Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids

Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification

Atmospheric Pressure Photoionization Tandem Mass Spectrometry of Androgens in Prostate Cancer

Androgen deprivation therapy is the most common treatment option for advanced prostate cancer. Almost all prostate cancers recur during androgen deprivation therapy, and new evidence suggests that androgen receptor activation persists despite castrate levels of circulating androgens. Quantitation of tissue levels of androgens is critical to understanding the mechanism of recurrence of prostate cancer during androgen deprivation therapy. A liquid chromatography atmospheric pressure photoionization tandem mass spectrometric method was developed for quantitation of tissue levels of androgens. Quantitation of the saturated keto-steroids dihydrotestosterone and 5-α-androstanedione required detection of a novel parent ion, [M + 15]+. The nature of this parent ion was explored and the method applied to prostate tissue and cell culture with comparison to results achieved using electrospray ionization

Aphrodisiac property of aqueous and methanolic extracts of Raphia vinifera (Arecaceae) in sexually experienced male rats

Background: Raphia vinifera (Arecaceae) is a medicinal plant commonly used as a sexual enhancer. Objective: To investigate the aphrodisiac potential of aqueous extract (AE) and methanolic extract (ME) of R. vinifera in sexually experienced male rats. Materials and Methods: Thirty male Wistar rats were randomly distributed into six groups (5 rats per group) and administered for 14 days with distilled water (10 ml/kg), sildenafil citrate (1.44 mg/kg), and AE or ME of R. vinifera (100 or 500 mg/kg). The copulatory activity was tested on days 0, 7, and 14 using receptive females. Further, on day 14, rats were sacrificed and biochemical analyses (testosterone, total protein, and acid phosphatase) were performed. Results: Sildenafil citrate significantly decreased the intromission latency (day 14, p = 0.04) and frequency (days 7 and 14, p = 0.03) but increased the mount frequency (day 14, p = 0.04), compared with control. Remarkably, R. vinifera enhanced the sexual activity by significantly decreasing the intromission latency (AE and ME, 500 mg/kg, day 14, p = 0.04) and increasing the mount frequency (AE and ME, 100 mg/kg, day 7, p = 0.02) compared with control. Moreover, R. vinifera improved plasmatic (AE, 100 mg/kg, p = 0.03; AE, 500 mg/kg, p = 0.001; ME, 100 mg/kg, p = 0.01) and testicular (AE, 100 mg/kg, p = 0.001; AE, 500 mg/kg, p = 0.01; ME, 100 mg/kg, p = 0.001; ME, 500 mg/kg, p = 0.01) testosterone levels as well as plasmatic total proteins concentration (ME, 500 mg/kg, p = 0.04). Conclusion: These findings showed that R. vinifera possesses an aphrodisiac property which could further justify its folkloric use in traditional medicine as a sexual enhancer

Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity

Intimal smooth muscle cells are a source but not a sensor of anti-inflammatory CYP450 derived oxylipins

AbstractVascular pathologies are associated with changes in the presence and expression of morphologically distinct vascular smooth muscle cells. In particular, in complex human vascular lesions and models of disease in pigs and rodents, an intimal smooth muscle cell (iSMC) which exhibits a stable epithelioid or rhomboid phenotype in culture is often found to be present in high numbers, and may represent the reemergence of a distinct developmental vascular smooth muscle cell phenotype. The CYP450-oxylipin - soluble epoxide hydrolase (sEH) pathway is currently of great interest in targeting for cardiovascular disease. sEH inhibitors limit the development of hypertension, diabetes, atherosclerosis and aneurysm formation in animal models. We have investigated the expression of CYP450-oxylipin-sEH pathway enzymes and their metabolites in paired intimal (iSMC) and medial (mSMC) cells isolated from rat aorta. iSMC basally released significantly larger amounts of epoxy-oxylipin CYP450 products from eicosapentaenoic acid > docosahexaenoic acid > arachidonic acid > linoleic acid, and expressed higher levels of CYP2C12, CYP2B1, but not CYP2J mRNA compared to mSMC. When stimulated with the pro-inflammatory TLR4 ligand LPS, epoxy-oxylipin production did not change greatly in iSMC. In contrast, LPS induced epoxy-oxylipin products in mSMC and induced CYP2J4. iSMC and mSMC express sEH which metabolizes primary epoxy-oxylipins to their dihydroxy-counterparts. The sEH inhibitors TPPU or AUDA inhibited LPS-induced NFκB activation and iNOS induction in mSMC, but had no effect on NFκB nuclear localization or inducible nitric oxide synthase in iSMC; effects which were recapitulated in part by addition of authentic epoxy-oxylipins. iSMCs are a rich source but not a sensor of anti-inflammatory epoxy-oxylipins. Complex lesions that contain high levels of iSMCs may be more resistant to the protective effects of sEH inhibitors

Activation of the Androgen Receptor by Intratumoral Bioconversion of Androstanediol to Dihydrotestosterone in Prostate Cancer

The androgen receptor (AR) mediates the growth of benign and malignant prostate in response to dihydrotestosterone (DHT). In patients undergoing androgen deprivation therapy for prostate cancer, AR drives prostate cancer growth despite low circulating levels of testicular androgen and normal levels of adrenal androgen. In this report we demonstrate the extent of AR transactivation in the presence of 5α-androstane-3α,17β-diol (androstanediol) in prostate-derived cell lines parallels the bioconversion of androstanediol to DHT. AR transactivation in the presence of androstanediol in prostate cancer cell lines correlated mainly with mRNA and protein levels of 17β-hydroxysteroid dehydrogenase 6 (17β-HSD6), one of several enzymes required for the interconversion of androstanediol to DHT and the inactive metabolite, androsterone. Levels of retinol dehydrogenase 5, and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9, which also convert androstanediol to DHT, were lower than 17β-HSD6 in prostate-derived cell lines, and higher in the castration-recurrent human prostate cancer xenograft. Measurements of tissue androstanediol using mass spectrometry demonstrated androstanediol metabolism to DHT and androsterone. Administration of androstanediol dipropionate to castration-recurrent CWR22R tumor bearing athymic castrated male mice produced a 28-fold increase in intratumoral DHT levels. AR transactivation in prostate cancer cells in the presence of androstanediol resulted from the cell-specific conversion of androstanediol to DHT, and androstanediol increased LAPC-4 cell growth. The ability to convert androstanediol to DHT provides a mechanism for optimal utilization of androgen precursors and catabolites for DHT synthesis

Cytochrome P450-derived epoxyeicosatrienoic acids and coronary artery disease in humans: a targeted metabolomics study

Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) exhibit potent cardiovascular protective effects in preclinical models, and promoting the effects of EETs has emerged as a potential therapeutic strategy for coronary artery disease (CAD). The relationship between circulating EET levels and CAD extent in humans, however, remains unknown. A panel of free (unesterified) plasma eicosanoid metabolites was quantified in 162 patients referred for coronary angiography, and associations with extent of CAD [no apparent CAD (N = 39), nonobstructive CAD (N = 51), and obstructive CAD (N = 72)] were evaluated. A significant relationship between free EET levels and CAD extent was observed (P = 0.003) such that the presence of obstructive CAD was associated with lower circulating EET levels. This relationship was confirmed in multiple regression analysis where CAD extent was inversely and significantly associated with EET levels (P = 0.013), and with a biomarker of EET biosynthesis (P < 0.001), independent of clinical and demographic factors. Furthermore, quantitative enrichment analysis revealed that these associations were the most pronounced compared with other eicosanoid metabolism pathways. Collectively, these findings suggest that the presence of obstructive CAD is associated with lower EET metabolite levels secondary to suppressed EET biosynthesis. Novel strategies that promote the effects of EETs may have therapeutic promise for patients with obstructive CAD

Characterization of the Cytochrome P450 epoxyeicosanoid pathway in non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is an emerging public health problem without effective therapies. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into bioactive epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory and protective effects. However, the functional relevance of the CYP epoxyeicosanoid metabolism pathway in the pathogenesis of NASH remains poorly understood. Our studies demonstrate that both mice with methionine-choline deficient (MCD) diet-induced NASH and humans with biopsy-confirmed NASH exhibited significantly higher free EET concentrations compared to healthy controls. Targeted disruption of Ephx2 (the gene encoding for soluble epoxide hydrolase) in mice further increased EET levels and significantly attenuated MCD diet-induced hepatic steatosis, inflammation and injury, as well as high fat diet-induced adipose tissue inflammation, systemic glucose intolerance and hepatic steatosis. Collectively, these findings suggest that dysregulation of the CYP epoxyeicosanoid pathway is a key pathological consequence of NASH in vivo, and promoting the anti-inflammatory and protective effects of EETs warrants further investigation as a novel therapeutic strategy for NASH

Deficiency of Soluble Epoxide Hydrolase Protects Cardiac Function Impaired by LPS-Induced Acute Inflammation

Lipopolysaccharide (LPS) is a bacterial wall endotoxin producing many pathophysiological conditions including myocardial inflammation leading to cardiotoxicity. Linoleic acid (18:2n6, LA) is an essential n-6 PUFA which is converted to arachidonic acid (20:4n6, AA) by desaturation and elongation via enzyme systems within the body. Biological transformation of PUFA through CYP-mediated hydroxylation, epoxidation, and allylic oxidation produces lipid mediators, which may be subsequently hydrolyzed to corresponding diol metabolites by soluble epoxide hydrolase (sEH). In the current study, we investigate whether inhibition of sEH, which alters the PUFA metabolite profile, can influence LPS induced cardiotoxicity and mitochondrial function. Our data demonstrate that deletion of soluble epoxide hydrolase provides protective effects against LPS-induced cardiotoxicity by maintaining mitochondrial function. There was a marked alteration in the cardiac metabolite profile with notable increases in sEH-derived vicinal diols, 9,10- and 12,13-dihydroxyoctadecenoic acid (DiHOME) in WT hearts following LPS administration, which was absent in sEH null mice. We found that DiHOMEs triggered pronounced mitochondrial structural abnormalities, which also contributed to the development of extensive mitochondrial dysfunction in cardiac cells. Accumulation of DiHOMEs may represent an intermediate mechanism through which LPS-induced acute inflammation triggers deleterious alterations in the myocardium in vivo and cardiac cells in vitro. This study reveals novel research exploring the contribution of DiHOMEs in the progression of adverse inflammatory responses toward cardiac function in vitro and in vivo

Dual modulation of cyclooxygenase and CYP epoxygenase metabolism and acute vascular inflammation in mice

Cyclooxygenase (COX)-derived prostaglandins and cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids are important regulators of inflammation; however, functional interactions between these pathways in the regulation of vascular inflammation in vivo have not been studied. We investigated the relative and additive effects of endothelial CYP2J2 overexpression (Tie2-CYP2J2-Tr), global sEH disruption (Ephx2−/−), and pharmacologic COX inhibition with indomethacin on the acute vascular inflammatory response to endotoxin in mice. Compared to vehicle-treated wild-type C57BL/6 controls, induction of myeloperoxidase (MPO) activity in lung and liver was similarly attenuated in Tie2-CYP2J2-Tr mice, Ephx2−/− mice and wild-type mice treated with moderate dose indomethacin. Dual modulation of both pathways, however, did not produce an additive anti-inflammatory effect. These findings demonstrate that both COX and CYP epoxygenase-mediated eicosanoid metabolism are important regulators of the acute vascular inflammatory response in vivo, and suggest that the anti-inflammatory effects of modulating each pathway may be mediated, at least in part, by overlapping mechanisms