Modulation of mitochondrial membrane potential and reactive oxygen species production by copper in astrocytes

In monolayers of cultured rat astrocytes a number of agents that induce oxidative stress act synergistically with exposure to copper leading to rapid depolarization of the mitochondrial membrane potential (Psi m) and increased reactive oxygen species (ROS) production. Copper sensitized astrocytes to the action of menadione, an intracellular generator of superoxide anion radical, exogenous hydrogen peroxide (H2O2) and rotenone, an inhibitor of mitochondrial electron transport chain complex I. However, significant differences were observed in the ability to modulate the copper-enhanced oxidative stress depending on which stressor was used. The inhibitor of mitochondrial permeability transition cyclosporin A attenuated the effect of copper and rotenone, but had no protective action in the case of H2O2/copper and menadione/copper combinations. The H2O2 scavenger pyruvate was effective at protecting mitochondria against damage associated with the combined exposure to H2O2/copper and menadione/copper but not to the rotenone/copper combination. The antioxidant Trolox was ineffective at protecting against any of these actions and indeed had a damaging effect when combined with copper. The membrane-permeable copper chelator neocuproine combined with sensitizing concentrations of menadione caused a decrease in Psi m, mimicking the action of copper. Penicillamine, a membrane-impermeable copper chelator, was effective at reducing copper sensitization. Endogenous copper, mobilized during periods of oxidative stress, may play a role in the pathophysiology of brain injury. Our results suggest that this might be particularly dangerous in dysfunctional conditions in which the mitochondrial electron transport chain is compromised.     

Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide-Induced Oxidative Stress in Nerve Terminals

Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.     

Tumour necrosis factor induced an early release of superoxide and a late mitochondrial membrane depolarization in L929 cells. Increase in the production of superoxide is not sufficient to mimic the action of TNF

Tumour necrosis factor alpha (TNF) cytotoxicity is mediated, at least in part, by oxidative stress. One of the post-receptor events shortly after the addition of TNF is the generation of the superoxide anion (O2-*). In the present study, we attempted to examine the role of O2-* in the regulation of mitochondrial membrane potential (Delta(Psi)m) and the release of cytochrome c (cyto c) in L929 cells after stimulation with TNF. Challenge of cells with TNF (50 ng/ml) resulted in an early (30 min after the addition of TNF) increase in the production of O2-*. The use of mitochondrial electron transport chain inhibitors such as antimycin A and rotenone could, respectively, potentiate or suppress the TNF-mediated release of O2-* and cytotoxicity. TNF also induced a late (>3 h after the addition of TNF) depolarization in the Delta(Psi)m. Reduction in the release of O2-* by rotenone (50 microM) or thenoyltrifluoroacetone (250 microM) suppressed both the TNF-mediated Delta(Psi)m depolarization and cyto c release. However, increase in the production of O2-* by antimycin A (25 microM) only slightly enhanced the TNF effect in altering the Delta(Psi)m and the release of cyto c. Treating cells with antimycin A alone could not induce a reduction in Delta(Psi)m nor a release of cyto c. Taken together, our results indicate that TNF induced damage in mitochondria in L929 cells. Our data also show that an increase in the production of O2-* was important in the TNF cytotoxicity, but was not sufficient to mimic the action of TNF.     

Reversible depolarization of in situ mitochondria by oxidative stress parallels a decrease in NAD(P)H level in nerve terminals

We have reported recently (Chinopoulos et al., 1999 J. Neurochem. 73, 220 228) that mitochondrial membrane potential (delta(psi)m) in isolated nerve terminals is markedly reduced by H2O2 in the absence of F0F1-ATPase working as a proton pump. Here we demonstrate that delta(psi)m reduced by H2O2 (0.5 mM) in the presence of oligomycin (10 mM), an inhibitor of the F0F1-ATPase, was able to recover by the addition of catalase (2000 U). Similarly, a decrease in the NAD(P)H level due to H2O2 can be reversed by catalase. In addition, H2O2 decreased the ATP level and the [ATP]:[ADP] ratio measured in the presence of oligomycin reflecting an inhibition of glycolysis by H2O2, but this effect was not reversible. The effect of H2O2 on delta(psi)m in the presence of the complex I inhibitor, rotenone, was also unaltered by addition of catalase. These results provide circumstantial evidence for a relationship between the decreased NAD(P)H level and the inability of mitochondria to maintain delta(psi)m during oxidative stress.     

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.     

Initiation of Neuronal Damage by Complex I Deficiency and Oxidative Stress in Parkinson's Disease

Oxidative stress and partial deficiencies of mitochondrial complex I appear to be key factors in the pathogenesis of Parkinson's disease. They are interconnected; complex I inhibition results in an enhanced production of reactive oxygen species (ROS), which in turn will inhibit complex I. Partial inhibition of complex I in nerve terminals is sufficient for in situ mitochondria to generate more ROS. H2O2 plays a major role in inhibiting complex I as well as a key metabolic enzyme, alpha-ketoglutarate dehydrogenase. The vicious cycle resulting from partial inhibition of complex I and/or an inherently higher ROS production in dopaminergic neurons leads over time to excessive oxidative stress and ATP deficit that eventually will result in cell death in the nigro-striatal pathway.     

Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells

3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2- generation in mitochondria respiring on the complex I substrates pyruvate+malate, an effect fully inhibited by rotenone. Antimycin A increased O2- production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2- production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2- formation driven with the complex II substrate succinate. At 0.6 microM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2- formation; however, at 40 microM myxothiazol (which completely inhibits both complexes I and III) eliminated O2- production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2- from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II.     

The Production of Reactive Oxygen Species in Intact Isolated Nerve Terminals Is Independent of the Mitochondrial Membrane Potential

Dependence on mitochondrial membrane potential (m) of hydrogen peroxide formation of in situ mitochondria in response to inhibition of complex I or III was studied in synaptosomes. Blockage of electron flow through complex I by rotenone or that through complex III by antimycin resulted in an increase in the rate of H₂O₂ generation as measured with the Amplex red assay. Membrane potential of mitochondria was dissipated by either FCCP (250nM) or DNP (50mM) and then the rate of H₂O₂ production was followed. Neither of the uncouplers had a significant effect on the rate of H₂O₂ production induced by rotenone or antimycin. Inhibition of the F₀F₁-ATPase by oligomycin, which also eliminates m in the presence of rotenone and antimycin, respectively, was also without effect on the ROS formation induced by rotenone and only slightly reduced the antimycin-induced H₂O₂ production. These results indicate that ROS generation of in situ mitochondria in nerve terminals in response to inhibition of complex I or complex III is independent of m. In addition, we detected a significant antimycin-induced H₂O₂ production when the flow of electrons through complex I was inhibited by rotenone, indicating that the respiratory chain of in situ mitochondria in synaptosomes has a substantial electron influx distal from the rotenone site, which could contribute to ROS generation when the complex III is inhibited.     

Hydrogen peroxide preconditioning protects PC12 cells against apoptosis induced by dopamine

Dopamine (DA), one of the major sources of reactive oxygen species (ROS), is implicated in neuronal death associated with Parkinson's disease (PD). Preconditioning with oxidative stress has been shown to provide cytoprotection similar to ischemic preconditioning (IPC), against cell apoptosis. In this study, using the model neurosecretory cell line, PC12, we investigated whether hydrogen peroxide (H(2)O(2)) at low concentration (10 microM) can protect PC12 cells against apoptosis induced by DA. PC12 cells were preconditioned with 10 microM H(2)O(2) for 90 min, followed by 24-h recovery and subsequent exposures to different concentrations (20, 50, 100 and 200 microM) of DA for 24-h, respectively. DA induced apoptotic cell death with significant morphological nuclear changes and DNA fragmentation as well as the dysfunction of mitochondria. Preconditioning with H(2)O(2) at 10 microM significantly reduced the percentage of apoptotic cells and partly blocked the decreases in 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction and mitochondrial membrane potential (MMP) induced by DA. These results suggest that preconditioning with low concentration of H(2)O(2) protected PC12 cells against DA-induced apoptosis, the part restoration of the damaged mitochondrial functions might be one of the underlying mechanisms of this cytoprotection.     

Role of mitochondria in ultraviolet-induced oxidative stress

The biological effects of ultraviolet radiation (UV), such as DNA damage, mutagenesis, cellular aging, and carcinogenesis, are in part mediated by reactive oxygen species (ROS). The major intracellular ROS intermediate is hydrogen peroxide, which is synthesized from superoxide anion ((*)O(2)(-)) and further metabolized into the highly reactive hydroxyl radical. In this study, we examined the involvement of mitochondria in the UV-induced H(2)O(2) accumulation in a keratinocyte cell line HaCaT. Respiratory chain blockers (cyanide-p-trifluoromethoxy-phenylhydrazone and oligomycin) and the complex II inhibitor (theonyltrifluoroacetone) prevented H(2)O(2) accumulation after UV. Antimycin A that inhibits electron flow from mitochondrial complex III to complex IV increased the UV-induced H(2)O(2) synthesis. The same effect was seen after incubation with rotenone, which blocks electron flow from NADH-reductase (complex I) to ubiquinone. UV irradiation did not affect mitochondrial transmembrane potential (DeltaPsi(m)). These data indicate that UV-induced ROS are produced at complex III via complex II (succinate-Q-reductase).     

Mitochondria are a major source of paraquat-induced reactive oxygen species production in the brain

Paraquat (PQ(2+)) is a prototypic toxin known to exert injurious effects through oxidative stress and bears a structural similarity to the Parkinson disease toxicant, 1-methyl-4-pheynlpyridinium. The cellular sources of PQ(2+)-induced reactive oxygen species (ROS) production, specifically in neuronal tissue, remain to be identified. The goal of this study was to determine the involvement of brain mitochondria in PQ(2+)-induced ROS production. Highly purified rat brain mitochondria were obtained using a Percoll density gradient method. PQ(2+)-induced hydrogen peroxide (H(2)O(2)) production was measured by fluorometric and polarographic methods. The production of H(2)O(2) was evaluated in the presence of inhibitors and modulators of the mitochondrial respiratory chain. The results presented here suggest that in the rat brain, (a) mitochondria are a principal cellular site of PQ(2+)-induced H(2)O(2) production, (b) PQ(2+)-induced H(2)O(2) production requires the presence of respiratory substrates, (c) complex III of the electron transport chain is centrally involved in H(2)O(2) production by PQ(2+), and (d) the mechanism by which PQ(2+) generates H(2)O(2) depends on the mitochondrial inner transmembrane potential. These observations were further confirmed by measuring PQ(2+)-induced H(2)O(2) production in primary neuronal cells derived from the midbrain. These findings shed light on the mechanism through which mitochondria may contribute to ROS production by other environmental and endogenous redox cycling agents implicated in Parkinson's disease.     

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

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

Endogenous glutamate contributes to the maintenance of glutathione level under oxidative stress in isolated nerve terminals

Exposure of isolated nerve terminals to hydrogen peroxide (25-500 microM) for 10 min produced a partially reversible decrease in the total and reduced glutathione level. No release and resynthesis of glutathione by the oxidant was involved in this effect. Loss of reduced glutathione was associated with elimination of H(2)O(2), which was very quick with >70% of the oxidant eliminated within 5 min. Recovery of both total and reduced glutathione was pronounced after 10 min when the majority of H(2)O(2) was eliminated. Previously we have reported that glutamate metabolism under oxidative stress contributes to the operation of the Krebs cycle, thus to the production of NAD(P)H [J. Neurosci. 20 (2000) 8972]. In the present study we addressed whether metabolism of endogenous glutamate plays a role in the maintenance of glutathione level in nerve terminals. Glutamine and beta-hydroxybutyrate (5mM), alternative metabolites in synaptosomes, were able to decrease the loss of total and reduced glutathione induced by hydrogen peroxide. Metabolic consumption of glutamate was reduced at the same time. In addition an increased demand on the glutathione system by the catalase inhibitor aminotriazole augmented the metabolic consumption of glutamate. It is concluded that under oxidative stress glutamate metabolism contributes to the maintenance of glutathione level, thus to the antioxidant capacity of nerve terminals.     

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.     

Reactivity of Hg(II) with superoxide: evidence for the catalytic dismutation of superoxide by Hg(II).

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O2-) and hydrogen peroxide (H2O2) production by rat kidney mitochondria, Hg(II) (1-6 microM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H2O2 production but an apparent decrease in mitochondrial O2- production. Hg(II) also inhibited O(2-)-dependent cytochrome c reduction (IC50 approximately 2-3 microM) when O2- was produced from xanthine oxidase. In contrast, Hg(I) did not react with O2- in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O2- by Hg(II). Hg(II) also inhibited the reactions of KO2 (i.e., O2-) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO2 in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO2. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O2- dismutation, leading to increased production of H2O2 by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.     

S-nitrosothiol inhibition of mitochondrial complex I causes a reversible increase in mitochondrial hydrogen peroxide production

We found that reversible inactivation of mitochondrial complex I by S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in isolated rat heart mitochondria resulted in a three-fold increase in H2O2 production, when mitochondria were respiring on pyruvate and malate, (but not when respiring on succinate or in the absence of added respiratory substrate). The inactivation of complex I and the increased H2O2 production were present in mitochondria washed free of SNAP or NO, but were partially reversed by light or dithiothreitol, treatments known to reverse S-nitrosation. Specific inhibition of complex I with rotenone increased H2O2 production to a similar extent as that caused by SNAP. The results suggest that S-nitrosation of complex I can reversibly increase oxidant production by mitochondria, which is potentially important in cell signalling and/or pathology.     

Hydrogen peroxide increases the activities of soxRS regulon enzymes and the levels of oxidized proteins and lipids in Escherichia coli

The effects of hydrogen peroxide treatments on Escherichia coli KS400 and AB1157 cells were assessed by monitoring the accumulation of oxidative damage products, carbonyl proteins and thiobarbituric acid-reactive substances (TBARS), as well as the activities of selected antioxidant enzymes. H(2)O(2) treatment stimulated increases in both TBARS and carbonyl protein levels in dose- and time-dependent manners in KS400 cells. The accumulation of TBARS was much more variable with H(2)O(2) treatment; TBARS content was significantly increased in response to 5 microM H(2)O(2), whereas a significant increase in carbonyl protein content occurred at 100 microM H(2)O(2). Similarly, treatment with 20 microM hydrogen peroxide for different lengths of time resulted in peak TBARS accumulation by 20 min, whereas carbonyl protein levels were significantly elevated only after 60 min. In AB1157 cells, treatment with 20 microM hydrogen peroxide for 20 min led to strong increases in both carbonyl protein and TBARS levels. This treatment also triggered increased activities of enzymes of the oxyR regulon (catalase, peroxidase, and glutathione reductase) in both strains. In the AB1157 strain, H(2)O(2) exposure also increased the activities of two enzymes of the soxRS regulon (superoxide dismutase and glucose-6-phosphate dehydrogenase) by 50-60%. The data show differential variability of lipids versus proteins to oxidative damage induced by H(2)O(2,) as well as strain-specific differences in the accumulation of damage products and the responses by antioxidant enzymes to H(2)O(2) stress.     

Topology of superoxide production from different sites in the mitochondrial electron transport chain

We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15%) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria.     

Cytosolic thioredoxin peroxidase I is essential for the antioxidant defense of yeast with dysfunctional mitochondria.

The specific role of cytosolic thioredoxin peroxidase I (cTPx I), encoded by TSA1 (thiol-specific antioxidant), was investigated in the oxidative stress response of Saccharomyces cerevisiae. In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity. However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident. All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H(2)O(2) in cells, which could be related to their elevated sensitivity to oxidative stress. In fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H(2)O(2). In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory-deficient cells.     

Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis

During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.