Ca2+-induced oxidative stress in brain mitochondria treated with the respiratory chain inhibitor rotenone

In this study we show that micromolar Ca(2+) concentrations (>10 microM) strongly stimulate the release of reactive oxygen species (ROS) in rotenone-treated isolated rat forebrain mitochondria. Ca(2+)-stimulated mitochondrial ROS release was associated with membrane lipid peroxidation and was directly correlated with the degree of complex I inhibition by rotenone. On the other hand, Ca(2+) did not increase mitochondrial ROS release in the presence of the complex I inhibitor 1-methyl-4-phenylpyridinium. Cyclosporin A had no effect on Ca(2+)-stimulated mitochondrial ROS release in the presence of rotenone, indicating that mitochondrial permeability transition is not involved in this process. We hypothesized that Ca(2+)-induced mitochondrial oxidative stress associated with partial inhibition of complex I may be an important factor in neuronal cell death observed in the neurodegenerative disorder Parkinson's disease.     

Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death

Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.     

Quantitative relationship between inhibition of respiratory complexes and formation of reactive oxygen species in isolated nerve terminals

In this study reactive oxygen species (ROS) generated in the respiratory chain were measured and the quantitative relationship between inhibition of the respiratory chain complexes and ROS formation was investigated in isolated nerve terminals. We addressed to what extent complex I, III and IV,respectively, should be inhibited to cause ROS generation. For inhibition of complex I, III and IV, rotenone, antimycin and cyanide were used, respectively, and ROS formation was followed by measuring the activity of aconitase enzyme. ROS formation was not detected until complex III was inhibited by up to 71 +/- 4%, above that threshold inhibition, decrease in aconitase activity indicated an enhanced ROS generation. Similarly, threshold inhibition of complex IV caused an accelerated ROS production. By contrast, inactivation of complex I to a small extent (16 +/- 2%) resulted in a significant increase in ROS formation, and no clear threshold inhibition could be determined. However, the magnitude of ROS generated at complex I when it is completely inhibited is smaller than that observed when complex III or complex IV was fully inactivated. Our findings may add a novel aspect to the pathology of Parkinson's disease, showing that a moderate level of complex I inhibition characteristic in Parkinson's disease leads to significant ROS formation. The amount of ROS generated by complex I inhibition is sufficient to inhibit in situ the activity of endogenous aconitase.     

Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production

Hepatitis C infection causes a state of chronic oxidative stress, which may contribute to fibrosis and carcinogenesis in the liver. Previous studies have shown that expression of the HCV core protein in hepatoma cells depolarized mitochondria and increased reactive oxygen species (ROS) production, but the mechanisms of these effects are unknown. In this study we examined the properties of liver mitochondria from transgenic mice expressing HCV core protein, and from normal liver mitochondria incubated with recombinant core protein. Liver mitochondria from transgenic mice expressing the HCV proteins core, E1 and E2 demonstrated oxidation of the glutathione pool and a decrease in NADPH content. In addition, there was reduced activity of electron transport complex I, and increased ROS production from complex I substrates. There were no abnormalities observed in complex II or complex III function. Incubation of control mitochondria in vitro with recombinant core protein also caused glutathione oxidation, selective complex I inhibition, and increased ROS production. Proteinase K digestion of either transgenic mitochondria or control mitochondria incubated with core protein showed that core protein associates strongly with mitochondria, remains associated with the outer membrane, and is not taken up across the outer membrane. Core protein also increased Ca(2+) uptake into isolated mitochondria. These results suggest that interaction of core protein with mitochondria and subsequent oxidation of the glutathione pool and complex I inhibition may be an important cause of the oxidative stress seen in chronic hepatitis C.     

Heavy metals do not induce ROS production by mitochondrial respirasome

Since the discovery of the respirasome constituted by complexes I, III₂, and IV, its precise participation in mitochondrial bioenergetics is poorly understood. We previously determined a higher NADH:DBQ oxidoreductase activity coupled to a lower ROS production by the respirasome than the free complex I. Toxicological studies suggest that respiratory complexes are heavy metals target during mitochondrial intoxication increasing ROS production, reducing ATP synthesis and cell viability; however, the inhibition of respiratory complexes activities by heavy metals is still unknown. Here we showed a putative deactivation of the respirasomal-complex I by seven of the most toxicologically relevant heavy metals, without increasing the ROS production. Contrastingly, the free complex I was more resistant to heavy metals but was 30 times more ROS-producing. These results underlie the preventive role of the respirasome in mitochondrial electron leak and ROS production and recall its disassembled in some pathologies which involve mitochondrial damage and oxidative stress.     

Production of reactive oxygen species by mitochondria: central role of complex III

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.     

Reverse electron flow-mediated ROS generation in ischemia-damaged mitochondria: Role of complex I inhibition vs. depolarization of inner mitochondrial membrane

Background

The reverse electron flow-induced ROS generation (RFIR) is decreased in ischemia-damaged mitochondria. Cardiac ischemia leads to decreased complex I activity and depolarized inner mitochondrial membrane potential (ΔΨ) that are two key factors to affect the RFIR in isolated mitochondria. We asked if a partial inhibition of complex I activity without alteration of the ΔΨ is able to decrease the RFIR.

Methods

Cardiac mitochondria were isolated from mouse heart (C57BL/6) with and without ischemia. The rate of H₂O₂ production from mitochondria was determined using amplex red coupled with horseradish peroxidase. Mitochondria were isolated from the mitochondrial-targeted STAT3 overexpressing mouse (MLS-STAT3E) to clarify the role of partial complex I inhibition in RFIR production.

Results

The RFIR was decreased in ischemia-damaged mouse heart mitochondria with decreased complex I activity and depolarized ΔΨ. However, the RFIR was not altered in the MLS-STAT3E heart mitochondria with complex I defect but without depolarization of the ΔΨ. A slight depolarization of the ΔΨ in wild type mitochondria completely eliminated the RFIR.

Conclusions

The mild uncoupling but not the partially decreased complex I activity contributes to the observed decrease in RFIR in ischemia-damaged mitochondria.

General significance

The RFIR is less likely to be a key source of cardiac injury during reperfusion.     

Chemical genetics analysis of an aniline mustard anticancer agent reveals complex I of the electron transport chain as a target

The antitumor agent 11β (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11β against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11β; flow cytometry studies showed that 11β exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11β inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11β blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11β enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11β, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11β, which supplements conventional DNA adduct formation to promote cancer cell death.     

Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by approximately 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2*- generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4-6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.     

Ca2+-induced permeabilization promotes free radical release from rat brain mitochondria with partially inhibited complex I

Mitochondrial complex I dysfunction has been implicated in a number of brain pathologies, putatively owing to an increased rate of reactive oxygen species (ROS) release. However, the mechanisms regulating the ROS burden are poorly understood. In this study we investigated the effect of Ca2+ loads on ROS release from rat brain mitochondria with complex I partially inhibited by rotenone. The addition of 20 nm rotenone to brain mitochondria increased ROS release. Ca2+ (100 microm) alone had no effect on ROS release, but greatly potentiated the effects of rotenone. The effect of Ca2+ was decreased by ruthenium red. Ca2+-challenged mitochondria lose about 88% of their glutathione and 46% of their cytochrome c under these conditions, although this depends only on Ca2+ loading and not complex I inhibition. ADP in combination with oligomycin decreased the loss of glutathione and cytochrome c and free radical generation. Cyclosporin A alone was ineffective in preventing these effects, but augmented the protection provided by ADP and oligomycin. Non-specific permeabilization of mitochondria with alamethicin also increased the ROS signal, but only when combined with partial inhibition of complex I. These results demonstrate that Ca2+ can greatly increase ROS release by brain mitochondria when complex I is impaired.     

Mitochondrial Complex II Can Generate Reactive Oxygen Species at High Rates in Both the Forward and Reverse Reactions

Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.     

Pancreatic mitochondrial complex I exhibits aberrant hyperactivity in diabetes

It is well established that NADH/NAD⁺ redox balance is heavily perturbed in diabetes, and the NADH/NAD⁺ redox imbalance is a major source of oxidative stress in diabetic tissues. In mitochondria, complex I is the only site for NADH oxidation and NAD⁺ regeneration and is also a major site for production of mitochondrial reactive oxygen species (ROS). Yet how complex I responds to the NADH/NAD⁺ redox imbalance and any potential consequences of such response in diabetic pancreas have not been investigated. We report here that pancreatic mitochondrial complex I showed aberrant hyperactivity in either type 1 or type 2 diabetes. Further studies focusing on streptozotocin (STZ)-induced diabetes indicate that complex I hyperactivity could be attenuated by metformin. Moreover, complex I hyperactivity was accompanied by increased activities of complexes II to IV, but not complex V, suggesting that overflow of NADH via complex I in diabetes could be diverted to ROS production. Indeed in diabetic pancreas, ROS production and oxidative stress increased and mitochondrial ATP production decreased, which can be attributed to impaired pancreatic mitochondrial membrane potential that is responsible for increased cell death. Additionally, cellular defense systems such as glucose 6-phosphate dehydrogenase, sirtuin 3, and NQO1 were found to be compromised in diabetic pancreas. Our findings point to the direction that complex I aberrant hyperactivity in pancreas could be a major source of oxidative stress and β cell failure in diabetes. Therefore, inhibiting pancreatic complex I hyperactivity and attenuating its ROS production by various means in diabetes might serve as a promising approach for anti-diabetic therapies.     

Mitochondrial metabolic suppression in fasting and daily torpor: consequences for reactive oxygen species production

Abstract Daily torpor results in an ～70% decrease in metabolic rate (MR) and a 20%-70% decrease in state 3 (phosphorylating) respiration rate of isolated liver mitochondria in both dwarf Siberian hamsters and mice even when measured at 37°C. This study investigated whether mitochondrial metabolic suppression also occurs in these species during euthermic fasting, when MR decreases significantly but torpor is not observed. State 3 respiration rate measured at 37°C was 20%-30% lower in euthermic fasted animals when glutamate but not succinate was used as a substrate. This suggests that electron transport chain complex I is inhibited during fasting. We also investigated whether mitochondrial metabolic suppression alters mitochondrial reactive oxygen species (ROS) production. In both torpor and euthermic fasting, ROS production (measured as H(2)O(2) release rate) was lower with glutamate in the presence (but not absence) of rotenone when measured at 37°C, likely reflecting inhibition at or upstream of the complex I ROS-producing site. ROS production with succinate (with rotenone) increased in torpor but not euthermic fasting, reflecting complex II inhibition during torpor only. Finally, mitochondrial ROS production was twofold more temperature sensitive than mitochondrial respiration (as reflected by Q(10) values). These data suggest that electron leak from the mitochondrial electron transport chain, which leads to ROS production, is avoided more efficiently at the lower body temperatures experienced during torpor.     

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.     

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.     

Disruption of alternative NAD(P)H dehydrogenases leads to decreased mitochondrial ROS in Neurospora crassa

Mitochondria are a main providers of high levels of energy, but also a major source of reactive oxygen species (ROS) during normal oxidative metabolism. The involvement of Neurospora crassa alternative NAD(P)H dehydrogenases in mitochondrial ROS production was evaluated. The growth responses of a series of respiratory mutants to several stress conditions revealed that disrupting alternative dehydrogenases leads to an increased tolerance to the redox cycler paraquat, with a mutant devoid of the external NDE1 and NDE2 enzymes being significantly more resistant. The nde1nde2 mutant mitochondria show a significant decrease in ROS generation in the presence and absence of paraquat, regardless of the respiratory substrate used, and an intrinsic increase in catalase activity. Analysis of ROS production by a complex I mutant (nuo51) indicates that, as in other organisms, paraquat-derived ROS in Neurospora mitochondria occur mainly at the level of complex I. We propose that disruption of the external NAD(P)H dehydrogenases NDE1 and NDE2 leads to a synergistic effect diminishing ROS generation by the mitochondrial respiratory chain. This, in addition to a robust increase in scavenging capacity, provides the mutant strain with an improved ability to withstand paraquat treatment.     

Vitamin E succinate protects hepatocytes against the toxic effect of reactive oxygen species generated at mitochondrial complexes I and III by alkylating agents.

The mechanism of alpha-tocopheryl succinate (TS) cytoprotection against mitochondria-derived oxidative stress was investigated. Incubation of isolated rat hepatocytes with ethyl methanesulfonate (EMS), a mitochondrial alkylating toxicant caused mitochondrial dysfunction and necrotic cell death that was dependent on the production of reactive oxygen species (ROS) and lipid peroxidation. Mitochondria isolated from these cells showed a 3-fold increase in lipid hydroperoxides and a selective depletion of alpha-tocopherol (T), which preceded cell death. The pretreatment of hepatocytes with TS dramatically enriched cells and mitochondria with alpha-tocopherol and provided these membranes with complete protection against EMS-induced oxidative damage. TS pretreatment suppressed EMS-induced cellular ROS production, generated from mitochondrial complex I and III sites. In addition, the treatment with either rotenone (ROT, a complex I inhibitor) or antimycin A (AA, a complex III inhibitor) potentiated EMS-induced lipid peroxidation and necrotic cell death which were again completely prevented by TS treatment. Surprisingly, TS did not protect hepatocytes against thenoyltrifluoroacetone (TTFA), a complex II inhibitor-induced enhancement of EMS-induced toxic oxidative damage. We conclude that the inhibition of mitochondrial ROS production and lipid peroxidation by T released from TS, are the critical events responsible for TS-mediated cytoprotection against toxic oxidative stress derived from both mitochondrial complexes I and III. Our findings suggest that TS treatment may prove useful in combating diseases associated with mitochondrial-derived oxidative stress.     

Testing the vicious cycle theory of mitochondrial ROS production: effects of H2O2 and cumene hydroperoxide treatment on heart mitochondria

Vicious cycle theories of aging and oxidative stress propose that ROS produced by the mitochondrial electron transport chain damage the mitochondria leading exponentially to more ROS production and mitochondrial damage. Although this theory is widely discussed in the field of research on aging and oxidative stress, there is little supporting data. Therefore, in order to help clarify to what extent the vicious cycle theory of aging is correct, we have exposed mitochondria in vitro to different concentrations of hydrogen peroxide or cumene-hydroperoxide (0, 30, 100 and 500 μM). We have found that 30 μM hydrogen peroxide (or higher concentrations) inhibit oxygen consumption in state 3 and increase ROS production with pyruvate/malate but not with succinate as substrate, indicating that these effects occur specifically at complex I. Similar levels of cumene-OOH inhibit state 3 respiration with both kinds of substrates, and increase ROS production in both state 4 and state 3 with pyruvate/malate and with succinate. The effects of cumene-OOH on ROS generation are due to action of the peroxide in the complex III or in the complex III plus complex I ROS generators. In all cases, the increase in ROS production occurred at a threshold level of peroxide exposure without further exponential increase in ROS generation. These results are consistent with the idea that ROS production can contribute to increase oxidative stress in old animals, but the results do not fit with a vicious cycle theory in which peroxide generation leads exponentially to more and more ROS production with age.     

The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I

Using rat dopaminergic and human neuroblastoma cell lines transduced with the NDI1 gene encoding the internal NADH dehydrogenase (Ndi1) from Saccharomyces cerevisiae, we investigated reactive oxygen species (ROS) generation caused by complex I inhibition. Incubation of non-transduced cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1-transduced cells showed a suppressed rate of ROS formation by the complex I inhibitors. We conclude that the Ndi1 enzyme is able to suppress ROS overproduction from defective complex I.