The effect of cis-diamminedichloroplatinum, selenite and a conjugate of cisplatin with selenite [(NH3)2Pt(SeO3)] on oxidative stress in blood platelets measured by chemiluminescence method.

The effects of cisplatin, sodium selenite and a conjugate of these compounds [(NH3)2Pt(SeO3)] on the generation of free radicals in blood platelets measured by chemiluminescence method were investigated in vitro. In platelets incubated with cisplatin (20 microM, 5 min) a dose-dependent increase of chemiluminescence was observed (p < 0.05). Contrary to the stimulatory action of cisplatin (20 microM) on the production of free radicals, selenite (1 microM) and its conjugate with cisplatin (20 microM) had only slight effects on the oxidative stress in platelets (p < 0.05). The observed increase of chemiluminescence after cisplatin action correlated with a decrease of platelet free thiols present in reduced glutathione. Pre-treatment of blood platelets with buthionine sulfoximine (50 microM, 1 h, 37 degrees C) leading to a decrease of glutathione reduced the cisplatin-induced generation of free radicals in these cells (p < 0.05).     

Inhibitory effect of resveratrol on free radical generation in blood platelets

Resveratrol (3,4',5-trihydroxystilbene), a compound found in many plants, has been shown to prevent coronary heart diseases and to exert a variety of antiinflammatory and anticancerogenic effects. It is effective in lowering the level of serum lipids and in inhibiting platelet aggregation. We evaluated the effect of trans-resveratrol on the production of free radicals in pig blood platelets and showed that resveratrol inhibited the production of different reactive oxygen species (O2*-, H2O2, singlet oxygen and organic radicals) measured by the luminol-dependent chemiluminescence in resting platelets (P < 0.05). Resveratrol inhibited also the generation of radicals in platelets activated by thrombin (P < 0.05). Treatment of platelets with resveratrol at concentrations of 6.25 and 12.5 microg/ml caused a statistically insignificant increase in the production of O2*- in these cells, as measured by reduction of cytochrome c; however, at higher doses (25, 50 and 100 microg/ml) resveratrol distinctly reduced the generation of O2*- in platelets (P < 0.05). We suggest that free radicals play an important role in the reduced reactivity of blood platelets induced by resveratrol.     

Free radicals as possible mediators of the actions of interferon

Interferons (IFNs), in addition to their antiviral action, have been shown to inhibit cell proliferation, induce differentiation of some tumor cells, activate NK cells and macrophages, and modulate phagocytosis. The exact mechanism(s) by which IFN can bring about these pleiotropic actions is not known. Recent studies, including our own (presented here), showed that IFN can augment free radical generation in the cells. Free radicals can stimulate lymphocytes mitogenically and activate macrophages and NK cells. It is also known that activated machophages and polymorphs produce oxidative metabolites, such as hydrogen peroxide, which is responsible for sterilizing action against microorganisms and cytotoxic activity against tumor cells. Free radicals are also known to inhibit cell division. Since IFN can augment free radical generation, it is suggested that free radicals mediate some of the actions of IFN.     

Effect of extracellularly generated free radicals on the plasma membrane permeability of isolated pancreatic B-cells

Previous experiments on alloxan diabetogenicity suggest that alloxan increases the permeability of B-cell plasma membranes by generation of noxious free radicals. Whether the radicals are generated intra- or extracellularly has however been disputed. To test if extracellularly generated free radicals could decrease trypan blue exclusion of dispersed islet cells, a radical-generating solution of xanthine oxidase/hypoxanthine was employed. The solution increased dye uptake by cells in the cell suspension. Superoxide dismutase and catalase but not scavengers of hydroxyl radicals protected against the increase in dye uptake. Both L- and D-glucose protected the cells from injury. It is concluded that extracellularly generated free radicals induce damage to the plasma membrane of islet cells. The result strengthens the hypothesis of plasma membrane damage by extracellularly generated free radicals as the primary event in alloxan diabetogenicity and may provide a link for explanation of damage caused by islet inflammation in juvenile diabetes.     

Superoxide dismutase activity and malonic dialdehyde level in blood platelets of patients with coronary disease

The investigations aimed at evaluating free superoxide radicals generation and the degree of blood platelets cell membrane lipids peroxidation on the base of superoxide dismutase activity and malonic dialdehyde level in patients with ischaemic heart disease. The obtained results have shown that blood platelets superoxide dismutase activity is markedly lower in patients with ischaemic heart disease than in healthy individuals whereas malonic dialdehyde levels are markedly higher.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

Abstract

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

Insulin-induced peroxynitrite production in human platelet-rich plasma

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 micromol/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. N(omega)-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.     

Role of Oxygen Free Radicals in Respiratory Distress Induced by Arachidonic Acid In the Rat

The purpose of our present study is the possible implication of oxygen free radicals in the respiratory distress induced in rats by intravenous administration of arachidonic acid (20mg/kg). The respiratory frequency was measured and plasma TXB2 concentration was assayed by RIA from blood withdrawn I min after arachidonic acid administration. The substances studied were: SOD, catalase, manifold, DMSO, BHT, imidazole. All the drugs, except imidazole, significantly protect the rats from the respiratory distress induced by arachidonic acid. SOD, catalase, BHT and imidatole inhibit whereas mannitol and DMSO increase the plasma levels of TXB2. We suggest that oxygen free radicals generated in the respiratory burst induced by arachidonic acid are mainly responsible for the consequent respiratory distress.     

Interaction(s) between essential fatty acids, eicosanoids, cytokines, growth factors and free radicals: Relevance to new therapeutic strategies in rheumatoid arthritis and other collagen vascular diseases

Eicosanoids, lymphokines, and free radicals are known to participate in the pathogenesis of inflammation. Tumour necrosis factor (TNF), interleukin-1 and 6 (IL-1 and IL-6) and colony stimulating factor -1 (CSF-1) are secreted mainly by activated macrophages, whereas T-cells secrete IL-2, IL-3, IL-4 and interferon-gamma (IFN-gamma). In addition, activated macrophages and lymphocytes can also produce eicosanoids and free radicals which have potent pro-inflammatory actions. Eicosanoids, lymphokines, and free radicals can modulate the immune response, cell proliferation, stimulate collagenase and proteases secretion and induce bone resorption; events which are known to be associated with various collagen vascular diseases. On the other hand transforming growth factor-beta (TGF-beta) produced by synovial tissue, platelets and lymphocytes can inhibit collagenase production, suppress T-cell and NK-cell proliferation and activation and block free radical generation and seems to be of benefit in rheumatoid arthritis. Drugs such as cyclosporine, 1,25,dihydroxycholecalciferol and pentoxyfylline can block lymphokine and TNF production and thus, may inhibit the inflammatory process. Essential fatty acids, the precursors of eicosanoids, are suppressors of T-cell proliferation, IL-1, IL-2 and TNF production and have been shown to be of benefit in rheumatoid arthritis, systemic lupus erythematosus and glomerulonephritis. Thus, the interactions between essential fatty acids, eicosanoids, lymphokines, TGF-beta and free radicals suggest that new therapeutic strategies can be devised to modify the course of collagen vascular diseases.     

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.     

Are indices of free radical damage related to exercise intensity

The possibility that plasma levels of malonaldehyde (MDA) are altered by exercise has been examined. The presence of MDA has been recognized to reflect peroxidation of lipids resulting from reactions with free radicals. Maximal exercise, eliciting 100% of maximal oxygen consumption (VO2max) resulted in a 26% increase in plasma MDA (P less than 0.005). Short periods of intermittent exercise, the intensity of which was varied, indicated a correlation between lactate and MDA (r2 = 0.51) (p less than 0.001). Blood lactate concentrations increased throughout this exercise regimen. A significant decrease (10.3%) in plasma MDA occurred at 40% VO2max. At 70% VO2max plasma MDA was still below resting values, however the trend to an increase in MDA with exercise intensity was evident. At exhaustion, plasma MDA and lactate were significantly greater than at rest. These results suggest, that exhaustive maximal exercise induces free radical generation while short periods of submaximal exercise (i.e. less than 70% VO2max) may inhibit it and lipid peroxidation.     

巨噬细胞产生NO.和O_2~-自由基的分子机理

建立了用顺磁共振（ＥＳＲ）和化学发光技术测定巨噬细胞产生ＮＯ和氧自由基的方法．捕捉到了巨噬细胞受佛波酯刺激产生的ＮＯ．和Ｏ－２自由基．测定了在不同浓度Ｌ－精氨酸存在时佛波酯刺激后巨噬细胞产生的ＮＯ自由基．研究了巨噬细胞产生的ＮＯ和氧自由基的分子机理．结果表明巨噬细胞不仅产生氧自由基而且产生ＮＯ自由基．ＮＡＤＰＨ氧化酶产生氧自由基的部位位于巨噬细胞膜的外侧．ＮＯ合成酶活化产生ＮＯ自由基比ＮＡＤＰＨ氧化酶活化产生氧自由基晚几分钟．     

DNA damage during glycation of lysine by methylglyoxal: assessment of vitamins in preventing damage

Summary. Amino acids react with methylglyoxal to form advanced glycation end products. This reaction is known to produce free radicals. In this study, cleavage to plasmid DNA was induced by the glycation of lysine with methylglyoxal in the presence of iron(III). This system was found to produce superoxide as well as hydroxyl radicals. The abilities of various vitamins to prevent damage to plasmid DNA were evaluated. Pyridoxal-5-phosphate showed maximum protection, while pyridoxamine showed no protection. The protective abilities could be directly correlated to inhibition of production of hydroxyl and superoxide radicals. Pyridoxal-5-phosphate exhibited low radical scavenging ability as evaluated by its TEAC, but showed maximum protection probably by interfering in free radical production. Pyridoxamine did not inhibit free radical production. Thiamine and thiamine pyrophosphate, both showed protective effects albeit to different extents. Tetrahydrofolic acid showed better antioxidant activity than folic acid but was found to damage DNA by itself probably by superoxide generation.     

Inhibition of protein hydroperoxide formation by protein thiols

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2' azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by beta-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

氧化磷脂与动脉粥样硬化

磷脂是构成生物膜和脂蛋白的重要成分,容易在自由基或非自由基以及酶促条件下发生氧化修饰,形成氧化磷脂(oxidized phospholipids,OxPLs),并进一步产生具有不同生物活性的氧化产物.临床证据表明,OxPLs在动脉粥样硬化(ath-erosclerosis,AS)发展过程中不断生成和转化,并在病变处积累...     

Response of Blood Platelets to Proteus mirabilis Lipopolysaccharide

The effects of the lipopolysaccharide (LPS) of Proteus mirabilis on the production of thiobarbituric acid reactive substances (TBARS) and the generation of superoxide radicals (O₂^?) by pig blood platelets were studied in vitro. The effect of LPS on TBARS formation in platelets was dependent on the concentration of endotoxin. LPS at concentrations above 0.1 μg/10⁸ platelets caused the production of TBARS concomitant with the generation of superoxide radicals. The responses of platelets to LPS suggest that endotoxin, like thrombin (a strong platelets agonist), stimulates an enzymatic cascade of platelet arachidonate via cyclooxygenase and produces thromboxane A₂ (TXA₂) concomitant with malonyldialdehyde (MDA).     

Susceptibility of plasma lipids to peroxidation

Lipid peroxidation is an old and yet novel subject. It induces membrane disturbance and damage and its products are known to induce the generation of various cytokines and cell signaling. In the present work, the susceptibility and specificity of human plasma lipids to oxidation were studied, aiming specifically at elucidating the effects of oxidation milieu and oxidants. Cholesteryl esters (CEs) and phosphatidylcholines (PCs) were more readily oxidized in plasma than in organic solution under similar conditions. The susceptibilities of PC and free cholesterol (FC) relative to CE to free radical-mediated lipid peroxidation induced by peroxyl radicals and peroxynitrite were smaller in plasma than in organic solution. The higher rate of CE oxidation by free radicals than PC may be accounted for by the physical effects as well as higher content of polyunsaturated lipids in CE than PC. On the contrary, PC was more readily oxidized than CE by lipoxygenases. The lipid hydroperoxides were stable in organic solution but reduced to the corresponding hydroxides in plasma, the rate being much faster for PC hydroperoxides than for CE and FC hydroperoxides. It was confirmed that free radical-mediated oxidation gave both cis,trans and trans,trans, racemic, random hydroperoxides, while that by lipoxygenase gave only regio- and stereo-specific cis,trans-hydroperoxide.     

Inhibition of protein hydroperoxide formation by protein thiols

Abstract

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

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.