Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

Free radical scavenging and antioxidant effects of lactate ion: an in vitro study.

Divergent literature data are found concerning the effect of lactate on free radical production during exercise. To clarify this point, we tested the pro- or antioxidant effect of lactate ion in vitro at different concentrations using three methods: 1) electron paramagnetic resonance (EPR) was used to study the scavenging ability of lactate toward the superoxide aion (O(2)(-).) and hydroxyl radical (.OH); 2) linoleic acid micelles were employed to investigate the lipid radical scavenging capacity of lactate; and 3) primary rat hepatocyte culture was used to study the inhibition of membrane lipid peroxidation by lactate. EPR experiments exhibited scavenging activities of lactate toward both O(2)(-). and.OH; lactate was also able to inhibit lipid peroxidation of hepatocyte culture. Both effects of lactate were concentration dependent. However, no inhibition of lipid peroxidation by lactate was observed in the micelle model. These results suggested that lactate ion may prevent lipid peroxidation by scavenging free radicals such as O(2)(-). and.OH but not lipid radicals. Thus lactate ion might be considered as a potential antioxidant agent.     

Degradation of vulcanized and nonvulcanized polyisoprene rubbers by lipid peroxidation catalyzed by oxidative enzymes and transition metals

Despite numerous reports concerning the biodegradation of rubber materials, there has been no report of rubber degradation by fully characterized enzymes. In the present paper, we presented a new method to decompose nonvulcanized and vulcanized polyisoprene rubbers by controlling the free radical chain reactions of lipids using oxidative enzymes, manganese peroxidase (MnP), laccase (Lac), and horseradish peroxidase (HRP). Nonvulcanized synthetic polyisoprene (IR) was degraded by the free radicals from unsaturated fatty acids produced by MnP, HRP, and a combination of Lac/1-hydroxybenzotriazole. In contrast, lipoxygenase caused no apparent degradation. Degradation of IR was also observed in lipid peroxidation initiated by the Fenton reaction (FR) and Mn(III), an oxidation product produced by MnP. Vulcanized polyisoprene rubber sheets were degraded by the lipid peroxidation initiated by HRP, MnP, Mn(III), and FR. Pyrolysis GC-MS analysis demonstrated that the lipid peroxidation liberated isoprenoid fragments from the vulcanized rubbers.     

Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max)

Inhibition of root elongation and modification of membrane properties are sensitive responses of plants to aluminium. The present paper reports on the effect of AI on lipid peroxidation and activities of enzymes related to production of activated oxygen species. Soybean seedlings (Glycine max L. cv. Sito) were precultured in solution culture for 3–5 days and then treated for 1–72 h with Al (AICI₃) concentrations ranging from 10 to 75 μM at a constant pH of 4.1. In response to Al supply, lipid peroxidation in the root tips (< 2 cm) was enhanced only after longer durations of treatment. Aluminium-dependent increase in lipid peroxidation was intensified by Fe²⁺ (FeSO₄). A close relationship existed between lipid peroxidation and inhibition of root-elongation rate induced by Al and/or Fe toxicity and/or Ca deficiency. Besides enhancement of lipid peroxidation in the crude extracts of root tips due to Al, the activities of superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) increased, whereas catalase (EC 1.11.1.6) activity decreased. This indicates a greater generation of oxygen free radicals and related tissue damage. The results suggest that lipid peroxidation is part of the overall expression of Al toxicity in roots and that enhanced lipid peroxidation by oxygen free radicals is a consequence of primary effects of Al on membrane structure.     

维生素C对黄瓜叶绿体膜脂过氧化的影响

研究了外源维生素对黄瓜叶绿体膜脂过氧化的影响。结果表明,黄瓜叶绿体内过量的维生素Ｃ导致了叶绿体膜的伤害,加剧了叶绿体膜脂过氧化程度,其原因可能是外源维生素Ｃ加快了叶绿体的电子传递速率,从而造成超氧物阴离子自由基的产生速率升高。     

The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membrane-lipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between alpha-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with alpha-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, alpha-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.     

Polymorphonuclear leukocytes-induced injury in hypoxic cardiac myocytes

Growing evidence suggests that free radicals derived from polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia-reperfusion injury. To elucidate the cellular mechanism by which activated PMNs exacerbate ischemic myocardial damage, we investigated the extent of cell injury, assessed by the morphological deterioration, free radical generation, and lipid peroxidation in mouse embryo myocardial cells coincubated with activated PMNs. The generation of PMN-derived free radicals was related to the extent of myocardial cell injury. When myocardial cell sheets were subjected to hypoxia and glucose-free media, myocardial cells were injured (cristalysis in the mitochondria and disruption of the sarcolemma) after adding various PMN activators, and the injury extended to the adjacent cells. Chemiluminescent emission and production of thiobarbituric acid-reactive substances in the coincubated cells increased markedly compared with myocardial cells or PMNs alone. The augmented lipid peroxidation coincided with the progression of myocardial cell injury. Catalase inhibited the myocardial cell injury by 52%, the chemiluminescence by 46%, and lipid peroxidation by 50%, whereas superoxide dismutase exhibited less pronounced inhibition. These results indicate that a chain reaction of lipid peroxidation in myocardial cells induced by PMN-derived free radicals closely correlates with membrane damage and contributes to the propagation of irreversible myocardial cell damage.     

Contribution of haemoglobin and membrane constituents modification to human erythrocyte damage promoted by peroxyl radicals of different charge and hydrophobicity

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2'-Azobis(2-amidinopropane) (AAPH) and 4,4'-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, as evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively.

AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin.

Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.     

Free radicals and their biological significance: present and future]

Free radicals are usually active species which have unpair electron (S) in molecules or Atomic groups. Of the free radicals, O2- and .OH could easily be produced in mammalian cells, by oxidation and reduction cycle catalyzed by fravoproteins and by iron + H2O2 reaction, respectively. Other free radicals would also be produced in mammalian cell, such as amino acid radicals, semiquinone radicals and flavine radicals etc. In general, free radicals cause cell injury though membrane lipid peroxidation and DNA strand cleavage and some other mechanisms.     

Role of glutathione S-transferases in oxidative stress-induced male germ cell apoptosis

Cellular apoptosis in a tissue may occur for the maintenance of proper ratio of cells or because of toxic effects of free radicals or other agents. Male germ cell apoptosis is pivotal in maintaining the proper functioning of the testis, but it is not clear how free radicals affect germ cells and what the defense mechanisms are that are used by these cells to combat the toxic effects of the products of oxidative stress. This study shows that male germ cells are susceptible to H(2)O(2)-induced stress and, upon exposure to H(2)O(2) in vitro, demonstrate a typical apoptotic phenotype that includes DNA fragmentation and formation of DNA ladders. Other changes include considerable accumulation of products of lipid peroxidation in the germ cells after exposure to H(2)O(2). Evidence is presented for the existence of multiple isoforms of glutathione S-transferases (GSTs) that possess both transferase and Se-independent peroxidase activity. Germ cell GST activity increases after H(2)O(2) exposure. If this increase in activity is inhibited with suitable inhibitors, the formation of products of lipid peroxidation is augmented, resulting in germ cell apoptosis. Also, when constitutive GST activity is inhibited, accumulation of products of lipid peroxidation occurs, resulting in increased cellular apoptosis. These data show that GSTs form a part of adaptive response of germ cells to oxidative stress and are important constituents in detoxifying the products of lipid peroxidation.     

Contribution of haemoglobin and membrane constituents modification to human erythrocyte damage promoted by peroxyl radicals of different charge and hydrophobicity

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2′-Azobis(2-amidinopropane) (AAPH) and 4,4′-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, as evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively.

AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin.

Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.     

Oxidative Processes Induced by <Emphasis Type="Italic">tert</Emphasis>-Butyl Hydroperoxide in Human Red Blood Cells: Chemiluminescence Studies

The erythrocyte is a good model for investigation of the mechanisms of cell damage induced by oxidizing agents. Oxidative damage to cell components and cellular metabolism results in impaired rheological properties of circulating red blood cells and is involved in the development of some pathologies. The aim of the present study was to elucidate further the oxidative processes induced by tert-butyl hydroperoxide (tBOOH) in erythrocytes, identify cellular targets damaged by the oxidant, as well as estimate the energy and stoichiometry of the reactions that occur. The generation of free radicals in the cell was registered using the chemiluminescence technique. The products of oxyhemoglobin (oxyHb) oxidation, changes in intracellular glutathione (GSH) pool, and accumulation of the stable products of membrane lipid peroxidation were concurrently measured. The oxidative processes induced by tBOOH in red blood cells can be described as follows: 1) rapid GSH oxidation (30-60 sec) by glutathione peroxidase; 2) formation of radicals in the reaction between tBOOH and cellular Hb, which are then immediately consumed in lipid peroxidation reactions; 3) generation of chemiluminescence by the radicals formed. Several stages of the oxidative processes can be revealed. The order of the chemiluminescence reaction (n) with respect to oxidant was estimated to be equal to 2.5 at oxidant concentrations less than 0.5 mM and equal to 1.0 at higher oxidant concentrations. The order of the reaction of membrane lipid peroxidation was found to be n = 2.2 at 0.25-0.6 mM tBOOH and n = 0.5 at higher oxidant concentrations. The apparent activation energy of membrane lipid peroxidation was 55.8 +/- 6.4 kJ/mol, and that of oxyHb oxidation was 108 +/- 16 kJ/mol. It is shown that the interaction of tBOOH and HOCl in erythrocytes is accompanied by changes in both the total number of radicals generated in the cell and the time corresponding to the maximal rate of radical generation.     

Protective effects of lazaroids against oxygen-free radicals induced lysosomal damage

Lipid peroxidation of membranes by oxygen free radicals has been implicated in various disease states. Different antioxidants and iron chelators have been used to reduce lipid peroxidation. Lazaroids have been used for the acute treatment of central nervous system disorders such as trauma and ischemia wherein lipid peroxidative processes take place.In this study we evaluated the effect of lazaroids (U-785 18F and U-74389F) on the release of acid phosphatase activity and formation of malondialdehyde (MDA) in rat liver lyosomes subjected to exogenously generated oxygen free radicals. There was a significant increase in the acid phosphatase release and MDA formation in the presence of oxygen free radicals. This was prevented by both the lazaroids. In a separate study the effect of lazaroid U-74389F was seen on the zymosan-stimulated polymorphonuclear (PMN) leukocyte-derived chemiluminescence. The PMN leukocyte chemiluminescent activity was attenuated by the lazaroid in a dose-dependent manner. These studies suggest that lazaroids may inhibit lipid peroxidation and stabilize the membrane.     

Lipid Free Radical Generation and Brain Cell Membrane Alteration Following Nitric Oxide Synthase Inhibition During Cerebral Hypoxia in the Newborn Piglet

Abstract: Nitric oxide (NO) is reported to cause neuronal damage through various mechanisms. The present study tests the hypothesis that NO synthase inhibition by N ^ω-nitro- l -arginine (NNLA) will result in decreased oxygen-derived free radical production leading to the preservation of cell membrane structure and function during cerebral hypoxia. Ten newborn piglets were pretreated with NNLA (40 mg/kg); five were subjected to hypoxia, whereas the other five were maintained with normoxia. An additional 10 piglets without NNLA treatment underwent the same conditions. Hypoxia was induced with a lowered FiO₂ and documented biochemically by decreased cerebral ATP and phosphocreatine levels. Free radicals were detected by using electron spin resonance spectroscopy with a spin trapping technique. Results demonstrated that free radicals, corresponding to alkoxyl radicals, were induced by hypoxia but were inhibited by pretreatment with NNLA before inducing hypoxia. NNLA also inhibited hypoxia-induced generation of conjugated dienes, products of lipid peroxidation. Na⁺,K⁺-ATPase activity, an index of cellular membrane function, decreased following hypoxia but was preserved by pretreatment with NNLA. These data demonstrate that during hypoxia NO generates free radicals via peroxynitrite production, presumably causing lipid peroxidation and membrane dysfunction. These results suggest that NO is a potentially limiting factor in the peroxynitrite-mediated lipid peroxidation resulting in membrane injury.     

水分胁迫对甘蔗叶片线粒体膜流动性的影响及其与膜脂过氧化的关系

用荧光探剂ANS对抗旱性不同的甘蔗品种在水分胁迫下叶片线粒体膜流动性的变化进行的研究表明,水分胁迫降低了线粒体膜的流动性,抗旱性强的甘蔗品种Co 617和F.Y.79-9的下降幅度分别小于抗旱性弱的Co 740和M.T.77-208;水分胁迫下线粒体膜流动性的下降与膜脂过氧化产物丙二醛含量的增加有密切关系。外源自由基处理试验也表明,甘蔗叶片线粒体膜流动性的下降与膜脂过氧化作用有关。     

Lipid peroxidation by peroxidase-catalyzed bioactivation of tyrosine

Tyrosyl free radicals generated by the peroxidase-catalyzed oxidation of peptide tyrosyl residues are known to yield the stable cross-linked product dityrosine. In the present report, horseradish peroxidase is used as a model of peroxidase to study oxidative modifications of non-protein cellular components. Tyrosyl free radicals promote, as many free radicals, the decay of β-phycoerythrin fluorescence emission, they oxidize NADH and ascorbic acid and initiate arachidonic acid peroxidation with formation of hydroperoxides and dienes. These results suggest that tyrosyl free radicals generated when tyrosine residues in protein and peptides are activated in vivo by peroxidase-H₂O₂ might undergo the peroxidation of membrane lipids.     

Evidence that free radical generation occurs during scorpion envenomation

Although it is well established that symptomatology, morbidity and death following scorpion envenomation are due to increases in neurotransmitter release secondary to toxins binding to voltage-sensitive sodium channels, the mechanism by which venom action is involved in damaging heart, liver, lungs and kidneys remains unclear. We hypothesized that scorpion toxins could induce the generation of high levels of free radicals responsible for membrane damage in organs targeted by venom action. We have investigated lipid peroxidation in different organs, through the evaluation of thiobarbituric acid reactive substances (TBARS), after experimental envenomation of rats by toxic fractions of Androctonus australis Hector venom. We have shown that scorpion toxins cause considerable lipid peroxidation in most vital organs. We also evaluated the protective effects of antioxidants in mice injected with lethal doses of toxins. Among the drugs tested, N-acetylcysteine (NAC) was effective in protecting the mice when injected prior to toxin application. However, the free radical scavenging properties of NAC seem less implicated in these protective effects than its ability to increase the fluidity of bronchial secretions. We therefore conclude that free radical generation only plays a minor role in the toxicity of scorpion venom.     

Effect of Al(III) on surface properties of Bifidobacterium thermophilum as a function of temperature

The effects of Al(III) on surface properties and lactate accumulation by Bifidobacterium thermophilum were investigated. Bacteria were treated with Al(III) at 37°C and 4°C, then exposed to free radicals or nisin. When exposed to Al(III) at 37°C, the organism exhibited spreading on hydrophobic surfaces and showed high susceptibility to free-radical alteration as indicated by Fe(III) binding, but showed little effect on lactate production in the presence or absence of nisin, even after washing with 2 mM EDTA. At 4°C, there was no increased surface spreading or binding of Fe(III), but protection against nisin action was present. This, however, was abolished after washing with EDTA. It was concluded that membrane fluidity is required to affect membrane lipid rearrangement, resulting in surface spreading and increased susceptibility to peroxidation, whereas only loose binding of Al(III) to membrane surfaces is sufficient to prevent transmembrane channel formation by nisin.     

丹参酮对心肌肌质网脂质过氧化过程中脂类自由基的清除作用

应用自旋捕集方法和化学发光方法研究天然抗氧化剂丹参酮（Ｔａｎｓｈｉｎｏｎｅ）对心肌肌质网膜脂质过氧化过程中产生的脂类自由基的清除作用。发现在一定的浓度范围内,丹参酮对脂质过氧化有较好的保护作用,在丹参酮浓度大于１ｍｇ／ｍｇ蛋白时,对脂类自由基清除率可达６０％。丹参酮对肌质网膜脂质过氧化的保护机理可能是通过清除脂类自由基而阻断脂质过氧化的链式反应,而不是清除氧自由基而防止脂质过氧化的启动。     

Lipid peroxidation as a possible cause of TCDD toxicity

The target tissues of TCDD, the dysfunctions that result in death in experimental animals, and the ultimate biochemical lesion(s) caused by TCDD are not known despite numerous studies. We have shown by the thiobarbituric acid and conjugated diene methods that TCDD induces hepatic lipid peroxidation in rats. The lipid peroxidation produced by TCDD is both dose and time dependent. A 5-6 fold increase in lipid peroxidation occurs within 6 days following the administration of 40 micrograms TCDD/kg body weight/day for 3 days. Thus, the toxicity of TCDD may be caused in part by free radical-mediated lipid peroxidation that leads to general cell membrane damage which can ultimately produce death in experimental animals at acutely toxic doses.