Antioxidant activity of resveratrol in endotoxin-stimulated blood platelets

Resveratrol (3,4′,5-trihydroxystilbene) is a natural molecule with antioxidant action. It is also considered to be a molecule with antiplatelet, anticancer and anti-inflammatory action. The effects of trans-resveratrol on the reactive oxygen species (ROS) generation and thiobarbituric acid-reactive substances (TBARS) in blood platelets induced by endotoxin (lipopolysaccharide, LPS) or thrombin were studiedin vitro. The production of superoxide radicals (O₂ ^.–) and other reactive oxygen species (H₂O₂, singlet oxygen, and organic radicals) in the presence of resveratrol was measured by a chemiluminescence method in resting blood platelets and platelets stimulated by LPS (0.3 μg/10⁸ platelets) or thrombin (2.5 U/10⁸ platelets). We have shown that resveratrol (6.25–100 μg/ml) inhibits chemiluminescence and generation of O₂ ^.– in blood platelets. It has an inhibitory effect on the production of ROS and TBARS in platelets caused by LPS or thrombin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Endotoxins stimulate generation of superoxide radicals and lipid peroxidation in blood platelets

Lipopolysaccharide (LPS, endotoxin) is an important structural constituent of the membrane of gram-negative bacteria with a wide range of biological effects. It can activate blood platelets. The purpose of present study was to determine the direct effect of endotoxins from Proteus mirabilis, differing significantly in their composition, on the generation of superoxide radicals and thiobarbituric acid reactive substances (TBARS) in blood platelets. Superoxide radicals were measured by means of superoxide dismutase-inhibitable reduction of cytochrome C. The TBARS determination (malonyldialdehyde) was used as a marker of endogenous arachidonate metabolism and thromboxane A2 synthesis. Results demonstrate that three endotoxins (LPS S1959, LPS R110, LPS R45) after 2 min of action, even at the lowest concentration (0.03 microg/10(8) platelets) stimulated the generation of TBARS and release of superoxide radicals. All LPS contain lipid A as a component but differ in their chemical composition in the polysaccharide part. It is suggested that the observed effects of LPS on blood platelets are attributable to their lipid A portion.     

The Effect of Lipopolysaccharide from <Emphasis Type="Italic">Proteus mirabilis</Emphasis> on the Level of the Stable End Metabolic Products of Nitric Oxide in Blood Platelets

Nitric oxide (^•NO) plays an important role in a number of physiologic processes. Evidence exists that ^•NO, which stimulates soluble guanylate cyclase and enhances cyclic guanosine monophosphate (cGMP) levels, may inhibit platelet activation. In contrast, during platelet activation induced by different agonists, synthesis of ^•NO in platelets occurs. In these studies, production of the stable end-products of ^•NO-nitrite and nitrate (NO_x) in human platelets, stimulated by different doses of lipopolysaccharide from Proteus mirabilis (LPS; endotoxin), has been evaluated. LPS is a weak platelet agonist that may activate various steps of platelet activation with the generation of reactive oxygen species. The mechanism of platelet activation induced by the endotoxin is not known. The aim of the present study was to measure the level of nitrite and NO_x in blood platelets treated with LPS and to examine the level of nitrotyrosine in platelet proteins caused by LPS. Our results show that LPS at a low concentration (6.8 ng/ml) caused a decrease (approximately 80%) in the NO_x level, whereas at higher concentrations (13.6 and 25 ng/ml) it induced an increase in the NO_x level (approximately 210% and 260%, respectively). Our results indicate that LPS, like other agonists (thrombin, platelet-activating factor), can stimulate ^•NO production in platelets. After incubating platelets with LPS, we also observed a distinct increase in platelet protein nitration (3-nitrotyrosine).     

<Emphasis Type="SmallCaps">l</Emphasis>-carnitine modulates blood platelet oxidative stress

The oxidative stress induced by acute exertion may interfere with blood platelet activation. The beneficial effect of l-carnitine (γ-trimethylamino-β-hydroxybutyric acid) on oxidative stress in blood platelets has not been fully investigated; however, different studies indicate that this compound modulates platelet functions. The aim of our study was to assess the effects of l-carnitine on platelet activation and oxidative/nitrative protein damage (determined by the levels of protein carbonyl groups, thiol groups, and 3-nitrotyrosine residues) in resting blood platelets or platelets treated with peroxynitrite (ONOO⁻, a strong physiological oxidant) in vitro. We also investigated the effects of l-carnitine on the level of platelet glutathione and on the formation of superoxide anion radicals ( O₂ ^{- ·} ) \left( {{\hbox{O}}_2^{ - \bullet }} \right) , lipid peroxidation measured by thiobarbituric acid reactive substances (TBARS) in blood platelets stimulated by thrombin (a strong physiological agonist), and platelet aggregation induced by adenosine diphosphate (a strong physiological stimulator). We have observed that carnitine decreases platelet activation (measured by platelet aggregation, the generation of O₂ ^{- ·} {\hbox{O}}_2^{ - \bullet } , and TBARS production). Moreover, our results in vitro demonstrate that carnitine may protect against oxidation of thiol groups induced by ONOO⁻. Thus, carnitine may have some protectory effects against oxidative changes induced in blood platelets.     

Resveratrol reduces oxidative stress induced by platinum compounds in blood platelets

The effects of resveratrol (trans-3,4',5-trihydroxystilbene) on the oxidative stress in blood platelets induced by platinum compounds [cisplatin and selenium-cisplatin conjugate] were studied in vitro. The production of thiobarbituric acid reactive substances (TBARS), the level of conjugate diene, the generation of superoxide anion radicals (O2-*) and other reactive oxygen species (O2-*, H2O2, singlet oxygen and organic radicals) were measured by chemiluminescence in blood platelets treated with platinum compounds. Cisplatin at the concentration of 10 microg/ml, as well as selenium-cisplatin conjugate (10 microg/ml) induced oxidative stress in blood platelets: an increase in TBARS, conjugate diene, chemiluminescence and generation of O2-*. In the presence of resveratrol (a natural compound with antioxidant activity) at the concentrations of 1-25 microg/ml, the chemiluminescence, the levels of O2-*, conjugate diene and TBARS were reduced (p < 0.05). We showed that resveratrol at different concentrations (1-25 microg/ml) had a protective effect against oxidative stress in platelets caused by platinum compounds (10 microg/ml) and it diminished platelet lipid peroxidation and reactive oxygen species generation induced by platinum compounds.     

Chemiluminescence response of the human polymorphonuclear neutrophil to lipopolysaccharides

Polymorphonuclear neutrophils (PMN) respond to a variety of stimuli with a sequence of reactions that lead to the production of “active oxygen” species, including H₂O₂, free radicals, such as superoxide (O₂ ⁻·) and hydroxyl (HO·), and singlet molecular oxygen (¹O₂). Some of these can oxidize (5-amino-2,3-dihydrophthalazine 1,4-dione) (luminol) to the ground state aminophthalate ion; this reaction sequence is accompanied by the generation of a photon and forms the basis for the chemiluminescence (CL) response. In this work we used a dedicated photon counting instrument to record CL from PMN incubated with bacterial lipopolysaccharide (LPS). We have studied the CL response to the LPS fromEscherichia coli strains 026:B6 and 055:B5, as well asSalmonella minnesota RE 595 and have determined that CL requires heat-labile serum factors, these most likely being intact components of the complement system.     

Superoxide free radicals are produced in glyoxysomes

The production of superoxide free radicals in pellet and supernatant fractions of glyoxysomes, specialized plant peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons, was investigated. Upon inhibition of the endogenous superoxide dismutase, xanthine, and hypoxanthine induced in glyoxysomal supernatants the generation of O₂⁻ radicals and this was inhibited by allopurinol. In glyoxysomal pellets, NADH stimulated the generation of superoxide radicals. Superoxide production by purines was due to xanthine oxidase, which was found predominantly in the matrix of glyoxysomes. The generation of O₂⁻ radicals in glyoxysomes by endogenous metabolites suggests new active oxygen-related roles for glyoxysomes, and for peroxisomes in general, in cellular metabolism.     

NADH Induces the Generation of Superoxide Radicals in Leaf Peroxisomes

In peroxisomes isolated from pea leaves (Pisum sativum L.) the production of superoxide free radicals (O₂⁻) by xanthine and NADH was investigated. In peroxisomal membranes, 100 micromolar NADH induced the production of O₂⁻ radicals. In the soluble fractions of peroxisomes, no generation of O₂⁻ radicals was observed by incubation with either NADH or xanthine, although xanthine oxidase was found located predominantly in the matrix of peroxisomes. The failure of xanthine to induce superoxide generation was probably due to the inability to fully suppress the endogenous Mn-superoxide dismutase activity by inhibitors which were inactive against xanthine oxidase. The generation of superoxide radicals in leaf peroxisomes together with the recently described production of these oxygen radicals in glyoxysomes (LM Sandalio, VM Fernández, FL Rupérez, LA del Río [1988] Plant Physiol 87: 1-4) suggests that O₂⁻ generation could be a common metabolic property of peroxisomes and further supports the existence of active oxygen-related rôles for peroxisomes in cellular metabolism.     

Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria

The effect in vivo of hexavalent chromium (Cr⁶⁺) on the respiratory electron transport activity and production of superoxide (O₂^–) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O₂ (NADH oxidase) and succinate to O₂ (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr⁶⁺ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr⁶⁺. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr⁶⁺, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr⁶⁺ increased the generation of O₂^– radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr⁶⁺ was the physiological impact of the metal‐induced enhanced generation of O₂^– radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr⁶⁺ towards enhanced production of O₂^– radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr⁶⁺. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O₂^– radicals suggest that root mitochondria are an important target of Cr⁶⁺‐induced oxidative stress in pea.     

Effects of Lipopolysaccharides from Gram-Negative Bacteria on the Level of Thiols in Blood Platelets

Lipopolysaccharide (endotoxin, LPS) activates blood platelets and stimulates generation of free radicals in these cells. The mechanism of platelet activation induced by LPS is not known. The aim of the present study was to examine how glutathione (GSH) and other thiol-containing compounds are involved in the oxidative stress in blood platelets caused by LPS. The HPLC technique has been used on the analysis of non-protein thiols from human blood platelets treated with lipopolysaccharides of different Gram-negative bacteria (Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa). Our results show that LPSs caused an increase (about 10%) of the level of reduced glutathione (GSH) and other nonprotein thiols such as cysteine (CSH) and cysteinylglycine (CGSH), whereas the total pool of these compounds was almost unchanged. LPS may react directly with thiols, since after incubation of LPSs with glutathione alone (in reduced form) we observed a distinct decrease of the level of platelet GSH.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Age-related increases in superoxide dismutase activity and thiobarbituric acid-reactive substances: Effect of bio-catalyzer in aged rat brain

This study describes, using electron spin resonance spectrometry/spin trapping technique, the increase superoxide dismutase (SOD) activity in the mitochondrial and cytosolic fraction of the cortex, midbrain, pons-medulla oblongata and cerebellum, and in thiobarbituric acid-reactive substances (TBARS) in the cortex, cerebellum and hippocampus of the aged rats. The results show that corresponding to the increased life span and improved physical conditions observed after peroral long-term treatment with Bio-catalyzer, a commercial natural fermented health food supplement marketed in Japan and in the Philippines and earlier reported to be a hydroxyl radical scavenger with weaker scavenging activity on superoxide radical (O ₂ ^– ), SOD which is involved in the metabolic degradation of O ₂ ^– was further increased, whereas TBARS decreased. These findings suggest that the increased SOD activity in the brain as a defense mechanism against age-related accumulation of reactive oxygen species, in particular superoxide radicals, was enhanced with Biocatalyzer treatment while age-related peroxidation of neuronal membrane, as measured by TBARS, was decreased.     

Free radical scavenging activity of the Japanese herbal medicine Toki-Shakuyaku-San (TJ-23) and its effect on superoxide dismutase activity,lipid peroxides,glutamate, and monoamine metabolites in aged rat brain

The free radical scavenging activity of the Japanese herbal medicine, Toki-Shakuyaku-San (TJ-23; TSUMURA & Co., Tokyo, Japan), was examined using electron spin resonance (ESR) spectrometry. TJ-23 scavenged 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), superoxide (O₂ ⁻), and hydroxyl radicals (·OH) dose-dependently. It also diminished carbon centered radicals (·C) generated by oxidative stress and inhibited thiobarbituric acid-reactive substances (TBARS) formation in mouse cortex homogenate. In addition, the effect of TJ-23 on the concentration of neurotransmitters and TBARS formation, and superoxide dismutase (SOD) activity in the cortex, hippocampus and striatum of the aged rat brain was studied. The concentrations of the metabolites of monoamines, glutamate and glutamine were decreased by 4 weeks of oral administration of TJ-23. The SOD activity of mitochondrial fraction was increased and TBARS formation was significantly suppressed. These results suggest that TJ-23 has an antioxidant action and would have a prophylactic effect against free radical-mediated neurological diseases associated with aging.     

Insulin-induced peroxynitrite production in human platelet-rich plasma

Abstract

Recent data support the possible role of nitric oxide (NO^?) in the development of insulin signalling. The aim of this study was to examine the effect of insulin on NO^? production by platelets. The chemiluminescence of platelet-rich plasma prepared from the blood of healthy volunteers was measured in the presence of luminol. Indirect detection of NO^? by luminol is possible in the form of peroxynitrite produced in the reaction of NO^? with a superoxide free radical. Luminol oxidation induced by hydroxyl free radical and lipid peroxidation was prevented by 150 µmol/l of desferrioxamine mesylate. Insulin, in the range of 0.084–840 nmol/l, induced a concentration-dependent increase in chemiluminescence, which was inhibited both by the competitive antagonist of the NO^? synthase enzyme, Nω-nitro-L-arginine methyl ester (at concentrations of 2.0–4.0 mmol/l, P <0.001), and by the elimination of superoxide free radicals using superoxide dismutase (72–144 IU/ml, P <0.001). In conclusion, we assume that the insulin-induced increase in chemiluminescence of platelet-rich plasma was due to increased production of NO^? and superoxide free radicals forming peroxynitrite. The data are consistent with production of peroxynitrite from human platelets under insulin stimulation.     

Mycotoxin Ochratoxin A-induced cell death and changes in oxidative metabolism of Arabidopsis thaliana

We evaluated the phytotoxicity of mycotoxin ochratoxin A (OTA) from Aspergillus and Penicillium strains on Arabidopsis thaliana. The results demonstrate that the growth of Arabidopsis thaliana on media containing OTA was inhibited significantly. Moreover, OTA induced necrotic lesions in detached leaves, which are reminiscent of hypersensitive response lesions that are activated during plant–pathogen interactions and other abiotic stress factors. From our study, we can see that OTA exposure stimulated a biphasic oxidative burst in the leaves, resulting in the generation of hydrogen peroxide (H₂O₂) and superoxide anion radicals (O₂^·−) and in the concomitant down-regulation of antioxidant enzyme defense responses and up-regulation of lipid peroxidation. These results suggested that OTA damage might result from reactive oxygen species pathways. Our experiments provide a useful model plant system for research on OTA-induced plant cell death.     

Anti-Angiogenesis Activities of Novel Peptide Complexes: Mitochondria-Disruptive 9mer Peptides Conjugated with the Integrin alpha V beta 3-Homing Cyclic RGD Motif

Chattonella is one of the most toxic red tide phytoplankton and causes severe damage to fish farming. Recent studies demonstrated that Chattonella sp. generates superoxide and hydroxyl radicals, which may be responsible for the toxicity of this plankton. However, little is known about the mechanism of the production of oxygen radicals by Chattonella, and the role of oxygen radicals in Chattonella themselves is also unclear. In this study, we found that superoxide dismutase (SOD) and catalase inhibited the growth of Chattonella marina concomitant with their morphological changes. In the presence of these enzymes, the shape of vegetative C. marina cells changed from spindle to round. Furthermore, the generation of oxygen radicals by C. marina depended on the growth phase; the rate of superoxide and hydrogen peroxide generation was the highest during exponentially growing phase and subsequently decreased to one-fifth of the maximal level in the stationary growth phase. These results suggest that oxygen radicals generated by C. marina play an essential role in their own survival, especially in cell division.     

Lipoxygenase-mediated glutathione oxidation and superoxide generation

Soybean lipoxygenase-mediated cooxidation of reduced glutathione (GSH) and concomitant superoxide generation was examined. The oxidation of GSH was dependent on the concentration of linoleic acid (LA), GSH, and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 0.42 mM LA, 2 mM GSH, and 50 pmole of enzyme/mL. The GSH oxidation was linear up to 10 minutes and exhibited a pH optimum of 9.0. The reaction displayed a K_m of 1.49 mM for GSH and V_max of 1.35 ± 0.02 μmoles/min/nmole of enzyme. Besides LA, arachidonic and γ-linolenic acids also supported the lipoxygenase-mediated GSH oxidation. Hydrogen peroxide and 13-hydroperoxylinoleic acid supported GSH cooxidation, but to a very limited extent. Oxidized glutathione (GSSG) was identified as the major product of the reaction based on the depletion of nicotinamide-adenine dinucleotide 3′-phosphate (NADPH) in the presence of glutathione reductase. The GSH oxidation was accompanied by the reduction of ferricytochrome c, which can be completely abolished by superoxide dismutase (SOD), suggesting the generation of superoxide anion radicals. Under optimal conditions, the rate of superoxide generation (measured as the SOD-inhibitable reduction of ferricytochrome c) was 10 ± 1.0 nmole/min/nmole of enzyme. These results clearly suggest that lipoxygenase is capable of oxidizing GSH to GSSG and simultaneously generating superoxide anion radicals, which may contribute to oxidative stress in cells under certain conditions.     

UV‐irradiation provokes generation of superoxide on cell wall polygalacturonic acid

We examined the redox effects of UV irradiation on cell wall isolates from Pisum sativum leaves, and polygalacturonic and galacturonic acid, in the presence of hydrogen peroxide. For this purpose, electron paramagnetic resonance spectroscopy and two spin‐traps (DEPMPO and BMPO), capable of differentiating between various free radicals, were applied. Systems were exposed to UV‐B (maximum emission at 312 nm) and UV‐A (352 nm) for 10 min (6 J m^–2 s^–1). Cell wall isolates exposed to UV in the presence of hydrogen peroxide, produced hydroxyl radical, carbon dioxide radical and superoxide. The production of superoxide was observed for cell wall isolates, polygalacturonic acid (in the presence and in the absence of calcium) and galacturonic acid, and it was diminished upon superoxide dismutase supplementation. The production is at least partially based on the reaction of hydroxyl radicals with (poly)galacturonic acid having carbon dioxide radicals as a products. Acting as a strong reducing agent, carbon dioxide radical reacts with molecular oxygen to produce superoxide. The results presented here shed a new light on: (1) the redox‐modulating role of cell wall; (2) the production of superoxide in the extracellular compartment; (3) the mechanisms involved in translating UV stress into molecular signaling and (4) some other UV‐related phenomena in plants, such as CO₂ emission.     

Effects of <Emphasis Type="SmallCaps">L</Emphasis>-mono Methyl-arginine, <Emphasis Type="Italic">N</Emphasis>-Acetyl-cysteine and Diphenyleniodonium on Free Radical Release in C6 Glial Cells Enriched in Hexacosenoic Acid

Previously, we have shown that C6 glial cells enriched in hexacosenoic acid (HA) incubated with oxidative stressors released higher amounts of nitric oxide (NO) products and superoxide (O^–₂), compared to native C6 cells. In the present study, we examined the effects of pre-treatment with some of free radical release inhibitors. The aim was to determine the origin of the enhanced generation of NO and superoxide, and to test the possibility of preventing it. Pre-treatment with L-mono-methyl-arginine and N-acetyl-cysteine in oxidized low-density lipoprotein (ox-LDL) exposed HA cells, inhibited not only nitrite but also superoxide production suggesting that (O^–₂) anion could partially derive from inducible NO synthase. We also observed that ox-LDL treatment of HA cells reduced the intracellular glutathione levels and activated extracellular signal-related kinases. Since this signalling is related to neurotoxic effect, our data substantiate the role of the free radicals in X-linked adrenoleukodystrophy pathogenesis, as HA cells have been used as an in vitro model for this disease.