Vascular hepoxilin and trioxilins mediate vasorelaxation through TP receptor inhibition in mouse arteries.

AIM: 12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12-LO activity. METHODS: Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A3 , trioxilin A3 and trioxilin C3 by mass spectrometry. Activities of these natural compounds were tested on isometric tension and intracellular calcium release. The role of thromboxane (TP) receptor was determined in HEK293 cells overexpressing TPα receptor (TPα -HEK). RESULTS: All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A3 , C3 and hepoxilin A3 (3 µm) relaxed arteries constricted with the thromboxane mimetic, U46619-constricted arteries (maximum relaxations of 78.9 ± 3.2, 29.7 ± 4.6, 82.2 ± 5.0 and 88.0 ± 2.4% respectively), but not phenylephrine-constricted arteries. In TPα-HEK cells, trioxilin A3 , C3 and hepoxilin A3 (10 µm) inhibited U46619 (10 nM)-induced increases in intracellular calcium by 53.0 ± 7.2%, 32.8 ± 5.0% and 37.9 ± 13.5% respectively. In contrast, trioxilin B3 and hepoxilin B3 were not synthesized in arteries and exhibited little biological activity. CONCLUSION: Trioxilin A3 and C3 and hepoxilin A3 are endogenous vascular relaxing factors. They are not endothelium-derived hyperpolarizing factors but mediate vascular relaxation by inhibiting TP agonist-induced increases in intracellular calcium. Thus, they regulate vascular homeostasis by acting as endogenous TP antagonists.

Hyperresistance to cholesterol hydroperoxide-induced peroxidative injury and apoptotic death in a tumor cell line that overexpresses glutathione peroxidase isotype-4.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX; GPX4) plays a key role in eukaryotic defense against potentially lethal peroxidative injury and also regulation of physiological peroxide tone. In this work we focused on the cytoprotective antiperoxidant effects of GPX4, using a breast tumor epithelial cell line that over-expresses the enzyme. Wild-type COH-BR1 cells, which exhibit little (if any) GPX4 activity, were transfected with a construct encoding the mitochondrion-targeted long (L) form of the enzyme. Several transfectant clones were selected which expressed relatively large amounts of GPX4, as determined by both Northern and Western analysis. Enzyme activity ranged from 15-fold to 190-fold greater than that of wild-type or null-transfected cells. The functional ramifications of GPX4 overexpression were tested by challenging cells with photochemically generated cholesterol hydroperoxides (ChOOHs) in liposomal form. Compared with vector controls, overexpressing clones were found to be substantially more resistant to ChOOH-induced killing, as determined by annexin-V (early apoptotic) and thiazolyl blue (mitochondrial dehydrogenase) reactivity. Concomitantly, the clones exhibited a striking hyper-resistance to free radical-mediated lipid peroxidation, as assessed by labeling cell membranes with [(14)C]cholesterol and measuring a family of radiolabeled oxidation products (ChOX). L-form GPX4's antiperoxidant and cytoprotective effects could reflect its ability to detoxify ChOOHs as they enter cells and/or cell-derived lipid hydroperoxides arising from ChOOH one-electron turnover.

Quantitative studies on the respiratory burst generated in peritoneal macrophages.

The luminol-dependent chemiluminescence of macrophages during the zymosan-stimulated respiratory burst has been studied both in the absence and in the presence of the radical inhibitor 3,5 di-tert-butyl-4-hydroxyphenyl propionic acid. In addition, the consumption of luminol and of the inhibitor has been followed analytically. Based on the rates of the consumption of the inhibitor, an iteration procedure yields a value of 2.2 x 10(-7) M for the steady-state concentration of radicals generated by cells at the maximum of the chemiluminescence in the presence of inhibitor. Approximate calculations have indicated that under the experimental conditions applied, additional formation of superoxide anion radicals by the oxidation of luminol is negligible. By assuming that in an inhibitor-free system the disappearance of radicals takes place via their combination process as well as by their interaction with luminol and/or with luminol-derived species, numerical integration yields a calculated curve of radical concentration versus time in fair agreement with experimental data and a rate-constant value for the combination of radicals of approximately 10(6) M-1 s-1, supporting literature findings according to which primarily superoxide anion radicals are formed.

Mechanism of volatile compound production during storage of sunflower Oil.

Volatile compounds formed in the course of the thermal decomposition of hydroperoxides during storage of sunflower oil were analyzed by headspace solid-phase microextraction sampling followed by gas chromatographic separation and mass spectral detection. The determining role of alkoxyl radicals in the process has been proven by electron spin resonance spectroscopic measurements. On the basis of analytical results, the reaction networks and mechanisms were constructed by computer modeling to describe the formation of volatile products of radical decomposition of hydroperoxides. We established that off-flavor aliphatic aldehydes are originated from only the alkoxyl radicals derived from trigliceride of linoleic acid. To find a specific additive, which redirects the formation of these radicals toward production of more stable species, is suggested.

In vivo experimental studies on the role of free radicals in photodynamic therapy. III. Photodynamic effect on free radicals generated in cell cultures.

The effect of excited Photofrin II molecules on the zymosan stimulated and luminol dependent chemiluminescence (CL) of macrophages, known as respiratory burst, has been studied. Excitation was carried out by light irradiation in the vicinity of the maximal CL by varying light doses. In separate experiments potassium salt of 3,5-di-tert-butyl-4-hydroxyphenyl propionic acid radical inhibitor was added to the system and its consumption measured spectroscopically. Based on the experimental results the decrease in the steady state radical concentrations under the influence of radical inhibitor and of triplet sensitizer generated in situ has been calculated. It has been established that the corresponding photodynamic effect is an inhibition-like process proceeding by triplet-doublet interactions.

Physico-chemical modeling of the role of free radicals in photodynamic therapy. III. Interactions of stable free radicals with excited photosensitizers studied by kinetic ESR spectroscopy.

The loss of paramagnetism of the stable free radical 4,4'-di-tert.-butyl- diphenyl-1-nitroxy during illumination of the photosensitizer meso-tetrahydroxyphenyl chlorine by light above 620 nm in the absence of oxygen has been followed by kinetic ESR spectroscopy. Addition of the spin trap alpha-(4-pyridyl-1-oxide)- N-tert.-butyl-nitrone reduces the initial rate of the disappearance of the free radical enabling to separate triplet-doublet interactions from processes between radicals stemming from the triplet sensitizer and the stable free radical. Kinetic treatment of the mechanism suggested yielded the rate constant of the triplet-doublet interaction proceeding via electron transfer being about 6% of the rate constant of the overall interaction including both energy transfer and electron transfer.

In vivo experimental studies on the role of free radicals in photodynamic therapy. II. Photodynamic effect on free radical concentration in mice tumors measured by ESR spectroscopy.

Changes in free radical concentrations of solid tumors of mice after photodynamic treatment using Photofrin II sensitizer has been measured by ESR spectroscopy. Decrease in the free radical concentrations by 46% in S180 and by 35% in P388 tumors induced photodynamically was found. As expected, the effect was proportional to the steady state concentrations of the free radicals in the malignant cells of mice prior to photodynamic treatment if comparing either different types of tumors or data referring to various stages of the development of identical types of tumors. Results support the contribution of the interactions between triplet excited sensitizer molecules and native free radicals to photodynamic effects as assumed earlier.

Physicochemical modeling of the role of free radicals in photodynamic therapy.

A new kinetic approach is suggested and experimentally supported for quantification of the spin-trapping procedure. Accordingly, the concentration of the spin adduct formed in the interaction between the spin-trap DMPO and various free radicals (cyanopropylperoxy, cumylperoxy, phenylethylperoxy, and hydroperoxy radicals) generated by the initiated oxidation of the parent molecules is followed by kinetic ESR spectrometry. The initial sections of the corresponding kinetics are linear indicating that during this period the consumption of the adduct is negligible and thus the rate of accumulation (W(A)) approximates the rate of formation (W(f)): W(A) approximately W(f) = k(ST)[Rad(*)][DMPO], supported also by the fact that the rate of initiation of oxidation equals W(A) at high [DMPO]. In addition, the circulatory experimental apparatus enables calculation of the rate of molecular decomposition of the adduct by stopping circulation (W(f) becomes negligible) and following the decrease of the ESR signal. Corresponding rate constants are summarized.

Physico-chemical modeling of the role of free radicals in photodynamic therapy. IV. Quantitative aspects of photodynamic effects on free radicals generated in cell cultures.

Production and the mechanism of the interactions of free radicals generated by stimulated macrophages in the presence of luminol and a free radical inhibitor was investigated to determine the possibility of using luminol-dependent chemiluminescence for studying photodynamic effects in biology. Earlier measurements have been revisited and additional experiments performed indicating that oxidation products of luminol neither inhibit the in vitro formation of radicals nor quench CL. Simulation based on the mechanism suggested revealed that the likely value for the rate constant of the primary step between luminol and superoxide anion radicals producing luminol radicals is 5x10(2)-1x10(3) M-1s-1. It has been established that the ratio of the concentration of radicals generated by the biological system to that formed by oxidation of luminol exceeds 10(3); that is, the contribution of the latter is negligible and the system is appropriate to measure quantitatively the effect of excited photosensitizers on free radicals.