Reactive oxygen species generated by the mitochondrial respiratory chain affect the complex III activity via cardiolipin peroxidation in beef-heart submitochondrial particles

The aim of this study was to investigate the effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of complex III and on the cardiolipin content in bovine-heart submitochondrial particles (SMP). ROS were produced by treatment of nicotinamide adenine dinucleotide (NADH) respiring SMP with rotenone. This treatment resulted in a production of superoxide anion, detected by the epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial-mediated ROS generation resulted in a marked loss of complex III activity and in a parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD + catalase. Exogenous added cardiolipin was able to almost completely prevent the ROS-mediated loss of complex III activity. No effect was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, or with peroxidized cardiolipin. The results demonstrate that mitochondrial-mediated ROS generation affects the activity of complex III via peroxidation of cardiolipin, which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those physiopathological conditions characterized by an increase in the basal production of ROS such as aging, ischemia/reperfusion and chronic degenerative diseases.     

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.     

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.     

Xanthohumol induces generation of reactive oxygen species and triggers apoptosis through inhibition of mitochondrial electron transfer chain complex I

Xanthohumol is a prenylflavonoid extracted from hops (Humulus lupulus). It possesses anti-cancer and anti-inflammatory activities in vitro and in vivo, and offers therapeutic benefits for treatment of metabolic syndromes. However, the precise mechanisms underlying its pharmacological effects remain to be elucidated, together with its cellular target. Here, we provide evidence that xanthohumol directly interacts with the mitochondrial electron transfer chain complex I (NADH dehydrogenase), inhibits the oxidative phosphorylation, triggers the production of reactive oxygen species, and induces apoptosis. In addition, we show that as a result of the inhibition of the mitochondrial oxidative phosphorylation, xanthohumol exposure causes a rapid decrease of mitochondrial transmembrane potential. Furthermore, we showed that xanthohumol up-regulates the glycolytic capacity in cells, and thus compensates cellular ATP generation. Dissection of the multiple steps of aerobic respiration by extracellular flux assays revealed that xanthohumol specifically inhibits the activity of mitochondrial complex I, but had little effect on that of complex II, III and IV. Inhibition of complex I by xanthohumol caused the overproduction of reactive oxygen species, which are responsible for the induction of apoptosis in cancer cells. We also found that isoxanthohumol, the structural isomer of xanthohumol, is inactive to cells, suggesting that the reactive 2-hydroxyl group of xanthohumol is crucial for its targeting to the mitochondrial complex I. Together, the remodeling of cell metabolism revealed here has therapeutic potential for the use of xanthohumol.     

Reactive oxygen species and oxidative stress

Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) and the antioxidant capacity of the cell. For long, ROS have been considered as harmful by-products of the normal aerobic metabolism process of the mitochondria, implicated in a large variety of diseases. But there are now growing evidences that controlled ROS production also play physiological roles especially in regulating cell redox homeostasis and cell signaling. Biological ROS effects are now well documented. Data show that living organisms have not only adapted themselves to coexist with free radicals but have also developed mechanisms to use them advantageously. However their main sources and mechanisms of action remain poorly described. This review focuses on the main properties of ROS and their paradoxical effects.     

Reactive oxygen species and mitochondrial diseases

A variety of diseases have been associated with excessive reactive oxygen species (ROS), which are produced mostly in the mitochondria as byproducts of normal cell respiration. The interrelationship between ROS and mitochondria suggests shared pathogenic mechanisms in mitochondrial and ROS-related diseases. Defects in oxidative phosphorylation can increase ROS production, whereas ROS-mediated damage to biomolecules can have direct effects on the components of the electron transport system. Here, we review the molecular mechanisms of ROS production and damage, as well as the existing evidence of mitochondrial ROS involvement in human diseases.     

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis.

Cytochrome c release from mitochondria is a critical event in the apoptosis induction. Dissociation of cytochrome c from the mitochondrial inner membrane (IMM) is a necessary first step for cytochrome c release. In the present study, the effect of reactive oxygen species (ROS) on the dissociation of cytochrome c from beef-heart submitochondrial particles (SMP) and on the cardiolipin content was investigated. Exposure of SMP to mitochondrial-mediated ROS generation resulted in a large dissociation of cytochrome c from SMP and in a parallel loss of cardiolipin. Both these effects were directly and significantly correlated and also abolished by superoxide dismutase+catalase. These results demonstrate that ROS generation induces the dissociation of cytochrome c from IMM via cardiolipin peroxidation. The data may prove useful in clarifying the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol.     

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.     

Reactive oxygen species differentially affect T cell receptor-signaling pathways

Oxidative stress plays an important role in the induction of T lymphocyte hyporesponsiveness observed in several human pathologies including cancer, rheumatoid arthritis, leprosy, and AIDS. To investigate the molecular basis of oxidative stress-induced T cell hyporesponsiveness, we have developed an in vitro system in which T lymphocytes are rendered hyporesponsive by co-culture with oxygen radical-producing activated neutrophils. We have observed a direct correlation between the level of T cell hyporesponsiveness induced and the concentration of reactive oxygen species produced. Moreover, induction of T cell hyporesponsiveness is blocked by addition of N-acetyl cysteine, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, and catalase, confirming the critical role of oxidative stress in this system. The pattern of tyrosine-phosphorylated proteins was profoundly altered in hyporesponsive as compared with normal T cells. In hyporesponsive T cells, T cell receptor (TCR) ligation no longer induced phospholipase C-gamma1 activation and caused reduced Ca(2+) flux. In contrast, despite increased levels of ERK1/2 phosphorylation, TCR-dependent activation of mitogen-activated protein kinase ERK1/2 was unaltered in hyporesponsive T lymphocytes. A late TCR-signaling event such as caspase 3 activation was as well unaffected in hyporesponsive T lymphocytes. Our data indicate that TCR-signaling pathways are differentially affected by physiological levels of oxidative stress and would suggest that although "hyporesponsive" T cells have lost certain effector functions, they may have maintained or gained others.     

The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles

The effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of cytochrome c oxidase and on the cardiolipin content in bovine heart submitochondrial particles (SMP) was studied. ROS were produced by treatment of succinate-respiring SMP with antimycin A. This treatment resulted in a large production of superoxide anion, measured by epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial mediated ROS generation, led to a marked loss of cytochrome c oxidase activity and to a parallel loss of cardiolipin content. Both these effects were completely abolished by SOD+catalase. Added cardiolipin was able to almost completely restore the ROS-induced loss of cytochrome c oxidase activity. No restoration was obtained with peroxidized cardiolipin. These results demonstrate that mitochondrial mediated ROS generation affects the activity of cytochrome c oxidase via peroxidation of cardiolipin which is needed for the optimal functioning of this enzyme complex. These results may prove useful in probing molecular mechanism of ROS-induced peroxidative damage to mitochondria which have been proposed to contribute to aging, ischemia/reperfusion and chronic degenerative diseases.     

Reactive oxygen species production by forward and reverse electron fluxes in the mitochondrial respiratory chain

Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD(+) reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.     

Enhancement of oxidative damage to cultured cells and Caenorhabditis elegans by mitochondrial electron transport inhibitors

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in primary and cultured cells as well as in the nematode Caenorhabditis elegans (C. elegans) treated simultaneously with electron transport inhibitors and oxygen gas. Oxygen loading enhanced the damage of PC 12 cells by thenoyltrifluoroacetone (TTFA, a complex II inhibitor), but did not by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and sodium azide (a complex IV inhibitor). In primary hepatocytes, the enhancement was observed with the addition of sodium azide and rotenone, but not by TTFA or antimycin. In the nematode, only rotenone and TTFA enhanced the sensitivity under hyperoxia. These results demonstrate that highly specific inhibitors of electron transport can induce oxygen hypersensitivity in cell levels such as PC 12 cells and primary hepatocytes, and animal level of C. elegans. In addition the cell damage is different dependent on cell type and organism.     

Reaction of complex I of the mitochondrial electron transport chain with artificial oxidizers

The steady-state kinetics of oxidation of the mitochondrial NADH: ubiquinone oxidoreductase (complex I, EC 1.6.99.3) by artificial electron acceptors--p-quinones and inorganic complexes has been investigated. A limiting stage in the NADH: ferricyanide reductase reaction is a reductive half-reaction. Ferricyanide interacts with negative-charged protein groups taking part in the NADH binding. The rate constants of the quinone reduction by complex I vary from 1.10(6) to 4.10(3) M-1s-1. The NADH, NAD+ and ADP-ribose inhibition data indicate that oxidizers in the rotenono-insensitive reaction interact with the redox centre near the NAD+/NADH binding site, most probably with FMN.     

Reactive oxygen species regulate autophagy through redox-sensitive proteases

Starvation induces autophagy through a signal transduction pathway that is not fully understood. In a recent issue of The EMBO Journal, Scherz-Shouval and colleagues (Scherz-Shouval et al., 2007) show that reactive oxygen species (ROS) occurring during starvation serve as signaling molecules that initiate autophagy.     

Reactive oxygen species and DNA damage after ultrasound exposure

The aim of this work was to detect the formation of hydrogen peroxide and hydroxyl radicals after ultrasound (US) exposure and test the hypothesis that reactive oxygen species induced by ultrasound can contribute to DNA damage. Formation of reactive oxygen species was observed in incubated medium after sonication with 1 MHz continuous ultrasound at the intensities of 0.61-2.44 W/cm2. Free radicals and hydrogen peroxide produced by ultrasound exposure of cells can lead to DNA damage. Comet assay was used to assess the effect of ultrasound on the level of nuclear DNA damage. The nucleated erythrocytes from fish were exposed in vitro to ultrasound at the same intensities and frequency. It was noticed that ultrasound in all used intensities induced DNA damage. The effect was not eliminated by the addition of catalase, which indicates that DNA damage was not caused by hydrogen peroxide only. The results showed that the DNA damage can be repair and this mechanism was the most effective after 30 and 60 min after sonication. Furthermore, the ultrasound-induced DNA damage in the presence of sonosensitizer (Zn- and AlCl-phthalocyanine) was studied. It was noticed that phthalocyaniens (Pcs) alone or with ultrasound did not induce significant changes in the level of DNA damage.     

Shared evolutionary footprints suggest mitochondrial oxidative damage underlies multiple complex I losses in fungi

Oxidative phosphorylation is among the most conserved mitochondrial pathways. However, one of the cornerstones of this pathway, the multi-protein complex NADH : ubiquinone oxidoreductase (complex I) has been lost multiple independent times in diverse eukaryotic lineages. The causes and consequences of these convergent losses remain poorly understood. Here, we used a comparative genomics approach to reconstruct evolutionary paths leading to complex I loss and infer possible evolutionary scenarios. By mining available mitochondrial and nuclear genomes, we identified eight independent events of mitochondrial complex I loss across eukaryotes, of which six occurred in fungal lineages. We focused on three recent loss events that affect closely related fungal species, and inferred genomic changes convergently associated with complex I loss. Based on these results, we predict novel complex I functional partners and relate the loss of complex I with the presence of increased mitochondrial antioxidants, higher fermentative capabilities, duplications of alternative dehydrogenases, loss of alternative oxidases and adaptation to antifungal compounds. To explain these findings, we hypothesize that a combination of previously acquired compensatory mechanisms and exposure to environmental triggers of oxidative stress (such as hypoxia and/or toxic chemicals) induced complex I loss in fungi.     

Mitochondrial dysfunction in rat brain with aging Involvement of complex I, reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered a key factor in brain aging process. Mitochondrial respiration is an important site of ROS production and hence a potential contributor to brain functional changes with aging. In this study we examined the effect of aging on complex I activity, oxygen consumption, ROS production and phospholipid composition in rat brain mitochondria. The activity of complex I was reduced by 30% in brain mitochondria from 24 months aged rats relative to young animals. These changes in complex I activity were associated with parallel changes in state 3 respiration. H(2)O(2) generation was significantly increased in mitochondria isolated from aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 31% as function of aging, while there was a significant increase in the level of peroxidized cardiolipin. The age-related decrease in complex I activity in brain mitochondria could be reversed by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids. It is proposed that aging causes brain mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with brain aging.     

TNF-induced mitochondrial damage: a link between mitochondrial complex I activity and left ventricular dysfunction

Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction. This study was conducted to determine whether a TNF-induced increase in superoxide (O₂^?) contributes to mitochondrial damage in the left ventricle (LV) by impairing respiratory complex I activity. We employed an electron paramagnetic resonance (EPR) method to measure O₂^? and oxygen consumption in mitochondrial respiratory complexes, using an oxygen label. Adult male Sprague–Dawley rats were divided into four groups: control, TNF treatment (ip), TNF+ apocynin (APO; 200 μmol/kg bw, orally), and TNF+ Tempol (Temp; 300 μmol/kg bw, orally). TNF was injected daily for 5 days. Rats were sacrificed, LV tissue was collected, and mitochondria were isolated for EPR studies. Total LV ROS production was significantly higher in TNF animals than in controls; APO or Temp treatment ameliorated TNF-induced LV ROS production. Total mitochondrial ROS production was significantly higher in the TNF and TNF+ APO groups than in the control and TNF+ Temp groups. These findings suggest that TNF alters the cellular redox state, reduces the expression of four complex I subunits by increasing mitochondrial O₂^? production and depleting ATP synthesis, and decreases oxygen consumption, thereby resulting in mitochondrial damage and leading to LV dysfunction.