The effect of chronic alcohol feeding on lipid peroxidation in microsomes: lack of relationship to hydroxyl radical generation

Chronic alcohol feeding causes microsomal induction including increased generation of hydroxyl radicals. Ethanol induced liver injury may be mediated by lipid peroxidation for which hydroxyl radicals have been proposed as major mediators. Ethanol promotes lipid peroxidation when given acutely but also may serve as a hydroxyl radical scavenger. Therefore, we studied the acute and chronic effects of alcohol on microsomal lipid peroxidation and hydroxyl radical generation. Chronic alcohol feeding in rats increased microsomal generation of hydroxyl radicals but lipid peroxidation of endogenous lipid was inversely related to hydroxyl radical generation. Ethanol (50mM) had a slight inhibitory effect on hydroxyl radical production in peroxidizing microsomes, no effect on endogenous lipid peroxidation and enhanced the lysis of RBCs added as targets of peroxidation. Enhanced microsomal generation of hydroxyl radicals following chronic alcohol feeding is not an important mediator of lipid peroxidation.     

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.     

Temporal relationship of free radical-induced lipid peroxidation and loss of latent enzyme activity in highly enriched hepatic lysosomes

Loss of latency due to membrane lipid peroxidation induced in vitro was studied in highly purified rat liver lysosomes. Enriched fractions of lysosomes were isolated by free flow electrophoresis. Lipid peroxidation of lysosomes, assayed as malondialdehyde formation, was catalyzed by a radical generating system consisting of dihydroxyfumaric acid and Fe3+-ADP. The peroxidation reaction occurred readily at 37 degrees C and reached a plateau at 10 min; however, the loss of lysosomal latency, determined as increased percentage free beta-N-acetylglucosaminidase activity, occurred more gradually and reached a maximum after 30 min. Scavengers of superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radicals did not inhibit the peroxidation reaction nor prevent the loss of lysosomal latency. However, preincubation of the lysosomes with alpha-tocopherol effectively blocked the induction of peroxidation and substantially reduced the loss of lysosomal latency. These results indicate that the lysosomal membrane is susceptible to free radical-induced lipid peroxidation; further, this process may be the immediate cause of the subsequent disintegration of the lysosome. The nature of the protective effect of alpha-tocopherol is unclear but may be due to its interaction with the unsaturated membrane lipids and the subsequent interruption of the chain-reaction initiated by free radicals.     

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.     

Peroxidation of proteins and lipids in suspensions of liposomes, in blood serum, and in mouse myeloma cells

There is growing evidence that proteins are early targets of reactive oxygen species, and that the altered proteins can in turn damage other biomolecules. In this study, we measured the effects of proteins on the oxidation of liposome phospholipid membranes, and the formation of protein hydroperoxides in serum and in cultured cells exposed to radiation-generated hydroxyl free radicals. Lysozyme, which did not affect liposome stability, gave 50% protection when present at 0.3 mg/ml, and virtually completely prevented lipid oxidation at 10 mg/ml. When human blood serum was irradiated, lipids were oxidized only after the destruction of ascorbate. In contrast, peroxidation of proteins proceeded immediately. Protein hydroperoxides were also generated without a lag period in hybrid mouse myeloma cells, while at the same time no lipid peroxides formed. These results are consistent with the theory that, under physiological conditions, lipid membranes are likely to be effectively protected from randomly-generated hydroxyl radicals by proteins, and that protein peroxyl radicals and hydroperoxides may constitute an important hazard to biological systems under oxidative stress.     

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 insult to human red blood cells induced by free radical initiator AAPH and its inhibition by a commercial antioxidant mixture.

This study was carried out to investigate sequel of oxidative insult to human erythrocytes induced by a water-soluble radical initiator, 2,2'-azobis-(amidinopropane) dihydrochloride (AAPH) and the effect of a commercially available mixed antioxidant (Blackmores, BioAce Excel), containing alpha-tocopherol, ascorbic acid, beta-carotene and some herbal extracts (containing grape seed catechins and milk thistle derived silybin), on lipid peroxidation, degradation of membrane proteins and haemolysis. We performed this study in order firstly to clarify aspects of the mechanism of AAPH induced free radical damage in human erythrocytes and secondly to establish in vitro conditions by which the efficacy of mixed antioxidant preparations may fairly and objectively be compared. In the process of oxidation initiated by peroxyl radical, a rapid loss of reduced glutathione occurred in the first 60 min. Formation of thiobarbitric acid-reactive substances indicative of lipid peroxidation increased subsequently and almost reached maximal levels at 180 min before significant apparent degradation of membrane proteins was detected. At this point, a significant haemolysis occurred. This sequence of events is consistent with the idea that haemolysis is a consequence of lipid peroxidation and the degradation of membrane proteins. The mixed commercial antioxidant, which suppressed lipid peroxidation and protected membrane proteins against degradation induced by peroxyl radicals, also effectively delayed AAPH induced haemolysis. The system we describe provides a sound objective basis for the in vitro comparison of the potential efficacy of the hundreds of antioxidant nutritional supplements currently available in the market place.     

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.     

Drought acclimation reduces O2*- accumulation and lipid peroxidation in wheat seedlings

Abiotic stresses cause ROS accumulation, which is detrimental to plant growth. It is well known that acclimation of plants under mild or sub-lethal stress condition leads to development of resistance in plants to severe or lethal stress condition. The generation of ROS and subsequent oxidative damage during drought stress is well documented in the crop plants. However, the effect of drought acclimation treatment on ROS accumulation and lipid peroxidation has not been examined so far. In this study, the effect of water stress acclimation treatment on superoxide radical (O(2)(-z.rad;)) accumulation and membrane lipid peroxidation was studied in leaves and roots of wheat (Triticum aestivum) cv. C306. EPR quantification of superoxide radicals revealed that drought acclimation treatment led to 2-fold increase in superoxide radical accumulation in leaf and roots with no apparent membrane damage. However under subsequent severe water stress condition, the leaf and roots of non-acclimated plants accumulated significantly higher amount of superoxide radicals and showed higher membrane damage than that of acclimated plants. Thus, acclimation-induced restriction of superoxide radical accumulation is one of the cellular processes that confers enhanced water stress tolerance to the acclimated wheat seedlings.     

Generation of Hydroxyl Radicals and Its Relation to Cellular Oxidative Damage in Plants Subjected to Water Stress

The hydroxyl radical ('OH) is one of the roost reactive mdieales known to chemistry and is believed to be a major active free radicle responsible for modifications of macmmolecules and cellular damage. Two lines of evidence strongly indicate that 'OH radicals are generated in a Fenton-type Haber-Weiss reactions in plants subjected to water stress. Firstly, water stress causes an increase in the concentration of catalytic metals, which are critical for Fenton-like reactions to proceed in vivo. Furthermore, subrmillimolar concentrations of H2O2 and ascorbic acid(or O2－ ) in the drought-stressed plants are large enough to support the Fentontype Haber-Weiss reactions. Secondly, there is oxidation of proteins and lipids in the drought-stressed plants; a process that requires a catalytic metal and that, at least for protein oxidation, is mediated by the 'OH radicals. Protein oxidation is thought to involve binding of metal ions to the proteins and subsequent site-specific attack by the 'OH radicals arising from the roetal-catalysed decomposition of H2O2. It has been proposed that protein oxidation may be a better index than lipid peroxidation because the latter fields many different products and these only appear after a lag period. The validity of malondialdehyde (MDA), an early product of lipid peroxidation, as an index of lipid peroxidation has been argued by the non-specific method of its measurement. The 'OH radicals are not the only necessary initiator for lipid peroxidation and lipid peroxidation is not usually involved in plants exposed to water stress.     

Quinone formation from carcinogenic benzo[a]pyrene mediated by lipid peroxidation in phosphatidylcholine liposomes

The behavior of benzo[a]pyrene (B[a]P) during peroxidation of phosphatidylcholine (PC) liposomes initiated by an azo compound was investigated to examine the mechanism of quinone formation from carcinogenic B[a]P mediated by nonenzymatic lipid peroxidation occurring in vivo. B[a]P had a retarding effect on the peroxidation of polyunsaturated fatty acid moiety of PC. The major oxidation products which accumulated in the peroxidized liposomes were B[a]P 1,6-, 3,6-, and 6,12-quinone. Antioxidants acting as scavengers of chain-propagating lipid peroxy radicals effectively prevented not only lipid peroxidation but also B[a]P oxidation in the liposomal suspension. PC hydroperoxides, the primary products of PC oxidation, did not react with B[a]P in the absence of the azo compound, indicating that lipid peroxy radicals, not lipid hydroperoxides, are responsible for the formation of these quinones. The experiments using 18O2 gas and 18O-labeled methyl linoleate hydroperoxides demonstrated that B[a]P quinones are formed by incorporating molecular oxygen and their origin is partly due to the lipid peroxy radical. The mechanism proposed for the formation of B[a]P quinones mediated by peroxidation of membrane lipids involves a direct attack of the lipid peroxy radical on B[a]P and subsequent autocatalytic oxidation. Weak carcinogenic and noncarcinogenic pentacyclic aromatic hydrocarbons showed little reactivity to the lipid peroxy radical in the liposomes. Thus, the facility of the peroxidative attack on B[a]P may be related to the powerful carcinogenic activity of this substance.     

Enhanced lipid peroxidation by extracellular ATP in PC12 cells

Recently we have demonstrated that extracellular ATP acts as an excitatory neurotransmitter and enhances cell death in the presence of ferrous ions. By using a newly developed cis-parinaric acid fluorescence technique, we demonstrated that ATP, in a dose dependent manner, enhanced the increased membrane lipid peroxidation in PC12 cells when cells were incubated with micromolar FeCl₂/DTP. P₂ purinoceptor agonists, α,β-methylene ATP and 2-methylthio-ATP, induced PC12 cell lipid peroxidation, but to a lesser extent than ATP. ATP-induced Ca²⁺ influx via P₂ purinoceptor activation significantly increased the intracellular Ca²⁺ concentration, which may have triggered a free radical generating cascade(s), and led to membrane lipid peroxidation and cell death. Since oxidative stress has been implicated in certain neurodegenerative diseases such as aging, extracellular ATP may contribute to neuronal cell death by an oxidative mechanism involving lipid peroxidation.     

Effect of factors of favism on the protein and lipid components of rat erythrocyte membrane

Erythrocytes prepared from riboflavin- and tocopherol-deficient (RT^?) and from control rats were used to investigate the mechanism of oxidative hemolysis by the factors of favism. RT^? erythrocytes have a defense system against the oxidative stress which is blocked either where regeneration of GSH occurs or the scavenging of the radicals from the membrane is prevented. The oxidative factors used were isouramil, divicine and diamide. When RT^? erythrocytes were treated with isouramil, GSH decreased to undetectable levels and was not regenerated. Complete hemolysis occurred, but no oxidation of SH groups of membrane proteins or formation of spectrin polymers was detected. A similar effect was observed with diamide. However, SH groups of membrane proteins were completely oxidized and spectrin polymers were formed. Extensive lipid peroxidation was also detected together with a 30% fall in the arachidonic acid level. Control erythrocytes treated with either isouramil or diamide were not hemolyzed. When treated with isouramil, after a fall in the first few minutes, the GSH level was completely regenerated after 20 min. Incubation with diamide caused extensive oxidation of SH groups of membrane proteins and formation of spectrin polymers. No lipid peroxidation was detected after treatment with isouramil, but the same decrease of arachidonic acid occurred as in RT^? erythrocytes. These results support the hypothesis that oxidative hemolysis by the factors of favism is caused by uncontrolled peroxidation of membrane lipids.     

Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E

The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.     

脂质过氧化对人红细胞膜脂流动性的影响

研究枯稀过氧化氢/高铁血红素体系所产生的烷基过氧自由基对红细胞的损伤。测定了脂质过氧化的产物——丙二脂的生成,并证明阿魏酸钠对脂质过氧化的抑制。荧光偏振的结果指出,膜脂过氧化以后降低了膜脂的流动性。人红细胞用5DSA和16DSA标记并用ESR检测膜脂流动性,结果表明,序参数S几乎没有发生变化,旋转相关时间τ值的增加证明膜脂过氧化以后,疏水尾部的物理状态发生了改变。经脂质过氧化以后,红细胞膜中的不饱和脂防酸的减少,可能是降低膜脂流动性的原因之一。     

Fluorescence imaging of lipid peroxidation in isolated rat lungs during nonhypoxic lung ischemia

We have shown previously that ischemia results in reactive oxygen species production by lung endothelium that occurs within 3-5 s after flow cessation and is followed by lipid peroxidation at 15-30 min as determined by assay of thiobarbituric acid-reactive substances, conjugated dienes, and protein carbonyls in lung homogenate. The present study evaluated membrane lipid peroxidation in isolated, ventilated rat lungs using a fluorescence imaging method that permits continuous observation of pulmonary subpleural microvascular endothelial cells in situ. Diphenyl-1-pyrenylphosphine (DPPP), a fluorescent probe which localizes in the plasma membrane and shows increased fluorescence emission after its oxidation by lipid hydroperoxides, was used for detection of membrane lipid peroxidation. Compared to continuously perfused control lungs, endothelial cell DPPP fluorescence increased significantly within 1 min of ischemia (i.e., flow cessation); these changes were prevented by pretreatment with 0.5 mM alpha-tocopherol succinate (vitamin E) added to the perfusate. Increased DPPP fluorescence was confirmed by spectrofluorometry of lipid extracts of lung homogenates. These data indicate that DPPP can be used for the real-time detection of lipid peroxidation in an intact organ. Ischemia results in peroxidation of the pulmonary microvascular endothelial cell membrane and this insult can be detected as early as 1 min after the onset of ischemia compatible with a radical-mediated process.     

Characterization of the cytolytic reaction mechanism of the human natural killer (NK) lymphocyte: resolution into binding, programming, and killer cell-independent steps

Various parameters of the cytolytic reaction mechanisms of the human natural killer (NK) lymphocyte were studied to characterize the lytic cycle. NK cytolysis was determined to occur in three definable steps. 1) Binding of PBL to the NK-sensitive targets Molt-4 or K562 was rapid (less than 1 min), occurred at temperatures below 37 degrees C, was Mg++3-dependent, Ca++3-independent, and was prevented by dispersion of the cells into 10% dextran. 2) Subsequent to binding, programming for lysis as determined by a Ca++ pulse method was more protracted, requiring up to 2 hr to occur and was strictly dependent on Ca++ for cytolysis to proceed. In standard cytotoxicity assays, however, programming for lysis was more rapid occurring in 10 to 30 min. Programming was inhibited by EDTA, EGTA/Mg++ and by temperatures below 37 degrees C. Furthermore, after binding but in the absence of initiation of programming for lysis, the frequency of target binding cells did not change and the NK cell did not lose its lytic potential. 3) Killer cell-independent cytolysis (KCIL) was determined by the addition of EDTA to "programmed" targets and dispersion of these cells into dextran-containing medium, which resulted in virtually 100% dissociation of conjugated cells. KCIL was Ca++ and Mg++-independent and was blocked at reduced temperatures only if the dextran was prechilled to 4 degrees C before addition. The kinetics of 51Cr release during KCIL was rapid and complete 30 min after dispersion. Interferon-activated NK cells expressed an increased rate of cytolysis in Ca++ pulse experiments. This was due to an increased rate of the Ca++-dependent step(s) during the programming events. The rate of the Ca++-independent steps, however, were similar with control and IFN-activated cells.     

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.     

Mitochondrial damage and its role in causing hepatocyte injury during stimulation of lipid peroxidation by iron nitriloacetate.

Incubation of isolated rat hepatocytes with 0.1 mM iron nitrilotriacetic acid (FeNTA) caused a rapid rise in lipid peroxidation followed by a substantial increase in trypan blue staining and lactate dehydrogenase release, but did not affect the protein and non-protein thiol content of the cells. Hepatocyte death was preceded by the decline of mitochondrial membrane potential, as assayed by rhodamine 123 uptake, and by the depletion of cellular ATP. Chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid or inhibition of Ca2+ cycling within the mitochondria by LaCl3 or cyclosporin A did not prevent the decline of rhodamine 123 uptake. On the other hand, a dramatic increase in the conjugated diene content was observed in mitochondria isolated from FeNTA-treated hepatocytes. Oxidative damage of mitochondria was accompanied by the leakage of matrix enzymes glutamic oxalacetic aminotransferase (GOT) and glutamate dehydrogenase (GLDH). The addition of the antioxidant N,N'-diphenylphenylene diamine (DPPD) completely prevented GOT and GLDH leakage, inhibition of rhodamine 123 uptake, and ATP depletion induced by FeNTA, indicating that Ca(2+)-independent alterations of mitochondrial membrane permeability consequent to lipid peroxidation were responsible for the loss of mitochondrial membrane potential. DPPD addition also protected against hepatocyte death. Similarly hepatocytes prepared from fed rats were found to be more resistant than those obtained from starved rats toward ATP depletion and cell death caused by FeNTA, in spite of undergoing a comparable mitochondrial injury. A similar protection was also observed following fructose supplementation of hepatocytes isolated from starved rats, indicating that the decline of ATP was critical for the development of FeNTA toxicity. From these results it was concluded that FeNTA-induced peroxidation of mitochondrial membranes impaired the electrochemical potential of these organelles and led to ATP depletion which was critical for the development of irreversible cell injury.     

Lipid peroxidation and changes in cytochrome c oxidase and xanthine oxidase activity in organophosphorus anticholinesterase induced myopathy

A possible role of radical oxygen species (ROS) initiated lipid peroxidation in diisopropylphosphorofluoridate (DFP)-induced muscle necrosis was investigated by quantifying muscle changes in F₂-isoprostanes, novel and extremely accurate markers of lipid peroxidation in vivo. A significant increase in F₂-isoprostanes of 56% was found in the diaphragm of rats 60 min after DFP-induced fasciculations. As possible source of ROS initiating lipid peroxidation, the cytocrome-c oxidase (Cyt-ox) and xanthine dehydrogenasexanthine oxidase (XD-XO) systems were investigated. Within 30 min of onset of fasciculations Cyt-ox activity was reduced by 50% from 0.526 to 0.263 μmol/mg prot/min and XO activity increased from 0.242 to 0.541 μmol/mg prot/min. Total XD-XO activity was unchanged, indicating a conversion from XD into XO. In rats pretreatment with the neuromuscular blocking agent d-tubocurarine, prevented DFP-induced fasciculations, increases in F₂-isoprostanes and changes in Cyt-ox or XD-XO. The decrease in Cyt-ox and increase in XO suggest that ROS are produced during DFP induced muscle fasciculations initiating lipid peroxidation and subsequent myopathy.