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Oxylipins are metabolites of polyunsaturated fatty acids that mediate cardiovascular health by attenuation of inflammation, vascular tone, hemostasis, and thrombosis. Very low-density lipoproteins (VLDL) contain oxylipins, but it is unknown whether the liver regulates their concentrations. In this study, we used a perfused liver model to observe the effect of inflammatory lipopolysaccharide (LPS) challenge and soluble epoxide hydrolase inhibition (sEHi) on VLDL oxylipins. A compartmental model of deuterium-labeled linoleic acid and palmitic acid incorporation into VLDL was also developed to assess the dependence of VLDL oxylipins on fatty acid incorporation rates. LPS decreased the total fatty acid VLDL content by 30% [6%,47%], and decreased final concentration of several oxylipins by a similar amount (13-HOTrE, 35% [4%,55%], -1.3 nM; 9(10)-EpODE, 29% [3%,49%], -2.0 nM; 15(16)-EpODE, 29% [2%,49%], -1.6 nM; AA-derived diols, 32% [5%,52%], -2.4 nM; 19(20)-DiHDPA, 31% [7%,50%], -1.0 nM). However, the EPA-derived epoxide, 17(18)-EpETE, was decreased by 75% [49%,88%], (-0.52 nM) with LPS, double the suppression of other oxylipins. sEHi increased final concentration of DHA epoxide, 16(17)-EpDPE, by 99% [35%,193%], (2.0 nM). Final VLDL-oxylipin concentrations with LPS treatment were not correlated with linoleic acid kinetics, suggesting they were independently regulated under inflammatory conditions. We conclude that the liver regulates oxylipin incorporation into VLDL, and the oxylipin content is altered by LPS challenge and by inhibition of the epoxide hydrolase pathway. This provides evidence for delivery of systemic oxylipin signals by VLDL transport.

Epoxyoctadecamonoenoic acids (EpOMEs) are epoxide derivatives of linoleic acid (9,12-octadecadienoic acid) and include 9,10-EpOME and 12,13-EpOME. They are synthesized by cytochrome P450 monooxygenases (CYPs) and degraded by soluble epoxide hydrolase (sEH). Although EpOMEs are well known to play crucial roles in mediating various physiological processes in mammals, their role is not well understood in insects. This study chemically identified their presence in insect tissues: 941.8 pg/g of 9,10-EpOME and 2,198.3 pg/g of 12,13-EpOME in fat body of a lepidopteran insect, Spodoptera exigua. Injection of 9,10-EpOME or 12,13-EpOME into larvae suppressed the cellular immune responses induced by bacterial challenge. EpOME treatment also suppressed the expression of antimicrobial peptide (AMP) genes. Among 139 S. exigua CYPs, an ortholog (SE51385) to human EpOME synthase was predicted and its expression was highly inducible upon bacterial challenge. RNA interference (RNAi) of SE51385 prevented down-regulation of immune responses at a late stage (> 24 h) following bacterial challenge. A soluble epoxide hydrolase (Se-sEH) of S. exigua was predicted and showed specific expression in all development stages and in different larval tissues. Furthermore, its expression levels were highly enhanced by bacterial challenge in different tissues. RNAi reduction of Se-sEH interfered with hemocyte-spreading behavior, nodule formation, and AMP expression. To support the immune association of EpOMEs, urea-based sEH inhibitors were screened to assess their inhibitory activities against cellular and humoral immune responses of S. exigua. 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) was highly potent in suppressing the immune responses. The addition of AUDA to a pathogenic bacterium significantly increased bacterial pathogenicity by suppressing host immune defense. In sum, this study demonstrated that EpOMEs play a crucial role in facilitating anti-inflammatory respons

Verticillium dahliae is a broad host-range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved. Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed. Population genomics previously revealed that G-LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G-LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N-lauroylethanolamine (N-acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase. The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

Verticillium dahliae is a broad host-range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved. Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed. Population genomics previously revealed that G-LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G-LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N-lauroylethanolamine (N-acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase. The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

Epoxide hydrolases (EHs) are enzymes that play roles in metabolizing xenobiotic epoxides from the environment, and in regulating lipid signaling molecules, such as juvenile hormones in insects and epoxy fatty acids in mammals. In this study we fed mosquitoes with an epoxide hydrolase inhibitor AUDA during artificial blood feeding, and we found the inhibitor increased the concentration of epoxy fatty acids in the midgut of female mosquitoes. We also observed ingestion of AUDA triggered early expression of defensin A, cecropin A and cecropin B2 at 6 h after blood feeding. The expression of cecropin B1 and gambicin were not changed more than two fold compared to controls. The changes in gene expression were transient possibly because more than 99% of the inhibitor was metabolized or excreted at 42 h after being ingested. The ingestion of AUDA also affected the growth of bacteria colonizing in the midgut, but did not affect mosquito longevity, fecundity and fertility in our laboratory conditions. When spiked into the blood, EpOMEs and DiHOMEs were as effective as the inhibitor AUDA in reducing the bacterial load in the midgut, while EETs rescued the effects of AUDA. Our data suggest that epoxy fatty acids from host blood are immune response regulators metabolized by epoxide hydrolases in the midgut of female mosquitoes, inhibition of which causes transient changes in immune responses, and affects growth of microbes in the midgut.

In insects, epoxide hydrolases (EHs) play critical roles in the metabolism of xenobiotic epoxides from the food resources and in the regulation of endogenous chemical mediators, such as juvenile hormones. Using the baculovirus expression system, we expressed and characterized an epoxide hydrolase from Anopheles gambiae (AgEH) that is distinct in evolutionary history from insect juvenile hormone epoxide hydrolases (JHEHs). We partially purified the enzyme by ion exchange chromatography and isoelectric focusing. The experimentally determined molecular weight and pI were estimated to be 35 kD and 6.3 respectively, different than the theoretical ones. The AgEH had the greatest activity on long chain epoxy fatty acids such as 14,15-epoxyeicosatrienoic acids (14,15-EET) and 9,10-epoxy-12Z-octadecenoic acids (9,10-EpOME or leukotoxin) among the substrates evaluated. Juvenile hormone III, a terpenoid insect growth regulator, was the next best substrate tested. The AgEH showed kinetics comparable to the mammalian soluble epoxide hydrolases, and the activity could be inhibited by AUDA [12-(3-adamantan-1-yl-ureido) dodecanoic acid], a urea-based inhibitor designed to inhibit the mammalian soluble epoxide hydrolases. The rabbit serum generated against the soluble epoxide hydrolase of Mus musculus can both cross-react with natural and denatured forms of the AgEH, suggesting immunologically they are similar. The study suggests there are mammalian sEH homologs in insects, and epoxy fatty acids may be important chemical mediators in insects.

The ability of a nitroxyl fatty acid (NFA) to bind specifically to albumin is abolished when, in the absence of stabilizers, a 4% solution of this protein is heated above a critical temperature of 60 degrees C. This treatment leads to the formation of "albumin polymers" as classically evidenced by GPC. Since the bound fraction is evidenced in EPR spectroscopy by a large anisotropic component, the presence of this anisotropy can be used in the assessment of the quality of the pharmaceutical preparations of albumin, which are usually pasteurized in order to inactivate viruses. Moreover, in sharp contrast with the behavior of albumin dispersions, lyophilised albumin subjected to heat treatment at 70 degrees C for 24 h left the protein untouched regarding its NFA binding and GPC profile.

The ability of a nitroxyl fatty acid (NFA) to bind specifically to albumin is abolished when, in the absence of stabilizers, a 4% solution of this protein is heated above a critical temperature of 60 degrees C. This treatment leads to the formation of "albumin polymers" as classically evidenced by GPC. Since the bound fraction is evidenced in EPR spectroscopy by a large anisotropic component, the presence of this anisotropy can be used in the assessment of the quality of the pharmaceutical preparations of albumin, which are usually pasteurized in order to inactivate viruses. Moreover, in sharp contrast with the behavior of albumin dispersions, lyophilised albumin subjected to heat treatment at 70 degrees C for 24 h left the protein untouched regarding its NFA binding and GPC profile.

In this study, we report the synthesis and the evaluation as MRI contrast agent of a new compound (nitroxyl fatty acid, NFA), where a pyrrolidinoxyl radical (3-carboxy-proxyl, PCA) is linked to a fatty acid moiety. Fatty acids were selected as vector because they present a high affinity for the liver, their efficient cellular uptake being the result of a specific interaction with a transmembrane transporter (liver plasma membrane-fatty acid binding protein). The uptake of 3H-oleic acid is inhibited after the injection of 1 mmol/kg of NFA, suggesting that NFA recognizes the same transmembrane transporter as the natural fatty acids. The higher relaxivity R1 of NFA in albumin solutions, compared with PCA, was explained by the immobilization of the nitroxyl radical in the protein. MR imaging was performed using T1-weighted images on mice in order to compare the contrast effect obtained after the injection of 1 mmol/kg of radical. The % signal enhancement in the liver 5 min after intravenous injection was 49 +/- 4 and 14 +/- 5 for NFA and PCA, respectively. NFA allowed a better delimitation of some necrotic tumors (Novikoff hepatocarcinoma) due to its preferential uptake by the nontumorous tissue.

In this study, we report the synthesis and the evaluation as MRI contrast agent of a new compound (nitroxyl fatty acid, NFA), where a pyrrolidinoxyl radical (3-carboxy-proxyl, PCA) is linked to a fatty acid moiety. Fatty acids were selected as vector because they present a high affinity for the liver, their efficient cellular uptake being the result of a specific interaction with a transmembrane transporter (liver plasma membrane-fatty acid binding protein). The uptake of 3H-oleic acid is inhibited after the injection of 1 mmol/kg of NFA, suggesting that NFA recognizes the same transmembrane transporter as the natural fatty acids. The higher relaxivity R1 of NFA in albumin solutions, compared with PCA, was explained by the immobilization of the nitroxyl radical in the protein. MR imaging was performed using T1-weighted images on mice in order to compare the contrast effect obtained after the injection of 1 mmol/kg of radical. The % signal enhancement in the liver 5 min after intravenous injection was 49 +/- 4 and 14 +/- 5 for NFA and PCA, respectively. NFA allowed a better delimitation of some necrotic tumors (Novikoff hepatocarcinoma) due to its preferential uptake by the nontumorous tissue.

RIVM rapport:A derivatization procedure is described for the long chain fatty acid, lauric acid and metabolites using a fluorescent probe 4-(bromomethyl)-7-methoxycoumarin. The derivatives can be separated and detected in an isocratic high performance liquid chromatografic system using a fluorescence detector. The derivatization is rapid, simple and gives a good quantification by the use of an internal standard. The procedure has been applied on samples from a toxicity experiment with the plasticizer di (2-ethylhexyl)phthalate (DEHP). The activity of cytochrome P-450 IVA1 or lauric acid hydroxylase (LAH) in liver homogenates can be determined by the quantification of the 11- and 12-hydroxylated metabolites of lauric acid. The method turned out to be very sensitive and suitable to replace similar assays using radioactive compounds.

RIVM rapport:A derivatization procedure is described for the long chain fatty acid, lauric acid and metabolites using a fluorescent probe 4-(bromomethyl)-7-methoxycoumarin. The derivatives can be separated and detected in an isocratic high performance liquid chromatografic system using a fluorescence detector. The derivatization is rapid, simple and gives a good quantification by the use of an internal standard. The procedure has been applied on samples from a toxicity experiment with the plasticizer di (2-ethylhexyl)phthalate (DEHP). The activity of cytochrome P-450 IVA1 or lauric acid hydroxylase (LAH) in liver homogenates can be determined by the quantification of the 11- and 12-hydroxylated metabolites of lauric acid. The method turned out to be very sensitive and suitable to replace similar assays using radioactive compounds.

RIVM rapport:A derivatization procedure is described for the long chain fatty acid, lauric acid and metabolites using a fluorescent probe 4-(bromomethyl)-7-methoxycoumarin. The derivatives can be separated and detected in an isocratic high performance liquid chromatografic system using a fluorescence detector. The derivatization is rapid, simple and gives a good quantification by the use of an internal standard. The procedure has been applied on samples from a toxicity experiment with the plasticizer di (2-ethylhexyl)phthalate (DEHP). The activity of cytochrome P-450 IVA1 or lauric acid hydroxylase (LAH) in liver homogenates can be determined by the quantification of the 11- and 12-hydroxylated metabolites of lauric acid. The method turned out to be very sensitive and suitable to replace similar assays using radioactive compounds.