Soluble Epoxide Hydrolase Activity Research Articles

Abstract 531: Regulation of Plasma 20-HETE and Soluble Epoxide Hydrolase by Salt Balance in Normotensive Subjects

The tissue sources of circulating and urinary 20-HETE and EETs are not well known. We have previously shown urine 20-HETE abnormalities in salt-sensitive (SS) hypertension. We studied plasma 20-HETE and EETs in normotensive volunteers classified as SS or salt resistant (SR) with an acute protocol of salt loading (HS, 460 mmol diet and iv) and depletion (LS, 10 mmol and furosemide). Plasma 20-HETE and total (EET+DHET) or active EET did not differ between SR and SS during baseline (B, UNaV 146±17 mmol/24hr), HS (422±20) or LS (24±6). In normal SR subjects, HS did not affect the levels of either eicosanoid but reduced degradation of EETs into DHETs by decreasing the activity of soluble epoxide hydrolase (sEH or DHET/[EET+DHET]). No major clinical or biochemical correlates for plasma 20-HETE were identified in B, HS or LS. In SR but not SS, EET+DHET (r=0.57, p<0.003) and DHET (r=0.45, p<0.03) correlated with aldosterone during B and HS. LS stimulated 20-HETE in both SS and SR and also unexpectedly increased EET+DHET in SR subjects. This did not lead to increased EETs because of concomitant stimulation of sEH with increased degradation into DHETs. In SR, stimulation of 20-HETE by LS correlated with greater natriuresis (r=0.71, p<0.02), greater reduction of MAP (r=0.66, p<0.03) and lesser stimulation of aldosterone (r=0.64, p<0.04), relationships not observed in SS. Finally, in SR subjects only, during B and HS, plasma 20-HETE correlated with the activity of sEH (r=0.56, p<0.005) and levels of DHET (r=0.63, p<0.001), but not with total or active EETs. Our data on stimulation of plasma 20-HETE by LS and its correlates (markers of severity of sodium depletion) suggest recruitment of systemic vasoconstrictor 20-HETE for maintenance of BP. Plasma EET responses to HS and LS are not consistent with its renal natriuretic role but correlate with aldosterone responses to salt balance, perhaps reflecting modulation of aldosterone actions on extrarenal ENaC. Although no significant correlations were detected between blood pressure and EETs, there was a direct relationship between plasma 20-HETE, sEH activity and degradation of EETs into DHETs. This suggests coordinated vasoconstriction and vasodilation by these eicosanoids in normal SR humans, which is disrupted in normotensive SS.

Arg287Gln variant of EPHX2 and epoxyeicosatrienoic acids are associated with insulin sensitivity in humans

Epoxyeicosatrienoic acids (EETs) protect against the development of insulin resistance in rodents. EETs are hydrolyzed to less biologically active diols by soluble epoxide hydrolase (encoded for by EPHX2). Functional variants of EPHX2 encode for enzymes with increased (Lys55Arg) or decreased (Arg287Gln) hydrolase activity. This study tested the hypothesis that variants of EPHX2 are associated with insulin sensitivity or secretion in humans. Subjects participating in metabolic phenotyping studies were genotyped. Eighty-five subjects underwent hyperglycemic clamps. There was no relationship between the Lys55Arg genotype and insulin sensitivity or secretion. In contrast, the EPHX2 287Gln variant was associated with higher insulin sensitivity index (p=0.019 controlling for body mass index and metabolic syndrome). Also, there was an interactive effect of EPHX2 Arg287Gln genotype and body mass index on insulin sensitivity index (p=0.029). There was no relationship between EPHX2 Arg287Gln genotype and acute or late-phase glucose-stimulated insulin secretion, but disposition index was higher in 287Gln carriers compared with Arg/Arg (p=0.022). Plasma EETs correlated with insulin sensitivity index (r=0.64, p=0.015 for total EETs) and were decreased in the metabolic syndrome. A genetic variant that results in decreased soluble epoxide hydrolase activity is associated with increased insulin sensitivity, as are higher EETs.

Soluble epoxide hydrolase activity regulates inflammatory responses and seizure generation in two mouse models of temporal lobe epilepsy.

Neuroinflammation is known to be involved in epileptogenesis with unclear mechanisms. Inhibition of soluble epoxide hydrolase (sEH) seems to offer anti-inflammatory protection to ischemic brain injury in rodents. Thus, it is hypothesized that sEH inhibition might also affect the neuroinflammatory responses caused by epileptic seizures. In the present study, we investigated the involvement of sEH in neuroinflammation, seizure generation and subsequent epileptogenesis using two mouse models of temporal lobe epilepsy. Experimental epileptic seizures were induced by either pilocarpine or electrical amygdala kindling in both wild-type (WT) C57BL/6 mice and sEH knockout (sEH KO) mice. The sEH expression in the hippocampus was detected by immunohistochemistry and Western blot analysis. The effects of the sEH hydrolase inhibitors, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy) phenyl)-urea (TPPU), and of the genetic deletion of sEH on seizure-induced neuroinflammatory responses and the development of epilepsy were evaluated. In the hippocampus of WT mice, sEH was mainly expressed in astrocytes (GFAP(+)), neurons (NeuN(+)) and scattered microglia (Iba-1(+)) in the regions of CA1, CA3 and dentate gyrus. Expression of sEH was significantly increased on day 7, 14, 21 and 28 after pilocarpine-induced status epilepticus (SE). Administration with sEH inhibitors attenuated the SE-induced up-regulation of interleukin-1β (IL-1β) and interleukin-6 (IL-6), the degradation of EETs, as well as IκB phosphorylation. Following treatment with AUDA, the frequency and duration of spontaneous motor seizures in the pilocarpine-SE mice were decreased and the seizure-induction threshold of the fully kindled mice was increased. Up-regulation of hippocampal IL-1β and IL-6 was found in both WT and sEH KO mice after successful induction of SE. Notably, sEH KO mice were more susceptible to seizures than WT mice. Seizure related neuroinflammation and ictogenesis were attenuated by pharmacological inhibition of sEH enzymatic activity but not by sEH genetic deletion. Therefore, sEH may play an important role in the generation of epilepsy. Furthermore, the effectiveness of AUDA in terms of anti-inflammatory and anti-ictogenesis properties suggests that it may have clinical therapeutic implication for epilepsy in the future, particularly when treating temporal lobe epilepsy.

Melatonin prevents maternal fructose intake-induced programmed hypertension in the offspring: roles of nitric oxide and arachidonic acid metabolites.

Fructose intake has increased globally and is linked to hypertension. Melatonin was reported to prevent hypertension development. In this study, we examined whether maternal high fructose (HF) intake causes programmed hypertension and whether melatonin therapy confers protection against the process, with a focus on the link to epigenetic changes in the kidney using next-generation RNA sequencing (NGS) technology. Pregnant Sprague-Dawley rats received regular chow or chow supplemented with HF (60% diet by weight) alone or with additional 0.01% melatonin in drinking water during the whole period of pregnancy and lactation. Male offspring were assigned to four groups: control, HF, control+melatonin (M), and HF+M. Maternal HF caused increases in blood pressure (BP) in the 12-wk-old offspring. Melatonin therapy blunted the HF-induced programmed hypertension and increased nitric oxide (NO) level in the kidney. The identified differential expressed gene (DEGs) that are related to regulation of BP included Ephx2, Col1a2, Gucy1a3, Npr3, Aqp2, Hba-a2, and Ptgs1. Of which, melatonin therapy inhibited expression and activity of soluble epoxide hydrolase (SEH, Ephx2 gene encoding protein). In addition, we found genes in arachidonic acid metabolism were potentially involved in the HF-induced programmed hypertension and were affected by melatonin therapy. Together, our data suggest that the beneficial effects of melatonin are attributed to its ability to increase NO level in the kidney, epigenetic regulation of genes related to BP control, and inhibition of SEH expression. The roles of DEGs by the NGS in long-term epigenetic changes in the adult offspring kidney require further clarification.

Flaxseed consumption reduces blood pressure in patients with hypertension by altering circulating oxylipins via an α-linolenic acid-induced inhibition of soluble epoxide hydrolase.

In a randomized, double-blinded, controlled clinical trial, participants with peripheral arterial disease (75% hypertensive) consumed 30 g of milled flaxseed/d for 6 months. The flaxseed group exhibited significant reductions in systolic (-10 mm Hg) and diastolic (-7 mm Hg) blood pressure. Flaxseed contains the n3 fatty acid α-linolenic acid. Plasma α-linolenic acid increased with ingestion of flaxseed and was inversely associated with blood pressure. However, the antihypertensive mechanism was unclear. Oxylipins derived from polyunsaturated fatty acids regulate vascular tone. Therefore, the objective was to examine whether flaxseed consumption altered plasma oxylipins in a manner that influenced blood pressure. Plasma of FlaxPAD (Flaxseed for Peripheral Arterial Disease) participants underwent solid phase extraction and high-performance liquid chromatography-mass spectrometry/mass spectrometry analysis. The flaxseed group exhibited significant decreases in 8 plasma oxylipins versus control. Six of these (5,6-, 8,9-, 11,12-, 14,15-dihydroxyeicosatrienoic acid and 9,10- and 12,13-dihydroxyoctadecenoic acid) were products of soluble epoxide hydrolase, a pharmacological target for antihypertensive treatment. Patients exhibiting a decrease in total plasma soluble epoxide hydrolase-derived oxylipins, exhibited a significant decrease in systolic blood pressure (mean [95% confidence interval], -7.97 [-14.4 to -1.50] mm Hg) versus those who exhibited increased plasma soluble epoxide hydrolase-derived oxylipins (+3.17 [-4.78 to 11.13] mm Hg). These data suggest that a flaxseed bioactive may have decreased blood pressure via soluble epoxide hydrolase inhibition. Using a soluble epoxide hydrolase inhibitor screening assay, increasing concentrations of α-linolenic acid decreased soluble epoxide hydrolase activity (P=0.0048; ρ=-0.94). In conclusion, α-linolenic acid in flaxseed may have inhibited soluble epoxide hydrolase, which altered oxylipin concentrations that contributed to the antihypertensive effects in patients with peripheral arterial disease. http://www.clinicaltrials.gov. Unique identifier: NCT00781950.

A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: A randomized crossover trial

ObjectiveThe beneficial effects of fish and n-3 polyunsaturated fatty acids (PUFAs) consumption on atherosclerosis have been reported in numerous epidemiological studies. However, to the best of our knowledge, the effects of a fish-based diet intervention on endothelial function have not been investigated. Therefore, we studied these effects in postmenopausal women with type 2 diabetes mellitus (T2DM). Materials/MethodsTwenty-three postmenopausal women with T2DM were assigned to two four-week periods of either a fish-based diet (n-3 PUFAs≧3.0g/day) or a control diet in a randomized crossover design. Endothelial function was measured with reactive hyperemia using strain-gauge plethysmography and compared with the serum levels of fatty acids and their metabolites. Endothelial function was determined with peak forearm blood flow (Peak), duration of reactive hyperemia (Duration) and flow debt repayment (FDR). ResultsA fish-based dietary intervention improved Peak by 63.7%, Duration by 27.9% and FDR by 70.7%, compared to the control diet. Serum n-3 PUFA levels increased after the fish-based diet period and decreased after the control diet, compared with the baseline (1.49 vs. 0.97 vs. 1.19mmol/l, p<0.0001). There was no correlation between serum n-3 PUFA levels and endothelial function. An increased ratio of epoxyeicosatrienoic acid/dihydroxyeicosatrienoic acid was observed after a fish-based diet intervention, possibly due to the inhibition of the activity of soluble epoxide hydrolase. ConclusionsA fish-based dietary intervention improves endothelial function in postmenopausal women with T2DM. Dissociation between the serum n-3 PUFA concentration and endothelial function suggests that the other factors may contribute to this phenomenon.

Acute mercury toxicity modulates cytochrome P450, soluble epoxide hydrolase and their associated arachidonic acid metabolites in C57BL/6 mouse heart (652.5)

Mercury exposure is associated with increased risk of cardiovascular disease and profound cardiotoxicity. However, the correlation between Hg2+‐mediated toxicity and alteration in cardiac cytochrome P450s (Cyp) and their dependent arachidonic acid metabolites has never been investigated. Therefore, we investigated the effect of acute mercury toxicity on the expression of Cyp‐epoxygenases and Cyp‐ω‐hydroxylases; and their associated arachidonic acid metabolites in mice hearts. In addition, we examined the expression and activity of soluble epoxide hydrolase (sEH) as a key player in arachidonic acid metabolism pathway. Mercury toxicity was induced by a single intraperitoneal injection (IP) of 2.5 mg/kg of mercuric chloride (HgCl2). Our results showed that mercury treatment caused a significant induction of the cardiac hypertrophy markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP); in addition to Cyp1a1, Cyp1b1, Cyp2b9, Cyp2b10, Cyp2b19, Cyp2c29, Cyp2c38, Cyp4a10, Cyp4a12, Cyp4a14, Cyp4f13, Cyp4f15, Cyp4f16 and Cyp4f18 gene expression. Moreover, Hg2+ significantly increased sEH protein expression and activity, in hearts of mercury‐treated mice, with a consequent decrease in 14,15‐, and 11,12‐epoxyeicosatrienoic acids (EETs) formation. Whereas the formation of 14,15‐, 11,12‐, 8,9‐dihydroxyeicosatrienoic acids (DHETs) was significantly increased. In conclusion, acute Hg2+ toxicity modulates the expression of several Cyp and sEH enzymes with a consequent decrease in the cardioprotective EETs which could represent a novel mechanism by which mercury causes progressive cardiotoxicity. Furthermore, inhibiting sEH might represent a novel therapeutic approach to prevent Hg2+‐induced hypertrophy.Grant Funding Source: Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN 250139

Coordinated responses of plasma 20-HETE and soluble epoxide hydrolase to salt in normal subjects: alterations in salt-sensitive normotension

We measured plasma 20-HETE, total (EET+DHET) and active EET (isoforms 11-12 and 14-15) in response to acute changes in salt balance in normotensive volunteers classified as salt-resistant (SR, 13) or saltsensitive (SS, 8) by 24-hr salt loading (160 mmol diet, 300 iv) and 24-hr salt depletion (10 mmol diet + furosemide 40 mg x 3). 20-HETE and total (EET+DHET) or active EET were not different between SR and SS in any state of salt balance and did not change with salt-loading in either group. During baseline and salt loading (UNaV 146 17 and 422 20 mmol/day) 20-HETE correlated with the activity of soluble epoxide hydrolase (sEH1⁄4DHET/[DHET+EET], r1⁄40.56, p<.005) and the level of DHET (r1⁄40.63, p<.001) but not with EET+DHET or EET. These relationships were not present in SS. Salt depletion increased 20-HETE in both SR (0.32 0.23 ng/ml, p<.02), and SS (0.48 0.20, p<.02). D20-HETE correlated with the magnitude of natriuresis (r1⁄40.71, p<.02), reduction of mean arterial pressure (r1⁄40.66, p<0.03), and negatively with the change in aldosterone (r1⁄4-0.64, p<.04) produced by salt-depletion in SR, but not in SS. Salt depletion in SR increased EET+DHET (0.10 0.05 ng/ml, p<.04) but not EET (0.05 0.04), reflecting increased activity of sEH (0.02 0.01, p<.0.03) with 31% conversion of increased EET+DHET into DHET. In contrast, in SS, neither EET+DHET nor EET increased after salt depletion. Conversion of EET+DHET to DHET, in the absence of stimulated sEH in this group, was only 15%. Our data suggest coordinated regulation of vasoconstrictor and vasodilator eicosanoids in all states of salt balance in normal subjects (SR group). Higher levels of 20-HETE in a salt-replete state and their responses to salt depletion are associated with increased degradation of EETs by sEH. Whether this is a direct effect of 20-HETE on sEH synthesis or activity cannot be ascertained from our study. This physiologic coordinated regulation seems disrupted in SS, and may play a role in salt sensitivity of blood pressure. Finally, the correlates of the increase of 20-HETE in response to salt depletion in SR suggest that this eicosanoid is recruited, as other vasoactive agents are, for compensatory vasoconstriction during volume depletion in normal subjects.

Modulation of arachidonic Acid metabolism in the rat kidney by sulforaphane: implications for regulation of blood pressure.

Background. We investigated the effects of sulforaphane (SF), the main active isothiocyanate in cruciferous vegetables, on arachidonic acid (AA) metabolism in the kidney and its effect on arterial blood pressure, using spontaneously hypertensive rats (SHR) as models. Methods. Rats were treated for 8 weeks with either drinking water alone (control) or SF (20 or 40 mg/kg) added to drinking water. Mean arterial pressure (MAP) was measured at 7-day intervals throughout the study. At the end of treatment rats were euthanized, and kidneys were harvested to prepare microsomes and measure enzymes involved in regulation of vasoactive metabolites: CYP4A, the key enzyme in the formation of 20-hydroxyeicosatetraenoic acid, and the soluble epoxide hydrolase, which is responsible for the degradation of the vasodilator metabolites such as epoxyeicosatetraenoic acids. Effect of SF on kidney expression of CYP4A was investigated by immunoblotting. Results. We found that treatment with SF leads to significant reductions in both, the expression and activity of renal CYP4A isozymes, as well as the activity of soluble epoxide hydrolase (sEH). Consistent with these data, we have found that treatment with SF resisted the progressive rise in MAP in the developing SHR in a dose-dependent manner. Conclusion. This is the first demonstration that SF modulates the metabolism of AA by both P450 enzymes and sEH in SHR rats. This may represent a novel mechanism by which SF protects SHR rats against the progressive rise in blood pressure.

Acute mercury toxicity modulates cytochrome P450, soluble epoxide hydrolase and their associated arachidonic acid metabolites in C57Bl/6 mouse heart

Mercury exposure is associated with increased risk of cardiovascular disease and profound cardiotoxicity. However, the correlation between Hg2+-mediated toxicity and alteration in cardiac cytochrome P450s (Cyp) and their dependent arachidonic acid metabolites has never been investigated. Therefore, we investigated the effect of acute mercury toxicity on the expression of Cyp-epoxygenases and Cyp-ω-hydroxylases and their associated arachidonic acid metabolites in mice hearts. In addition, we examined the expression and activity of soluble epoxide hydrolase (sEH) as a key player in arachidonic acid metabolism pathway. Mercury toxicity was induced by a single intraperitoneal injection (IP) of 2.5mg/kg of mercuric chloride (HgCl2). Our results showed that mercury treatment caused a significant induction of the cardiac hypertrophy markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP); in addition to Cyp1a1, Cyp1b1, Cyp2b9, Cyp2b10, Cyp2b19, Cyp2c29, Cyp2c38, Cyp4a10, Cyp4a12, Cyp4a14, Cyp4f13, Cyp4f15, Cyp4f16 and Cyp4f18 gene expression. Moreover, Hg2+ significantly increased sEH protein expression and activity levels in hearts of mercury-treated mice, with a consequent decrease in 14,15-, and 11,12-epoxyeicosatrienoic acids (EETs) levels. Whereas the formation of 14,15-, 11,12-, 8,9-dihydroxyeicosatrienoic acids (DHETs) was significantly increased. In conclusion, acute Hg2+ toxicity modulates the expression of several Cyp and sEH enzymes with a consequent decrease in the cardioprotective EETs which could represent a novel mechanism by which mercury causes progressive cardiotoxicity. Furthermore, inhibiting sEH might represent a novel therapeutic approach to prevent Hg2+-induced hypertrophy.

Epoxygenated fatty acid profile and soluble epoxide hydrolase (sEH) activity in healthy and laminitic horses

Epoxygenated fatty acid profile and soluble epoxide hydrolase (sEH) activity in healthy and laminitic horses

Soluble Epoxide Hydrolase Inhibition Does Not Prevent Cardiac Remodeling and Dysfunction After Aortic Constriction in Rats and Mice

Epoxyeicosatrienoic acids, substrates for soluble epoxide hydrolase (sEH), exhibit vasodilatory and antihypertrophic activities. Inhibitors of sEH might therefore hold promise as heart failure therapeutics. We examined the ability of sEH inhibitors GSK2188931 and GSK2256294 to modulate cardiac hypertrophy, fibrosis, and function after transverse aortic constriction (TAC) in rats and mice. GSK2188931 administration was initiated in rats 1 day before TAC, whereas GSK2256294 treatment was initiated in mice 2 weeks after TAC. Four weeks later, cardiovascular function was assessed, plasma was collected for drug and sEH biomarker concentrations, and left ventricle was isolated for messenger RNA and histological analyses. In rats, although GSK2188931 prevented TAC-mediated increases in certain genes associated with hypertrophy and fibrosis (α-skeletal actin and connective tissue growth factor), the compound failed to attenuate TAC-induced increases in left ventricle mass, posterior wall thickness, end-diastolic volume and pressure, and perivascular fibrosis. Similarly, in mice, GSK2256294 did not reverse cardiac remodeling or systolic dysfunction induced by TAC. Both compounds increased the sEH substrate/product (leukotoxin/leukotoxin diol) ratio, indicating sEH inhibition. In summary, sEH inhibition does not prevent cardiac remodeling or dysfunction after TAC. Thus, targeting sEH seems to be insufficient for reducing pressure overload hypertrophy.

5,14-HEDGE, a 20-HETE mimetic, reverses hypotension and improves survival in a rodent model of septic shock: Contribution of soluble epoxide hydrolase, CYP2C23, MEK1/ERK1/2/IKKβ/IκB-α/NF-κB pathway, and proinflammatory cytokine formation

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 33mmHg and heart rate rose by 57beats/min in LPS (10mg/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 (30mg/kg, s.c.; 1h 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 (30mg/kg, s.c.; 1h 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.

Acute arsenic treatment alters cytochrome P450 expression and arachidonic acid metabolism in lung, liver and kidney of C57Bl/6 mice

1. Arsenic (As(III)) toxicity has received increasing attention as human exposure to arsenic is associated with pulmonary, hepatic and renal toxicities. Therefore, in the present study, we investigated the effect of acute As(III) treatment on pulmonary, hepatic and renal cytochrome (CYP) P450-mediated arachidonic acid metabolism.2. Our results demonstrated that acute As(III) treatment (12.5 mg/kg) altered CYP epoxygenases, CYP ω-hydroxylases and EPHX2 mRNA levels that were isozyme and tissue specific.3. Furthermore, As(III) increased the formation of epoxyeicosatrienoic acids (EETs) in the kidney without affecting their levels in the lung or liver. In addition, acute As(III) treatment increased dihydroxyeicosatrienoic acid (DHETs) formation in the lung, while it did not affect liver DHETs formation and decreased kidney DHETs formation.4. As(III) also increased total epoxygenases activity in the lung while it decreased its levels in the kidney and had no effect on the liver. Furthermore, As(III) increased 20-hydroxyeicosatetraenoic acid formation in the liver while it decreased its formation in the kidney.5. Lastly, As(III) increased soluble epoxide hydrolase activity in the lung, while it decreased its levels in the kidney and had no effect on the liver. In conclusion, this is the first demonstration that As(III) alters arachidonic acid metabolism in a tissue specific manner.

Alleviation of lung injury by glycyrrhizic acid in benzo(a)pyrene exposed rats: Probable role of soluble epoxide hydrolase and thioredoxin reductase

Benzo(a)pyrene [B(a)P] is known to alter lung physiology by interfering in various intracellular pathways including alterations in NF-κB activities, cytokine release and cell survival. NF-κB suppression/activation plays a major role in cell survival status. Present investigation deals with such kind of effects of B(a)P on lungs in relation with soluble epoxide hydrolase (sEH) and thioredoxin reductase (TrxR) activities. Glycyrrhizic acid (GA), an active principle of Glycyrrhiza glabra (Licorice), is known to modulate various molecular processes. In the present study, we investigated the protective effects of GA against B(a)P induced debilities in lungs of Wistar rats. Intratracheal instillation of B(a)P significantly suppressed NF-κB translocation, sEH, TrxR and catalase activities in lung tissue. A marked induction of H2O2 levels along with caspases activation (caspases-2, -3, -6, -8, and -9) in lung tissue after B(a)P exposure was observed. Lung injury was assessed by measuring lactate dehydrogenase (LDH), alkaline phosphatase (ALP), total cell count, total protein, neutrophil elastase activity in bronchoalveolar lavage fluid (BALF). Reduction in phospholipid content further potentiated these parameters. GA oral administration (50 and 100mg/kg b.wt.) significantly showed protection of lung epithelium by suppression of caspases activities in lung tissue and reduction of total protein, total cells, elastase activity, LDH and ALP activities along with fortification of phospholipids in BALF. Histological observations also confirm the findings in above mentioned parameters. Results indicate a strong correlation between amelioration of sEH and TrxR activities, and NF-κB activation. The present investigation gives an insight into probable mechanisms of lung injuries induced by short term exposures of B(a)P and prevention by glycyrrhizic acid.

Complementary Screening Techniques Yielded Fragments that Inhibit the Phosphatase Activity of Soluble Epoxide Hydrolase

Complementary Screening Techniques Yielded Fragments that Inhibit the Phosphatase Activity of Soluble Epoxide Hydrolase

Pharmacological Inhibition of Soluble Epoxide Hydrolase Ameliorates Diet-Induced Metabolic Syndrome in Rats

The signs of metabolic syndrome following chronic excessive macronutrient intake include body weight gain, excess visceral adipose deposition, hyperglycaemia, glucose and insulin intolerances, hypertension, dyslipidaemia, endothelial damage, cardiovascular hypertrophy, inflammation, ventricular contractile dysfunction, fibrosis, and fatty liver disease. Recent studies show increased activity of soluble epoxide hydrolase (sEH) during obesity and metabolic dysfunction. We have tested whether sEH inhibition has therapeutic potential in a rat model of diet-induced metabolic syndrome. In these high-carbohydrate, high-fat-fed rats, chronic oral treatment with trans-4-[4-(3-adamantan-1-ylureido)-cyclohexyloxy]-benzoic acid (t-AUCB), a potent sEH inhibitor, alleviated the signs of metabolic syndrome in vivo including glucose, insulin, and lipid abnormalities, changes in pancreatic structure, increased systolic blood pressure, cardiovascular structural and functional abnormalities, and structural and functional changes in the liver. The present study describes the pharmacological responses to this selective sEH inhibitor in rats with the signs of diet-induced metabolic syndrome.

Development of a semi-automated LC/MS/MS method for the simultaneous quantitation of 14,15-epoxyeicosatrienoic acid, 14,15-dihydroxyeicosatrienoic acid, leukotoxin and leukotoxin diol in human plasma as biomarkers of soluble epoxide hydrolase activity in vivo

Substrates and products of soluble epoxide hydrolase (sEH) such as 14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), leukotoxin, and leukotoxin diol are potential biomarkers for assessing sEH activity in clinical trial subjects. To quantify them, we have developed and validated a semi-automated and relatively high-throughput assay in a 96-well plate format using liquid chromatography–mass spectrometry. 14,15-EET, 14,15-DHET, leukotoxin and leukotoxin diol, as well as their deuterium labeled internal standards were extracted from human plasma by liquid–liquid extraction using ethyl acetate. The four analytes were separated from other endogenous lipid isomers using liquid chromatography coupled with tandem mass spectrometry. The method was validated over a concentration range of 0.05–50 ng/mL. The validation results show that the method is precise, accurate and well-suited for analysis of clinical samples. The turn-around rate of the assay is approximately 200 samples per day.

Soluble epoxide hydrolase in the generation and maintenance of high blood pressure in spontaneously hypertensive rats

We hypothesized that perinatal inhibition of soluble epoxide hydrolase (SEH), which metabolizes epoxyeicosatrienoic acids in the arachidonic acid (AA) cascade, with an orally active SEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), would persistently reduce blood pressure (BP) in adult SHR despite discontinuation of AUDA at 4 wk of age. Renal cytoplasmic epoxide hydrolase-2 (Ephx2) gene expression was enhanced in SHR vs. WKY from 2 days to 24 wk. Effects of perinatal treatment with AUDA, supplied to SHR dams until 4 wk after birth, on BP in female and male offspring and renal oxylipin metabolome in female offspring were observed and contrasted to female SHR for direct effects of AUDA (8-12 wk). Briefly, inhibition of SEH was effective in persistently reducing BP in female SHR when applied during the perinatal phase. This was accompanied by marked increases in major renal AA epoxides and decreases in renal lipoxygenase products of AA. Early inhibition of SEH induced a delayed increase in renal 5-HETE at 24 wk, in contrast to a decrease at 2 wk. Inhibition of SEH in female SHR from 8 to 12 wk did not reduce BP but caused profound decreases in renal 15(S)-HETrE, LTB4, TBX2, 5-HETE, and 20-HETE and increases in TriHOMEs. In male SHR, BP reduction after perinatal AUDA was transient. Thus, Ephx2 transcription and SEH activity in early life may initiate mechanisms that eventually contribute to high BP in adult female SHR. However, programmed BP-lowering effects of perinatal SEH inhibition in female SHR cannot be simply explained by persistent reduction in renal SEH activity but rather by more complex and temporally dynamic interactions between the renal SEH, lipoxygenase, and cyclooxygenase pathways.

Examining the potency of suggested inhibitors for the phosphatase activity of the human soluble epoxide hydrolase by molecular dynamics simulations

The N-terminal domain of human soluble epoxide hydrolase ( EH) has been recently reported as phosphatase. In this work, molecular dynamics (MD) simulations were performed to study the binding modes of three inhibitors ( A, B and C, Scheme 1) of the phosphatase activity. Recently, compound A has been tested as inhibitor of the phosphatase activity of the EH enzyme with IC 50 = 0.84 μM. Results reveal that the hydrophobic tail of each inhibitor resides in the hydrophobic cleft of the N-terminal domain while the polar head interacts with the Mg 2+-aspertate active site and the nearby polar and charged amino acid residues. The obtained MD trajectories were analyzed to study and discuss the hydrogen bonds formed between enzyme and each inhibitor and the role of Mg 2+ ion in recognition. MM-PBSA method was used to estimate the binding free energy of each EH/inhibitor complex. Results indicate that guest–host electrostatic interactions have the largest contribution to the complex stability. Also, a significant contribution of van der Waals interactions was found. The suggested inhibitor ( B) exhibits much higher affinity toward the enzyme compared to the experimentally tested inhibitor A.