Diphenyleneiodonium acutely inhibits reactive oxygen species production by mitochondrial complex I during reverse, but not forward electron transport

We investigated the effects of diphenyleneiodonium (DPI) on superoxide production by complex I in mitochondria isolated from rat skeletal muscle. Superoxide production was measured indirectly as hydrogen peroxide production. In a conventional medium containing chloride, DPI strongly inhibited superoxide production by complex I driven by reverse electron transport from succinate. In principle, this inhibition could be explained by an observed decrease in the mitochondrial pH gradient caused by the known chloride-hydroxide antiport activity of DPI. In a medium containing gluconate instead of chloride, DPI did not affect the pH gradient. In this gluconate medium, DPI still inhibited superoxide production driven by reverse electron transport, showing that the inhibition of superoxide production was not dependent on changes in the pH gradient. It had no effect on superoxide production during forward electron transport from NAD-linked substrates in the presence of rotenone (to maximise superoxide production from the flavin of complex I) or antimycin (to maximise superoxide production from complex III), suggesting that the effects of DPI were not through inhibition of the flavin. We conclude that DPI has the novel and potentially very useful ability to prevent superoxide production from the site in complex I that is active during reverse electron transport, without affecting superoxide production during forward electron transport.     

Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism

The recent knowledge on mitochondria as the substantial source of reactive oxygen species, namely superoxide and hydrogen peroxide efflux from mitochondria, is reviewed, as well as nitric oxide and subsequent peroxynitrite generation in mitochondria and their effects. The reactive oxygen species formation in extramitochondrial locations, in peroxisomes, by cytochrome P450, and NADPH oxidase reaction, is also briefly discussed. Conditions are pointed out under which mitochondria represent the major ROS source for the cell: higher percentage of non-phosphorylating and coupled mitochondria, in vivo oxygen levels leading to increased intensity of the reverse electron transport in the respiratory chain, and nitric oxide effects on the redox state of cytochromes. We formulate hypotheses on the crucial role of ROS generated in mitochondria for the whole cell and organism, in concert with extramitochondrial ROS and antioxidant defense. We hypothesize that a sudden decline of mitochondrial ROS production converts cells or their microenvironment into a “ROS sink” represented by the instantly released excessive capacity of ROS-detoxification mechanisms. A partial but immediate decline of mitochondrial ROS production may be triggered by activation of mitochondrial uncoupling, specifically by activation of recruited or constitutively present uncoupling proteins such as UCP2, which may counterbalance the mild oxidative stress.     

Peroxisomes and reactive oxygen species,a lasting challenge

Oxidases generating and enzymes scavenging H₂O₂ predestine peroxisomes (PO) to a pivotal organelle in oxygen metabolism. Catalase, the classical marker enzyme of PO, exhibits both catalatic and peroxidatic activity. The latter is responsible for the staining with 3,3′-diamino-benzidine, which greatly facilitated the visualization of the organelle and promoted further studies on PO. d-Amino acid oxidase catalyzes with strict stereospecificity the oxidative deamination of d-amino acids. The oxidase is significantly more active in the kidney than in liver and more in periportal than pericentral rat hepatocytes. Peroxisomes in these tissues differ in their enzyme activity and protein concentration not only in adjacent cells but even within the same one. Moreover, the enzyme appears preferentially concentrated in the central region of the peroxisomal matrix compartment. Urate oxidase, a cuproprotein catalyzing the oxidation of urate to allantoin, is confined to the peroxisomal core, yet is lacking in human PO. Recent experiments revealed that cores in rat hepatocytes appear in close association with the peroxisomal membrane releasing H₂O₂ generated by urate oxidase to the surrounding cytoplasma. Xanthine oxidase is exclusively located to cores, oxidizes xanthine thereby generating H₂O₂ and O₂ ^– radicals. The latter are converted to O₂ and H₂O₂ by CuZn superoxide dismutase, which has been shown recently to be a bona fide peroxisomal protein. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1-4, 2008.     

In vitro reactive oxygen species production by histatins and copper(I,II)

The ability of the histidine-rich peptides, histatin-5 (Hst-5) and histatin-8 (Hst-8), to support the generation of reactive oxygen species during the Cu-catalyzed oxidation of ascorbate and cysteine has been evaluated. High levels of hydrogen peroxide (70–580 mol/mol Cu/h) are produced by aqueous solutions containing Cu(II), Hst-8 or Hst-5, and a reductant, either ascorbate or cysteine, as determined by the postreaction Amplex Red assay. When the reactions are conducted in the presence of superoxide dismutase, the total hydrogen peroxide produced is decreased, more so in the presence of the peptides (up to 50%), suggesting the intermediacy of superoxide in these reactions. On the other hand, the presence of sodium azide or sodium formate, traps for hydroxyl radicals, has no appreciable effect on the total hydrogen peroxide production for the Cu–Hst systems. EPR spin-trapping studies using 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) in the cysteine–Cu(II) reactions reveal the formation of the CYPMPO–hydroperoxyl and CYPMPO–hydroxyl radical adducts in the presence of Hst-8, whereas only the latter was observed with Cu alone.     

Interplay between bax, reactive oxygen species production, and cardiolipin oxidation during apoptosis

Bax/Bak activation and cardiolipin peroxidation are essential for cytochrome c release during apoptosis, yet, the link between them remains elusive. We report that sequence of events after exposure of mouse embryonic fibroblast (MEF) cells to actinomycin D followed the order: Bax translocation → superoxide production → cardiolipin peroxidation. Genetic ablation of Bax/Bak inhibited actinomycin D induced superoxide production and cardiolipin peroxidation. Rotenone caused robust superoxide generation but did not trigger cardiolipin peroxidation in Bax/Bak double knockout MEF cells. This suggests that, in addition to participating in ROS generation, Bax/Bak play another specific role in cardiolipin oxidation. In isolated mitochondria, recombinant Bax enhanced succinate induced cardiolipin oxidation and cytochrome c release. Mitochondrial peroxidase activity, likely involved in cardiolipin peroxidation, was enhanced upon incubation with recombinant Bax. Thus, cardiolipin peroxidation may be causatively and time-dependently related to Bax/Bak effects on ROS generation and peroxidase activation of cytochrome c.     

Polyglutamine expansion inhibits respiration by increasing reactive oxygen species in isolated mitochondria

Huntington’s disease results from expansion of the polyglutamine (PolyQ) domain in the huntingtin protein. Although the cellular mechanism by which pathologic-length PolyQ protein causes neurodegeneration is unclear, mitochondria appear central in pathogenesis. We demonstrate in isolated mitochondria that pathologic-length PolyQ protein directly inhibits ADP-dependent (state 3) mitochondrial respiration. Inhibition of mitochondrial respiration by PolyQ protein is not due to reduction in the activities of electron transport chain complexes, mitochondrial ATP synthase, or the adenine nucleotide translocase. We show that pathologic-length PolyQ protein increases the production of reactive oxygen species in isolated mitochondria. Impairment of state 3 mitochondrial respiration by PolyQ protein is reversed by addition of the antioxidants N-acetyl-l-cysteine or cytochrome c. We propose a model in which pathologic-length PolyQ protein directly inhibits mitochondrial function by inducing oxidative stress.     

Generation of reactive oxygen species by the mitochondrial electron transport chain

Generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain (ETC), which is composed of four multiprotein complexes named complex I-IV, is believed to be important in the aging process and in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. Previous studies have identified the ubiquinone of complex III and an unknown component of complex I as the major sites of ROS generation. Here we show that the physiologically relevant ROS generation supported by the complex II substrate succinate occurs at the flavin mononucleotide group (FMN) of complex I through reversed electron transfer, not at the ubiquinone of complex III as commonly believed. Indirect evidence indicates that the unknown ROS-generating site within complex I is also likely to be the FMN group. It is therefore suggested that the major physiologically and pathologically relevant ROS-generating site in mitochondria is limited to the FMN group of complex I. These new insights clarify an elusive target for intervening mitochondrial ROS-related processes or diseases.     

Phosphate increases mitochondrial reactive oxygen species release

The effects of inorganic phosphate (P_i), the main intracellular membrane permeable anion capable of altering mitochondrial pH gradients (ΔpH), were measured on mitochondrial H₂O₂ release. As expected, P_i decreased ΔpH and increased the electric membrane potential (ΔΨ). Mitochondrial H₂O₂ release was stimulated by P_i and also by its structural analogue arsenate. However, acetate, another membrane-permeable anion, did not stimulate mitochondrial H₂O₂ release. The stimulatory effect promoted by P_i was prevented by CCCP, which decreases transport of P_i across the inner mitochondrial membrane, indicating that P_i must be in the mitochondrial matrix to stimulate H₂O₂ release. In conclusion, we found that P_i and arsenate stimulate mitochondrial reactive oxygen release, an effect that may contribute towards oxidative stress under conditions such as ischemia/reperfusion, in which high-energy phosphate bonds are hydrolyzed.     

Molecular identification of the enzyme responsible for the mitochondrial NADH-supported ammonium-dependent hydrogen peroxide production

A homogeneous protein with a subunit apparent molecular mass of ∼50 kDa that catalyzes the previously described mitochondrial NADH-supported ammonium-stimulated hydrogen peroxide production (Grivennikova, V.G., Gecchini, G. and Vinogradov, A.D. (2008) FEBS Lett. 583, 1287–1291) was purified from the mitochondrial matrix of bovine heart. Chromatography of partially purified protein showed that the peaks of ammonium-stimulated NADH-dependent H₂O₂ production and that of NADH:lipoamide oxidoreductase activity coincided. The catalytic properties and mass spectrometry of the trypsin-digested protein revealed peptides that allowed identification of the protein as the Bos taurus dihydrolipoyl dehydrogenase.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Heat shock augments angiotensin II-induced vascular contraction through increased production of reactive oxygen species

A temporal increase in temperature triggers a series of stress responses and alters vascular smooth muscle (VSM) contraction induced by agonist stimulation. Here we examined the role of reactive oxygen species (ROS) in heat shock-dependent augmentation of angiotensin II (AngII)-induced VSM contraction. Endothelium-denuded rat aortic rings were treated with heat shock for 45 min at 42 °C and then subjected to assays for the production of force, ROS, and the expression of ROS-related enzymes. AngII-induced contraction was enhanced in heat shock-treated aorta. AngII-induced production of hydrogen peroxide and superoxide were elevated in response to the heat shock treatment. Pre-treatment with superoxide dismutases (SOD) mimetic and inhibitors for glutathione peroxidase and NADPH oxidase but not for xanthine oxidase eliminated an increase in the AngII-induced contraction in the heat shock-treated aorta. Heat shock increased the expression of p47phox, a cytosolic subunit of NADPH oxidase, but not Cu-Zn-SOD and Mn-SOD. In addition, heat shock increased contraction that was evoked by hydrogen peroxide and pyrogallol. These results suggest that heat shock causes an elevation of ROS as well as a sensitization of ROS signal resulting in an augmentation of VSM contraction in response to agonist.     

Fatty acids as modulators of the cellular production of reactive oxygen species

Long-chain nonesterified ("free") fatty acids (FFA) and some of their derivatives and metabolites can modify intracellular production of reactive oxygen species (ROS), in particular O(2)(-) and H(2)O(2). In mitochondria, FFA exert a dual effect on ROS production. Because of slowing down the rate of electron flow through Complexes I and III of the respiratory chain due to interaction within the complex subunit structure, and between Complexes III and IV due to release of cytochrome c from the inner membrane, FFA increase the rate of ROS generation in the forward mode of electron transport. On the other hand, due to their protonophoric action on the inner mitochondrial membrane ("mild uncoupling effect"), FFA strongly decrease ROS generation in the reverse mode of electron transport. In the plasma membrane of phagocytic neutrophils and a number of other types of cells, polyunsaturated FFA stimulate O(2)(-) generation by NADPH oxidase. These effects of FFA can modulate signaling functions of ROS and be, at least partly, responsible for their proapoptotic effects in several types of cells.     

线粒体电子传递链电子漏的化学发光测定

本实验用差速离心法分离正常大鼠肝脏和心肌线粒体 ,以lucigenin (探测超氧阴离子 )与luminol (探测过氧化氢 )为探剂 ,用化学发光法测定METC电子漏的生成。在反应体系中加入外源底物 ,其发光强度明显高于空白对照 (体系中无线粒体 )。在肝线粒体体系中 ,无论是lucigenin还是luminol诱发的发光 ,琥珀酸底物引起的发光强均要高于丙酮酸 /苹果酸引起的发光强度。在心肌线粒体 luminol体系中也有与肝线粒体相似的结果 ,在心肌线粒体 lucigenin体系中 ,加入外源底物丙酮酸 /苹果酸诱发的发光强度高于琥珀酸诱发的发光强度     

Arabidopsis AAL-toxin-resistant mutant atr1 shows enhanced tolerance to programmed cell death induced by reactive oxygen species

The fungal AAL-toxin triggers programmed cell death (PCD) through perturbations of sphingolipid metabolism in AAL-toxin-sensitive plants. While Arabidopsis is relatively insensitive to the toxin, the loh2 mutant exhibits increased susceptibility to AAL-toxin due to the knockout of a gene involved in sphingolipid metabolism. Genetic screening of mutagenized loh2 seeds resulted in the isolation of AAL-toxin-resistant mutant atr1.Atr1 displays a wild type phenotype when grown on soil but it develops less biomass than loh2 on media supplemented with 2% and 3% sucrose. Atr1 was also more tolerant to the reactive oxygen species-generating herbicides aminotriazole (AT) and paraquat. Microarray analyses of atr1 and loh2 under AT-treatment conditions that trigger cell death in loh2 and no visible damage in atr1 revealed genes specifically regulated in atr1 or loh2. In addition, most of the genes strongly downregulated in both mutants were related to cell wall extension and cell growth, consistent with the apparent and similar AT-induced cessation of growth in both mutants. This indicates that two different pathways, a first controlling growth inhibition and a second triggering cell death, are associated with AT-induced oxidative stress.     

The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.     

Effect of phenolic acids of microbial origin on production of reactive oxygen species in mitochondria and neutrophils

Cardiovascular death is frequently associated with atherosclerosis, a chronic multifactorial disease and a leading cause of death worldwide. Genetically engineered mouse models have proven useful for the study of the mechanisms underlying cardiovascular diseases. The apolipoprotein E-deficient mouse has been the most widely used animal model of atherosclerosis because it rapidly develops severe hypercholesterolemia and spontaneous atherosclerotic lesions similar to those observed in humans. In this review, we provide an overview of the cardiac and vascular phenotypes and discuss the interplay among nitric oxide, reactive oxygen species, aging and diet in the impairment of cardiovascular function in this mouse model.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified ("free") fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Modulation of mitochondrial K permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K⁺ current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K⁺ in the assay medium. The effects of p13 on ROS are mimicked by the K⁺ ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K⁺ vs. H⁺ currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K⁺ permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.     

Ionizing radiation accelerates Drp1-dependent mitochondrial fission, which involves delayed mitochondrial reactive oxygen species production in normal human fibroblast-like cells

Ionizing radiation is known to increase intracellular level of reactive oxygen species (ROS) through mitochondrial dysfunction. Although it has been as a basis of radiation-induced genetic instability, the mechanism involving mitochondrial dysfunction remains unclear. Here we studied the dynamics of mitochondrial structure in normal human fibroblast like cells exposed to ionizing radiation. Delayed mitochondrial production was peaked 3 days after irradiation, which was coupled with accelerated mitochondrial fission. We found that radiation exposure accumulated dynamin-related protein 1 (Drp1) to mitochondria. Knocking down of Drp1 expression prevented radiation induced acceleration of mitochondrial fission. Furthermore, knockdown of Drp1 significantly suppressed delayed production of mitochondrial . Since the loss of mitochondrial membrane potential, which was induced by radiation was prevented in cells knocking down of Drp1 expression, indicating that the excessive mitochondrial fission was involved in delayed mitochondrial dysfunction after irradiation.