New Alkoxy- Analogues of Epoxyeicosatrienoic Acids Attenuate Cisplatin Nephrotoxicity In Vitro via Reduction of Mitochondrial Dysfunction, Oxidative Stress, Mitogen-Activated Protein Kinase Signaling, and Caspase Activation.

The usage of cisplatin, a highly potent chemotherapeutic, is limited by its severe nephrotoxicity. Arachidonic acid (ARA)-derived epoxyeicosatrienoic acids (EETs) and soluble epoxide hydrolase (sEH) inhibitors were shown to ameliorate this dose-limiting side effect, but both approaches have some pharmacological limitations. Analogues of EETs are an alternative avenue with unique benefits, but the current series of analogues face concerns regarding their structure and mimetic functionality. Hence, in this study, regioisomeric mixtures of four new ARA alkyl ethers were synthesized, characterized, and assessed as EET analogues against the concentration- and time-dependent toxicities of cisplatin in porcine proximal tubular epithelial cells. All four ether groups displayed bioisostere activity, ranging from marginal for methoxy- (1), good for n-propoxy- (4), and excellent for ethoxy- (2) and i-propoxy- (3). Compounds 2 and 3 displayed cytoprotective effects comparable to that of an EET regioisomeric mixture (5) against high, acute cisplatin exposures but were more potent against low to moderate, chronic exposures. Compounds 2 and 3 (and 5) acted through stabilization of the mitochondrial transmembrane potential and attenuation of reactive oxygen species, leading to reduced phosphorylation of mitogen-activated protein kinases p38 and JNK and decreased activation of caspase-9 and caspase-3. This study demonstrates that alkoxy- groups are potent and more metabolically stable bioisostere alternatives to the epoxide within EETs that enable sEH-independent activity. It also illustrates the potential of ether-based mimics of EETs and other epoxy fatty acids as promising nephroprotective agents to tackle the clinically relevant side effect of cisplatin without compromising its antineoplastic function.

Serum Nonesterified Fatty Acids and Incident Stroke: The CHS.

Background Significant associations between total nonesterified fatty acid (NEFA) concentrations and incident stroke have been reported in some prospective cohort studies. We evaluated the associations between incident stroke and serum concentrations of nonesterified saturated, monounsaturated, polyunsaturated, and trans fatty acids. Methods and Results CHS (Cardiovascular Health Study) participants (N=2028) who were free of stroke at baseline (1996-1997) and had an archived fasting serum sample were included in this study. A total of 35 NEFAs were quantified using gas chromatography. Cox proportional hazards regression models were used to evaluate associations of 5 subclasses (nonesterified saturated, monounsaturated, omega (n)-6 polyunsaturated, n-3 polyunsaturated, and trans fatty acids) of NEFAs and individual NEFAs with incident stroke. Sensitivity analysis was conducted by excluding cases with hemorrhagic stroke (n=45). A total of 338 cases of incident stroke occurred during the median 10.5-year follow-up period. Total n-3 (hazard ratio [HR], 0.77 [95% CI, 0.61-0.97]) and n-6 (HR, 1.32 [95% CI, 1.01-1.73]) subclasses of NEFA were negatively and positively associated with incident stroke, respectively. Among individual NEFAs, dihomo-γ-linolenic acid (20:3n-6) was associated with higher risk (HR, 1.29 [95% CI, 1.02-1.63]), whereas cis-7-hexadecenoic acid (16:1n-9c) and arachidonic acid (20:4n-6) were associated with a lower risk (HR, 0.67 [95% CI, 0.47-0.97]; HR, 0.81 [95% CI. 0.65-1.00], respectively) of incident stroke per standard deviation increment. After the exclusion of cases with hemorrhagic stroke, these associations did not remain significant. Conclusions A total of 2 NEFA subclasses and 3 individual NEFAs were associated with incident stroke. Of these, the NEFA n-3 subclass and dihomo-γ-linolenic acid are diet derived and may be potential biomarkers for total stroke risk.

Kidney-Targeted Epoxyeicosatrienoic Acid Analog, EET-F01, Reduces Inflammation, Oxidative Stress, and Cisplatin-Induced Nephrotoxicity.

Although epoxyeicosatrienoic acid (EET) analogs have performed well in several acute and chronic kidney disease models, targeted delivery of EET analogs to the kidney can be reasonably expected to reduce the level of drug needed to achieve a therapeutic effect and obviate possible side effects. For EET analog kidney-targeted delivery, we conjugated a stable EET analog to folic acid via a PEG-diamine linker. Next, we compared the kidney targeted EET analog, EET-F01, to a well-studied EET analog, EET-A. EET-A or EET-F01 was infused i.v. and plasma and kidney tissue collected. EET-A was detected in the plasma but was undetectable in the kidney. On the other hand, EET-F01 was detected in the plasma and kidney. Experiments were conducted to compare the efficacy of EET-F01 and EET-A for decreasing cisplatin nephrotoxicity. Cisplatin was administered to WKY rats treated with vehicle, EET-A (10 mg/kg i.p.) or EET-F01 (20 mg/kg or 2 mg/kg i.p.). Cisplatin increased kidney injury markers, viz., blood urea nitrogen (BUN), N-acetyl-ß-(D)-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1), and thiobarbituric acid reactive substances (TBARS). EET-F01 was as effective as EET-A in decreasing BUN, NAG, KIM-1, TBARS, and renal histological injury caused by cisplatin. Despite its almost 2×-greater molecular weight compared with EET-A, EET-F01 was comparably effective in decreasing renal injury at a 10-fold w/w lower dose. EET-F01 decreased cisplatin nephrotoxicity by reducing oxidative stress and inflammation. These data demonstrate that EET-F01 targets the kidney, allows for a lower effective dose, and combats cisplatin nephrotoxicity. In conclusion, we have developed a kidney targeted EET analog, EET-F01, that demonstrates excellent potential as a therapeutic for kidney diseases.

Bioconversion of arachidonic acid into human 14,15-hepoxilin B3 and 13,14,15-trioxilin B3 by recombinant cells expressing microbial 15-lipoxygenase without and with epoxide hydrolase.

OBJECTIVE: To produce high concentrations of 13-hydroxy-14,15-epoxy-eicosatrienoic acid (14,15-hepoxilin B3, 14,15-HXB3) and 13,14,15-trihydroxy-eicosatrienoic acid (13,14,15-trioxilin B3, 13,14,15-TrXB3) from arachidonic acid (ARA) using microbial 15-lipoxygenase (15-LOX) without and with epoxide hydrolase (EH), respectively. RESULTS: The products obtained from the bioconversion of ARA by recombinant Escherichia coli cells containing Archangium violaceum 15-LOX without and with Myxococcus xanthus EH were identified as 14,15-HXB3 and 13,14,15-TrXB3, respectively. Under the optimal conditions of 30 g cells L-1, 200 mM ARA, 25 °C, and initial pH 7.5, the cells converted 200 mM ARA into 192 mM 14,15-HXB3 and 100 mM 13,14,15-TrXB3 for 150 min, with conversion yields of 96 and 51% and productivities of 77 and 40 mM h-1, respectively. CONCLUSION: These are the highest concentrations, productivities, and yields of hepoxilin and trioxilin from ARA reported thus far.

Specific delivery of delta-5-desaturase siRNA via RNA nanoparticles supplemented with dihomo-γ-linolenic acid for colon cancer suppression.

We have previously demonstrated that DGLA treatment along with Delta-5-Desaturase (D5D) siRNA in various types of cancer cells enhances the formation of 8-HOA from COX-2-catalyzed DGLA peroxidation, which in turn inhibits cancer cell growth and migration. However, delivery of naked siRNA remains a formidable challenge due to its "off-target" effect. In this study, we employed RNA nanotechnology for specific delivery of D5D-siRNA to xenograft colon tumors using 3WJ RNA nanoparticles. When a targeting module, i.e., the EpCAM aptamer, was incorporated, the 3WJ pRNA nanoparticles were able specifically deliver D5D siRNA to human colon cancer HCA-7 cells both in vitro and in vivo, resulting in significant downregulation of D5D expression. Co-treatment with DGLA in combination with 3WJ-EpCAM-siRNA induced a higher DGLA/AA ratio and enhanced formation of 8-HOA at a threshold level, and in HCA-7 tumor-bearing mice, induced significant tumor suppression. We further confirmed that 8-HOA formation, promoted by COX-2-catalyzed DGLA peroxidation, inhibited HDAC and consequently induced apoptosis in tumor cells. Therefore, the 3WJ RNA nanoparticle system holds great promise as a suitable therapeutic delivery platform for colon cancer therapy.

Biotransformation of polyunsaturated fatty acids to bioactive hepoxilins and trioxilins by microbial enzymes.

Hepoxilins (HXs) and trioxilins (TrXs) are involved in physiological processes such as inflammation, insulin secretion and pain perception in human. They are metabolites of polyunsaturated fatty acids (PUFAs), including arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, formed by 12-lipoxygenase (LOX) and epoxide hydrolase (EH) expressed by mammalian cells. Here, we identify ten types of HXs and TrXs, produced by the prokaryote Myxococcus xanthus, of which six types are new, namely, HXB5, HXD3, HXE3, TrXB5, TrXD3 and TrXE3. We succeed in the biotransformation of PUFAs into eight types of HXs (>35% conversion) and TrXs (>10% conversion) by expressing M. xanthus 12-LOX or 11-LOX with or without EH in Escherichia coli. We determine 11-hydroxy-eicosatetraenoic acid, HXB3, HXB4, HXD3, TrXB3 and TrXD3 as potential peroxisome proliferator-activated receptor-γ partial agonists. These findings may facilitate physiological studies and drug development based on lipid mediators.

Effects of dronedarone, amiodarone and their active metabolites on sequential metabolism of arachidonic acid to epoxyeicosatrienoic and dihydroxyeicosatrienoic acids.

Cardiac enzymes such as cytochrome P450 2J2 (CYP2J2) metabolize arachidonic acid (AA) to cardioprotective epoxyeicosatrienoic acids (EETs), which in turn are metabolized by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs). As EETs and less potent DHETs exhibit cardioprotective and vasoprotective functions, optimum levels of cardiac EETs are paramount in cardiac homeostasis. Previously, we demonstrated that dronedarone, amiodarone and their main metabolites, namely N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA), potently inhibit human cardiac CYP2J2-mediated astemizole metabolism in vitro. In this study, we investigated the inhibition of recombinant human CYP450 enzymes (rhCYP2J2, rhCYP2C8, rhCYP2C9)-mediated AA metabolism and human recombinant sEH (rhsEH)-mediated EET metabolism by dronedarone, amiodarone, NDBD and NDEA. A static model describing sequential metabolism was further developed to predict the aggregate effect of dual-inhibition of rhCYP2J2 and rhsEH on the fold-of 14,15-EET level (CEET'/CEET). Dronedarone, amiodarone and NDBD inhibit rhCYP2J2-mediated metabolism of AA to 14,15-EET with Ki values of 3.25, 5.48, 1.39µM respectively. Additionally, dronedarone, amiodarone, NDBD and NDEA inhibit rhsEH-mediated metabolism of 14,15-EET to 14,15-DHET with Ki values of 5.10, 13.08, 2.04, 1.88µM respectively. Based on static sequential metabolism modelling, dronedarone (CEET'/CEET=0.85), amiodarone (CEET'/CEET=0.48) and NDBD (CEET'/CEET=0.76) were predicted to decrease cardiac 14,15-EET level whereas NDEA (CEET'/CEET>35.5) was predicted to elevate it. Based on our novel findings, we postulate the differential cardiac exacerbation potential of dronedarone and amiodarone is partly associated with their differential inhibition potencies of cardiac CYP2J2 and sEH.

Orally Active Epoxyeicosatrienoic Acid Analogs.

Biologically active epoxyeicosatrienoic acid (EET) regioisomers are synthesized from arachidonic acid by cytochrome P450 epoxygenases of endothelial, myocardial, and renal tubular cells. EETs relax vascular smooth muscle and decrease inflammatory cell adhesion and cytokine release. Renal EETs promote sodium excretion and vasodilation to decrease hypertension. Cardiac EETs reduce infarct size after ischemia-reperfusion injury and decrease fibrosis and inflammation in heart failure. In diabetes, EETs improve insulin sensitivity, increase glucose tolerance, and reduce the renal injury. These actions of EETs emphasize their therapeutic potential. To minimize metabolic inactivation, 14,15-EET agonist analogs with stable epoxide bioisosteres and carboxyl surrogates were developed. In preclinical rat models, a subset of agonist analogs, termed EET-A, EET-B, and EET-C22, are orally active with good pharmacokinetic properties. These orally active EET agonists lower blood pressure and reduce cardiac and renal injury in spontaneous and angiotensin hypertension. Other beneficial cardiovascular actions include improved endothelial function and cardiac antiremodeling actions. In rats, EET analogs effectively combat acute and chronic kidney disease including drug- and radiation-induced kidney damage, hypertension and cardiorenal syndrome kidney damage, and metabolic syndrome and diabetes nephropathy. The compelling preclinical efficacy supports the prospect of advancing EET analogs to human clinical trials for kidney and cardiovascular diseases.

Structural determinants of 5',6'-epoxyeicosatrienoic acid binding to and activation of TRPV4 channel.

TRPV4 cation channel activation by cytochrome P450-mediated derivatives of arachidonic acid (AA), epoxyeicosatrienoic acids (EETs), constitute a major mechanisms of endothelium-derived vasodilatation. Besides, TRPV4 mechano/osmosensitivity depends on phospholipase A2 (PLA2) activation and subsequent production of AA and EETs. However, the lack of evidence for a direct interaction of EETs with TRPV4 together with claims of EET-independent mechanical activation of TRPV4 has cast doubts on the validity of this mechanism. We now report: 1) The identification of an EET-binding pocket that specifically mediates TRPV4 activation by 5',6'-EET, AA and hypotonic cell swelling, thereby suggesting that all these stimuli shared a common structural target within the TRPV4 channel; and 2) A structural insight into the gating of TRPV4 by a natural agonist (5',6'-EET) in which K535 plays a crucial role, as mutant TRPV4-K535A losses binding of and gating by EET, without affecting GSK1016790A, 4α-phorbol 12,13-didecanoate and heat mediated channel activation. Together, our data demonstrates that the mechano- and osmotransducing messenger EET gates TRPV4 by a direct action on a site formed by residues from the S2-S3 linker, S4 and S4-S5 linker.

Enzymatic and free radical formation of cis- and trans- epoxyeicosatrienoic acids in vitro and in vivo.

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid (AA) oxidation that have important cardioprotective and signaling properties. AA is an ω-6 polyunsaturated fatty acid (PUFA) that is prone to autoxidation. Although hydroperoxides and isoprostanes are major autoxidation products of AA, EETs are also formed from the largely overlooked peroxyl radical addition mechanism. While autoxidation yields both cis- and trans-EETs, cytochrome P450 (CYP) epoxygenases have been shown to exclusively catalyze the formation of all regioisomer cis-EETs, on each of the double bonds. In plasma and red blood cell (RBC) membranes, cis- and trans-EETs have been observed, and both have multiple physiological functions. We developed a sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay that separates cis- and trans- isomers of EETs and applied it to determine the relative distribution of cis- vs. trans-EETs in reaction mixtures of AA subjected to free radical oxidation in benzene and liposomes in vitro. We also determined the in vivo distribution of EETs in several tissues, including human and mouse heart, and RBC membranes. We then measured EET levels in heart and RBC of young mice compared to old. Formation of EETs in free radical reactions of AA in benzene and in liposomes exhibited time- and AA concentration-dependent increase and trans-EET levels were higher than cis-EETs under both conditions. In contrast, cis-EET levels were overall higher in biological samples. In general, trans-EETs increased with mouse age more than cis-EETs. We propose a mechanism for the non-enzymatic formation of cis- and trans-EETs involving addition of the peroxyl radical to one of AA's double bonds followed by bond rotation and intramolecular homolytic substitution (SHi). Enzymatic formation of cis-EETs by cytochrome P450 most likely occurs via a one-step concerted mechanism that does not allow bond rotation. The ability to accurately measure circulating EETs resulting from autoxidation or enzymatic reactions in plasma and RBC membranes will allow for future studies investigating how these important signaling lipids correlate with heart disease outcomes.

Epoxyeicosatrienoic acid ameliorates cerebral ischemia-reperfusion injury by inhibiting inflammatory factors and pannexin-1.

Epoxyeicosatrienoic acid (EET) has wide applications due to the unique biological effects of anti­hyperlipidemia, inhibition of platelet aggregation, anti­inflammation, anti­cancer, anti­lipid oxidation and the promotion of brain tissue development. The present study investigated whether EET ameliorates cerebral ischemia­reperfusion injury (CIRI) by inhibiting inflammatory factors and pannexin. Specific pathogen­free 7­week­old male Sprague­Dawley rats were randomly divided into three groups: Sham, CIRI and EET. Neurological deficit scores, cerebral infarct volume and cerebral edema were assessed in CIRI rats. Enzyme­linked immunosorbent assays were performed to detect tumor necrosis factor­α, interleukin­6, nuclear factor­κB and inducible nitric oxide synthase (iNOS) levels, and western blot analysis was performed also used to assess cleaved caspase­3, phospholipase A2 (PLA2), cyclooxygenase­2 and prostaglandin E2 (PGE2) protein expression levels. EET ameliorated cerebral injury and CIRI­induced cleaved caspase­3 protein expression levels in rats. EET additionally suppressed CIRI­induced inflammation reactions and iNOS protein expression in rats. Furthermore, the protein expression levels of PLA2, PGE2 and pannexin­1 in CIRI rats were inhibited by treatment with EET. These results indicated that EET reduces CIRI by inhibiting inflammation and levels of cleaved caspase­3, PLA2, PGE2 and pannexin-1.

Synthesis of cyclooxygenase metabolites of 8,9-epoxyeicosatrienoic acid (EET): 11- and 15-hydroxy 8,9-EETs.

COX metabolites of 8,9-EET, previously observed as potent mitogenic lipid mediators, were synthesized for the first time by using two synthetic approaches. These synthetic materials allow for structural confirmation of COX metabolites of 8,9-EET and further study of their biological roles.

Vasodilatory responses of renal interlobular arteries to epoxyeicosatrienoic acids analog are not enhanced in Ren-2 transgenic hypertensive rats: evidence against a role of direct vascular effects of epoxyeicosatrienoic acids in progression of experimental heart failure.

Pathophysiological mechanisms underlying the development of renal dysfunction and progression of congestive heart failure (CHF) remain poorly understood. Recent studies have revealed striking differences in the role of epoxyeicosatrienoic acids (EETs), active products of cytochrome P-450-dependent epoxygenase pathway of arachidonic acid, in the progression of aorto-caval fistula (ACF)-induced CHF between hypertensive Ren-2 renin transgenic rats (TGR) and transgene-negative normotensive Hannover Sprague-Dawley (HanSD) controls. Both ACF TGR and ACF HanSD strains exhibited marked intrarenal EETs deficiency and impairment of renal function, and in both strains chronic pharmacologic inhibition of soluble epoxide hydrolase (sEH) (which normally degrades EETs) normalized EETs levels. However, the treatment improved the survival rate and attenuated renal function impairment in ACF TGR only. Here we aimed to establish if the reported improved renal function and attenuation of progression of CHF in ACF TGR observed after she blockade depends on increased vasodilatory responsiveness of renal resistance arteries to EETs. Therefore, we examined the responses of interlobar arteries from kidneys of ACF TGR and ACF HanSD rats to EET-A, a new stable 14,15-EET analog. We found that the arteries from ACF HanSD kidneys rats exhibited greater vasodilator responses when compared to the ACF TGR arteries. Hence, reduced renal vasodilatory responsiveness cannot be responsible for the lack of beneficial effects of chronic sEH inhibition on the development of renal dysfunction and progression of CHF in ACF HanSD rats.

PGC-1 alpha regulates HO-1 expression, mitochondrial dynamics and biogenesis: Role of epoxyeicosatrienoic acid.

BACKGROUND/OBJECTIVES: Obesity is a risk factor in the development of type 2 diabetes mellitus (DM2), which is associated with increased morbidity and mortality, predominantly as a result of cardiovascular complications. Increased adiposity is a systemic condition characterized by increased oxidative stress (ROS), increased inflammation, inhibition of anti-oxidant genes such as HO-1 and increased degradation of epoxyeicosatrienoic acids (EETs). We previously demonstrated that EETs attenuate mitochondrial ROS. We postulate that EETs increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which controls mitochondrial function, oxidative metabolism and induction of HO-1. METHODS: Cultured murine adipocytes and mice fed a high fat (HF) diet were used to assess functional relationship between EETs, HO-1 and (PGC-1α) using an EET analogue (EET-A) and lentivirus to knock down the PPARGC1A gene. RESULTS: EET-A increased PGC-1α and HO-1 in cultured adipocytes and increased the expression of genes involved in thermogenesis and adipocyte browning (UCP1 and PRDM16, respectively). PGC-1α knockdown prevented EET-A-induced HO-1expression, suggesting that PGC-1α is upstream of HO-1. MRI data obtained from fat tissues showed that EET-A administration to mice on a HF diet significantly reduced total body fat content, subcutaneous and visceral fat deposits and reduced the VAT: SAT ratio. Moreover EET-A normalized the VO2 and RQ (VCO2/VO2) in mice fed a HF diet, an effect that was completely prevented in PGC-1α deficient mice. In addition, EET-A increased mitochondrial biogenesis and function as measured by OPA1, MnSOD, Mfn1, Mfn2, and SIRT3, an effect that was inhibited by knockdown of PGC-1α. CONCLUSION: Taken together, our findings show that EET-A increased PGC-1α thereby increasing mitochondrial viability, increased fusion potential thereby providing metabolic protection and increased VO2 consumption in HF-induced obesity in mice, thus demonstrating that the EET-mediated increase in HO-1 levels require PGC-1α expression.

Epoxyeicosatrienoic acids (EETs) form adducts with DNA in vitro.

Epoxyeicosatrienoic acids (EETs) are potent lipid mediators formed by cytochrome P450 epoxygenases from arachidonic acid. They consist of four regioisomers of cis-epoxyeicosatrienoic acids: 5,6-, 8,9-, 11,12- and 14,15-EET. Here we investigated whether these triene epoxides are electrophilic enough to form covalent adducts with DNA in vitro. Using the thin-layer chromatography (TLC) (32)P-postlabelling method for adduct detection we studied the reaction of individual deoxynucleoside 3'-monophosphates and calf thymus DNA with the four racemic EETs. Under physiological conditions (pH 7.4) only ±11,12-EET11,12-EET formed adducts with DNA in a dose dependent manner detectable by the (32)P-postlabelling method. However, when pre-incubated at pH 4 all four racemic EETs were capable to bind to DNA forming several adducts. Under these conditions highest DNA adduct levels were found with ±11,12-EET followed by ±5,6-EET, ±8,9-EET, and ±14,15-EET, all of them two orders of magnitude higher (between 3 and 1 adducts per 10(5) normal nucleotides) than those obtained with ±11,12-EET at pH 7.4. Similar DNA adduct patterns consisting of up to seven spots were observed with all four racemic EETs the most abundant adducts being derived from the reaction with deoxyguanosine and deoxyadenosine. In summary, when analysed by the (32)P-postlabelling method all four racemic EETs formed multiple DNA adducts after activation by acidic pH, only ±11,12-EET produced DNA adducts in aqueous solution at neutral pH. Therefore, we conclude from our in vitro studies that EETs might be endogenous genotoxic compounds.

Epoxides Derived from Dietary Dihomo-Gamma-Linolenic Acid Induce Germ Cell Death in C. elegans.

Dietary fats are not created equally, slight differences in structure lead to crucial differences in function. Muticellular organisms use polyunsaturated fatty acid as substrates to produce potent signaling molecules crucial for many physiological processes, including reproduction. Here we explored the mechanism responsible for germ cell loss induced by dietary supplementation of dihomo-gamma-linolenic acid (DGLA, 20:3n-6) in the roundworm Caenorhabditis elegans. In this study we found that C. elegans CYP-33E2 activity produces a range of epoxy and hydroxy metabolites from dietary DGLA. Knockdown of cyp-33E2 suppressed the DGLA-induced sterility phenotype. Additionally, direct exposure of two specific DGLA-derived epoxy products, 8,9- and 14,15-epoxyeicosadienoic acids, produced germ cell abnormalities in the C. elegans gonad. We propose that sterility is mediated by the production of toxic DGLA-derived epoxides that trigger germ cell destruction. These studies are the first to establish a biological activity for a CYP-produced metabolite of DGLA.

A lipid mediator hepoxilin A3 is a natural inducer of neutrophil extracellular traps in human neutrophils.

Pulmonary exacerbations in cystic fibrosis airways are accompanied by inflammation, neutrophilia, and mucous thickening. Cystic fibrosis sputum contains a large amount of uncleared DNA contributed by neutrophil extracellular trap (NET) formation from neutrophils. The exact mechanisms of the induction of NETosis in cystic fibrosis airways remain unclear, especially in uninfected lungs of patients with early cystic fibrosis lung disease. Here we show that Hepoxilin A3, a proinflammatory eicosanoid, and the synthetic analog of Hepoxilin B3, PBT-3, directly induce NETosis in human neutrophils. Furthermore, we show that Hepoxilin A3-mediated NETosis is NADPH-oxidase-dependent at lower doses of Hepoxilin A3, while it is NADPH-oxidase-independent at higher doses. Together, these results demonstrate that Hepoxilin A3 is a previously unrecognized inducer of NETosis in cystic fibrosis lungs and may represent a new therapeutic target for treating cystic fibrosis and other inflammatory lung diseases.

Plasma phospholipids, non-esterified plasma polyunsaturated fatty acids and oxylipids are associated with BMI.

The obese lipid profile is associated with increased free fatty acids and triacylglycerides. Currently, little is known about the plasma lipid species associated with obesity. In this study, we compared plasma lipid fatty acid (FA) profiles as a function of BMI. Profiling phospholipid (PL) FAs and their respective oxylipids could predict which obese individuals are more likely to suffer from diseases associated with chronic inflammation or oxidative stress. We investigated the relationship between BMI and plasma PL (PPL) FA composition in 126 men using a quantitative gas chromatography analysis. BMI was inversely associated with both PPL nervonic and linoleic acid (LA) but was positively associated with both dihomo-γ-linolenic and palmitoleic acid. Compared to lean individuals, obese participants were more likely to have ω-6 FAs, except arachidonic acid and LA, incorporated into PPLs. Obese participants were less likely to have EPA and DHA incorporated into PPLs compared to lean participants. Non-esterified plasma PUFA and oxylipid analysis showed ω-6 oxylipids were more abundant in the obese plasma pool. These ω-6 oxylipids are associated with increased angiogenesis (i.e. epoxyeicosatrienoates), reactive oxygen species (i.e. 9-hydroxyeicosatetraenoate), and inflammation resolution (i.e. Lipoxin A4). In summary, BMI is directly associated with specific PPL FA and increased ω-6 oxylipids.

Pathophysiology of the hepoxilins.

There is increasing evidence from various scientific groups that hepoxilins represent novel inflammatory mediators. In vitro studies have shown that the hepoxilins cause mobilization of intracellular calcium in human neutrophils, cause plasma leakage, and potently stimulate chemotaxis of human neutrophils. In vivo, the hepoxilin pathway is activated in conditions of inflammation, e.g. after pathogen infection, in inflamed conditions (psoriasis, arthritis), and hepoxilins promote inflammatory hyperalgesia and allodynia. Although much work has demonstrated an effect of hepoxilins on neutrophils, the hepoxilin pathway has been demonstrated in a variety of tissues, including the lung, brain, pituitary, pancreatic islets, skin, etc. A genetic defect linked to a deficiency in hepoxilin formation has been described and believed to be responsible for the scaly skin observed in ichthyosis. Despite their biological and chemical instability, the involvement of the hepoxilin pathway in pathology has been demonstrated in vitro and in vivo through either isolation of the hepoxilins themselves (or their metabolites) or implied through the use of stable hepoxilin analogs. These analogs have additionally shown efficacy in animal models of lung fibrosis, cancer, thrombosis and diabetes. Research on these compounds has merely scratched the surface, but results published to date have suggested that the hepoxilin pathway is a distinct and novel pathway leading to inflammation and hepoxilin antagonists may provide the means of controlling early aspects of the acute inflammatory phase. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Whatever happened to the epoxyeicosatrienoic Acid-like endothelium-derived hyperpolarizing factor? The identification of novel classes of lipid mediators and their role in vascular homeostasis.

SIGNIFICANCE: Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid (AA) to generate epoxyeicosatrienoic acids (EETs). The latter are biologically active and reported to act as an endothelium-derived hyperpolarizing factor as well as to affect angiogenic and inflammatory signaling pathways. RECENT ADVANCES: In addition to AA, the CYP enzymes also metabolize the ω-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid to generate bioactive lipid epoxide mediators. The latter can be more potent than the EETs, but their actions are under investigated. The ω3-epoxides, like the EETs, are metabolized by the soluble epoxide hydrolase (sEH) to corresponding diols, and epoxide hydrolase inhibition increases epoxide levels and demonstrates anti-hypertensive as well as anti-inflammatory effects. CRITICAL ISSUES: It seems that the overall consequences of CYP activation largely depend on enzyme substrate preference and the endogenous ω-3/ω-6 PUFA ratio. FUTURE DIRECTIONS: More studies combining PUFA profiling with cell signaling and disease studies are required to determine the spectrum of molecular pathways affected by the different ω-6 and ω-3 PUFA epoxides and diols. Such information may help improve dietary studies aimed at promoting health via ω-3 PUFA supplementation and/or sEH inhibition.