Roles of catalase and cytochrome C in hydroperoxide-dependent lipid peroxidation and chemiluminescence in rat heart and kidney mitochondria

A recent report (Radi et al., J. Biol. Chem. 266:22028–22034, 1991) showed that rat heart mitochondria contain catalase. The protective role of mitochondrial catalase was tested by exposing heart or kidney mitochondria and mitoplasts to two oxidants (H₂O₂) or tert-butyl hydroperoxide, t-BOOH), estimating lipid peroxidation (as thiobarbituric acid-reactive substances, TBARS) and overall oxidative stress (as chemiluminescence). Additional controls included heart and kidney preparations from aminotriazole-treated (catalase-depleted) rats. Both oxidants increased TBARS in catalase-free preparations to similar extents over their respective controls (between 200 to 350%). In catalase-containing preparations, H₂O₂ lipid peroxidation increased by only 40 to 96% over controls. Similar qualitative results were obtained when measuring chemiluminescence. The catalytic role of cytochrome c in mitochondrial lipid peroxidation was investigated by exposing either control or cytochrome-c-depleted kidney mitoplasts (catalase free) to either H₂O₂ or t-BOOH. Hydrogen-peroxide-dependent mitochondrial lipid peroxidation varied with cytochrome c concentrations, remaining close to controls when cytochrome c concentration decreased by 66%, even though there was no catalase present. Tert-butyl hydroperoxide-dependent lipid peroxidation was less affected by cytochrome c remaining 2.3-fold above controls under the same conditions, suggesting that organic peroxides are more likely to remain in the less polar membrane environment being decomposed by heme or nonheme iron imbedded in the inner mitochondrial membrane. Chemiluminescence was less affected by cytochrome c depletion. Comparing control and cytochrome-c-deficient mitochondria, chemiluminescence was 1.7-fold and 2.8-fold higher when control preparations were challenged with t-BOOH or H₂O₂, respectively.     

tert–butylhydroperoxide—Induced toxicity in isolated hepatocytes: Contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also, occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide–induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

Characterization of a putative thioredoxin peroxidase prx1 of <Emphasis Type="Italic">Candida albicans</Emphasis>

In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H₂O₂ scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H₂O₂, menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H₂O₂. These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H₂O₂, but its cellular function is specified to t-BOOH.     

Resolution of renal sulfhydryl oxidase from gamma-glutamyltransferase by covalent chromatography on cysteinylsuccinamidopropyl-glass

Sulfhydryl oxidase from bovine kidney cortex was purified 2500-fold by covalent chromatography using cysteinylsuccinamidopropyl-glass. GSH oxidation catalyzed by the resulting preparation was found to be totally enzymatic, as judged by the inability of the preparation to reduce nitro blue tetrazolium, and H₂O₂ was found to be a product, as had been previously observed with milk sulfhydryl oxidase. No GSH peroxidase activity could be detected, using either H₂O₂ or t-butylhydroperoxide. The chromatographically purified renal sulfhydryl oxidase was resolved from γ-glutamyltransferase as evidenced by a 12,000-fold increase in ratio of the two enzymatic activities over that exhibited by crude kidney homogenates, and antibodies raised against purified milk sulfhydryl oxidase cross-reacted with the kidney oxidase, but not the kidney transferase.     

Extreme differences between Hemoglobins I and II of the Clam Lucina pectinalis in their reactions with nitrite

The clam Lucina pectinalis supports its symbiotic bacteria by H₂S transport in the open and accessible heme pocket of Lucina Hb I and by O₂ transport in the narrow and crowded heme pocket of Lucina Hb II. Remarkably, air-equilibrated samples of Lucina Hb I were found to be more rapidly oxidized by nitrite than any previously studied Hb, while those of Lucina Hb II showed an unprecedented resistance to oxidation induced by nitrite. Nitrite-induced oxidation of Lucina Hb II was enabled only when O₂ was removed from its active site. Structural analysis revealed that O₂ “clams up” the active site by hydrogen bond formation to B10Tyr and other distal-side residues. Quaternary effects further restrict nitrite entry into the active site and stabilize the hydrogen-bonding network in oxygenated Lucina Hb II dimers. The dramatic differences in nitrite reactivities of the Lucina Hbs are not related to their O₂ affinities or anaerobic redox potentials, which were found to be similar, but are instead a result of differences in accessibility of nitrite to their active sites; i.e. these differences are due to a kinetic rather than thermodynamic effect. Comparative studies revealed heme accessibility to be a factor in human Hb oxidation by nitrite as well, as evidenced by variations of rates of nitrite-induced oxidation that do not correlate with R and T state differences and inhibition of oxidation rate in the presence of O₂. These results provide a dramatic illustration of how evolution of active sites with varied heme accessibility can moderate the rates of inner-sphere oxidative reactions of Hb and other heme proteins.     

Hydrogen peroxide-induced oxidative damages in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H₂O₂-treated and non-treated control cells. It was observed that increased H₂O₂ concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells.     

Spontaneous and radical-induced plasma membrane lipid peroxidation in differently oxidant-sensitive yeast species and its suppression by antioxidants

Formation of thiobarbituric acid-reactive substances (TBRS; nmol/mg lipids) indicative of lipid peroxidation was measured in whole cells and in isolated plasma membrane lipids from three yeast species differing in oxidant sensitivity (Schizosaccharomyces pombe, Saccharomyces cerevisiae andRhodotorula glutinis) after exposure to the Fenton reagent, Fe^II, H₂O₂,tert-butyl hydroperoxide (TBHP) and azo compounds (AAPH, ACHN). In whole cells, spontaneous TBRS formation rose in the sequenceS. pombe<S. cerevisiae<R. glutinis (1:∼5:∼7). Oxidants increased the TBRS production 13–18 fold in the sequence Fe^II∼TBHP>AAPH∼ACHN∼Fe-Fenton>H₂O₂. This increase need not be solely due to increased lipid peroxidation. In isolated plasma membrane lipids from all three species, the spontaneous TBRS production referred to 1 mg lipids was 9–13-fold higher than in whole cells. InS. pombe lipids, only TBHP increased the TBRS production. In lipids fromS. cerevisiae andR. glutinis, all added oxidants increased the spontaneous TBRS production 2–3 times in the sequence TBHP>ACHN>AAPH>Fe^II>Fe-Fenton>H₂O₂. Oxidant-induced TBRS production in both whole cells and isolated membrane lipids was partially suppressed by the lipid peroxidation inhibitors 2,6-di-tert-butyl-4-methylphenol (“butylated hydroxytoluene”; BHT) and the newly synthesized PYA12 compound. Both agents were more effective in isolated lipids than in whole cells and against OH-producing than against ROO-or RO-producing oxidants. Yeast membrane lipids, which are generally poor in polyunsaturated fatty acids, are thus subject to perceptible lipid peroxidation.     

Triosephosphate isomerase tyrosine nitration induced by heme–NaNO2–H2O2 or peroxynitrite: Effects of different natural phenolic compounds

Peroxynitrite and heme peroxidases (or heme)–H₂O₂–NaNO₂ system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways‐induced protein nitration and oxidation are controversial. Both nitrating systems can dose‐dependently induce triosephosphate isomerase (TIM) nitration, however, heme–H₂O₂–NaNO₂ was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3‐morpholinosydnonimine hydrochloride (SIN‐1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme–H₂O₂–NaNO₂ and SIN‐1 and protein oxidation induced by SIN‐1, but promoted heme–H₂O₂–NaNO₂‐induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN‐1‐induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme–H₂O₂–NaNO₂‐induced nitration. Our results indicated that peroxynitrite and heme–H₂O₂–NaNO₂‐induced protein nitration was different, and the later one could be a better model for anti‐nitration compounds screening.     

Hydrogen peroxide pretreatment induces osmotic stress tolerance by influencing osmolyte and abscisic acid levels in maize leaves

Hydrogen peroxide (H₂O₂) functions as a signal molecule in plants under abiotic and biotic stresses. Leaves of detached maize (Zea mays L.) seedlings were used to study the function of H₂O₂ pretreatment in osmotic stress resistance. Low H₂O₂ concentration (10 mM) which did not cause a visual symptom of water deficit (leaf rolling) was applied to the seedlings. Exogenous H₂O₂ alone increased leaf water potential, endogenous H₂O₂ content, abscisic acid (ABA) concentration, and metabolite levels including soluble sugars, proline, and polyamines while it decreased lipid peroxidation and stomatal conductance. Osmotic stress induced by polyethylene glycol (PEG 6000) decreased leaf water potential and stomatal conductance but enhanced lipid peroxidation, endogenous H₂O₂ content, the metabolite levels, and ABA content. H₂O₂ pretreatment also induced the metabolite accumulation and improved water status, stomatal conductance, lipid peroxidation, ABA, and H₂O₂ levels under osmotic stress. These results indicated that H₂O₂ pretreatment may alleviate water loss and induce osmotic stress resistance by increasing the levels of soluble sugars, proline, and polyamines thus ABA and H₂O₂ production slightly decrease in maize seedlings under osmotic stress.     

Oxygen binding to partially nitrosylated hemoglobin

Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb–NO). Many aspects of the formation and persistence of Hb–NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O₂. Partially nitrosylated Hb samples had predominantly hexacoordinate NO–heme geometry and resisted oxidation when exposed to O₂ in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO–heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO–heme geometry also affected O₂ binding equilibria. O₂-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O₂ affinity T-state of the Hb by increasing percentages of NO–heme, much as occurs with increasing levels of CO–heme. Samples containing IHP showed small decreases in O₂ affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O₂-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO–heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O₂ transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Response to oxidative stress as a welfare parameter in swine

Summary

Pro-oxidant effects of hemoglobin-derived heme and iron contribute to the progressive damage observed in β thalassemic and sickle (HbS) red blood cells. Agents that prevent heme/iron release and inhibit their redox activity might diminish such injury. Consequently, the inhibitory effects of chloroquine (CQ), a heme-binding antimalarial drug, and a novel dichloroquine compound (CQ-D2) on iron release and lipid peroxidation were investigated. In contrast to normal hemoglobin, significant amounts of iron were released from both purified hemin and α-hemoglobin chains during incubations with exogenous reduced glutathione (GSH) and/or H₂O₂. Addition of either CQ or CQ-D2 effectively inhibited GSH- and GSH/H₂O₂-mediated iron release from hemin (P<0.001). During prolonged incubations (6 h), both CQ and CQ-D2 significantly decreased the release of heme-free iron from both purified hemoglobin and α-hemoglobin chains. Interestingly, CQ and CQ-D2 differentially affected the redox availability of the heme-bound iron. The CQ: heme complex significantly enhanced membrane lipid peroxidation whereas CQ-D2 dramatically (P<0.001) inhibited heme-dependent peroxidation to almost baseline levels. In summary, CQ-derivatives which render heme redox inert and prevent the release of free iron from heme might be beneficial in the treatment of certain hemoglobinopathies and, perhaps, other pathologies promoted by delocalized heme/iron.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

Myoglobin-catalyzed epoxidation of styrene in organic solvents accelerated by bioimprinting

Myoglobin imprinted in aqueous solution with ligands binding to its heme iron, followed by lyophilization, catalyzed the epoxidation of styrene with H₂O₂or tert-butyl hydroperoxide in organic solvents much faster than the non-imprinted protein under otherwise the same conditions.     

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.     

Sickle Cell Hemoglobin in the Ferryl State Promotes βCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10)

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H₂O₂) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H₂O₂ molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H₂O₂ at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized βCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.     

Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

The deleterious effects of H₂O₂ on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H₂O₂ resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H₂O₂ on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H₂O₂ on respiratory complexes was almost completely recovered by β-mercaptoethanol treatment. H₂O₂ treatment resulted in full resistance to Q_O site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Q_O) of complex III is the target for H₂O₂. H₂O₂ did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H₂O₂ addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.     

Distinct roles of peroxynitrite and hydroxyl radical in triggering stunned myocardium-like impairment of cardiac myocytes in vitro

Myocardial stunning is characterized by the impairment of excitation-contraction coupling via a decrease in myofilament Ca²⁺ responsiveness, thought to be triggered by hydroxyl radicals (·OH) generated upon reperfusion. Since peroxynitrite is also expected to be produced during reperfusion, we examined whether it can induce a stunned myocardium-like impairment of cardiac myocytes. Its effect on cultured cardiac myocytes was compared with that of hydrogen peroxide (H₂O₂), ·OH source. Infusion of peroxynitrite (0.2 mM) induced a decrease in cell motion and a complete arrest in diastole at 2.9 ± 0.3 min, which coincided with an elevation in [Ca²⁺]_i. Arrest induced by infusion of H²O² (10 mM) was not associated with an increase in [Ca²⁺]_i. The ATP content was unaffected by peroxynitrite (control, 34.3 ± 3.4: + peroxynitrite, 32.9 ± 3.5 nmol/mg protein) and the cells remained viable. Sulfhydryl (SH) content was decreased by peroxynitrite, but not by H₂O₂. The membrane fluidity (a measure of peroxidation of the membrane lipids) was not affected by peroxynitrite, but was decreased by H₂O₂. Onset time of arrest was unaffected by deferoxamine (0.2 mM), but was delayed by DTT (10 mM) (from 2.9 ± 0.3 to 19.2 ± 1.6 min). Nitrotyrosine content was unchanged by peroxynitrite, and its augmentation with Fe³⁺/EDTA (1 mM) was not associated with a shortened onset time of arrest. The function of the Na⁺/Ca²⁺ exchanger was impaired by peroxynitrite, but not by H₂O₂. Peroxynitrite and H₂O₂ each induce arrest, but only the former increases [Ca²⁺]_i. One of the mechanisms of the increase in [Ca²⁺]_i is Na⁺/Ca²⁺ exchanger dysfunction. The impairments were induced through SH oxidation by peroxynitrite, but through lipid peroxidation by H₂O₂. Myocardial stunning may be induced by both species in concert.     

Effect of Cadmium Ions on Antioxidant Defense System in Sunflower Cotyledons

Sunflower (Helianthus annuus L.) seeds were germinated and grown in the presence of 50, 100 and 200 μM CdCl₂. The lower concentration (50 μM) of Cd² ions produced slight decrease in reduced glutathione (GSH) content and overall increase (except superoxide dismutase) in antioxidant enzyme activities, and in H₂O₂ concentration. Chlorophyll content, lipid peroxidation and protein oxidation were not affected under 50 μM CdCl₂. GSH content was diminished under 100 and 200 μM CdCl₂, and except for superoxide dismutase, which activity remained unaltered, overall decreases in the antioxidant enzyme activities (catalase, ascorbate peroxidase, dehydroascorbate peroxidase, glutathione reductase) and in guaiacol peroxidase were observed. These Cd² concentrations caused a decrease in chlorophyll content as well as an increase in lipid peroxidation, protein oxidation and H₂O₂ concentration. All the observed effects were more evident with the highest concentration of cadmium chloride used. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synthesis and antioxidative activity of metalloporphyrins bearing 2,6-di-tert-butylphenol pendants

The novel metalloporphyrins (M = HH, Fe, Mn, Co, Cu, Zn) bearing 2,6-di-tert-butylphenol pendants as antioxidant substituents, and a long chain hydrocarbon palmitoyl group have been synthesized. The oxidation of compounds by PbO₂ leads to the formation of the corresponding 2,6-di-tert-butylphenoxyl radicals studied by EPR. The activity of porphyrins in lipid peroxidation has been examined using (1) in vitro lipid peroxidation induced by tert-butylhydroperoxide in respiring rat liver mitochondria, (2) in vitro lipid peroxidation in liver homogenates of Wistar strain rats, and (3) a model process of peroxidation of (Z)-octadec-9-enic (oleic) acid as a structural fragment of lipids. The activity of these compounds depends dramatically on the nature of metal and might be changed from antioxidative (M = HH, Mn, Cu, Zn) to indifferent (M = Co), and to pro-oxidative one (M = Fe). The anti- or pro-oxidative action of these compounds may be derived from the concurrence between the involvement of 2,6-di-tert-butylphenol pendants acting as radical scavengers and redox active metal center promoting oxidation processes. The results of this study suggest that the polytopic compounds combining in one molecule 2,6-di-tert-butylphenol pendants, metalloporphyrin moiety, and a palmitoyl group, are membrane active compounds and might be studied in an effort to find novel pharmaceutical agents.     

Nitric oxide acts as an antioxidant and delays methyl jasmonate-induced senescence of rice leaves

In the present study, we evaluate the protective effect of nitric oxide (NO) against senescence of rice leaves promoted by methyl jasmonate (MJ). Senescence of rice leaves was determined by the decrease of protein content. MJ treatment resulted in (1) induction of leaf senescence, (2) increase in H₂O₂ and malondialdehyde (MDA) contents, (3) decrease in reduced form glutathione (GSH) and ascorbic acid (AsA) contents, and (4) increase in antioxidative enzyme activities (ascorbate peroxidase, glutathione reductase, peroxidase and catalase). All these MJ effects were reduced by free radical scavengers such as sodium benzoate and GSH. NO donors [N-tert-butyl-α-phenylnitrone (PBN), sodium nitroprusside, 3-morpholinosydonimine, and AsA+NaNO₂] were effective in reducing MJ-induced leaf senescence. PBN prevented MJ-induced increase in the contents of H₂O₂ and MDA, decrease in the contents of GSH and AsA, and increase in the activities of antioxidative enzymes. The protective effect of PBN on MJ-promoted senescence, MJ-increased H₂O₂ content and lipid peroxidation, MJ-decreased GSH and AsA, and MJ-increased antioxidative enzyme activities was reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, a NO-specific scavenger, suggesting that the protective effect of PBN is attributable to NO released. Reduction of MJ-induced senescence by NO in rice leaves is most likely mediated through its ability to scavenge active oxygen species including H₂O₂