 expression is downregulated by the fibrates treatment, the results reveal that changes in CYP2J2/sEH ratio are closely associated with cell proliferation and could explain the differing proliferation response of cells to different concentrations of fibrates., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Previous preclinical evidence has suggested that the elevation of epoxyeicosatrienoic acids (EETs) derived from the cytochrome P450 (CYP) epoxygenases-dependent metabolism of arachidonic acid has important anti-inflammatory effects. However, the levels of EETs and their synthetic and metabolic enzymes in human ulcerative colitis has not been evaluated., Method: To evaluate EETs and the expression of relevant CYP isoforms and the metabolizing enzyme, soluble epoxide hydrolase (sEH), tissue biopsies were collected from 16 pairs of ulcerative colitis patients' tissues and matched with adjacent non-inflamed tissues. EETs were extracted from tissue homogenates and analyzed by liquid chromatography coupled with tandem mass spectrometry., Results: The concentration of EETs was higher in ulcerative colitis tissues compared with matched adjacent non-inflamed tissues (1.91 ± 0.98 ng/mg vs. 0.96 ± 0.77 ng/mg, mean ± SD, P < 0.01). As shown by immunohistochemistry, sEH was present in the cytoplasm and intestinal mucosa and showed a decline in ulcerative colitis tissues compared with matched adjacent non-inflamed tissues. Western blot analyses showed reduced sEH expression in ulcerative colitis tissues compared with matched adjacent non-inflamed tissues, whereas CYP2J2 increased in ulcerative colitis tissues (P < 0.05). However, there was no statistically significant difference observed in CYP2C8 and CYP2C9 protein expression between them (P > 0.05)., Conclusion: Our data suggest that the increase in EET levels may be part of a protective mechanism in ulcerative colitis. Furthermore, the concentration of EETs could be a key factor for drug therapy for ulcerative colitis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Genetic variants at PTK2B-CLU locus pose as high-risk factors for many age-related disorders. However, the role of these variants in disease progression is less characterized. In this study, we aimed to investigate the functional significance of a clusterin intronic SNP, rs2279590, that has been associated with pseudoexfoliation, Alzheimer's disease (AD) and diabetes. We have previously shown that the alleles at rs2279590 differentially regulate clusterin (CLU) gene expression in lens capsule tissues. This polymorphism resides in an active regulatory region marked by H3K27Ac and DNase I hypersensitive site and is an eQTL for CLU expression. Here, we report the presence of an enhancer element in surrounding region of rs2279590. Deletion of a 115 bp intronic region flanking the rs2279590 variant through CRISPR-Cas9 genome editing in HEK293 cells demonstrated a decreased clusterin gene expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that rs2279590 with allele 'A' constitutes a transcription factor binding site for heat shock factor-1 (HSF1) but not with allele 'G'. By binding to allele 'A', HSF1 abrogates the enhancer effect of the locus as validated by reporter assays. Interestingly, rs2279590 locus has a widespread enhancer effect on two nearby genes, protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase-2 (EPHX2); both of which have been previously associated with AD as risk factors. To summarize, our study unveils a mechanistic role of the common variant rs2279590 that can affect a variety of aging disorders by regulating the expression of a specific set of genes., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Leukotriene A4 hydrolase (LTA4H), a bifunctional zinc metallo-enzyme, is reportedly overexpressed in several human cancers. Our group has focused on LTA4H as a potential target for cancer prevention and/or therapy. In the present study, we report that LTA4H is a key regulator of cell cycle at the G0/G1 phase acting by negatively regulating p27 expression in skin cancer. We found that LTA4H is overexpressed in human skin cancer tissue. Knocking out LTA4H significantly reduced skin cancer development in the 7,12-dimethylbenz(a)anthracene (DMBA)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted two-stage skin cancer mouse model. LTA4H depletion dramatically decreased anchorage-dependent and -independent skin cancer cell growth by inducing cell cycle arrest at the G0/G1 phase. Moreover, our findings showed that depletion of LTA4H enhanced p27 protein stability, which was associated with decreased phosphorylation of CDK2 at Thr160 and inhibition of the CDK2/cyclin E complex, resulting in down-regulated p27 ubiquitination. These findings indicate that LTA4H is critical for skin carcinogenesis and is an important mediator of cell cycle and the data begin to clarify the mechanisms of LTA4H's role in cancer development., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

An effective epoxide hydrolase (EH) production strain was mutagenized using 60 Co gamma and UV irradiation. Among positive mutant strains, the EH activity of C2-44 reached 33.7 U/g, which was 267% as much as that of the original Aspergillus niger ZJB-09103. Compared with the wild type, there were significant changes in morphology for C2-44, including the color of mycelia on the slants and the shape of conidial head. In addition, glucose and soybean cake were the optimal carbon and nitrogen source in terms of EH activity for the mutant C2-44 instead of soluble starch and peptone for the wild-type strain. The reaction time required to reach 99% enantiomeric excesses of (S)-epichlorohydrin from racemic substrate was shortened significantly by the mutant C2-44. This phenomenon was probably explained by the higher V max for hydrolysis of racemic epichlorohydrin by C2-44 compared with Aspergillus niger ZJB-09103., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

The concentration of soluble epoxide hydrolase (sEH) protein was studied in renal medulla of adult rats from hypertensive ISIAH strain and normotensive WAG strain. The sEH is a key enzyme in metabolism of epoxyeicosatrienoic acids capable of activating endothelial NO-synthase and nitrogen oxide formation, and therefore being vasodilators. An increase in the sEH protein concentration (that we found) allows one to assume that the oxidative stress is increased in the renal medulla of hypertensive rats, and the bloodflow is decreased.

Soluble epoxide hydrolase (sEH) converts epoxyeicosatrienoic acids that are endothelium-derived hyperpolarizing factors into less active dihydroxyeicosatrienoic acids. Previously, we reported a decrease in adenosine A 1 receptor (A 1 AR) protein levels in sEH knockout (sEH -/- ) and an increase in sEH and A 1 AR protein levels in A 2A AR -/- mice. Additionally, K ATP channels are involved in adenosine receptor (AR)-dependent vascular relaxation. Thus, we hypothesize that a potential relationship may exist among sEH over-expression, A 1 AR upregulation, inactivation of K ATP channels, and increased in vascular tone. We performed DMT myograph muscle tension measurements and western blot analysis in isolated mouse mesenteric arteries (MAs) from wild-type (WT) and endothelial over-expression of sEH (Tie2-sEH Tr) mice. Our data revealed that NECA (a non-selective adenosine receptors agonist)-induced relaxation was significantly reduced in Tie2-sEH Tr mice, and CCPA (A 1 AR agonist)-induced contraction was increased in Tie2-sEH Tr mice. A 1 AR-dependent contraction in Tie2-sEH Tr mice was significantly attenuated by pharmacological inhibition of CYP4A (HET0016, 10 µM), PKCα (GO6976, 1 µM), and ERK1/2 (PD58059, 1 µM). Our western blot analysis revealed significantly higher basal protein expression of CYP4A, A 1 AR, and reduced p-ERK in MAs of Tie2-sEH Tr mice. Notably, pinacidil (K ATP channel opener)-induced relaxation was also significantly reduced in MAs of Tie2-sEH Tr mice. Furthermore, K ATP channel-dependent relaxation in MAs was enhanced by inhibition of PKCα and ERK1/2 in WT but not Tie2-sEH Tr mice. In conclusion, our data suggest that over-expression of sEH enhances A 1 AR-dependent contraction and reduces K ATP channel-dependent relaxation in MAs. These results suggest a possible interaction between sEH, A 1 AR, and K ATP channels in regulating vascular tone.

Leukotriene B4 (LTB4 ) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8(+) T cells. The role of the LTB4 -BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8(+) T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1β. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8(+) T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1β. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8(+) T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4 -BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8(+) T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis., (© 2015 John Wiley & Sons Ltd.)

Evidence suggests that upregulation of soluble epoxide hydrolase (sEH) is associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac hypertrophy, and heart failure. However, the upregulation mechanism is still unknown. In this study, we treated H9C2 cells with buthionine sulfoximine (BSO) to explore whether oxidative stress upregulates sEH gene expression and to identify the molecular and cellular mechanisms behind this upregulatory response. Real-time PCR and Western blot analyses were used to measure mRNA and protein expression, respectively. We demonstrated that BSO significantly upregulated sEH at mRNA levels in a concentration- and time-dependent manner, leading to a significant increase in the cellular hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Furthermore, BSO significantly increased the cytosolic phosphorylated IκB-α and translocation of NF-κB p50 subunits, as measured by Western blot analysis. This level of translocation was paralleled by an increase in the DNA-binding activity of NF-κB P50 subunits. Moreover, our results demonstrated that pretreatment with the NF-κB inhibitor PDTC significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression in a dose-dependent manner. Additionally, mitogen-activated protein kinases (MAPKs) were transiently phosphorylated by BSO treatment. To understand further the role of MAPKs pathway in BSO-mediated induction of sEH mRNA, we examined the role of extracellular signal-regulated kinase (ERK), c-JunN-terminal kinase (JNK), and p38 MAPK. Indeed, treatment with the MEK/ERK signal transduction inhibitor, PD98059, partially blocked the activation of IκB-α and translocation of NF-κB p50 subunits induced by BSO. Moreover, pretreatment with MEK/ERK signal transduction inhibitors, PD98059 and U0126, significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression. These results clearly demonstrated that the NF-κB signaling pathway is involved in BSO-mediated induction of sEH gene expression, and appears to be associated with the activation of the MAPK pathway. Furthermore, our findings provide a strong link between sEH-induced cardiac dysfunction and involvement of NF-κB in the development of cellular hypertrophy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Epoxide hydrolases (EHs), are enantioselective enzymes as they catalyze the kinetic resolution of racemic epoxides into the corresponding enantiopure vicinal diols, which are useful precursors in the synthesis of chiral pharmaceutical compounds. Here, we have identified and cloned two putative epoxide hydrolase genes (cpeh and sneh) from marine bacteria, Candidatus pelagibacter ubique and terrestrial bacteria, Stackebrandtia nassauensis, respectively and overexpressed them in pET28a vector in Escherichia coli BL21(DE3). The CPEH protein (42kDa) was found to be overexpressed as inactive inclusion bodies while SNEH protein (40kDa) was found to form soluble aggregates. In this study, the recombinant CPEH was successfully transformed from insoluble aggregates to the soluble and functionally active form, using pCold TF vector, though with low EH activity. To prevent the soluble aggregate formation of SNEH, it was co-expressed with GroEL/ES chaperone and was also fused with trigger factor (TF) chaperone at its N-terminus. The TF chaperone-assisted correct folding of SNEH led to a purified active EH with a specific activity of 3.85μmol/min/mg. The pure enzyme was further used to biocatalyze the hydrolysis of 10mM benzyl glycidyl ether (BGE) and α-methyl styrene oxide (MSO) with an enantiomeric excess of the product (eep) of 86% and 73% in 30 and 15min, respectively. In conclusion, this is the first report about the heterologous expression of epoxide hydrolases using TF as a molecular chaperone in pCold TF expression vector, resulting in remarkable increase in the solubility and activity of the otherwise improperly folded recombinant epoxide hydrolases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Insulin, elevated during obesity, regulates xenobiotic biotransformation enzymes, potentially through phosphatidylinositol 3-kinase (PI3K) signaling, in extraovarian tissues. PI3K regulates oocyte viability, follicular activation, and ovarian chemical biotransformation. 7,12-Dimethylbenz[a]anthracene (DMBA), a carcinogen and ovotoxicant, destroys all stages of follicles, leading to premature ovarian failure. Obesity has been reported to promote DMBA-induced tumors, but it remains unknown whether obesity affects ovarian xenobiotic metabolism. Therefore, we investigated ovarian expression of xenobiotic metabolism genes-microsomal epoxide hydrolase (Ephx1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1), and PI3K-signaling members (protein kinase B [AKT] alpha [Akt1], beta [Akt2], and the forkhead transcription factor subfamily 3 [Foxo3])-in lean and obese female mice after DMBA exposure (1 mg/kg; intraperitoneal injection for 14 days). Relative to lean, obese mice had decreased (P < 0.05) healthy primordial and primary follicle numbers but increased (P < 0.05) secondary and preovulatory follicles numbers. Obesity increased (P < 0.05) Akt1, Akt2, Gstm1, and Ephx1 mRNA and pAKT(Ser473/Thr308), GSTM1, GSTP1, and EPHX1 protein levels. DMBA decreased (P < 0.05) ovarian weight in lean and obese mice, however, obese DMBA-treated females had a greater reduction (P < 0.05) in ovarian weight. In both lean and obese mice, DMBA decreased (P < 0.05) all stages of healthy follicle numbers, increased Gstp1 and Ephx1 mRNA as well as GSTM1, GSTP1, and EPHX1 protein levels, and decreased Akt1 and Akt2 mRNA as well as pAKT(Ser473) or pAKT(Thr308), FOXO3, and pFOXO3(Ser253) protein expression. There was an additive effect between obesity and DMBA exposure for increased Gstm1 and Ephx1 mRNA as well as GSTM1 and EPHX1 protein expression.

Phenethyl isothiocyanate (PEITC) is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin). The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT), N-acetyltransferase (NAT), glucuronosyl transferase (UDP), and epoxide hydrolase (EH) following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake), 0.6 µmol/g (medium dose), and 6.0 µmol/g (high dose), and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention.

Epoxyeicosatrienoic acids (EETs) have antihypertensive properties and play a part in the maintenance of renal microvascular function. EETs mediate vasodilation of rat preglomerular microvessels and activate ion channels. Ephx2 is coding for the soluble epoxide hydrolase (sEH) which catalyze the degradation of EETs. Renal cortex and renal medulla were tested for Ephx2 mRNA level in hypertensive ISIAH and normotensive WAG rats at rest and emotional stress conditions. The microarray analysis and real-time PCR were used to assess the transcriptional activity of Ephx2. Enhanced transcriptional activity of Ephx2 in both renal structures of ISIAH rats was found at rest and stress conditions. The emotional stress caused elevation of Ephx2 mRNA level in renal medulla of ISIAH rats and opposite response--a decrease in Ephx2 expression in the renal medulla and cortex of WAG rats.The results suggest Ephx2 participation in the control of the vascular tone changes in kidney promoting the hypertensive state in the ISIAH rats.

Hyperglycemia worsens stroke, yet rigorous glycemic control does not improve neurologic outcome. An alternative is to target downstream molecular mediator(s) triggered by hyperglycemia but independent of prevailing glycemia. Soluble epoxide hydrolase (sEH) is a potential mediator of injury via its metabolism of neuroprotective epoxyeicosatrienoic acids (EETs). We tested whether hyperglycemia exacerbates cerebral injury by upregulating sEH and decreasing brain EET levels. Type 1 diabetes mellitus was modeled by streptozotocin (STZ; 50 mg/kg per day intraperitoneally, 5 days) in male mice. At 4 weeks, STZ-treated and control mice underwent 45-minute middle cerebral artery occlusion (MCAO) with or without sEH blockade by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 1 mg/kg intraperitoneally daily for 6 days before MCAO). The STZ-treated mice had increased sEH mRNA expression in cerebral vessels and decreased EET concentrations in brain. There was no difference in cortical perfusion between groups. The STZ-treated mice sustained larger brain infarct than controls. Pretreatment with t-AUCB eliminated the difference in infarct size and EETs concentration between STZ-treated mice and controls, without altering glycemia. We conclude that type 1 diabetes mellitus upregulates sEH mRNA and decreases concentrations of neuroprotective EETs within the brain, leading to worse stroke outcome. The data indicate that sEH antagonism may be beneficial in the setting of hyperglycemic stroke.

We have previously demonstrated that a stable synthetic analog of 20-HETE, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), restores vascular reactivity, blood pressure, and heart rate in endotoxemic rats. The aim of this study was to determine whether decreased renal expression and activity of soluble epoxide hydrolase (sEH), MEK1, ERK1/2, IKKβ, IκB-α, and NF-κB as well as systemic and renal proinflammatory cytokine production associated with increased expression and activity of CYP2C23 contributes to the effect of 5,14-HEDGE to prevent hypotension, tachycardia, inflammation, and mortality in response to systemic administration of lipopolysaccharide (LPS). Blood pressure fell by 33 mmHg and heart rate rose by 57 beats/min in LPS (10 mg/kg, i.p.)-treated rats. Administration of LPS also increased mRNA and protein expression of sEH associated with a decrease in CYP2C23 mRNA and protein expression. Increased activity of sEH and p-MEK1, p-ERK1/2, p-IκB-α, NF-κB, and p-NF-κB protein levels as well as TNF-α and IL-8 production by LPS were also associated with a decreased activity of AA epoxygenases. These effects of LPS were prevented by 5,14-HEDGE (30 mg/kg, s.c.; 1 h after LPS). Treatment of endotoxemic mice with 5,14-HEDGE also raised the survival rate of animals from 84% to 98%. A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, 20-HEDE (30 mg/kg, s.c.; 1 h after LPS) prevented the effects of 5,14-HEDGE on blood pressure, heart rate, expression and/or activity of sEH, CYP2C23, and ERK1/2 as well as TNF-α and IL-8 levels in rats treated with LPS. These results suggest that decreased expression and/or activity of sEH and MEK1/ERK1/2/IKKβ/IκB-α/NF-κB pathway as well as proinflammatory cytokine production associated with increased CYP2C23 expression and antiinflammatory mediator formation participate in the protective effect of 5,14-HEDGE against hypotension, tachycardia, inflammation, and mortality in the rodent model of septic shock., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: Glucosinolates/isothiocyanates are an established class of naturally occurring chemopreventive agents, a principal mechanism of action being to limit the generation of genotoxic metabolites of chemical carcinogens, as a result of modulation of cytochrome P450 and phase II detoxification enzymes. The objective of this study was to assess whether a glucosinolate-rich extract from Daikon sprouts, containing glucroraphasatin and glucoraphenin, is a potential chemopreventive agent by modulating such enzymes in the liver and lung of rats., Methods: Rats were exposed to the glucosinolate-rich Daikon extract through the diet, at three dose levels, for 14 days, so that the low dose simulates dietary intake., Results: At the low dose only, a modest increase was noted in the hepatic dealkylations of methoxy-, ethoxy-, pentoxyresorufin and benzyloxyquinoline that was accompanied by elevated expression of CYP1 and CYP3A2 apoprotein levels. In lung, only a modest increase in the dealkylation of pentoxyresorufin was observed. At higher doses, in both tissues, these increases were abolished. At the same low dietary dose, the Daikon extract elevated markedly glutathione S-transferase activity paralleled by rises in GSTα, GSTμ and GSTπ protein expression. An increase was also noted in quinone reductase activity and expression. Finally, glucuronosyl transferase and epoxide hydrolase activities and expression were also up-regulated, but necessitated higher doses., Conclusion: Considering the ability of Daikon glucosinolates to effectively enhance detoxification enzymes, in particular glutathione S-transferase, it may be inferred that consumption of this vegetable may possess significant chemopreventive activity and warrants further evaluation through epidemiology and studies in animal models of cancer.

In response to endothelial cell activation, arachidonic acid can be converted by cytochrome P450 (CYP) epoxygenases to epoxyeicosatrienoic acids (EETs), which have potent vasodilator and anti-inflammatory properties. EETs are rapidly degraded in vivo to the less active dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). Since the beginning of the 2000s, the role of EET pathway in human health and its alteration in diseases has been shown by measuring EETs/DHET levels in blood, by evaluating the relationship between CYP/sEH gene polymorphisms, which modify enzyme activity and thus EETs/DHET level, and by assessing the inhibitory effect of the local administration of CYP epoxygenase inhibitor on endothelium-dependent dilatation. By combining these functional and biological approaches, we demonstrated that EETs are the endothelial factors released by CYP epoxygenases involved in the flow-mediated dilatation of conduit arteries in healthy subjects, together with the impairment of EET availability in essential hypertensive patients at this level. The modulation of EET pathway now emerges as a new promising pharmacological target that may improve the clinical management of patients at high cardiovascular risk. In this respect, the restoration of EET availability using a new class of agents, the inhibitors of sEH, gave promising results in various animal models of cardiovascular diseases, reducing blood pressure and target organ damage, and a first product has entered clinical evaluation.

Enediyne natural product biosynthesis is characterized by a convergence of multiple pathways, generating unique peripheral moieties that are appended onto the distinctive enediyne core. Kedarcidin (KED) possesses two unique peripheral moieties, a (R)-2-aza-3-chloro-β-tyrosine and an iso-propoxy-bearing 2-naphthonate moiety, as well as two deoxysugars. The appendage pattern of these peripheral moieties to the enediyne core in KED differs from the other enediynes studied to date with respect to stereochemical configuration. To investigate the biosynthesis of these moieties and expand our understanding of enediyne core formation, the biosynthetic gene cluster for KED was cloned from Streptoalloteichus sp. ATCC 53650 and sequenced. Bioinformatics analysis of the ked cluster revealed the presence of the conserved genes encoding for enediyne core biosynthesis, type I and type II polyketide synthase loci likely responsible for 2-aza-l-tyrosine and 3,6,8-trihydroxy-2-naphthonate formation, and enzymes known for deoxysugar biosynthesis. Genes homologous to those responsible for the biosynthesis, activation, and coupling of the l-tyrosine-derived moieties from C-1027 and maduropeptin and of the naphthonate moiety from neocarzinostatin are present in the ked cluster, supporting 2-aza-l-tyrosine and 3,6,8-trihydroxy-2-naphthoic acid as precursors, respectively, for the (R)-2-aza-3-chloro-β-tyrosine and the 2-naphthonate moieties in KED biosynthesis.

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.

Hypertension is sexually dimorphic and modified by removal of endogenous sex steroids. This study tested the hypothesis that endogenous gonadal hormones exert differential effects on protein expression in the kidney and mesentery of SHR. At ~5 weeks of age male and female SHR underwent sham operation, orchidectomy, or ovariectomy (OVX). At 20-23 weeks of age, mean arterial pressure (MAP) was measured in conscious rats. The mesenteric arterial tree and kidneys were collected, processed for Western blots, and probed for Cu Zn superoxide dismutase (SOD1), soluble epoxide hydrolase (sEH), and Alpha 2A adrenergic receptor (A2AR) expression. MAP was unaffected by ovariectomy (Sham 164 ± 4: Ovariecttomy 159 ± 3 mm Hg). MAP was reduced by orchidectomy (Sham 189 ± 5:Orchidectomy 167 ± 2 mm Hg). In mesenteric artery, SOD1 expression was greater in male versus female SHR. Orchidectomy increased while ovariectomy decreased SOD1 expression. The kidney exhibited a different pattern of response. SOD1 expression was reduced in male compared to female SHR but gonadectomy had no effect. sEH expression was not significantly different among the groups in mesenteric artery. In kidney, sEH expression was greater in males compared to females. Ovariectomy but not orchidectomy increased sEH expression. A2AR expression was greater in female than male SHR in mesentery artery and kidney. Gonadectomy had no effect in either tissue. We conclude that sexually dimorphic hypertension is associated with regionally specific changes in expression of three key proteins involved in blood pressure control. These data suggest that broad spectrum inhibition or stimulation of these systems may not be the best approach for hypertension treatment. Instead regionally targeted manipulation of these systems should be investigated.

To examine the hepatoprotective activities of Nigella sativa (Ns) and thymoquinone (TQ) against carbon tetrachloride (CCl(4))-induced hepatotoxicity, the effects of water extract of Ns seeds (50 mg/kg) or TQ (5 mg/kg in corn oil) by gavage for 5 days on detoxifying enzymes and glutathione were compared in healthy and CCl(4)-challenged (1 mL/kg in corn oil, intraperitoneally [ip], a single dose) rats. Both Ns and TQ countered the elevations in serum alanine aminotransferase activity, oxidized glutathione level, and stress ratio caused by CCl(4). Both Ns and TQ ameliorated the reductions in the activities and messenger RNA (mRNA) levels of glutathione S-transferase, NAD(P)H-quinone oxidoreductase, and microsomal epoxide hydrolase, as well as the reductions in reduced glutathione and cysteine levels produced by CCl(4). In many instances, Ns was much superior to TQ in providing protection against the damaging effects caused by CCl(4). This protection could be attributed to the induction of chemoprotective enzymes probably through increasing transcription.

Microarray technology was used to explore differences in brain gene expression under basal conditions in two strains of psychogenetically selected rats which differ in anxiety/stress responses, the inbred Roman High-(RHA-I) and Roman Low-(RLA-I) Avoidance rats. Microarray analysis detected 14 up-regulated and 24 down-regulated genes in RLA-I vs. RHA-I rats functionally related to neurobiological processes. The differentially expressed genes CAMKK2, CRHBP, EPHX2, HOMER3, NDN, PRL and RPL6 were selected for microarray validation using qRT-PCR. EPHX2, CAMKK2 (both up-regulated in RLA-I vs. RHA-I rats) and HOMER3 (down-regulated in RLA-I vs. RHA-I rats) showed a similar tendency and fold-change both in microarray and RT-PCR analyses; PRL (up-regulated in RLA-I vs. RHA-I rats), CRHBP and RPL6 (both down-regulated in RLA-I vs. RHA-I animals) showed a similar tendency but a different order of magnitude of change among experiments; finally, NDN was validated neither in tendency nor in magnitude of change., (Published by Elsevier Ireland Ltd.)

The potential of three isothiocyanates, namely R,S-sulforaphane, erucin and phenethyl isothiocyanate, of two naturally occurring glucosinolates, namely glucoerucin and glucoraphanin, and of the enantiomers of sulforaphane to modulate glucuronosyl transferase and epoxide hydrolase, two major carcinogen-metabolising enzyme systems, was investigated in precision-cut rat liver slices. Following exposure of the slices to the isothiocyanates (0-25 μM), erucin and phenethyl isothiocyanate, but not R,S-sulforaphane, elevated glucuronosyl transferase and epoxide hydrolase activities and expression, determined immunologically. Of the two enantiomers of sulforaphane, the R-enantiomer enhanced, whereas the S-enantiomer impaired, glucuronosyl transferase activity and only the former increased protein expression; furthermore, R-sulforaphane was more effective than the S-enantiomer in up-regulating microsomal epoxide hydrolase. When precision-cut rat liver slices were exposed to the same concentrations of glucoerucin and glucoraphanin, both glucosinolates caused a marked increase in the activity and expression of the microsomal epoxide hydrolase but had no effect on glucuronosyl transferase activity. It may be inferred that the ability of isothiocyanates to enhance hepatic microsomal epoxide hydrolase and glucuronosyl transferase activities is dependent on the nature of the side chain. Moreover, in the case of sulforaphane, the naturally occurring R-enantiomer increased both activities, whereas, in contrast, activities were impaired in the case of the S-enantiomer. Finally, intact glucosinolates are potent inducers of epoxide hydrolase and can thus contribute directly to the chemopreventive potential associated with cruciferous vegetable consumption.

We recently demonstrated that benzo(a)pyrene (BaP) causes cardiac hypertrophy by altering arachidonic acid metabolism through the induction of the expression of CYP ω-hydroxylases and soluble epoxide hydrolase (sEH) enzymes. The inhibition of CYP ω-hydroxylase enzymes partially reversed the BaP-induced cardiac hypertrophy. Therefore, it is important to examine whether the inhibition of sEH also confers cardioprotection. For this purpose, male Sprague-Dawley rats were injected intraperitoneally daily with either the sEH inhibitor 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS; 0.65 mg/kg), BaP (20 mg/kg), or the combination of BaP (20 mg/kg) and TUPS (0.65 mg/kg) for 7 days. Thereafter, the heart, liver, and kidney were harvested, and the heart to body weight ratio was measured. The expression of the hypertrophic markers, sEH, heme oxygenase-1, and CYP450 enzymes was determined. Our results demonstrate that BaP alone significantly induced the expression of sEH and CYP ω-hydroxylases in the heart, liver, and kidney tissues. Treatment with TUPS significantly reversed the BaP-mediated induction of the hypertrophic markers, completely prevented the increase in the heart to body weight ratio, and reduced the BaP-induced CYP1A1, CYP1B1, CYP4F4, and CYP4F5 genes in the heart. The current study demonstrates the cardioprotective effect of sEH inhibitor, TUPS, against BaP-induced cardiac hypertrophy and further confirms the role of sEH and CYP450 enzymes in the development of cardiac hypertrophy.

Background: The interactions between luteal, vascular endothelial, immune cells and its products: steroids, peptide hormones, prostaglandins (PGs), growth factors and cytokines play a pivotal role in the regulation of corpus luteum (CL) function. Luteal endothelial cells undergo many dynamic morphological changes and their action is regulated by cytokines. The aims are: (1) to establish in vitro model for bovine luteal endothelial cells examination; (2) to study the effect of cytokines: tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) on cell viability, leukotrienes (LTs) and PG synthases, and endothelin-1 (EDN-1) mRNA, protein expression and their secretion in bovine immortalized luteal endothelial (EnCL-1) cells., Methods: The primary cultures of bovine luteal endothelial cells were immortalized by transfection with vector carrying the Simian virus 40 T-antigen (SV40 T-ag) sequence. Expression of SV40 T-ag gene in EnCL-1 cells was confirmed by RT-PCR and immunofluorescence staining showed the presence of endothelial cell markers: VE-cadherin and von Willebrand factor. EnCL-1 cells were stimulated by TNFalpha with IFNgamma (50 ng/ml each) for 24 h. Cell viability, mRNA expression (real time RT-PCR), protein expression (western blotting) for LTC4 synthase (LTC4S), LTA4 hydrolase (LTA4H), PGE2 and PGF2alpha synthases and endothelin-1 (EDN-1), and levels of LTs (B4 and C4) and PGs (E2 and F2alpha) and EDN-1 in the medium (EIA) were evaluated., Results: We received immortalized luteal endothelial cell line (EnCL-1). Cytokines did not change EnCL-1 cell viability but increased mRNA expression of LTC4S, LTA4H, PGE2 and PGF2alpha synthases and EDN-1. EDN-1/2/3, LTC4 and PGF2alpha synthases protein expression were elevated in the presence of TNFalpha/IFNgamma, and accompanied by increased EDN-1, LTC4 and PGF2alpha secretion. Cytokines had no effect on PGES and LTA4H protein expression, and PGE2 and LTB4 release., Conclusions: TNFalpha and IFNgamma modulate EnCL-1 cell function. Moreover, established EnCL-1 cell line appears to be a good model for investigating the molecular mechanisms related to cytokines action and aa metabolites production in cattle.

As a continuous effort of developing highly enantioselective epoxide hydrolase from marine microorganisms, it was found that Maritimibacter alkaliphilus KCCM 42376 [corrected] was highly enantioselective toward racemic glycidyl phenyl ether (GPE). An open reading frame (ORF) encoding a putative epoxide hydrolase (EHase) was cloned from the genome of Maritimibacter alkaliphilus KCCM 42376 [corrected], followed by expression and purification in Escherichia coli. The purified EHase (REH) hydrolyzed (S)-GPE preferentially over (R)-GPE. Enantiopure (R)-GPE from kinetic resolution of 29.2 mM racemic GPE using the purified REH could be obtained with enantiopurity of more than 99.9% enantiomeric excess (ee) and 38.4% yield (theoretical, 50%) within 20 min (enantiomeric ratio (E-value): 38.4). The enantioselective activity of REH toward GPE was also confirmed by the analysis of the vicinal diol, 3-phenoxy-1,2-propanediol. To our knowledge, this study demonstrates the highest enantioselective resolution of racemic GPE using a purified biocatalyst among the known native EHases., ((c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We have shown previously that high expression levels of microsomal epoxide hydrolase (mEH) correlate with a poor prognosis of breast cancer patients receiving tamoxifen, suggesting that enhanced mEH expression could lead to antiestrogen resistance (Fritz et al. in J Clin Oncol 19:3-9, 2001). Thus, the purpose of this study was to gain insights into the role of mEH in hormone-responsive tissues. We analyzed biopsy samples of the endometrium by immunohistochemical staining, pointing to a regulation of mEH during the menstrual cycle: during the first half mEH expression was low, increased during the second half and reached highest levels during pregnancy. Additionally, the progesterone receptor (PR) positive human endometrial cell lines IKPRAB-36 (estrogene receptor alpha [ERalpha] negative) and ECC1-PRAB72 (ERalpha positive) were chosen to further investigate the hormonal regulation of mEH expression. Western Blot and quantitative RT-PCR analysis revealed an increase of mEH expression after treatment with medroxy-progesterone 17-acetate (MPA) in the ERalpha containing ECC1-PRAB72 cells. In contrast our results suggest that MPA had no influence on the mEH protein level in the ERalpha- IKPRAB-36 cells. In conclusion, mEH expression is regulated by progesterone in the presence of both PRs and ERalpha.

Both in-house human genetic and literature data have converged on the identification of leukotriene 4 hydrolase (LTA(4)H) as a key target for the treatment of cardiovascular disease. We combined fragment-based crystallography screening with an iterative medicinal chemistry effort to optimize inhibitors of LTA(4)H. Ligand efficiency was followed throughout our structure-activity studies. As applied within the context of LTA(4)H inhibitor design, the chemistry team was able to design a potent compound 20 (DG-051) (K(d) = 26 nM) with high aqueous solubility (>30 mg/mL) and high oral bioavailability (>80% across species) that is currently undergoing clinical evaluation for the treatment of myocardial infarction and stroke. The structural biology-chemistry interaction described in this paper provides a sound alternative to conventional screening techniques. This is the first example of a gene-to-clinic paradigm enabled by a fragment-based drug discovery effort.

Soluble epoxide hydrolase (sEH) is a key enzyme involved in the metabolism of epoxy fatty acid mediators such as epoxyeicosatrienoic acids with emerging roles in the regulations of hypertension and inflammation. Inhibitors of human sEH (hsEH) are effective drug candidates for the treatment of cardiovascular diseases. Preparation of hsEH for enzyme inhibition studies has been carried out by using baculovirus expression system. We herein explored the feasibility of expression of hsEH in Escherichia coli cells for the study of high-throughput screening assays of enzyme inhibitors, because the bacterial expression system is easier to handle and more cost-effective than the baculovirus expression system. The functional target enzyme was successfully produced in prokaryotic expression system by an auto-induction method and exhibited comparable enzyme activity to that yielded in baculovirus expression system. The bacterial-hsEH showed similar sensitivity to the baculovirus-hsEH against six reported inhibitors. Overalls indicate that bacterial expression of hsEH employed in the present study is useful for preparing enzymatically active hsEH, leading to effective performance of high-throughput screening assay of hsEH inhibitors and to rapid identification of novel drug candidates for the treatment of cardiovascular diseases.

Microsomal epoxide hydrolase (EPHX1) biotransforms epoxide derivatives of pharmaceuticals, including metabolites of certain antiepileptic medications, such as phenytoin and carbamazepine, and many environmental epoxides, such as those derived from butadiene, benzene, and carcinogenic polyaromatic hydrocarbons. We previously identified a far upstream promoter region, designated E1-b, in the EPHX1 gene that directs expression of an alternatively spliced EPHX1 mRNA transcript in human tissues. In this investigation, we characterized the structural features and expression character of the E1-b promoter region. Results of quantitative real-time polymerase chain reaction analyses demonstrated that the E1-b variant transcript is preferentially and broadly expressed in most tissues, such that it accounts for the majority of total EPHX1 transcript in vivo. Comparative genomic sequence comparisons indicated that the human EPHX1 E1-b gene regulatory region is primate-specific. Direct sequencing and genotyping approaches in 450 individuals demonstrated that the E1-b promoter region harbors a series of transposable element cassettes, including a polymorphic double Alu insertion. Results of reporter assays conducted in several human cell lines demonstrated that the inclusion of the Alu(+/+) insertion significantly decreases basal transcriptional activities. Furthermore, using haplotype block analyses, we determined that the E1-b polymorphic promoter region was not in linkage disequilibrium with two previously identified nonsynonomous single nucleotide polymorphisms (SNPs) in the coding region or with functional SNPs previously identified in the proximal promoter region of the gene. These results demonstrate that the upstream E1-b promoter is the major regulator of EPHX1 expression in human tissues and that polymorphism in this region may contribute an interindividual risk determinant to xenobiotic-induced toxicities.

A gene encoding a putative epoxide hydrolase (EHase) was identified by analyzing an open reading frame of the genome sequence of Novosphingobium aromaticivorans, retaining the conserved catalytic residues such as the catalytic triad (Asp177, Glu328, and His355) and the oxyanion hole. The enantioselective EHase gene (neh) was cloned, and the recombinant EHase could be purified to apparent homogeneity by one step of metal affinity chromatography and further characterized. The purified N. aromaticivorans enantioselective epoxide hydrolase (NEH) showed enantioselective hydrolysis toward styrene oxide, glycidyl phenyl ether, epoxybutane, and epichlorohydrin. The optimal EHase activity toward styrene oxide occurred at pH 6.5 and 45 degrees C. The purified NEH could preferentially hydrolyze (R)-styrene oxide with enantiomeric excess of more than 99% and 11.7% yield after 20-min incubation at an optimal condition. The enantioselective hydrolysis of styrene oxide was also confirmed by the analysis of the vicinal diol, 1-phenyl-1,2-ethanediol. The hydrolyzing rates of the purified NEH toward epoxide substrates were not affected by as high as 100 mM racemic styrene oxide.

The polycyclic aromatic hydrocarbon 7, 12-dimethylbenz[a]anthracene, (DMBA), targets and destroys all follicle types in rat and mouse ovaries. DMBA requires bioactivation to DMBA-3,4-diol-1,2-epoxide for ovotoxicity via formation of the intermediate, DMBA-3,4-diol (catalyzed by microsomal epoxide hydrolase; mEH). mEH was shown to be involved in DMBA bioactivation for ovotoxicity induction in B6C3F(1) mouse ovaries. The current study compared DMBA and DMBA-3,4-diol mediated ovotoxicity, and investigated mEH involvement in DMBA-3,4-diol bioactivation in Fischer 344 (F344) rat ovary. F344 postnatal day (PND) 4 rat ovaries were cultured in vehicle control or media containing 1) DMBA or DMBA-3,4-diol (12.5 nM - 1 muM; 15 days); 2) DMBA (1 muM; 6 h - 15 days); and 3) DMBA (1 muM) or DMBA-3,4-diol (75 nM)+/-the mEH activity inhibitor cyclohexene oxide (CHO; 2 mM; 4 days). Ovaries were histologically evaluated and mEH mRNA and protein were measured by reverse transcriptase PCR or Western blotting, respectively. Ovotoxicity following 15 days of culture occurred (P<0.05) at lower concentrations of DMBA-3,4-diol (12.5 nM - primordial; 75 nM - primary) than DMBA (75 nM - primordial; 375 nM - primary). The temporal pattern of mEH expression following DMBA exposure showed mRNA up-regulation (P<0.05) on day 2, with increased protein (P<0.05) on day 4, the earliest time of observed follicle loss (P<0.05). mEH inhibition prevented DMBA-induced, but not DMBA-3,4-diol-induced ovotoxicity. These results demonstrate a conserved response in mice and rats for ovarian mEH involvement in DMBA bioactivation to its ovotoxic, 3,4-diol-1,2-epoxide form.

Soluble epoxide hydrolase (sEH) is a xenobiotic-metabolizing enzyme that metabolizes epoxides to produce vicinal diols. Diabetes is a common pathological condition which effects drug metabolism. This study, investigates changes in the levels of sEH in mice with diabetes induced by streptozotocin (STZ). Diabetes reduced the amount of sEH protein in the liver and insulin restored the level of protein. The kidneys are a target of diabetes. Diabetes significantly decreased levels of sEH protein and also mRNA. The distribution of sEH in the kidney was studied with immunostaining. There was distinct staining in the proximal tubules but not in the glomerulus or other regions. Diabetes is characterized by high glucose concentrations that lead to increased production of reactive oxygen species (ROS). High glucose suppressed sEH mRNA and protein expression in Hep3B cells. NADPH oxidase is the main source of ROS generation in high glucose condition. The NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPIC), inhibited decrease in sEH expression at high glucose and hydrogen peroxide suppressed sEH expression. These findings indicate that diabetes reduces sEH expression by inducing ROS and may have important effects on the metabolism of xenobiotics and endogenous substrates of sEH.

The metabolism of arachidonic acid by the cyclooxygenase (COX) or lipoxygenase (LO) pathways generates eicosanoids that have been implicated in the pathogenesis of a variety of human diseases, including cancer. In this study, we examined the expression and significance of components within the 5-LO pathway in human neuroblastoma, an embryonal tumor of the sympathetic nervous system. High expression of 5-LO, 5-LO-activating protein (FLAP), leukotriene A(4) hydrolase, leukotriene C(4) synthase, and leukotriene receptors was detected in a majority of primary neuroblastoma tumors and all cell lines investigated. Expression of 5-LO and FLAP was evident in tumor cells but not in nonmalignant adrenal medulla where neuroblastomas typically arise. Moreover, neuroblastoma cells produce leukotrienes, and stimulation of neuroblastoma cells with leukotrienes increased neuroblastoma cell viability. Inhibitors of 5-LO (AA-861), FLAP (MK-886), or the leukotriene receptor antagonist montelukast inhibited neuroblastoma cell growth by induction of G(1)-cell cycle arrest and apoptosis. Similarly, specific 5-LO and leukotriene receptor silencing by small interfering RNA decreased neuroblastoma cell growth. These findings provide new insights into the pathobiology of neuroblastoma, and the use of leukotriene pathway inhibitors as a novel adjuvant therapy for children with neuroblastoma warrants further consideration.

Polyether metabolites are an important class of natural products. Although their biosynthesis, especially construction of polyether skeletons, attracted organic chemists for many years, no experimental data on the enzymatic polyether formation has been obtained. In this study, a putative epoxide hydrolase gene lsd19 found on the biosynthetic gene cluster of an ionophore polyether lasalocid was cloned and successfully overexpressed in Escherichia coli. Using the purified Lsd19, a proposed substrate, bisepoxyprelasalocid, and its synthesized analogue were successfully converted into lasalocid A and its derivative via a 6-endo-tet cyclization mode. On the other hand, treatment of the bisepoxide with trichloroacetic acid gave isolasalocid A via a 5-exo-tet cyclization mode. Therefore, the enzymatic conversion observed in this study unambiguously showed that the bisepoxyprelasalocid is an intermediate of the lasalocid biosynthesis and that Lsd19 catalyzes the sequential cyclic ether formations involving an energetically disfavored 6-endo-tet cyclization. This is the first example of the enzymatic epoxide-opening reactions leading to a polyether natural product.

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid, which function in the brain to regulate cerebral blood flow and protect against ischemic brain injury. EETs are converted by soluble epoxide hydrolase (sEH) to the corresponding inactive diol metabolites. Previous animal studies have indicated that sEH gene deletion or treatment with sEH inhibitors results in increased levels of EETs and protection against stroke-induced brain damage. To begin elucidating the underlying mechanism for these effects, we sought to determine the distribution, expression, and activity of sEH in human brain samples obtained from patients with no neurological changes/pathologies. Immunohistochemical analyses showed the distribution of sEH mainly in the neuronal cell bodies, oligodendrocytes, and scattered astrocytes. Surprisingly, in the choroid plexus, sEH was found to be highly expressed in ependymal cells. Vascular localization of sEH was evident in several regions, where it was highly expressed in the smooth muscles of the arterioles. Western blot analysis and enzyme assays confirmed the presence of sEH in the normal brain. Our results indicate differential localization of sEH in the human brain, thus suggestive of an essential role for this enzyme in the central nervous system. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in approximately 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of approximately 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.

We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P450 (CYP)1A expression for up to 45 days by mechanisms other than persistence of the parent MC (Moorthy, J. 2000. Pharmacology. Exp. Ther. 294, 313-322). The CYP1A genes are members of the Ah gene battery that also encode CYP1B1 and phase II enzymes such as glutathione S-transferase (GST-alpha), UDP glucuronyl transferase (UGT)1A, NAD(P)H (nicotinamide adenine dinucleotide phosphate, reduced):quinone oxidoreductase I (NQO1), aldehyde dehydrogenase (ALDH), etc. Therefore, in this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1B1 and phase II genes, which are in part regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 mumol/kg), ip, once daily for 4 days, and expression of CYP1B1 and several phase II (e.g., GST-alpha, NQO1) genes and their corresponding proteins were determined in lung and liver. The major finding was that MC persistently induced (3- to 10-fold) the expression of several phase II enzymes, including GST-alpha, NQO1, UGT1A1, ALDH, and epoxide hydrolase in both tissues for up to 28 days. However, MC did not elicit sustained induction of CYP1B1. Our results thus support the hypothesis that MC elicits coordinated and sustained induction of phase II genes presumably via persistent activation of the AHR, a phenomenon that may have implications for chemical-induced carcinogenesis and chemopreventive strategies in humans.

Genetic polymorphisms of microsomal epoxide hydrolase (mEH) have been associated with increased risk of lung cancer. However, expression of mEH and its clinical significance in non-small cell lung cancer (NSCLC) have not been investigated. In this study we investigated the expression and genetic polymorphism of mEH in non-small cell lung cancer (NSCLC) patients. Genetic polymorphism was determined by restriction fragment length polymorphism of polymerase chain reaction (PCR) products. The allelic expression pattern as well as expression level of mEH were determined by reverse transcription-PCR (RT-PCR), cDNA sequencing, sequence alignment, immunoblotting and immunohistochemistry. Genotype distributions of mEH in Taiwan's NSCLC patients were 44.4% of 340TAC/340TAC, 48.6% of 340TAC/340CAC, and 7.0% of 340CAC/340CAC in exon 3, and 80.6% of 418CAT/418CAT, 19.4% of 418CAT/418CGT and 0% of 418CGT/418CGT in exon 4. Of the 72 NSCLC biopsies analyzed, mEH was expressed in 60 (83%) surgical specimens, and the major allelic expression pattern was fast type (Tyr113) in exon 3 (90.3%) and slow type (His139) in exon 4 (100%). Immunohistochemical staining showed that mEH was expressed in 326 of 423 (77.0%) tumor (lung tissue) specimens and in 48 of 93 (51.6%) metastatic lymph nodes. A significant difference in patient survival was found when mEH expression and adriamycin-containing chemotherapy were used to group patients (p=0.0167). In conclusion, with the combination of fast type (Tyr113) and slow type (His139), the mEH enzyme expressed in most NSCLC patients may have intermediate activity. Our findings indicate that with respect to cancer risk and disease progression, the expression level of mEH is as important as genetic polymorphism. In addition, mEH expression in NSCLC could be involved in drug resistance and prognosis of patients.

Epoxide hydrolases (EHs), especially those of fungal origin, have the ability to catalyse the enantioselective hydrolysis of epoxides to their corresponding diols. Recombinant DNA technology has been used extensively to overproduce these catalysts for the efficient hydrolytic kinetic resolution of epoxides, which serve as high-value intermediates in the fine chemicals and pharmaceutical industries. Degenerate primers, based on data from available EH-encoding gene sequences, in conjunction with inverse PCR, were used to amplify the genomic EH-encoding gene from Rhodotorula mucilaginosa. The 2347 bp genomic sequence revealed a 1979 bp ORF containing nine introns. The cDNA sequence revealed an 1185 bp EH-encoding gene that translates into a 394 amino acid protein exhibiting low sequence homology towards the known EH proteins. The EH gene from R. mucilaginosa was functionally expressed in Yarrowia lipolytica using a constitutive integrative expression cassette. Whole-cell biotransformation of (2,3-epoxypropyl)benzene, using the recombinant EH, revealed activity and selectivity far superior to any other activity and selectivity reported in literature using wild-type organisms. The GenBank Accession No. for the R. mucilaginosa EH gene is AY627310.

Endogenous factors, including hormones, growth factors and cytokines, play an important role in the regulation of hepatic drug metabolizing enzyme expression in both physiological and pathophysiological conditions. Diabetes, fasting, obesity, protein-calorie malnutrition and long-term alcohol consumption produce changes in hepatic drug metabolizing enzyme gene and protein expression. This difference in expression alters the metabolism of xenobiotics, including procarcinogens, carcinogens, toxicants and therapeutic agents, potentially impacting the efficacy and safety of therapeutic agents, and/or resulting in drug-drug interactions. Although the mechanisms by which xenobiotics regulate drug metabolizing enzymes have been studied intensively, less is known regarding the cellular signaling pathways and components which regulate drug metabolizing enzyme gene and protein expression in response to hormones and cytokines. Recent findings, however, have revealed that several cellular signaling pathways are involved in hormone- and growth factor-mediated regulation of drug metabolizing enzymes. Our laboratory has reported that insulin and growth factors regulate drug metabolizing enzyme gene and protein expression, including cytochromes P450 (CYP), glutathione S-transferases (GST) and microsomal epoxide hydrolase (mEH), through receptors which are members of the large receptor tyrosine kinase (RTK) family, and by downstream effectors such as phosphatidylinositol 3-kinase, mitogen activated protein kinase (MAPK), Akt/protein kinase B (PKB), mammalian target of rapamycin (mTOR), and the p70 ribosomal protein S6 kinase (p70S6 kinase). Here, we review current knowledge of the signaling pathways implicated in regulation of drug metabolizing enzyme gene and protein expression in response to insulin and growth factors, with the goal of increasing our understanding of how disease affects these signaling pathways, components, and ultimately gene expression and translational control.

Primary effusion lymphoma (PEL) is a herpesvirus-8-associated lymphoproliferative disease characterized by migration of tumor cells to serous body cavities. PEL cells originate from postgerminal center B cells and share a remarkable alteration in B cell transcription factor expression and/or activation with classical Hodgkin's disease cells. Comparative analysis of gene expression by cDNA microarray of BCBL-1 cells (PEL), L-428 (classical Hodgkin's disease), and BJAB cells revealed a subset of genes that were differentially expressed in BCBL-1 cells. Among these, four genes involved in cell migration and chemotaxis were strongly up-regulated in PEL cells: leukotriene A4 (LTA4) hydrolase (LTA4H), IL-16, thrombospondin-1 (TSP-1), and selectin-P ligand (PSGL-1). Up-regulation of LTA4H was investigated at the transcriptional level. Full-length LTA4H promoter exhibited 50% higher activity in BCBL-1 cells than in BJAB or L-428 cells. Deletion analysis of the LTA4H promoter revealed a positive cis-regulatory element active only in BCBL-1 cells in the promoter proximal region located between -76 and -40 bp. Formation of a specific DNA-protein complex in this region was confirmed by EMSA. Coculture of ionophore-stimulated primary neutrophils with BCBL-1 cells leads to an increased production of LTB4 compared with coculture with BJAB and L-428 cells as measured by enzyme immunoassay, demonstrating the functional significance of LTA4H up-regulation.

Leukotrienes (LT) are a group of proinflammatory lipid mediators that are implicated in the pathogenesis and progression of atherosclerosis. Here we report that mRNA levels for the three key proteins in LTB4 biosynthesis, namely 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), and LTA4 hydrolase (LTA4H), are significantly increased in human atherosclerotic plaque (n = 72) as compared with healthy controls (n = 6). Neither LTC4 synthase nor any of the LT receptors exhibits significantly increased mRNA levels. Immunohistochemical staining revealed abundant expression of 5-LO, FLAP, and LTA4H protein, colocalizing in macrophages of intimal lesions. Human lesion tissue converts arachidonic acid into significant amounts of LTB4, and a selective, tight-binding LTA4H inhibitor can block this activity. Furthermore, expression of 5-LO and LTA4H, but not FLAP, is increased in patients with recent or ongoing symptoms of plaque instability, and medication with warfarin correlates with increased levels of FLAP mRNA. In contrast to human plaques, levels of 5-LO mRNA are not significantly increased in plaque tissues from two atherosclerosis-prone mouse strains, and mouse plaques exhibit segregated cellular expression of LTA4H and 5-LO as well as strong increases of CysLT1 and CysLT2 mRNA. These discrepancies indicate that phenotypic changes in the synthesis and action of LT in specific mouse models of atherosclerosis should be cautiously translated into human pathology. The abundant expression of LTA4H and correlation with plaque instability identify LTA4H as a potential target for pharmacological intervention in treatment of human atherosclerosis.

We reported previously that insulin elevated alpha-class glutathione S-transferase (GSTs) protein levels in primary cultured rat hepatocytes (Kim et al., 2003b). In contrast, glucagon down-regulated alpha- and pi-class GST expression, and mechanistic research implicated cAMP and protein kinase A in this process (Kim et al., 2003b). The present study examines the signaling pathways involved in the regulation of alpha-class GST in response to insulin in primary cultured rat hepatocytes. Protein levels of GSTA1/2 and GSTA3/5 and activity of GST toward 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were increased in an insulin concentration-dependent manner. Treatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or rapamycin, an inhibitor of mammalian target of rapamycin and ribosomal p70 S6 kinase (p70S6K) phosphorylation, or with an adenovirus containing green fluorescent protein and a dominant-negative and kinase-dead Akt, effectively inhibited the insulin-mediated increase in alpha-class GST expression and GST activity toward NBD. In contrast, PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of mitogen-activated protein kinase kinase, SP600125 (1,9-pyrazoloanthrone), an inhibitor of c-Jun N-terminal kinase, SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imadazole], an inhibitor of p38 mitogen-activated protein kinase, or bisindolylmaleimide, a broad spectrum inhibitor of protein kinase C, did not inhibit the insulin-mediated increase in alpha-class GST protein levels in hepatocytes. These results show that PI3K/Akt/p70S6K signaling is active in the insulin-mediated up-regulation of the antioxidant defense system and that low insulin levels, as encountered in diabetes, potentially increase the susceptibility of hepatocytes to xenobiotic-mediated and/or oxidative stress-mediated damage.

Mycobacterium tuberculosis epoxide hydrolases A and B, corresponding to open reading frames Rv3617 and Rv1938, are detoxification enzymes against epoxides. The recombinant forms of these enzymes have been expressed in Escherichia coli and purified to homogeneity. Diffraction-quality crystals of Rv3617 and Rv1938 were obtained by the hanging-drop vapour-diffusion technique. Crystals of Rv3617 and Rv1938 diffracted to 3.0 and 2.1 A resolution, respectively, at the ALS synchrotron at Berkeley, CA, USA.