leukotoxin and 9-10-dihydroxy-12-octadecenoic-acid

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid found in the Western diet. Cytochrome P450-derived LA metabolites 9,10-epoxyoctadecenoic acid (9,10-EpOME), 12,13-epoxyoctadecenoic acid (12,13-EpOME), 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME) have been studied for their association with various disease states and biological functions. Previous studies of the EpOMEs and DiHOMEs have focused on their roles in cytotoxic processes, primarily in the inhibition of the neutrophil respiratory burst. More recent research has suggested the DiHOMEs may be important lipid mediators in pain perception, altered immune response and brown adipose tissue activation by cold and exercise. The purpose of this review is to summarize the current understanding of the physiological and pathophysiological roles and modes of action of the EpOMEs and DiHOMEs in health and disease.

Topics: Adipose Tissue, Brown; Analgesics; Animals; Cytochrome P-450 Enzyme System; Endocrine System; Epoxide Hydrolases; Exotoxins; Humans; Immune System; Inflammation; Linoleic Acid; Lipids; Lung; Mice; Neutrophils; Oleic Acids; Oxidation-Reduction; Pain Management; Respiratory Burst; Stearic Acids

LC/MS quantification of leukotoxin (LTX) and leukotoxin diol (LTXdiol) in plasma has been previously reported, however large sample volumes are required for achieving stated assay Lower Limit of Quantification (LLOQ). Reported here is a fit-for-purpose LC/MS method that reduces plasma volume from 700 to 25 µL and omits pre-concentration steps. These improvements make for a method with increased utility in mouse studies offering limited sample volumes. Additionally, omitting pre-concentration steps streamlines sample processing, which can now be completed in under 10 min. This method can be used to quickly answer if the ratio of LTX to LTXdiol changes with the dose of the therapeutic drug so this could be used as a potential biomarker for correlating PK/PD effects. No extensive assay characterization was performed before application to an exploratory in-life study. Basal levels of LTX and LTXdiol in plasma were quantified by LC-MRM across 10 individual mice, and the average signal-to-noise was 36 for LTX and 3039 for LTXdiol, with CVs of 29.4% and 15.2%, respectively. Addition of LTX and LTXdiol reference standard at 5, 25, and 75 ng/mL into pooled mouse plasma was quantifiable within 30% relative error using a surrogate matrix calibration curve ranging from 0.8 to 200 ng/mL. The average ratio of LTX to LTXdiol across the 10 mice was 0.32, consistent with previous reports. Finally, the method was applied to a mouse PK/PD study to monitor LTX/LTXdiol kinetics after a single oral dose of a soluble epoxide hydrolase inhibitor. The mean plasma ratio of LTX to LTXdiol increased up to 10-fold by 3 h post-dose followed by a decrease to near pre-dose levels by 24 h, consistent with transient inhibition of sEH-mediated conversion of LTX to LTXdiol. The method improvements described here will make subsequent quantification of LTX and LTXdiol in mouse studies significantly easier.

Topics: Animals; Biomarkers; Chromatography, Liquid; Exotoxins; Female; Mice; Mice, Inbred BALB C; Reproducibility of Results; Stearic Acids; Tandem Mass Spectrometry

This study documents that chlorinated analogs of leukotoxin diol 1, in which the vic-diol has been replaced with vic-chlorides (2), induce caspase 3 activity and apoptosis on HepG2 cells in a dose-dependent manner in analogy to the parent diol. This suggests that chlorides may substitute for hydroxyls in certain lipids as bioisosteres in defined biological settings.

Topics: Apoptosis; Exotoxins; Halogenation; Humans; Molecular Structure; Stearic Acids

Polyunsaturated fatty acids such as linoleic acid are well known dietary lipids that may be atherogenic by activating vascular endothelial cells. In the liver, fatty acids can be metabolized by cytochrome P450 (CYP) enzymes, but little is known about the role of these enzymes in the vascular endothelium. CYP 2C9 is involved in linoleic acid epoxygenation, and the major product of this reaction is leukotoxin (LTX). We investigated the role of CYP-mediated mechanisms of linoleic acid metabolism in endothelial cell activation by examining the effects of linoleic acid or its oxidized metabolites such as LTX and leukotoxin diol (LTD).. The effect of linoleic acid on CYP 2C9 gene expression was studied by RT-PCR. Oxidative stress was monitored by measuring DCF fluorescence and intracellular glutathione levels, and electrophoretic mobility shift assay was carried out to study the activation of oxidative stress sensitive transcription factors. Analysis of oxidized lipids was carried out by liquid chromatography/mass spectrometry.. Linoleic acid treatment for six hours increased the expression of CYP 2C9 in endothelial cells. Linoleic acid-mediated increase in oxidative stress and activation of AP-1 were blocked by sulfaphenazole, a specific inhibitor of CYP 2C9. The linoleic acid metabolites LTX and LTD increased oxidative stress and activation of transcription factors only at high concentrations.. Our data show that CYP 2C9 plays a key role in linoleic acid-induced oxidative stress and subsequent proinflammatory events in vascular endothelial cells by possibly causing superoxide generation through uncoupling processes.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP2C9; Dose-Response Relationship, Immunologic; Electrophoretic Mobility Shift Assay; Endothelial Cells; Endothelium, Vascular; Exotoxins; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation; Glutathione; Humans; Immunosuppressive Agents; Inflammation Mediators; Linoleic Acid; NF-kappa B; Oxidative Stress; Pulmonary Artery; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Stearic Acids; Swine; Transcription Factor AP-1; Umbilical Veins

Leukotoxin is clinically associated with acute respiratory distress syndrome (ARDS). Recently, we found that leukotoxin-diol, the hydrated product of leukotoxin, is more toxic than the parent leukotoxin in vitro (Moghaddam and colleagues, Nature Med. 1997;3:562-566). To test if this difference in the toxicity of leukotoxin and leukotoxin-diol exists in vivo, Swiss Webster mice were administered leukotoxin or leukotoxin-diol. All mice treated with leukotoxin-diol died of ARDS-like respiratory distress, whereas the animals exposed to leukotoxin at the same dose survived. Histopathologic evaluation of the lungs revealed massive alveolar edema and hemorrhage with interstitial edema around blood vessels in the lungs of mice treated with leukotoxin-diol, whereas the lungs of mice treated with identical doses of leukotoxin had perivascular edema only and little change in alveolar spaces. Immunohistochemistry showed that the soluble epoxide hydrolase responsible for the hydrolysis of leukotoxin to its diol is concentrated in the vascular smooth muscle of small and medium-sized pulmonary vessels. In addition, 4-phenylchalcone oxide, an inhibitor of soluble epoxide hydrolase, was found to decrease the mortality induced by leukotoxin but had no effect on mortality induced by leukotoxin-diol. These studies provide strong in vivo evidence that leukotoxin may act as a protoxicant and that the corresponding diol is a putative toxic mediator involved in the development of ARDS.

Topics: Animals; Chalcone; Chalcones; Dose-Response Relationship, Drug; Edema; Enzyme Inhibitors; Epoxide Hydrolases; Exotoxins; Lung; Male; Mice; Respiratory Distress Syndrome; Stearic Acids

Leukotoxin (Lx), an epoxide derivative of linoleic acid, has been suggested to be a toxic mediator of multiple organ failure in burn patients and of acute respiratory distress syndrome. Lx production was recently shown during myocardial ischemia/reperfusion. However, a recent study suggested that to be toxic Lx must be metabolized to Lx-diol. In the present study, isolated adult rat ventricular myocytes were studied with the whole-cell patch-clamp technique to determine the effects of these compounds on cardiac electrical activity. Measurements of action potentials showed that neither linoleic acid nor Lx (100 microM) caused any significant changes in action potential properties. However, Lx-diol in the range of 10-100 microM produced a dose dependent increase in duration and a decrease in overshoot of the action potential. Subsequent voltage clamp experiments isolating Na current (INa) and transient outward K current (Ito) revealed that Lx-diol inhibited INa and Ito by about 80% at 100 microM, while linoleic acid and Lx had no effect on these currents at the same concentration. While Lx-diol produced the same inhibition of INa and Ito at 100 microM, its effects were more potent on Ito with significant inhibition at 10 microM. Lx-diol also hastened the activation kinetics of Ito but not INa. The action of Lx-diol was rapid (reaching steady state in 3-5 min) and was reversible in 5-10 min following washout. Thus, Lx-diol could favor arrhythmias or cardiac arrest in intact heart and may be responsible for the cardiac problems seen in systemic inflammatory response syndrome. These results further support the suggestion that Lx is not toxic in the heart but rather must be metabolized to Lx-diol to produce toxic effects on cardiac muscle.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Cells, Cultured; Dose-Response Relationship, Drug; Exotoxins; Heart Ventricles; Linoleic Acid; Mass Spectrometry; Myocardium; Patch-Clamp Techniques; Potassium; Rats; Sodium; Stearic Acids