Oxidative modification of human low-density lipoprotein by horseradish peroxidase in the absence of hydrogen peroxide

Heme-peroxidases, such as horseradish peroxidase (HRP), are among the most popular catalysts of low density lipoprotein (LDL) peroxidation. In this model system, a suitable oxidant such as H₂O₂ is required to generate the hypervalent iron species able to initiate the peroxidative chain. However, we observed that traces of hydroperoxides present in a fresh solution of linoleic acid can promote lipid peroxidation and apo B oxidation, substituting H₂O₂.

Spectral analysis of HRP showed that an hypervalent iron is generated in the presence of H₂O₂ and peroxidizing linoleic acid. Accordingly, careful reduction of the traces of linoleic acid lipid hydroperoxide prevented formation of the ferryl species in HRP and lipid peroxidation. However, when LDL was oxidized in the presence of HRP, the ferryl form of HRP was not detectable, suggesting a Fenton-like reaction as an alternative mechanism. This was supported by the observation that carbon monoxide, a ligand for the ferrous HRP, completely inhibited peroxidation of LDL.

These results are in agreement with previous studies showing that myoglobin ferryl species is not produced in the presence of phospholipid hydroperoxides, and emphasize the relevance of a Fenton-like chemistry in peroxidation of LDL and indirectly, the role of pre-existing lipid hydroperoxides.     

Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation

Mouse and human spermatozoa, but not rabbit spermatozoa, have long been known to be sensitive to loss of motility induced by exogenous H₂O₂. Recent work has shown that loss of sperm motility in these species correlates with the extent of spontaneous lipid peroxidation. In this study, the effect of H₂O₂ on this reaction in sperm of the three species was investi gated. The rate of spontaneous lipid peroxidation in mouse and human sperm is markedly enhanced in the presence of 1-5 mM H₂O₂, while the rate in rabbit sperm is unaffected by H₂O₂. The enhancement of lipid peroxidation, the rate of reaction of H₂O₂ with the cells, the activity of sperm glutathione peroxidase, and the endogenous glutathione content are highest in mouse sperm, intermediate in human sperm, and very low in rabbit sperm. Inac tivation of glutathione peroxidase occurs in the presence of H₂O₂ due to complete conver sion of endogenous glutathione to GSSG: No GSH is available as electron donor substrate to the peroxidase. Inactivation of glutathione peroxidase by the inhibitor mercaptosucci nate has the same effect on rate of lipid peroxidation and loss of motility in mouse and human sperm as does H₂O₂. This implies that H₂O₂ by itself at 1-5 mM is not intrinsically toxic to the cells. With merceptosuccinate, the endogenous glutathione is present as GSH in mouse and human sperm, indicating that the redox state of intracellular glutathione by itself plays little role in protecting the cell against spontaneous lipid peroxidation. Mouse and human sperm also have high rates of superoxide production. We conclude that the key intermediate in spontaneous lipid peroxidation is lipid hydroperoxide generated by a chain reaction initiated by and utilizing superoxide. Removal of this hydroperoxide by gluta thione peroxidase protects these sperm against peroxidation; inactivation of the peroxidase allows lipid hydroperoxide to increase and so increases the peroxidation rate. Rabbit sperm have low rates of superoxide reaction due to high activity of their superoxide dismutase; lack of endogenous glutathione and low peroxidase activity does not affect their rate or lipid peroxidation. As a result, these sperm are not affected by either H₂O₂ or mercapto-succinate. These results lead us to postulate a mechanism for spontaneous lipid peroxida tion in mammalian sperm which involves reaction of lipid hydroperoxide and O₂ as the rate-determining step.     

Ability of Ferric Nitrilotriacetate Complex with Three pH-dependent Conformations to Induce Lipid Peroxidation

This study examined the generation of reactive oxygen species (ROS) and the induction of lipid peroxidation by carcinogenic iron(III)-NTA complex (1:1), which has three conformations with two pKa values (pKa₁≈4, pKa₂≈8). These conformations are type (a) in acidic conditions of pH 1-6, type (n) in neutral conditions of pH 3-9, and type (b) in basic conditions of pH 7-10. The iron(III)-NTA complex was reduced to iron(II) complex under cool-white fluorescent light without the presence of any reducer. The reduction rates of three species of iron(III)-NTA were in the order type (a)?type (n) ? type (b). Iron(III)-NTA-dependent lipid peroxidation was induced in the presence and absence of preformed lipid peroxides (L-OOH) through processes associated with and without photoreduction of iron(III). The order of the abilities of the three species of iron(III)-NTA to initiate the three mechanisms of lipid peroxidation was: (1) type (a) ? type (n) ? type (b) in lipid peroxidation that is induced L-OOH- and H₂O₂-dependently and mediated by the photoreduction of iron(III); (2) type (b) ? type (n) ? type (a) in lipid peroxidation that is induced L-OOH- and H₂O₂-dependently but not mediated by the photoreduction of iron(III); (3) type (n) ? type (b) ? type (a) in lipid peroxidation that is induced peroxide-independently and mediated by the photoactivation but not by the photoreduction of iron(III). The rate of lipid peroxidation induced L-OOH-dependently is faster than that induced H₂O₂-dependently in the mechanism (1), but the rate of lipid peroxidation induced H₂O₂-dependently is faster than that induced L-OOH-dependently in the mechanism (2). In the lag process of mechanism (3), L-OOH and/or some free radical species, not ¹O₂, were generated by photoactivation of iron(III)-NTA. These multiple pro-oxidant properties that depend on the species of iron(III)-NTA were postulated to be a principal cause of its carcinogenicity.     

Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and Root Nodules of Medicago sativa

Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 have been analyzed for the presence of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC 1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol of nodules possess CN⁻-insensitive SOD activities. Studies of SOD inactivation with H₂O₂ indicate that, very likely, a Mn-SOD is present in the bacteroids, and suggest that the cytosol contain both Mn-SOD and Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By contrast, the nodule cytosol exhibits an elevated peroxidase activity as compared with the foliar tissue; this activity was completely inhibited by 50 to 100 micromolar KCN. The significantly lower contents of H₂O₂ and malondialdehyde (a product of lipid peroxidation) in nodules with respect to those in leaves reveal that the above-mentioned bacteroid and cytosol enzymes act in an efficient and combined manner to preserve integrity of nodule cell membranes and to keep leghemoglobin active.     

Strobilanthes crispus attenuates renal carcinogen, iron nitrilotriacetate (Fe-NTA)-mediated oxidative damage of lipids and DNA

This study was aimed to evaluate the effect of Strobilanthes crispus extract for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Incubation of postmitochondrial supernatant and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces lipid peroxidation and DNA damage to about 2.3-fold and 2.9-fold, respectively, as compared to control (P < 0.05). In lipid peroxidation protection studies, S. crispus treatment showed a dose-dependent inhibition (45–53% inhibition, P < 0.05) of Fe-NTA and H₂O₂ induced lipid peroxidation. Similarly, in DNA damage protection studies, S. crispus treatment also showed a dose-dependent inhibition (18–30% inhibition, P < 0.05) of DNA damage. In addition, the protection was closely related to the content of phenolic compounds as evident by S. crispus extract showing the value of 124.48 mg/g total phenolics expressed as gallic acid equivalent (GAE, mg/g of extract). From these studies, it is concluded that S. crispus inhibits peroxidation of membrane lipids and DNA damage induced by Fe-NTA and H₂O₂ and possesses the potential to be used to treat or prevent degenerative diseases where oxidative stress is implicated.     

Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins

The effects of all-zinc metallothionein (Zn-metallothionein) and predominantly cadmium metallothionein (Cd/Zn-metallothionein) on free radical lipid peroxidation have been investigated, using erythrocyte ghosts as the test system. When treated with xanthine and xanthine oxidase, Zn-metallothionein and Cd/Zn-metallothionein underwent thiolate group oxidation and metal ion release that was catalase-inhibitable, but superoxide dismutase-non-inhibitable. Similar treatment in the presence of ghosts and added Fe(III) resulted in metallothioneen oxidation that was significantly inhibited by superoxide dismutase. Ghosts incubated with xanthine/xanthine oxidase/Fe(III) underwent H₂O₂- and O₂⁻-dependent lipid peroxidation, as measured by thiobarbituric acid reactivity. Neither type of metallothionein had any effect on xanthine oxidase activity, but both strongly inhibited lipid peroxidation when added to the membranes concurrently with xanthine/xanthine oxidase/iron. This inhibition was far greater and more sustained than that caused by dithiothreitol at a concentration equivalent to that of metallothionein thiolate. Significant protection was also afforded when ghosts plus Cd/Zn-metallothionein or Zn/metallothionein were preincubated with H₂O₂ and Fe(III), and then subjected to vigorous peroxidation by the addition of xanthine and xanthine oxidase. These results could be mimicked by using Cd(II) or Zn(II) alone. Previous studies suggested that Zn(II) inhibits xanthine/xanthine oxidase/iron-driven lipid peroxidation in ghosts by interfering with iron binding and redox cycling. Therefore, the primary determinant of metallothionein proteciion appears to be metal release and subsequent uptake by the membranes. These results have important implications concerning the antioxidant role of metallothionein, a protein known to be induced by various prooxidant conditions.     

An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation

A model lipid peroxidation system dependent upon the hydroxyl radical, generated by Fenton's reagent, was compared to another model system dependent upon the enzymatic generation of superoxide by xanthine oxidase. Peroxidation was studied in detergent-dispersed linoleic acid and in phospholipid liposomes. Hydroxyl radical generation by Fenton's reagent (FeCl₂ + H₂O₂) in the presence of phospholipid liposomes resulted in lipid peroxidation as evidenced by malondialdehyde and lipid hydroperoxide formation. Catalase, mannitol, and Tris-Cl were capable of inhibiting activity. The addition of EDTA resulted in complete inhibition of activity when the concentration of EDTA exceeded the concentration of Fe²⁺. The addition of ADP resulted in slight inhibition of activity, however, the activity was less sensitive to inhibition by mannitol. At an ADP to Fe²⁺ molar ratio of 10 to 1, 10 mm mannitol caused 25% inhibition of activity. Lipid peroxidation dependent on the enzymatic generation of superoxide by xanthine oxidase was studied in liposomes and in detergent-dispersed linoleate. No activity was observed in the absence of added iron. Activity and the apparent mechanism of initiation was dependent upon iron chelation. The addition of EDTA-chelated iron to the detergent-dispersed linoleate system resulted in lipid peroxidation as evidenced by diene conjugation. This activity was inhibited by catalase and hydroxyl radical trapping agents. In contrast, no activity was observed with phospholipid liposomes when iron was chelated with EDTA. The peroxidation of liposomes required ADP-chelated iron and activity was stimulated upon the addition of EDTA-chelated iron. The peroxidation of detergent-dispersed linoleate was also enhanced by ADP-chelated iron. Again, this peroxidation in the presence of ADP-chelated iron was not sensitive to catalase or hydroxyl radical trapping agents. It is proposed that initiation of superoxide-dependent lipid peroxidation in the presence of EDTA-chelated iron occurs via the hydroxyl radical. However, in the presence of ADP-chelated iron, the participation of the free hydroxyl radical is minimal.     

Evidence for the accumulation of peroxidized lipids in membranes of senescing cotyledons

Fluorescent products of lipid peroxidation accumulate with age in microsomal membranes from senescing cotyledons of Phaseolus vulgaris. The temporal pattern of accumulation is closely correlated with a rise in the lipid phase transition temperature reflecting the formation of gel phase lipid. Increased levels of fluorescent peroxidation products are also detectable in total lipid extracts of senescent cotyledons. Lipoxygenase activity increases with advancing age by about 3-fold on a fresh weight basis and 4-fold on a dry weight basis indicating that the tissue acquires elevated levels of lipid hydroperoxides. As well, levels of glutathione and superoxide dismutase activity decline on a dry weight basis as the cotyledons age, rendering the tissue more susceptible to oxidative damage. Catalase activity rises initially and then declines during senescence, but peroxidase activity rises steeply. Thus, apart from this increase in peroxidase, which would scavenge H₂O₂ only if appropriate cosubstrates were available, the defense mechanisms for coping with activated oxygen species (O₂⁻, H₂O₂, OH) are less effective in the older tissue. The observations support the contention that formation of gel phase lipid in senescing membranes is attributable to lipid peroxidation and suggest that the reactions of lipid peroxidation are utilized by the cotyledons to mediate deteriorative changes accompanying the mobilization and transport of metabolites from the storage tissue to the developing embryo.     

Participation of superoxide,hydrogen peroxide and hydroxyl radicals in NADPH-cytochrome P-450 reductase-catalyzed peroxidation of methyl linolenate

• 1.1. NADPH-cytochrome P-450 reductase-catalyzed peroxidation of methyl linolenate is inhibited by superoxide dismutase, catalase, ethanol and mannitol and is potentiated by H₂O₂.
• 2.2. H₂O₂ is shown to be generated in the incubation mixture in the presence of NADPH and NADPH-cytochrome P-450 reductase. If the system contains Fe-EDTA complex, H₂O₂ is not formed. In the presence of the enzyme and Fe-EDTA complex, added H₂O₂ is consumed.
• 3.3. In the presence of Fe-EDTA complex, NADPH-cytochrome P-450 reductase is shown to generate O_2⁻ at a slow rate.These results suggest that H₂O₂ produced from O_2⁻ is decomposed to form OH· by the action of Fe-EDTA complex in the lipid peroxidation system and that OH· is a trigger of lipid peroxidation.

     

Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): Lethal peroxidative membrane injury

Oxidative stress induced by hydrogen peroxide (H₂O₂) may contribute to the pathogenesis of ischemic-reperfusion injury in the heart. For the purpose of investigating directly the injury potential of H₂O₂ on heart muscle, a cellular model of H₂O₂-induced myocardial oxidative stress was developed. This model employed primary monolayer cultures of intact, beating neonatal-rat cardiomy-ocytes and discrete concentrations of reagent H₂O₂ in defined, supplement-free culture medium. Cardiomyocytes challenged with H₂O₂ readily metabolized it such that the culture content of H₂O₂ diminished over time, but was not depleted. The consequent H₂O₂-induced oxidative stress caused lethal sarcolemmal disruption (as measured by lactate dehydrogenase release), and cardiomyocyte integrity could be preserved by catalase. During oxidative stress, a spectrum of cellular derangements developed, including membrane phospholipid peroxidation, thiol oxidation, consumption of the major chain-breaking membrane antiperoxidant (α-tocopherol), and ATP loss. No net change in the protein or phospholipid contents of cardiomyocyte membranes accompanied H₂O₂-induced oxidative stress, but an increased turnover of these membrane constituents occurred in response to H₂O₂. Development of lethal cardiomyocyte injury during H₂O₂-induced oxidative stress did not require the presence of H₂O₂ itself; a brief “pulse” exposure of the cardiomyocytes to H₂O₂ was sufficient to incite the pathogenic mechanism leading to cell disruption. Cardiomyocyte disruption was dependent upon an intracellular source of redox-active iron and the iron-dependent transformation of internalized H₂O₂ into products (e.g., the hydroxyl radical) capable of initiating lipid peroxidation, since iron chelators and hydroxyl-radical scavengers were cytoprotective. The accelerated turnover of cardiomyocyte-membrane protein and phospholipid was inhibited by antiperoxidants, suggesting that the turnover reflected molecular repair of oxidized membrane constituents. Likewise, the consumption of α-tocopherol and the oxidation of cellular thiols appeared to be epiphenomena of peroxidation. Antiperoxidant interventions coordinately abolished both H₂O₂-induced lipid peroxidation and sarcolemmal disruption, demonstrating that an intimate pathogenic relationship exists between sarcolemmal peroxidation and lethal compromise of cardiomyocyte integrity in response to H₂O₂-induced oxidative stress. Although sarcolemmal peroxidation was causally related to cardiomyocyte disruption during H₂O₂-induced oxidative stress, a nonperoxidative route of H₂O₂ cytotoxicity was also identified, which was expressed in the complete absence of cardiomyocyte-membrane peroxidation. The latter mode of H₂O₂-induced cardiomyocyte injury involved ATP loss such that membrane peroxidation and cardiomyocyte disruption on the one hand and cellular de-energization on the other could be completely dissociated. The cellular pathophysiology of H₂O₂ as a vectorial signal for cardiomyocyte necrosis that “triggers” irreversible peroxidative disruption of the sarcolemma has implications regarding potential mechanisms of oxidative injury in the postischemic heart.     

Desferrioxamine as an Electron Donor. Inhibition of Membranal Lipid Peroxidation Initiated by H202 –Activated Metmyoglobin and Other Peroxidizing Systems

Desferoxamine (DFO) involvement in several peroxidative systems was studied. These sytems included: a) membranal lipid peroxidation initiated by H₂O₂-activated metmyoglobin (or methemoglobin); b) phenol-red oxidation by activated metmyoglobin or horseradish peroxidase (HRP): c) β-carotene-linoleate couple oxidation stimulated by lipoxygenase or hemin. Desferrioxamine was found to inhibit all these systems but not ferrioxamine (FO). Phenol-red oxidation by H₂0₂-horseradish peroxidase was inhibited competitively with DFO. Kinetic studies using the spectra changes in the Soret region of metmyoglobin suggest a mechanism by which H₂0₂ reacts with the iron-heme to form an intermediate of oxy-ferryl myoglobin that subsequently reacts with DFO to return the activated compound to the resting state. These activities of DFO resemble the reaction of other electron donors.     

Experimental approaches to free radicals and ageing

Abstract

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10–34% inhibition; P <0.05) of Fe-NTA plus H₂O₂-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36–71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H₂O₂. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.     

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H₂O₂, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H₂O₂-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H₂O₂-induced liposome leakage was abolished by cyanide presumably competing with H₂O₂ for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H₂O₂ permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H₂O₂, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.     

Involvement of Molecular Oxygen in the Enzyme-Catalyzed NADH Oxidation and Ferric Leghemoglobin Reduction

Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb⁺³), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O₂ was removed from the reaction system, but all were restored upon readdition of O₂. In the absence of exogenous electron carriers and in the presence of O₂ and excess NADH, FLbR catalyzed NADH oxidation with the generation of H₂O₂ functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction. The formation of H₂O₂ during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H₂O₂. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction were not constant but changed with time and with concentrations of NADH and O₂ in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb⁺³ by reaction intermediates. A study of the affinity of FLbR for O₂ showed that the enzyme required at least micromolar levels of dissolved O₂ for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.     

Effect of Arbuscular Mycorrhizal Inoculation on Salt-induced Nodule Senescence in <Emphasis Type="Italic">Cajanus cajan</Emphasis> (Pigeonpea)

Many physiological and biochemical plant processes affected by salt stress trigger premature nodule senescence and decrease their ability to fix nitrogen. The objective of this study was to evaluate the role of arbuscular mycorrhiza (AM) in moderating salt-induced premature nodule senescence in Cajanus cajan (L.) Millsp. Greenhouse experiments were conducted in which the plants were exposed to salinity stress of 4, 6, and 8 dSm⁻¹. Various parameters linked to nodule senescence were assessed at 80 days after sowing. Nodulation, leghemoglobin content, and nitrogenase enzyme activity measured as acetylene-reducing activity (ARA) were evaluated. Two groups of antioxidant enzymes were studied: (1) enzymes involved in the detoxification of O₂⁻ radicals and H₂O₂, namely, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), and (2) enzymes that are important components of the ascorbate glutathione pathway responsible for the removal of H₂O₂, namely, glutathione reductase (GR) and ascorbate peroxidase (APOX). Exposure of plants to salinity stress enhanced nodule formation; however, nodule growth suffered remarkably and a marked decline in nodule biomass, relative permeability, and lipid peroxidation was observed. Leghemoglobin content and ARA were reduced under saline conditions. AM significantly improved nodulation, leghemoglobin content, and nitrogenase activity under salt stress. Activities of SOD, CAT, APOX, POX, and GR increased markedly in mycorrhizal-stressed plants. A synthesis of the evidence obtained in this study suggests a correlation between enhanced levels of antioxidant enzyme activities, reduced membrane permeability, reduced lipid peroxidation, and improved nitrogen-fixing efficiency of AM plants under stressed and unstressed conditions. These factors could be responsible for the protective effects of mycorrhiza against stress-induced premature nodule senescence.     

Paraquat and iron-dependent lipid peroxidation

The aim of this work was to study the effect of paraquat (P²⁺) on NADPH iron-dependent lipid peroxidation (basal peroxidation) either in the presence of NADPH or in the presence of NADPH-generating systems. When NADPH is present, P²⁺ potentiates NADPH iron-dependent lipid peroxidation, but use of NADPH-generating systems cancels this effect. This may be attributed to certain components in NADPH-generating systems such as glucose-6-phosphate and sodium isocitrate, which act as iron chelators. The binding of iron by these molecules facilitates its reduction and enhances its reactivity toward dioxygen molecules, leading to the formation of reactive species capable of initiating lipid peroxidation, such as Fe³⁺-O ₂ ⁻ . Under these conditions of rapid basal peroxidation, any additional reduction of iron(III) by a reduced form of P²⁺ (P^+.) has no apparent effect on the peroxidation itself, probably because the initial reaction between iron(II) and O₂ followed by initiation of the peroxidation are both rate-limiting steps in the process. Consequently, any alteration of the composition of the reacting mixture (e.g., buffers or the generating system) must be taken into consideration because the formation of new iron chelates can change the rate of basal peroxidation and will modify the effect of redoxcycling molecules.     

Cytochrome P450 oxidase activity and its role in NADPH dependent lipid peroxidation

A comparison is made between microsomal NADPH-dependent H₂O₂ production and malondialdehyde (MDA) formation in rat liver microsomes, obtained from phenobarbital pretreated rats. An increase in H₂O₂ formation was observed during NADPH-dependent disposition (10 min) of 100 μM diazepam (33%) and 2 mM hexobarbital (69%). In contrast orphenadrine (100 μM) and its mono-N-demethylated metabolite tofenacine (100 μM) decreased the H₂O₂ formation (35% and 55%, respectively). However, all these substrates were found to inhibit NADPH-dependent lipid peroxidation (60 min), estimated by measuring MDA formation, to various extents. These data strongly suggest that the oxidase activity of cytochrome P450 (H₂O₂ production) is not involved in a rate-limiting step in NADPH-dependent lipid peroxidation.     

Aerobic hydrogenase activity in Anacystis Nidulans The oxyhydrogen reaction

1. The oxyhydrogen reaction of Anacystis nidulans was studied manometrically and polarographically in whole cells and in cell-free preparations; the activity was found to be associated with the particulate fraction.2. Besides O₂, the isolated membranes reduced artificial electron acceptors of positive redox potential; the reactions were unaffected by O₂ levels <10–15%; aerobically the artificial acceptors were reduced simultaneously with O₂.3. H₂-supported O₂ uptake was inhibited by CO, KCN and 2-n-heptyl-8-hydroxyquinoline-N-oxide. Inhibition by CO was partly reversed by strong light. Uncouplers stimulated the oxyhydrogen reaction.4. The kinetic properties of O₂ uptake by isolated membranes were the same in presence of H₂ and of other respiratory substrates.5. Low rates of H₂ evolution by the membrane preparations were found in presence of dithionite; methyl viologen stimulated the reaction.6. The results indicate that under certain growth conditions Anacystis synthesizes a membrane-bound hydrogenase which appears to be involved in phosphorylative electron flow from H₂ to O₂ through the respiratory chain.     

Protein oxidation: examination of potential lipid-independent mechanisms for protein carbonyl formation

Previous data indicated that diquat-mediated protein oxidation (protein carbonyl formation) occurs through multiple pathways, one of which is lipid dependent, and the other, lipid independent. Studies reported here investigated potential mechanisms of the lipid-independent pathway in greater detail, using bovine serum albumin as the target protein. One hypothesized mechanism of protein carbonyl formation involved diquat-dependent production of H₂O₂, which would then react with site-specifically bound ferrous iron as proposed by Stadtman and colleagues. This hypothesis was supported by the inhibitory effect of catalase on diquat-mediated protein carbonyl formation. However, exogenous H₂O₂ alone did not induce protein carbonyl formation. Hydroxyl radical-generating reactions may result from the H₂O₂-catalyzed oxidation of ferrous iron, which normally is bound to protein in the ferric state. Therefore, the possible reduction of site-specifically bound Fe³⁺ to Fe²⁺ by the diquat cation radical (which could then react with H₂O₂) was also investigated. The combination of H₂O₂ and an iron reductant, ascorbate, however, also failed to induce significant protein carbonyl formation. In a phospholipid-containing system, an ADP:Fe²⁺ complex induced both lipid peroxidation and protein carbonyl formation; both indices were largely inhibitable by antioxidants. There was no substantial ADP:Fe²⁺-dependent protein carbonyl formation in the absence of phospholipid under otherwise identical conditions. Based on the lipid requirement and antioxidant sensitivity, these data suggest that ADP:Fe²⁺-dependent protein carbonyl formation occurs through reaction of BSA with aldehydic lipid peroxidation products. The precise mechanism of diquat-mediated protein carbonyl formation remains unclear, but it appears not to be a function of H₂O₂ generation or diquat cation radical-dependent reduction of bound Fe³⁺. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 185–190, 1998