Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.     

The quantitative measurement of H2O2 generation in isolated mitochondria

Adequate methods to measure the rate of mitochondrial oxygen radical generation are needed since oxygen radicals are involved in many pathologies. A fluorometric method appropriate to measure the rate of generation of H₂O₂ in intact mitochondria is described. Just after isolation of functional mitochondria from fresh tissues, rates of generation of H₂O₂ are kinetically measured by fluorometry in the presence of homovanillic acid and horseradish peroxidase. The method is specific for H₂O₂ and is sensitive enough to assay mitochondrial H₂O₂ generation in the presence of respiratory substrate without inhibitors of the respiratory chain. Simultaneous measurement of mitochondrial oxygen consumption allows calculation of the free radical leak: the percentage of electrons out of sequence which reduce oxygen to oxygen radicals along the mitochondrial respiratory chain instead of reducing oxygen to water at the terminal cytochrome oxidase. The method shows instantaneous response to H₂O₂. This makes it appropriate to study the quick effects of different inhibitors and modulators on the rate of mitochondrial oxygen radical production. Its application to the localization of the sites where caloric restriction decreases mitochondrial oxygen radical generation in heart mitochondria is described.     

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.     

H2O2 production and cytochrome c peroxidase activity in mitochondria isolated from the trypanosomatid hemoflagellate Crithidia fasciculata

H₂O₂ production by coupled mitochondrial fractions from the protozoan, Crithidia fasciculata, has been measured spectrophotometrically by the formation of the stable enzyme-substrate complex with yeast cytochrome c peroxidase. H₂O₂ formation was observed with succinate, l-α-glycerophosphate, l-proline, α-ketoglutarate, and with endogenous substrate. The maximum rate of H₂O₂ generation obtained with each substrate in the presence of antimycin A was about 10% of the state 4 rate of O₂ respiration, and only 1–2% of the carbonylcyanide m-fluorophenylhydrazone-uncoupled respiratory rate. Therefore, excess O₂ uptake due to the formation of H₂O₂ cannot satisfactorily account for the low ADP:O ratios previously reported.Cytochrome c peroxidase activity was measured in mitochondrial preparations by recording the decrease in absorbance at 550 nm during the oxidation of horse heart ferrocytochrome c which was observed after addition of H₂O₂. The distribution of activity after sonic disruption of mitochondrial preparations was that expected for a soluble enzyme. The activity was proportional to the amount of enzyme protein added, and was abolished by heating at 100 °C for 3 min. Total cytochrome c peroxidase activity in mitochondrial fractions isolated from C. fasciculata was calculated to be 0.3% that of isolated yeast mitochondria, but it is suggested that the in vivo activity may be considerably higher than this estimate.     

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H₂O₂ emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H₂O₂ emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.     

Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria

Rat and pigeon heart mitochondria supplemented with antimycin produce 0.3–1.0nmol of H₂O₂/min per mg of protein. These rates are stimulated up to 13-fold by addition of protophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chloromethoxyphenylhydrazone and pentachlorophenol). Ionophores, such as valinomycin and gramicidin, and Ca²⁺ also markedly stimulated H₂O₂ production by rat heart mitochondria. The enhancement of H₂O₂ generation in antimycin-supplemented mitochondria and the increased O₂ uptake of the State 4-to-State 3 transition showed similar protophore, ionophore and Ca²⁺ concentration dependencies. Thenoyltrifluoroacetone and N-bromosuccinimide, which inhibit succinate–ubiquinone reductase activity, also decreased mitochondrial H₂O₂ production. Addition of cyanide to antimycin-supplemented beef heart submitochondrial particles inhibited the generation of O₂⁻, the precursor of mitochondrial H₂O₂. This effect was parallel to the increase in cytochrome c reduction and it is interpreted as indicating the necessity of cytochrome c₁³⁺ to oxidize ubiquinol to ubisemiquinone, whose autoxidation yields O₂⁻. The effect of protophores, ionophores and Ca²⁺ is analysed in relation to the propositions of a cyclic mechanism for the interaction of ubiquinone with succinate dehydrogenase and cytochromes b and c₁ [Wikstrom & Berden (1972) Biochim. Biophys. Acta 283, 403–420; Mitchell (1976) J. Theor. Biol. 62, 337–367]. A collapse in membrane potential, increasing the rate of ubisemiquinone formation and O₂⁻ production, is proposed as the molecular mechanism for the enhancement of H₂O₂ formation rates observed on addition of protophores, ionophores and Ca²⁺.     

Dependence of H2O2 Formation by Rat Heart Mitochondria on Substrate Availability and Donor Age

We have examined the substrate specificity and inhibitor sensitivity of H₂O₂ formation by rat heart mitochondria. Active H₂O₂ production requires both a high fractional reduction of Complex I (indexed by NADH/NAD⁺ + NADH ratio) and a high membrane potential, . These conditions are achieved with supraphysiological concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of H₂O₂ formation are much lower (less than 0.1% of respiratory chain electron flux) but may be stimulated by the Complex III inhibitor antimycin A, but not by myxothiazol. Addition of Mn²⁺ to give 10 nmol/mg of mitochondrial protein enhances H₂O₂ production with all substrate combinations, possibly by repleting mitochondrial superoxide dismutase with this cation. Contrary to previously published work, no increased activity of H₂O₂ production was found with heart mitochondria from senescent (24 month) rats, relative to young adults (6 month).     

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.     

Antioxidant enzymes, hydrogen peroxide metabolism, and respiration in rat heart during experimental hyperammonemia

The effects of toxic ammonia doses on H₂O₂ metabolism, energy metabolism, and antioxidant enzyme activities in rat heart were studied. Ammonium acetate administration to animals proved to increase total superoxide dismutase (SOD), catalase, and glutathione peroxidase activities in the heart cytoplasmic fraction as well as Mn-SOD, catalase, and glutathione reductase in heart mitochondria. Conversely, ammonia inhibited the same activities in the brain, liver, and erythrocytes. Hyperammonemia had no effect on the levels of ATP, ADP and total adenine nucleotides in the heart but decreased them in the brain. Ammonia impaired oxidative phosphorylation and increased the rate of H₂O₂ production in heart and brain mitochondria. The ammonia concentration inhibiting antioxidant enzymes in the liver and brain can be insufficient for such effect in the heart.     

Pyridine nucleotides and H2 as electron donors to the respiratory and photosynthetic electron-transfer chains and to nitrogenase in Anabaena heterocysts

The pathways through which NADPH, NADH and H₂ provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O₂ uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H₂ supported cyanide-sensitive, respiratory O₂ uptake and light-enhanced, cyanide-insensitive uptake of O₂ resulting from electron donation to O₂ at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O₂ or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H₂ supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H₂ as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented.     

Effects of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> extract on heart and liver mitochondrial functions: mechanism(s) of action

Though extracts of Ginkgo biloba leaves (GBE) have a wide pharmacological application, little is known about GBE effects on mitochondria. In this work, effects of ethanolic GBE on the respiration of isolated rat heart and liver mitochondria were investigated. We found that GBE stimulates the pyruvate + malate-dependent State 2 respiration of heart mitochondria and decreases mitochondrial membrane potential. Uncoupling effect of GBE was found to be due to its protonophoric action and is likely to be mediated by the ATP/ADP-translocator and uncoupling proteins. The effect of GBE was less in liver than in heart mitochondria. State 3 respiration of heart mitochondria was slightly stimulated at low and depressed at higher GBE concentrations. Inhibition of State 3 respiration of heart mitochondria was not relieved by uncoupler indicating that GBE may inhibit the respiratory chain complexes or the substrate transport. However, Complex IV of the respiratory chain was not inhibited by GBE. H₂O₂ generation was attenuated by low concentration of GBE probably due to mild uncoupling. The data suggest that mild but not severe uncoupling activity of GBE may be important in providing pharmacological protection of cellular functions in pathological situations.     

Lipoperoxide radical production during oxidation of cardiolipin in the complex with cytochrome c

The key stage of apoptosis is lipid peroxidation which causes cytochrome c efflux from mitochondria. Cardiolipin-bound cytochrome c on the surface of the inner mitochondrial membrane is supposed to be a main lipoperoxidation catalyst. In this work, lipoperoxide radical (LOO·) production in the complex of cytochrome c (Cyt C) with bovine heart cardiolipin (BCL) was investigated with the method of chemiluminescence (CL) in the presence of a physical activator, coumarin dye C-525. It was shown that a CL flash with a half quenching time of 1.12 min was observed after the addition of Cyt C to a BCL+C-525 solution in the absence of hydrogen peroxide. At H₂O₂ concentrations of 0.1–0.5 mM, quenching time reduced at constant CL flash amplitude and at H₂O₂ concentrations of 1–5 mM, the amplitude of CL increased with the growth of peroxide concentration. It testifies to different mechanisms of BCL oxidation: the lipoxygenase mechanism in the absence of H₂O₂ and at low H₂O₂ concentrations, and the peroxidase mechanism at higher H₂O₂ concentrations. When small H₂O₂ amounts were added, another CL flash was observed in the course of a lipoxygenase reaction whose light sum increased with time in parallel with the extent of the following inhibition of CL. Iron chelators EDTA and o-phenanthroline made no significant effect on the CL associated with cytochrome c lipoxygenase action, while desferal, a well-known peroxidase and lipoxygenase inhibitor, inhibited CL by half in a concentration of 18 μM. A scheme of reactions resulting in LOO· radical production on BCL oxidation by the Cyt C-cardiolipin complex in the absence and in the presence of H₂O₂ was suggested.     

Heat-shock response protects peripheral blood mononuclear cells (PBMCs) from hydrogen peroxide-induced mitochondrial disturbance

The present study was designed to investigate ex vivo the protective mechanisms of heat-shock response against H₂O₂-induced oxidative stress in peripheral blood mononuclear cells (PBMCs) of rats. Twenty-four hours later, heat-shock treatment was executed in vivo; rat PBMCs were collected and treated with H₂O₂. The accumulation of reactive oxygen species and the mitochondrial membrane potential were evaluated by intracellular fluorescent dHE and JC-1 dye staining, respectively, and expression of HSP72 and cytochrome c was detected by Western blot analysis. Cellular apoptosis was assayed by TUNEL staining and double staining of Annexin V and PI. The results showed that H₂O₂-induced oxidative stress leads to intracellular superoxide accumulation and collapse of the mitochondrial membrane potential in rat PBMCs. Moreover, cellular apoptosis was detected after H₂O₂ treatment, and the release of mitochondrial cytochrome c from mitochondria to cytosol was significantly enhanced. Heat-shock pretreatment decreases the accumulation of intracellular superoxide in PBMCs during H₂O₂-induced oxidative stress. Moreover, heat-shock treatment prevents the collapse of the mitochondrial membrane potential and cytochrome c release from mitochondria during H₂O₂-induced oxidative stress. In conclusion, mitochondria are critical organelles of the protective effects of heat-shock treatment. Cellular apoptosis during H₂O₂-induced oxidative stress is decreased by heat-shock treatment through a decrease in superoxide induction and preservation of the mitochondrial membrane potential.     

Mitochondrial Biosvnthesis Controls the Sensitivitvy of Chinese Hamster Cells to Hydrogen Peroxide

The mechanism of H₂O₂-resistance of Hpr-4, a variant of Chinese hamster V79 cells, was investigated. The effect of H₂O₂ on the mitochondria of the parental and Hpr-4 cells was compared. First, both biochemical and ultrastructural results showed that mitochondria in the parental cells were damaged by exposure to H₂O₂, while those in Hpr-4 cells recovered from the damage. Second, the H₂O₂ resistance of Hpr-4 cells was reversibly reduced or recovered by the addition or removal of inhibitors of mitochondrial biosynthesis, respectively. Third, the parental cells were auxotrophic to pyruvate after exposure to H₂O₂. Fourth, H₂O₂-sensitivity of the parental cells was also enhanced by the inhibition of mitochondrial biosynthesis. From these results, it was concluded that the mitochondria of Hpr-4 cells apparently had a greater resistance to H₂O₂ than those of the parental cells and that functional mitochondria were involved in the recovery of Chinese hamster V79 cells from H₂O₂-induced damage.     

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (H₂O₂). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O₂⁻/H₂O₂ formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O₂⁻/H₂O₂ formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H₂O₂. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O₂⁻/H₂O₂ production by KGDHC, induced a ~ 86% and ~ 84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O₂⁻/H₂O₂ formation by ~ 45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O₂⁻/H₂O₂ production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H₂O₂.     

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K<Superscript>+</Superscript> channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H₂O₂ release using different substrates and ATP-sensitive K⁺ transport activities are increased in mitochondria from animals on high fat diets. The increase in H₂O₂ release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K⁺ channels, indicating it was not related to an observed increase in K⁺ transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K⁺ transport in mitochondria can be modulated by diet.     

Selective cytochemical localization of peroxidase,cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine

Summary In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H₂O₂. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H₂O₂. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Parthenolide regulates oxidative stress-induced mitophagy and suppresses apoptosis through p53 signaling pathway in C2C12 myoblasts

Muscle redox disturbances and oxidative stress have emerged as a common pathogenetic mechanism and potential therapeutic intervention in some muscle diseases. Parthenolide (PTL), a sesquiterpene lactone found in large amounts in the leaves of feverfew, possesses anti-inflammatory, anti-migraine, and anticancer properties. Although PTL was reported to alleviate cancer cachexia and improve skeletal muscle characteristics in a cancer cachexia model, its actions on oxidative stress-induced damage in C2C12 myoblasts have not been reported and the regulatory mechanisms have not yet been defined. In our study, PTL attenuated H₂O₂-induced growth inhibition and morphological changes. Furthermore, PTL exhibited scavenging activity against reactive oxygen species and protected C2C12 cells from apoptosis in response to H₂O₂. Meanwhile, PTL suppressed collapse of the mitochondrial membrane potential, thereby contributing to normalizing H₂O₂-induced autophagy flux and mitophagy, correlating with inhibiting degradation of mitochondrial marker protein TIM23, the increase in LC3-II expression and the reduction of mitochondria DNA. Besides its protective effect on mitochondria, PTL also prevented H₂O₂-induced lysosomes damage in C2C12 cells. In addition, the phosphorylation of p53, cathepsin B, and Bax/Bcl-2 protein levels, and the translocation of Bax from the cytosol to mitochondria induced by H₂O₂ in C2C12 cells was significantly reduced by PTL. In conclusion, PTL modulates oxidative stress-induced mitophagy and protects C2C12 myoblasts against apoptosis, suggesting a potential protective effect against oxidative stress-associated skeletal muscle diseases.     

Nicotinamide Nucleotide Transhydrogenase (Nnt) Links the Substrate Requirement in Brain Mitochondria for Hydrogen Peroxide Removal to the Thioredoxin/Peroxiredoxin (Trx/Prx) System

Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H₂O₂) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H₂O₂ catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP⁺, provides the link between mitochondrial respiration and H₂O₂ detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H₂O₂ in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H₂O₂, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H₂O₂ catabolism, (b) decreased NADPH and increased NADP⁺ levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H₂O₂ and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H₂O₂ antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases.     

Modulation of peroxynitrite produced via mitochondrial nitric oxide synthesis during Ca2+ and succinate-induced oxidative stress in cardiac isolated mitochondria

We hypothesized that NO^• is generated in isolated cardiac mitochondria as the source for ONOO⁻ production during oxidative stress. We monitored generation of ONOO⁻ from guinea pig isolated cardiac mitochondria subjected to excess Ca²⁺ uptake before adding succinate and determined if ONOO⁻ production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl₂ and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O₂^•−. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO⁻ to form dityrosine (diTyr) in proportion to the ONOO⁻ present. We found that exposing mitochondria to excess CaCl₂ before succinate resulted in an increase in diTyr and amplex red fluorescence (H₂O₂) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa²⁺ and succinate, increased mitochondrial ONOO⁻ production via NO^• and O₂^•−. Changes in mitochondrial ONOO⁻ production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO^• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO⁻ production, PTIO decreased production of ONOO⁻, and TEMPOL decreased ONOO⁻ levels by converting more O₂^•− to H₂O₂. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO⁻ into the buffer is dependent both on O₂^•− released from mitochondria and NO^• derived from a mtCa²⁺-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.