The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.     

Active Oxygen Damage Effect of Chlorophyll Degradation in Rice Seedlings Under Osmotic Stress

The changes of chlorophyll (Chl) content and contents of protochlorophyllide (Pchl), superoxide radical (O2-) and hydrogen peroxide (H2O2), malondialdehyde (MDA), ascorbic acid (ASA), glutathione (GSH), carotenoid (CAR) and the binding capacity of chlorophyll-protein (Chl-Pro) in rice (Oryza sativa L. ) seedlings exposed to osmotic stress induced by PEG 6000 (–0. 5 MPa, –0.8 MPa) were investigated to explore the relationship between Chl degradation and active oxygen effect. Under osmotic stress, Chl degradation was accompanied by the increase of contents of O2-, H2O2 and MDA and the decrease of contents of antioxidants AsA, GSH and CAR. The binding of Chl-Pro was loosened with the change of time and intensity of osmotic stress. Pretreatment with scavengers for active oxygen, such as AsA, α-tocopherol and mannitol retarded lipid peroxidation and reduced the oxidative injury of Chl, but Fe2+, H2O2 and Fenton reaction promoted the formation of MDA. The Fenton reaction accelerated the degradation of Chl. The results indicate that Chl degradation in rice seedlings induced by osmotic stress may be mainly due to the formation of more active hydroxyl radicals ('OH) through Fenton reaction and Haber-Weiss reaction.     

Effect of 24-epibrassinolide on drought stress-induced changes in Chorispora bungeana

Brassinosteroids (BRs) have been proposed to increase the resistance of plants to drought stress. The effect of foliar application of 0.1 μM 24-epibrassinolide (EBR) on chlorophyll (Chl) content, photosystem 2 (PS 2) photochemistry, membrane permeability, lipid peroxidation, relative water content (RWC), proline content, and the antioxidant system in drought-stressed Chorispora bungeana plants was investigated. The results showed that polyethylene glycol (PEG) induced water stress decreased RWC, Chl content and variable to maximum Chl fluorescence ratio (F_v/F_m) less in plants pretreated with EBR than in non-pretreated plants. In addition, lipid peroxidation, measured in terms of malondialdehyde content, membrane permeability and proline content in drought-stressed plants were less increased in EBR pretreated plants, while antioxidative enzyme activities and reduced ascorbate and glutathione contents were more increased in EBR pretreated than in non-pretreated plants. These results suggested that EBR could improve plant growth under drought stress     

Gamma radiolysis as a tool to study lipoprotein oxidation mechanisms

Well-defined quantities of *OH, O2*-,HO2* or RO2*)radicals (reactive oxygen species) can be specifically produced by radiolysis of water or ethanol. Such radical species can initiate one-electron oxidation or one-electron reduction reactions on numerous biological systems. The oxidative hypothesis of atherosclerosis classically admits the involvement of the oxidation of low density lipoproteins (LDLs) but also of high density lipoproteins (HDLs) in the development of the atherosclerotic process. The initiation mechanisms of this oxidation are still incompletely defined, although free radicals are likely involved. Therefore, gamma-radiolysis appears as a method of choice for the in vitro study of the mechanisms of oxidation of LDLs and HDLs by oxygen-centred free radicals (*OH, O2*-,HO2* and RO2*). Radiolytically oxidized lipoproteins exhibited a very well defined oxidation status (radiation dose-dependent quantification of vitamin E, beta-carotene, lipid peroxidation, protein carbonylation ...). gamma-Radiolysis is a less drastic method than other oxidation procedures such as for example copper ions. Moreover, gamma-radiolysis is also especially suitable for studying the reducing properties of antioxidant compounds with regard to their scavenging capacity.     

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.     

The Role of Oxygen in the Regulation of Nitrogenase Activity in Drought-Stressed Soybean Nodules

The aim of this study was to investigate the mechanism of nitrogenase inhibition in drought-stressed soybean (Glycine max L.) nodules to determine whether this stress was similar to other inhibitory treatments (e.g. detopping) known to cause an O2 limitation of nodule metabolism. Nodulated soybean plants were either detopped or subjected to mild, moderate, or severe drought stress by growth in different media and by withholding water for different periods. All treatments caused a decline in nitrogenase activity, and in the drought-stressed nodules, the decline was correlated with more negative nodule water potentials. Increases in rhizosphere O2 concentration stimulated nitrogenase activity much more in detopped plants than in drought-stressed plants, reflecting a greater degree of O2 limitation with the detopped treatment than with the drought-stressed treatment. These results indicated that drought stress differs from many other inhibitory treatments, such as detopping, in that its primary cause is not a decrease in nodule permeability and a greater O2 limitation of nodule metabolism. Rather, drought stress seems to cause a decrease in the maximum O2-sufficient rate of nodule respiration or nitrogenase activity, and the changes in nodule permeability reported to occur in drought-stressed nodules may be a response to elevated O2 concentrations in the infected cell that may occur as nodule respiration declines.     

The effects of La(III) on the peroxidation of membrane lipids in wheat seedling leaves under osmotic stress

The physiological effects of the rare earth ion La³⁺ on the peroxidation of membrane lipids in wheat (Triticum aestivum L.) seedling leaves under osmotic stress were determined. With the passage of time under osmotic stress, the inhibition ability of lanthanum ions to the relative membrane permeability and concentration of malondialdehyde, Superoxide radicals, and hydrogen peroxide caused by osmotic stress increased substantially, but no changes were noted in ferrous and relative water content. It indicated that lanthanum ions could not retain the water content because of osmotic stress. However, La³⁺ appears to decrease the production of OH by reducing the content of O₂ and H₂O₂ of Haber-Weiss and Fenton reactions, which efficiently alleviated peroxidation of membrane lipids under osmotic stress and, to some degree, protected the membrane from injury of free radicals. Thus, La³⁺ increased the tolerance ability of plant to osmotic stress, which could assure the function of membrane normal temporally after stressed.     

Myeloperoxidase-catalyzed phenoxyl radicals of vitamin E homologue, 2,2,5,7,8-pentamethyl- 6-hydroxychromane, do not induce oxidative stress in live HL-60 cells

We used myeloperoxidase-containing HL-60 cells to generate phenoxyl radicals from nontoxic concentrations of a vitamin E homologue, 2,2, 5,7,8-pentamethyl-6-hydroxychromane (PMC) to test whether these radicals can induce oxidative stress in a physiological intracellular environment. In the presence of H(2)O(2), we were able to generate steady-state concentrations of PMC phenoxyl radicals readily detectable by EPR in viable HL-60 cells. In HL-60 cells pretreated with succinylacetone, an inhibitor of heme synthesis, a greater than 4-fold decrease in myeloperoxidase activity resulted in a dramatically decreased steady-state concentrations of PMC phenoxyl radicals hardly detectable in EPR spectra. We further conducted sensitive measurements of GSH oxidation and protein sulfhydryl oxidation as well as peroxidation in different classes of membrane phospholipids in HL-60 cells. We found that conditions compatible with the generation and detection of PMC phenoxyl radicals were not associated with either oxidation of GSH, protein SH-groups or phospholipid peroxidation. We conclude that PMC phenoxyl radicals do not induce oxidative stress under physiological conditions in contrast to their ability to cause lipid peroxidation in isolated lipoproteins in vitro.     

Oxygen-dependent antagonism of lipid peroxidation

Measurements of the rates for formation of conjugated dienes, malonylaldehyde, and lipid hydroperoxides show that increasing the concentration of O2 from 0.11 mM to 0.35 mM or 0.69 mM can slow the rate of linoleic acid peroxidation in a xanthine oxidase/hypoxanthine system. This effect is seen at pH 7.0 but not 7.4 and depends on the presence of monounsaturated fatty acids (oleic, cis, or trans vaccenic acid). Oxygen antagonism of ascorbic acid-iron-EDTA mediated lipid peroxidation is similarly dependent on fatty acid mixtures and occurs at pH 5.0 and 6.0 but not 7.0. The efficiency of initiation of peroxidation in the xanthine oxidase system is unaffected by monounsaturated fatty acids and O2 concentration. Increasing the O2 concentration increases the rate of superoxide radical production, but there is no change in salicylate hydroxylation (e.g., OH. production) or ferrous ion concentration. Oxygen-mediated slower rates of lipid peroxidation are associated with either increased H2O2 production or, based on an indirect assay, singlet O2 production. Increased O2 concentrations increase the rate of azobisisobutyronitrile-initiated lipid peroxidation as expected but addition of exogenous superoxide radicals slows the rate. Under similar conditions superoxide reacts with fatty acids to produce singlet O2. Overall, the data suggest that O2-mediated antagonism occurs because of termination reactions between hydroperoxyl (HO2.) and organic radicals, and singlet O2 or H2O2 are products of these reactions.     

Lipid peroxidation during the oxidation of haemoproteins by hydroperoxides. Relation to electronically excited state formation

The formation of electronically excited states during hydroperoxide metabolism is analysed in terms of recombination reactions involving secondary peroxyl radicals and scission of the O? O bond of peroxides by haemoproteins, mainly myoglobin. Both processes may be sequentially interrelated, for the cleavage of H₂O₂ by metmyoglobin leads to the formation of a strong oxidizing equivalent with the capability to promote peroxidation of polyunsaturated fatty acids. The decomposition of lipid hydroperoxides by ferryl-hydroxo complexes, as that formed during the oxidation of metmyoglobin by H₂O₂, is a source of peroxyl radicals, the recombination of which proceeds with elimination of a conjugated triplet carbonyl or singlet oxygen.     

Enhanced oxidation of flavan-3-ols and proanthocyanidin accumulation in water-stressed tea plants

(-)-Epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two major tea flavan-3-ols, have received attention in food science and biomedicine because of their potent antioxidant properties. In plants, flavan-3-ols serve as proanthocyanidin (PA) building blocks, and although both monomeric flavan-3-ols and PAs show antioxidant activity in vitro, their antioxidant function in vivo remains unclear. In the present study, EC quinone (ECQ) and EGCG quinone (EGCGQ), the oxidation products of EC and EGCG, increased up to 100- and 30-fold, respectively, in tea plants exposed to 19 days of water deficit. Oxidation of EC and EGCG preceded PAs accumulation in leaves, which increased from 35 to 53 mg gDW(-1) after 26 days of water deficit. Aside from the role monomeric flavan-3-ols may play in PAs biosynthesis, formation of ECQ and EGCGQ strongly negatively correlated with the extent of lipid peroxidation in leaves, thus supporting a protective role for these compounds in drought-stressed plants. Besides demonstrating flavonoid accumulation in drought-stressed tea plants, we show for the first time that EC and EGCG are oxidized to their respective quinones in plants in vivo.     

Peroxidase catalysed formation of cytotoxic prooxidant phenothiazine free radicals at physiological pH

The antipsychotic phenothiazines may have other therapeutic applications because of their ability to kill bacteria, plasmids and tumor cells. They are also known to undergo a peroxidase-catalysed oxidation to form cation radicals that are stable at acid pH, but are not detected at a neutral pH. The objective of this project was to determine whether phenothiazine cation radical metabolites could cause oxidative stress at a neutral pH resulting in cytotoxicity. At a neutral pH, catalytic amounts of phenothiazines were found to be oxidised by a peroxidase/H2O2 system and also caused ascorbate, GSH and NADH cooxidation. NADH and GSH co-oxidation was accompanied by oxygen uptake and was increased by the addition of catalytic amounts of superoxide dismutase, indicating that the superoxide radical was formed. The phenothazines were different from other peroxidase substrates in that the NADH, ascorbate or GSH cooxidation was faster at pH 6.0 than pH 7.4, thereby partly reflecting the cation radical stability. The order of catalytic effectiveness found was promazine > chlorpromazine > trifluoperazine. Peroxidase/H2O2 also markedly increased phenothiazine cytotoxicity towards isolated rat hepatocytes at nontoxic phenothiazine concentrations. At both pH 6.0 and 7.4, the same order of phenothiazine catalytic effectiveness was observed as seen in the co-oxidation experiments. Cytotoxicity to hepatocytes could be attributed to oxidative stress as most hepatocyte glutathione oxidation and lipid peroxidation preceded phenothiazine induced cytotoxicity and that cytotoxicity was prevented by the antioxidant butylated hydroxyanisole. This hepatocyte/peroxidase/H2O2 system could be a useful model for studying drug induced idiosyncratic hepatic injury enhanced by inflammation.     

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.     

金属离子胁迫产生植物膜脂过氧化的能力与离子的极化能力正相关

金属离子胁迫导致植物产生多种生理损伤,其中包括膜脂的过氧化。不同的金属离子诱导植物产生膜脂过氧化差异很大,迄今对这些差异与金属离子性质之间的具体关系所知甚少。金属离子对阴离子和其他分子的作用主要取决于其电荷数量、半径大小和电子层结构,亦即取决于其极化作用。作者以水培拟南芥与油菜为材料,以不同极化作用的金属离子（Li^＋、Na^＋、K^＋、ca^2＋、Fe^2＋与Fe^3＋）为研究对象,检测了极化作用不同的金属离子胁迫拟南芥与油菜1、3、5 d后膜脂过氧化产物丙二醛（Malondialdehyde,MDA）的含量变化。结果发现,相同浓度的一价离子Li^＋、Na^＋与K^＋’胁迫5 d后,离子半径较小的Li^＋诱导拟南芥与油菜产生的丙二醛比Na^＋与K^＋高2倍以上;相同浓度下电子层结构相同的Fe^3＋与Fe^2＋胁迫5 d后,电荷高的Fe^3＋诱导拟南芥产生的丙二醛比Fe^2＋高2倍以上,而Fe^3＋诱导油菜产生的丙二醛比Fe^2＋高5倍以上;相同浓度的二离子Fe^2＋与ca^2＋胁迫,电子层结构复杂的Fe^2＋比ca^2＋诱导更多的丙二醛产生。运用离子势综合表征离子极化能力,则丙二醛的含量与离子势大小显著正相关。上述结果说明,金属离子诱导膜脂过氧化的胁迫能力与金属极化作用正相关,即电荷越高、离子半径越小及电子层越复杂,产生氧化胁迫程度越强。     

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.     

Environmental stress causes oxidative damage to plant mitochondria leading to inhibition of glycine decarboxylase

A cytotoxic product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), rapidly inhibited glycine, malate/pyruvate, and 2-oxoglutarate-dependent O2 consumption by pea leaf mitochondria. Dose- and time-dependence of inhibition showed that glycine oxidation was the most severely affected with a K(0.5) of 30 microm. Several mitochondrial proteins containing lipoic acid moieties differentially lost their reactivity to a lipoic acid antibody following HNE treatment. The most dramatic loss of antigenicity was seen with the 17-kDa glycine decarboxylase complex (GDC) H-protein, which was correlated with the loss of glycine-dependent O2 consumption. Paraquat treatment of pea seedlings induced lipid peroxidation, which resulted in the rapid loss of glycine-dependent respiration and loss of H-protein reactivity with lipoic acid antibodies. Pea plants exposed to chilling and water deficit responded similarly. In contrast, the damage to other lipoic acid-containing mitochondrial enzymes was minor under these conditions. The implication of the acute sensitivity of glycine decarboxylase complex H-protein to lipid peroxidation products is discussed in the context of photorespiration and potential repair mechanisms in plant mitochondria.     

Effect of K-ATP channel opener-pinacidil on the liver mitochondria function in rats with different resistance to hypoxia during stress

We have examined the influence of ATP-sensitive potassium (KATP) channel opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) on the changes of energy metabolism in the liver of rats under the stress conditions. The rats were divided in two groups with high and low resistance to hypoxia. The stress was modeled by placing the rats in a cage filled with water and closed with a net. The distance from water to the net was only 5 cm. The effects of KATP opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) on ADP-stimulating mitochondrial respiration by Chance, calcium capacity of organellas and processes of lipid peroxidation in the liver of rats with different resistance to hypoxia under the stress condition have been investigated. We have used the next substrates of oxidation: 0.35 mM succinate and 1 mM alpha-ketoglutarate. The additional analyses were conducted with the use of inhibitors: mitochondrial enzyme complex I 10 mM rotenone and succinate dehydrohenase 2 mM malonic acid. It was shown that the stress condition evoked the succinate oxidation and the decrease of alpha-ketoglutarate efficacy, the increase of calcium mitochondrial capacity and the intensification of lipid peroxidation processes. Under the presence of succinate, the increase of O2 uptake with simultaneous decrease of ADP/O ratio in rats with high resistance under stress was observed. Simultaneously, oxidation of alpha-ketoglutarate, a NAD-dependent substrate, was inhibited. Pinacidil caused the reorganization of mitochondrial energy metabolism in favour of NAD-dependent oxidation and the improvment of the protection against stress. The decrease of the efficacy of mitochondrial energy processes functioning was shown in animals with low resistance to hypoxia. KATP channel opener pinacidil has a protective effect on the processes of mitochondrial liver energy support under stress. These changes deal with the increase of alpha-ketoglutarate oxidation (respiratory rate and ADP/O) and the decrease of lipid peroxidation processes. We concluded about protective effect ofpinacidil on mitochondrial functioning under stress.     

Role of Antioxidant Systems in Wheat Genotypes Tolerance to Water Stress

The role of plant antioxidant systems in stress tolerance was studied in leaves of three contrasting wheat genotypes. Drought imposed at two different stages after anthesis resulted in an increase in H2O2 accumulation and lipid peroxidation and decrease in ascorbic acid content. Antioxidant enzymes like superoxide dismutase, ascorbate peroxidase and catalase significantly increased under water stress. Drought tolerant genotype C 306 which had highest ascorbate peroxidase and catalase activity and ascorbic acid content also showed lowest H2O2 accumulation and lipid peroxidation (malondialdehyde content) under water stress in comparison to susceptible genotype HD 2329 which showed lowest antioxidant enzyme activity and ascorbic acid content and highest H2O2 content and lipid peroxidation. HD 2285 which is tolerant to high temperature during grain filling period showed intermediate behaviour. Superoxide dismutase activity, however, did not show significant differences among the genotypes under irrigated as well as water stress condition. It seems that H2O2 scavenging systems as represented by ascorbate peroxidase and catalase are more important in imparting tolerance against drought induced oxidative stress than superoxide dismutase alone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Oxidative damage shifts from lipid peroxidation to thiol arylation by catechol-containing antioxidants

Catechol-containing antioxidants are able to protect against lipid peroxidation by nonenzymatic scavenging of free radicals with their catechol moiety. During their antioxidant activity, catechol oxidation products such as semiquinone radicals and quinones are formed. These oxidation products of 4-methylcatechol inactivate the GSH-dependent protection against lipid peroxidation and the calcium sequestration in liver microsomes. This effect is probably due to arylation by oxidation products of 4-methylcatechol of free thiol groups of the enzymes responsible for the GSH-dependent protection and calcium sequestration, i.e. the free radical reductase and calcium ATPase. It is concluded that a catechol-containing antioxidant might shift radical damage from lipid peroxidation to sulfhydryl arylation.