Nitric oxide and hydrogen peroxide in tomato resistance. Nitric oxide modulates hydrogen peroxide level in o-hydroxyethylorutin-induced resistance to Botrytis cinerea in tomato.

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for activation of disease resistance reactions of plants to infection with a pathogen or elicitor treatment. However, biochemical mechanisms by which ROS and NO participate in these reactions are still under intensive study and controversial debate. We previously demonstrated that o-hydroxyethylorutin when applied on tomato leaves (Lycopersicon esculentum Mill. cv. "Perkoz") restricted Botrytis cinerea infection development. In this research we investigated ROS and NO generation in tomato plants treated with o-hydroxyethylorutin, non-treated and infected ones. The NO content was enhanced or decreased in the studied plants by supplying them with NO generator-SNP or scavenger-cPTIO. NO detection was carried out using diaminofluorescein diacetate (DAF-DA) in conjunction with confocal laser scanning microscopy. The influence of elevated and decreased levels of NO on B. cinerea infection development and ROS generation was studied. The elevated NO concentration in tomato leaves strongly decreased hydrogen peroxide concentration without affecting other studied ROS (superoxide anion and hydroxyl radical) levels. H2O2 concentrations in NO-supplied leaves were low regardless of further treatment of tomato leaves with o-hydroxyethylorutin or inoculation with B. cinerea. The low H2O2 concentration coincided with quick and severe infection development in NO-supplied leaves. As activities of enzymes generating (SOD EC 1.15.1.1)) and removing (APX EC 1.11.1.11, CAT EC 1.11.1.6) H2O2 were unchanged in the studied plants, the decrease in H2O2 concentration was probably due to a direct NO-H2O2 interaction.     

Ammonium-dependent hydrogen peroxide production by mitochondria

NADH-supported generation of H2O2 by permeabilized rat heart mitochondria was partially prevented by the specific complex I-directed inhibitor, NADH-OH, and was significantly stimulated by ammonium. Ammonium did not affect H2O2 production by complex I in coupled submitochondrial particles. The soluble mitochondrial matrix protein fraction catalyzed NADH-dependent H2O2 production, which was greatly (approximately 10-fold) stimulated by ammonium. We conclude that complex I is not the major contributor to mitochondrial superoxide (hydrogen peroxide) generation and that there are specific ammonium-sensitive NADH:oxygen oxidoreductase(s) in the mitochondrial matrix which are responsible for mitochondrial H2O2 production.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Sites of superoxide and hydrogen peroxide production during fatty acid oxidation in rat skeletal muscle mitochondria

H₂O₂ production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H₂O₂ production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H₂O₂ production was dominated by complex II (44% from site II_F in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site I_F). With added carnitine, H₂O₂ production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site III_Qo) and 25% from site I_F. Thus complex II (site II_F in the forward reaction) is a major source of H₂O₂ production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H₂O₂ production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site II_F in the reverse reaction). About half the rest was from site I_F, but a significant portion, ∼40 pmol H₂O₂·min⁻¹·mg protein⁻¹, was not from complex I, II, or III and was attributed to the proteins of β-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was ∼200 pmol H₂O₂·min⁻¹·mg protein⁻¹ under conditions of compromised antioxidant defense and reduced ubiquinone pool. Thus complex II and the ETF system both contribute to H₂O₂ productionduring fatty acid oxidation under appropriate conditions.     

Superoxide and hydrogen peroxide production by Drosophila mitochondria

Drosophila melanogaster is a key model organism for genetic investigation of the role of free radicals in aging, but biochemical understanding is lacking. Superoxide production by Drosophila mitochondria was measured fluorometrically as hydrogen peroxide, using its dependence on substrates, inhibitors, and added superoxide dismutase to determine sites of production and their topology. Glycerol 3-phosphate dehydrogenase and center o of complex III in the presence of antimycin had the greatest maximum capacities to generate superoxide on the cytosolic side of the inner membrane. Complex I had significant capacity on the matrix side. Center i of complex III, cytochrome c, and complex IV produced no superoxide. Native superoxide generation by isolated mitochondria was also measured without added inhibitors. There was a high rate of superoxide production with sn-glycerol 3-phosphate as substrate; two-thirds mostly from glycerol 3-phosphate dehydrogenase on the cytosolic side and one-third on the matrix side from complex I following reverse electron transport. There was little superoxide production from any site with NADH-linked substrate. Superoxide production by complex I following reverse electron flow from glycerol 3-phosphate was particularly sensitive to membrane potential, decreasing 70% when potential decreased 10 mV, showing that mild uncoupling lowers superoxide production in the matrix very effectively.     

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.     

Effect of beta-lapachone on superoxide anion and hydrogen peroxide production in Trypanosoma cruzi.

Addition of beta-lapachone, an o-naphthoquinone endowed with trypanocidal properties to respiring Trypanosoma cruzi epimastigotes induced the release of O2- and H2O2 from the whole cells to the suspending medium. The same beta-lapachone concentration (4 micron) that released H2O2 at maximal rate completely inhibited T. cruzi growth in a liquid medium. The position isomer, alpha-lapachone, did not stimulate O2- and H2O2 release, and did not inhibit epimastigote growth. beta-Lapachone was able to stimulate H2O2 production by the epimastigote homogenate in the presence of NADH as reductant. The same effect was observed with the mitochondrial fraction supplemented with NADH, where beta-lapachone enhanced the generation of O2- and H2O2 4.5- and 2.5-fold respectively. beta-Lapachone also increased O2- and H2O2 production (2.5 and 2-fold respectively) by the microsomal fraction with NADPH as reductant. Cyanide-insensitive NADH and NADPH oxidation by the mitochondrial and microsomal fractions (quinone reductase activity) was stimulated to about the same extent by beta-lapachone. alpha-Lapachone was unable to increase O2- and H2O2 production and quinone reductase activity of the mitochondrial and microsomal fractions.     

Nitric oxide inhibits superoxide production by inflammatory polymorphonuclear leukocytes

Nitric oxide(NO ·) has a complex role in the inflammatory response. Inthis study, we modified the levels of endogenous NO · in vivoin an acute model of inflammation and evaluated the interactionsbetween NO · and superoxide anion() produced bypolymorphonuclear leukocytes (PMNs) accumulated in the inflamed area.We injected phosphate-buffered saline (control group), 6 µmol ofL-N⁵-(1-iminoethyl)ornithine(L-NIO group), or 6 µmol ofL-arginine (L-arginine group) into thegranuloma pouch induced by carrageenan in rats. plus (indicative of NO · generation) was 188 nmol in the exudate of the control group, but itdecreased in the L-NIO group(P < 0.05) and increased in theL-arginine group(P < 0.05). When PMNsfrom treated rats were incubated in vitro, the productionof superoxide anion () decreased by ~46% in theL-arginine group. Furthermore, was inhibited in PMNs whenL-arginine was addedto the incubation medium before phorbol 12-myristate 13-acetatestimulation but not when added simultaneously. Our results suggest aprotective role for NO · in inflammation, through theinactivation of NADPH oxidase and the consequent impairment of production for cell-mediatedinjury.

     

Superoxide and hydrogen peroxide production in cyanide resistant Arum maculatum mitochondria

Submitochondrial particles from Arum maculatum containing a powerful cyanide insensitive oxidase were assayed by various methods to determine the end product of its interaction with oxygen. Using cytochrome c peroxidase to assay the production of H₂O₂ it was possible to detect H₂O₂ formation by Arum submitochondrial particles oxidizing NADH but not when oxidizing succinate. The rate of production of H₂O₂, however, was insufficient to account for the rate of oxygen uptake due to the alternate oxidase. The production of superoxide was determined using the luminol and adrenochrome assays. It was found that some superoxide was produced when Arum submitochondrial particles oxidized NADH but not when they oxidized succinate and again at insufficient rates to account for the rate of oxygen uptake by the alternate oxidase. stoichiometric determination of the ratio of NADH oxidized to oxygen taken up in the presence of 1 mM KCN, sufficient to inhibit catalase activity such that added peroxide remains stable, showed H₂O to be the only detectable product.It is suggested that although both H₂O₂ and superoxide are produced by A. maculatum submitochondrial particles this is not due to the alternate oxidase but may be due to another component of the respiratory chain possibly at the level of the NADH dehydrogenase.     

Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress

We have determined the underlying sites of H(2)O(2) generation by isolated rat brain mitochondria and how these can shift depending on the presence of respiratory substrates, electron transport chain modulators and exposure to stressors. H(2)O(2) production was determined using the fluorogenic Amplex red and peroxidase system. H(2)O(2) production was higher when succinate was used as a respiratory substrate than with another FAD-dependent substrate, alpha-glycerophosphate, or with the NAD-dependent substrates, glutamate/malate. Depolarization by the uncoupler p-trifluoromethoxyphenylhydrazone decreased H(2)O(2) production stimulated by all respiratory substrates. H(2)O(2) production supported by succinate during reverse transfer of electrons was decreased by inhibitors of complex I (rotenone and diphenyleneiodonium) whereas in glutamate/malate-oxidizing mitochondria diphenyleneiodonium decreased while rotenone increased H(2)O(2) generation. The complex III inhibitors antimycin and myxothiazol decreased succinate-induced H(2)O(2) production but stimulated H(2)O(2) production in glutamate/malate-oxidizing mitochondria. Antimycin and myxothiazol also increased H(2)O(2) production in mitochondria using alpha-glycerophosphate as a respiratory substrate. In substrate/inhibitor experiments maximal stimulation of H(2)O(2) production by complex I was observed with the alpha-glycerophosphate/antimycin combination. In addition, three forms of in vitro mitochondrial stress were studied: Ca(2+) overload, cold storage for more than 24 h and cytochrome c depletion. In each case we observed (i) a decrease in succinate-supported H(2)O(2) production by complex I and an increase in succinate-supported H(2)O(2) production by complex III, (ii) increased glutamate/malate-induced H(2)O(2) generation by complex I and (iii) increased alpha-glycerophosphate-supported H(2)O(2) generation by complex III. Our results suggest that all three forms of mitochondrial stress resulted in similar shifts in the localization of sites of H(2)O(2) generation and that, in both normal and stressed states, the level and location of H(2)O(2) production depend on the predominant energetic substrate.     

Nitric oxide, mitochondria and neurological disease

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurological disorders, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, stroke and amyotrophic lateral sclerosis. There is also a growing body of evidence to implicate excessive or inappropriate generation of nitric oxide (NO) in these disorders. It is now well documented that NO and its toxic metabolite, peroxynitrite (ONOO-), can inhibit components of the mitochondrial respiratory chain leading, if damage is severe enough, to a cellular energy deficiency state. Within the brain, the susceptibility of different brain cell types to NO and ONOO- exposure may be dependent on factors such as the intracellular reduced glutathione (GSH) concentration and an ability to increase glycolytic flux in the face of mitochondrial damage. Thus neurones, in contrast to astrocytes, appear particularly vulnerable to the action of these molecules. Following cytokine exposure, astrocytes can increase NO generation, due to de novo synthesis of the inducible form of nitric oxide synthase (NOS). Whilst the NO/ONOO- so formed may not affect astrocyte survival, these molecules may diffuse out to cause mitochondrial damage, and possibly cell death, to other cells, such as neurones, in close proximity. Evidence is now available to support this scenario for neurological disorders, such as multiple sclerosis. In other conditions, such as ischaemia, increased availability of glutamate may lead to an activation of a calcium-dependent nitric oxide synthase associated with neurones. Such increased/inappropriate NO formation may contribute to energy depletion and neuronal cell death. The evidence available for NO/ONOO--mediated mitochondrial damage in various neurological disorders is considered and potential therapeutic strategies are proposed.     

Redox cycling of anthracyclines by cardiac mitochondria. II. Formation of superoxide anion, hydrogen peroxide, and hydroxyl radical

In the accompanying paper (Davies, K. J. A., and Doroshow, J. A. (1986) J. Biol. Chem. 261, 3060-3067), we have demonstrated that anthracycline antibiotics are reduced to the semiquinone form at Complex I of the mitochondrial electron transport chain. In the experiments presented in this study we examined the effects of doxorubicin (Adriamycin), daunorubicin, and related quinonoid anticancer agents on superoxide, hydrogen peroxide, and hydroxyl radical production by preparations of beef heart submitochondrial particles. Superoxide anion formation was stimulated from (mean +/- S.E.) 1.6 +/- 0.2 to 69.6 +/- 2.7 or 32.1 +/- 1.5 nmol X min-1 X mg-1 by the addition of 90 microM doxorubicin or daunorubicin, respectively. However, the anthracycline 5-iminodaunorubicin, in which an imine group has been substituted in the C ring quinone moiety, did not increase superoxide production over control levels. In the presence of rotenone, initial rates of oxygen consumption and superoxide formation were identical under comparable experimental conditions. Furthermore, H2O2 production increased from undetectable control levels to 2.2 +/- 0.3 nmol X min-1 X mg-1 after treatment of submitochondrial particles with doxorubicin (200 microM). The hydroxyl radical, or a related chemical oxidant, was also detected after the addition of an anthracycline to this system by both ESR spectroscopy using the spin trap 5,5-dimethylpyrroline-N-oxide and by gas chromatographic quantitation of CH4 produced from dimethyl sulfoxide. Hydroxyl radical production, which was iron-dependent in this system, occurred in a nonlinear fashion with an initial lag phase due to a requirement for H2O2 accumulation. We also found that two quinonoid anti-cancer agents which produce less cardiotoxicity than the anthracyclines, mitomycin C, and mitoxantrone, stimulated significantly less or no hydroxyl radical production by submitochondrial particles. These experiments suggest that injury to cardiac mitochondria which is produced by anthracycline antibiotics may result from the generation of the hydroxyl radical during anthracycline metabolism by NADH dehydrogenase.     

Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψ_m) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψ_m and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψ_m was detected via safranin fluorescence and a TPP⁺ electrode, the rate of H₂O₂ formation was measured by Amplex UltraRed, the intramitochondrial pH (pH_in) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K⁺/H⁺ exchanger, nigericin lowered pH_in and ΔpH, followed by a compensatory increase in Δψ_m that led to an augmented H₂O₂ production. Valinomycin, a K⁺ ionophore, at low [K⁺] increased ΔpH and pH_in, decreased Δψ_m, which resulted in a decline in H₂O₂ formation. It was concluded that Δψ_m is dominant over ?pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H₂O₂ release and a decreased ?pH. This phenomenon reveals that from the pH component not ?pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψ_m might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury.     

Proton leak and hydrogen peroxide production in liver mitochondria from energy-restricted rats

Energy restriction (ER), without malnutrition, is the only environmental intervention that consistently increases maximum life span in laboratory rodents. One theory proposes that a reduction in energy expenditure and reactive oxygen species production is the mechanism responsible for this action of ER. To further test this theory, proton leak, H2O2 production, lipid peroxidation, and protein carbonyls were measured in mitochondria from FBNF1 rats fed either a control or 40% ER diet (onset at 6 mo of age). Liver mitochondria were isolated at 7 and 12 mo of age. Liver weight decreased 25 and 36% at 1 and 6 mo of ER, respectively (P < 0.05). ER resulted in an increase (P < 0.05) in percent total polyunsaturates, n-6 polyunsaturates, and total unsaturates (6 mo only) in mitochondrial lipids. These changes, however, were not associated with significant alterations in mitochondrial function. State 4 respiration and membrane potential were not different (P > 0.05) between groups at either assessment period. Similarly, proton leak kinetics were not different between control and ER animals. Top-down metabolic control analysis and its extension, elasticity analysis, were used at the 6-mo assessment and revealed no difference in control of the oxidative phosphorylation system between control and ER rats. H2O2 production with either succinate or pyruvate/malate substrates was also not different (P > 0.05) between groups at either time point. In conclusion, ER did not alter proton leak or H2O2 production at this age or stage of restriction in liver.     

Cytochrome oxidase-catalyzed superoxide generation from hydrogen peroxide.

Superoxide dismutase is shown to affect spectral changes observed upon cytochrome c oxidase reaction with H2O2, which indicates a possibility of O2- radicals being formed in the reaction. Using DMPO as a spin trap, generation of superoxide radicals from H2O2 in the presence of cytochrome oxidase is directly demonstrated. The process is inhibited by cyanide and is not observed with a heat-denatured enzyme pointing to a specific reaction in the oxygen-reducing centre of cytochrome c oxidase. The data support a hypothesis on a catalase cycle catalyzed by cytochrome c oxidase in the presence of excess H2O2 (Vygodina and Konstantinov (1988) Ann. NY Acad. Sci., 550, 124-138): (formula: see text)     

Nitric oxide induces apoptosis via hydrogen peroxide, but necrosis via energy and thiol depletion

We investigated the mechanisms by which two nitric oxide (NO) donors, diethylenetriamine/NO adduct (DETA/NO) and S-nitrosoglutathione (GSNO), induced cell death in a J774 macrophage cell line. Both NO donors induced caspase activation within 6 h, but only DETA/NO-induced caspase activation was sensitive to inhibition of p38 and was completely prevented by antioxidants catalase, ascorbate, dehydroascorbate, or N-acetylcysteine, suggesting that DETA/NO-induced apoptosis may be mediated by H(2)O(2). Consistent with this, DETA/NO acutely stimulated reactive oxygen species (ROS) production by mitochondria and cells, and inhibited catalase-mediated H(2)O(2) breakdown in cells. After prolonged, 24 h exposure of cells to DETA/NO, inactivation of caspases occurred, which was accompanied by an increase in necrosis. DETA/NO-induced necrosis was insensitive to caspase inhibitors, but was partially prevented by catalase or N-acetylcysteine, and was preceded by inhibition of glyceraldehyde-3-phosphate dehydrogenase and a decrease in cellular adenosine triphosphate (ATP). GSNO was even more potent in inhibiting glycolysis and switching apoptosis to necrosis. In cells depleted of glutathione, GSNO and DETA/NO induced rapid necrosis, which resulted from rapid depletion of ATP due to inhibition of glycolysis. Glycolytic intermediate 3-phosphoglycerate decreased DETA/NO-induced necrosis and increased apoptosis. We conclude that: (i). NO-induced apoptosis is mediated by H(2)O(2); (ii). NO-induced necrosis is mediated by energy failure speeded by thiol depletion.