Inhibition of oxidative hemolysis by quercetin,but not other antioxidants

We previously reported that lipid-soluble quercetin, not water-soluble dihydroquercetin, protects human red blood cells against oxidative damage. The objectives of this study were to determine if an antihemolytic effect could be produced by other lipid-soluble antioxidants and if anti-inflammatory activity played a role in antihemolysis by quercetin. This study compared three lipid-soluble polyphenols, muscadine, curcumin and quercetin, and three lipid- (α-tocopherol and α-tocotrienol) or water-soluble (ascorbic acid) vitamins. Among the tested polyphenols, muscadine was the most potent in inhibiting superoxide and 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH)-generated peroxyl radicals, whereas ascorbic acid was the most potent inhibitor of hydrogen peroxide. Activities of the polyphenols after lipid extractions showed that curcumin inhibited superoxide production to a greater extent than quercetin and muscadine. All blood cells were tested 20 min after incubation with the selected compounds. All the polyphenols caused inhibition of N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced neutrophil oxidative bursts. Quercetin, but not other polyphenols, significantly reduced AAPH-induced oxidative hemolysis. No significant effect on neutrophil oxidative burst or oxidative hemolysis was found with any of the tested vitamins. These results suggest that quercetin enhances the resistance of membrane to destruction by free radicals. This effect of quercetin is not directly mediated through antioxidative or anti-inflammatory actions. Antioxidant or anti-inflammatory potency may not be used as a simple criterion to select polyphenols for cell protection benefits.     

Factors influencing variable oxidative hemolysis of inbred mouse erythrocytes

The hemolysis of erythrocytes from certain inbred mouse strains (e.g., BALB/c) in response to hydrogen peroxide stress has been shown to be correlated with the type of hemoglobin beta chain (Kruckeberg, W.C., et al. (1987) Blood 70, 909-914). The characteristic hemolytic response of BALB/c red cells to oxidative stress resembles that of human red cells in that carbon monoxide and iron chelators inhibit hemolysis of both. Gross hemoglobin oxidation rates were similar in hemolytic (BALB/c) and nonhemolytic (C57BL/6) strains. The rate and degree of in vitro catalase inhibition by sodium azide was also the same for the two strains. Even in the presence of this catalase inhibitor the assayable hydrogen peroxide disappeared within seconds of its addition, yet hemolysis was not observed for about 15 min. The mechanism underlying this delay between hydrogen peroxide addition and disappearance and subsequent hemolysis is under investigation.     

Effect of mitochondria-targeted antioxidant SkQ1 on programmed cell death induced by viral proteins in tobacco plants

Programmed cell death (PCD) is the main defense mechanism in plants to fight various pathogens including viruses. The best-studied example of virus-induced PCD in plants is Tobacco mosaic virus (TMV)-elicited hypersensitive response in tobacco plants containing the N resistance gene. It was previously reported that the animal mitochondrial protein Bcl-xL, which lacks a homolog in plants, effectively suppresses plant PCD induced by TMV p50 — the elicitor of hyper-sensitive response in Nicotiana tabacum carrying the N gene. Our studies show that the mitochondria-targeted antioxidant SkQ1 effectively suppresses p50-induced PCD in tobacco plants. On the other hand, SkQ1 did not affect Poa semilatent virus TGB3-induced endoplasmic reticulum stress, which is followed by PCD, in Nicotiana benthamiana epidermal cells. These data suggest that mitochondria-targeted antioxidant SkQ1 can be used to study molecular mechanisms of PCD suppression in plants.     

Brain aging and mitochondria-targeted plastoquinone antioxidants of SkQ-type

Normal brain aging leads to decrease in cognitive functions, shrink in brain volume, loss of nerve fibers and degenerating myelin, reduction in length and branching of dendrites, partial loss of synapses, and reduction in expression of genes that play central roles in synaptic plasticity, vesicular transport, and mitochondrial functioning. Impaired mitochondrial functions and mitochondrial reactive oxygen species can contribute to the damage of these genes in aging cerebral cortex. This review discusses the possibility of using mitochondria-targeted antioxidants to slow the processes of brain aging.     

Prevention by antioxidants of the hemolysis of erythrocytes of cattle, pigs and humans treated with t-butyl hydroperoxide

Urate, 3-ribosylurate, ascorbate, glutathione and plasma protected bovine, porcine and human erythrocytes from hemolysis caused by t-butyl hydroperoxide (t-BHP). Urate partially protected porcine erythrocytes from hemolysis by t-BHP when it was added 15 min after the addition of the t-BHP, but it did not protect when added 30 min after the t-BHP. Glutathione and ascorbate protected oxyhemoglobin from oxidation to methemoglobin by t-BHP; 3-ribosylurate gave only slight protection. Urate stimulated the formation of methemoglobin from oxyhemoglobin during treatment with t-BHP.     

Water-soluble polyol-methanofullerenes as mitochondria-targeted antioxidants: Mechanism of action

The mechanism of an antioxidant action of water-soluble polyol – methanofullerenes C₆₀[C₉H₁₀O₄(OH)₄]₆ and C₆₀[C₁₃H₁₈O₄(OH)₄]₆ as the mild uncouplers of an oxidative phosphorylation and respiration is postulated. According to this mechanism, hydroxyl group of methanofullerenols can be protonated under excess of protons in the intermembrane space of hyperpolarized mitochondria. Protonation of fullerene derivatives is confirmed by the decrease in their negative Zeta potential in the pH below 5.4. Heavily protonated methanofullerenols become positively charged and move into the mitochondrial matrix. As a consequence, the proton gradient is dissipated, which causes a decrease in mitochondrial transmembrane potential (ΔΨ_m) and reduction in ROS production.     

Hypoosmotic hemolysis of erythrocytes by active carbonyl forms

Low-molecular-weight dicarbonyls formed during free radical peroxidation of polyene lipids (malondialdehyde) and autooxidation (glyoxal) or other oxidative transformations of glucose (methylglyoxal) are able to modify the structure of lipid-protein supramolecular complexes of cells. We investigated changes in the erythrocyte membrane structure after an 18-h exposure of human red blood cells in the presence of various natural dicarbonyls. The changes in the mechanical properties of the membrane after membrane modification by carbonyls were evaluated by the susceptibility of erythrocytes to hypoosmotic hemolysis. It has been shown that treatment of red blood cells with malondialdehyde increases the resistance of these cells to hypoosmotic hemolysis, whereas the malondialdehyde isomer, methylglyoxal, in contrast, makes red blood cells more sensitive to the action of hypoosmotic solutions. Paradoxically, a homologue of malondialdehyde, glyoxal, has no effect on hemolysis of red blood cells in hypoosmotic solutions. The findings point to the possibility of the multidirectional effect of low-molecular-weight dicarbonyls with similar structures on the structure and function of biological membranes.     

The effect of peroxynitrite and some antioxidants on the rate of osmotic hemolysis of bovine erythrocytes

Bovine erythrocytes treated with peroxynitrite (ONOO(-)), a cytotoxic species formed in vivo via the reaction of nitric oxide (NO(.)) and the superoxide anion (O2(-.)), show an increased rate of hemolysis on sudden osmotic stress. The increase in the rate was peroxynitrite concentration dependent. In the presence of some antioxidants (uric acid, ascorbic acid, glutathione, melatonin and albumin), this effect was significantly lower, with ascorbic acid as the most efficient antioxidant.     

Supplementation of endothelial cells with mitochondria-targeted antioxidants inhibit peroxide-induced mitochondrial iron uptake, oxidative damage, and apoptosis

The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H(2)O(2) and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 microM) and MitoVit-E (1 microM) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H(2)O(2)- and lipid peroxide-induced 2',7'-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis.     

Effect of the mitochondria-targeted antioxidant SkQ1 on development of spontaneous tumors in BALB/c mice

The mitochondria-targeted antioxidant SkQ1 (10-(6′-plastoquinonyldecyl)triphenylphosphonium) is a new pharmaceutical substance with a wide spectrum of effects including increase in lifespan of laboratory animals (for example, of BALB/c mice males) and inhibition of development of some experimental tumors and also of tumor cell growth. In this work, the effects of SkQ1 on development of spontaneous tumors in female and male BALB/c mice housed in an SPF-class vivarium were studied. We found that the addition of SkQ1 to drinking water at the dose of 1 and 30 nmol/kg body weight per day throughout the lifespan modified the spectrum of spontaneous tumors in the female mice, decreasing the incidence of follicular lymphomas. SkQ1 at the dose of 1 nmol/kg per day also suppressed the dissemination of these neoplasms, but it did not significantly influence the overall incidence of benign and malignant tumors (including primary multiple tumors) or the lifespan of the tumor-bearing mice (both males and females). Hence, the previously described ability of SkQ1 to increase the lifespan of laboratory BALB/c mice is not related to its anticarcinogenic activity.     

Inhibition of thrombocyte aggregation by antioxidants

The effects of antioxidants (3-hydroxypyridines, 5-hydroxypyrimidines, hindered phenols) on platelet aggregation were studied. All the compounds under study possessed low anti-aggregation activity against indometacin-sensitive aggregation (activation with arachidonic acid, 50 M). Half-maximal inhibition of aggregation was achieved at a concentration similar to that of the compounds used (10(-3) M in cases of indomethacin-insensitive aggregation, platelet activation by thrombine 1.5 mu/ml and Ca2+-ionophore A23187 1.5 g/ml). 4-methyl-2.6-ditretbutyl phenol (BHT) in the concentration range of 10(-5)-4 X 10(-5) M inhibited and in the concentration range of 4 X 10(-5)-10(-4) M activated indomethacin-sensitive aggregation. The latter effect was not observed in the absence of Ca2+ ions in the incubation medium. It is concluded that the effects of the antioxidants studied on platelet aggregation were due to their non-specific action on platelet membranes.     

Studies on hemolysis of human erythrocytes by linoleic acid

The results presented in this paper show that lysis of human erythrocytes by linoleic acid is not caused by peroxidation of the fatty acid. Peroxidase, superoxide dismutase and scavengers of O ₂ ⁻ and OH had no effect on the lysis while catalase showed only marginal inhibition suggesting that O ₂ ⁻ , OH, O ₂ ⁻ and H₂O₂ do not play any direct role in hemolysis by linoleic acid. Generators of H₂O₂ inhibited the lysis completely and methemoglobin cells were more resistant to hemolysis by linoleic acid. The fatty acid did neither bind to nor fomed complex with red cell ghosts. Membrane oxidation of sulphydryl groups was also not involved in the lysis. Β-Carotene, retinol and bile salts enhanced the lysis, while, cholesterol but not cholesterol acetate, inhibited it. Taurocholate-pretreated cells were more susceptible to linoleic acid lysis. These observations suggested-that lysis by linoleic acid may be due to its detergent property.     

Induction and decay of thermotolerance in human erythrocytes determined by hemolysis

Hemolysis was used as an endpoint for the measurement of damage to the plasma membrane in human erythrocytes after a single or a double heat shock. The thermotolerance of erythrocytes is a transitional phenomenon, reaching its maximum at a 3-hour incubation at 37 degrees C between the heat shocks.     

Alpha-tocopherol protection of erythrocytes from hemolysis induced by thermal injury

It is shown that skin burn is accompanied by activation of lipid peroxidation (accumulation of TBA-reactive substances and of fluorescent end-products) in the blood of experimental animals. The decrease in red blood cell membrane stability was demonstrated exerting as increase in the rate of spontaneous hemolysis, content of extraerythrocyte++ haemoglobin and increased sensitivity to exogenous oleic acid. It is estimated that alpha-tocopherol possesses protective stabilizing effect on red blood cell membrane. This stabilizing action is observed when alpha-tocopherol was injected both before the skin burn and immediately after it. It is concluded that two different mechanisms are responsible for stabilizing effect of tocopherol, namely: 1) antiradical, realized via inhibition of lipid peroxidation, and 2) non-antioxidant, caused by interaction of tocopherol with phospholipid hydrolysis products by phospholipases A2 (free fatty acids and lysophospholipids).     

Suicidal oxidative stress induced by certain antioxidants

Well known antioxidants-coumarins (7,8-dihydroxy-4-methyl coumarin-DHMC and 7,8-diacetoxy-4-methyl coumarin-DAMC) and flavonoids (quercetin-Q and quercetin penta-acetate-QPA) were investigated for their pro-oxidant effects in two human tumor cell lines. The breast carcinoma cell line (MDA-MB-468) was found to be more sensitive to treatment by the drugs-DAMC, Q and QPA at 10 microM than the glioma cell line (U-87MG), while DHMC was non toxic in both cell lines at this concentration. In MDA-MB-468 distinct growth inhibition was observed by 48 hr post treatment. Paradoxically, an increase in the formazan production was revealed by MTT assay at this time indicating an increase in the production of free radicals. An increase in the levels of reactive oxygen species (ROS) was also confirmed by DCFH-DA assay. In cells treated with DAMC, Q and QPA an increase in the percentage of cells with the hypodiploid DNA content was suggestive of apoptotic cell death. Taken together, these results suggest that an increase in oxidative stress caused by the pro-oxidant action of these drugs is responsible for cell death.