Ubiquinol and a Coenzyme Q Reducing System Protect Platelet Mitochondrial Function of Transfusional Buffy Coats from Oxidative Stress

The conditions under which Coenzyme Q (CoQ) may protect platelet mitochondrial function of transfusional buffy coats from aging and from induced oxidative stress were investigated. The Pasteur effect, i.e. the enhancement of lactate production after inhibition of mitochondrial respiratory chain, was exploited as a marker of mitochondrial function as it allows to calculate the ratio of mitochondrial ATP to glycolytic ATP. Reduced CoQ 10 improves platelet mitochondrial function of transfusional buffy coats and protects the cells from induced oxidative stress. Oxidized CoQ is usually less effective, despite the presence, shown for the first time in this study, of quinone reductase activities in the platelet plasma membranes. The addition of a CoQ reducing system to platelets is effective in enhancing the protection of platelet mitochondrial function from the oxidative stress. The results support on one hand a possibility of protection of mitochondrial function in aging by exogenous CoQ intake, on the other a possible application in protection of transfusional buffy coats from storage conditions and oxidative deterioration.     

Increased oxidative stress and coenzyme Q10 deficiency in juvenile fibromyalgia: amelioration of hypercholesterolemia and fatigue by ubiquinol-10 supplementation

Abstract

Fibromyalgia (FM) is characterized by generalized pain and chronic fatigue of unknown etiology. To evaluate the role of oxidative stress in this disorder, we measured plasma levels of ubiquinone-10, ubiquinol-10, free cholesterol (FC), cholesterol esters (CE), and free fatty acids (FFA) in patients with juvenile FM (n = 10) and in healthy control subjects (n = 67). Levels of FC and CE were significantly increased in juvenile FM as compared with controls, suggesting the presence of hypercholesterolemia in this disease. However, plasma level of ubiquinol-10 was significantly decreased and the ratio of ubiquinone-10 to total coenzyme Q10 (%CoQ10) was significantly increased in juvenile FM relative to healthy controls, suggesting that FM is associated with coenzyme Q10 deficiency and increased oxidative stress. Moreover, plasma level of FFA was significantly higher and the content of polyunsaturated fatty acids (PUFA) in total FFA was significantly lower in FM than in controls, suggesting increased tissue oxidative damage in juvenile FM. Interestingly, the content of monoenoic acids, such as oleic and palmitoleic acids, was significantly increased in FM relative to controls, probably to compensate for the loss of PUFA. Next, we examined the effect of ubiquinol-10 supplementation (100 mg/day for 12 weeks) in FM patients. This resulted in an increase in coenzyme Q10 levels and a decrease in %CoQ10. No changes were observed in FFA levels or their composition. However, plasma levels of FC and CE significantly decreased and the ratio of FC to CE also significantly decreased, suggesting that ubiquinol-10 supplementation improved cholesterol metabolism. Ubiquinol-10 supplementation also improved chronic fatigue scores as measured by the Chalder Fatigue Scale.     

Isotope-reinforced polyunsaturated fatty acids protect mitochondria from oxidative stress

Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t-butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure–activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D₁-Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D₂-Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20–50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function.     

Effects of combined quercetin and coenzyme Q(10) treatment on oxidative stress in normal and diabetic rats

Reactive oxygen species may be actively involved in the genesis of various pathological states such as ischemia-reperfusion injury, cancer, and diabetes. Our objective was to determine if subacute treatment with combined antioxidants quercetin and coenzyme Q(10) (10 mg/kg/day ip for 14 days) affects the activities of antioxidant enzymes in normal and 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Quercetin treatment raised blood glucose concentrations in normal and diabetic rats, whereas treatment with coenzyme Q(10) did not. Liver, kidney, heart, and brain tissues were excised and the activities of catalase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and concentrations of oxidized and reduced glutathione were determined. In the liver of diabetic rats, superoxide dismutase, glutathione peroxidase, and levels of both oxidized and reduced glutathione were significantly decreased from the nondiabetic control, and these effects were not reversed when antioxidants were administered. In kidney, glutathione peroxidase activity was significantly elevated in the diabetic rats as compared to nondiabetic rats, and antioxidant treatment did not return the enzyme activity to nondiabetic levels. In heart, catalase activity was increased in diabetic animals and restored to normal levels after combined treatment with quercetin and coenzyme Q(10). Cardiac superoxide dismutase was lower than normal in quercetin- and quercetin + coenzyme Q(10)-treated diabetic rats. There were no adverse effects on oxidative stress markers after treatment with quercetin or coenzyme Q(10) singly or in combination. In spite of the elevation of glucose, quercetin may be effective in reversing some effects of diabetes, but the combination of quercetin + coenzyme Q(10) did not increase effectiveness in reversing effects of diabetes.     

Effects of benfotiamine and coenzyme Q10 on kidney damage induced gentamicin

Objective

Gentamicin (GM) is an effective antibiotic against severe infection but has limitations related to nephrotoxicity. In this study, we investigated whether benfotiamine (BFT) and coenzyme Q10 (CoQ10), could ameliorate the nephrotoxic effect of GM in rats.

Effect of mitochondrial dysfunction and oxidative stress on endogenous levels of coenzyme Q(10) in human cells

Little is known about the regulation of endogenous CoQ(10) levels in response to mitochondrial dysfunction or oxidative stress although exogenous CoQ(10) has been extensively used in humans. In this study, we first demonstrated that acute treatment of antimycin A, an inhibitor of mitochondrial complex III, and the absence of mitochondrial DNA suppressed CoQ(10) levels in human 143B cells. Because these two conditions also enhanced formation of reactive oxygen species (ROS), we further investigated whether oxidative stress or mitochondrial dysfunction primarily contributed to the decrease of CoQ(10) levels. Results showed that H(2)O(2) augmented CoQ(10) levels, but carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a chemical uncoupler, decreased CoQ(10) levels in 143B cells. However, H(2)O(2) and FCCP both increased mRNA levels of multiple COQ genes for biosynthesis of CoQ(10) . Our findings suggest that ROS induced CoQ(10) biosynthesis, whereas mitochondrial energy deficiency caused secondary suppression of CoQ(10) levels possibly due to impaired import of COQ proteins into mitochondria.     

辅酶Q10 对子痫前期中氧化应激的防治作用的研究进展

子痫前期是导致全球孕产妇和围生儿发病和死亡的主要原因之一.子痫前期的病因至今尚未明确,但是大量研究已证实多系统的氧化应激与子痫前期发病机制有关.辅酶Q10是目前受到广泛关注的一种抗氧化剂,并且已有辅酶Q10药品制剂问世.本文从细胞水平简要总结了氧化应激与子痫前期发病机制的关系,并讨论了辅酶Q10对予痫前期中氧化应激的防治作用.希望为子痫前期的早期治疗及改善预后提供新的思路.     

The yeast Coq4 polypeptide organizes a mitochondrial protein complex essential for coenzyme Q biosynthesis

Coenzyme Q is a redox active lipid essential for aerobic respiration. The Coq4 polypeptide is required for Q biosynthesis and growth on non-fermentable carbon sources, however its exact function in this pathway is not known. Here we probe the functional roles of Coq4p in a yeast Q biosynthetic polypeptide complex. A yeast coq4-1 mutant harboring an E226K substitution is unable to grow on nonfermentable carbon sources. The coq4-1 yeast mutant retains significant Coq3p O-methyltransferase activity, and mitochondria isolated from coq4-1 and coq4-2 (E₁₂₁K) yeast point mutants contain normal steady state levels of Coq polypeptides, unlike the decreased levels of Coq polypeptides generally found in strains harboring coq gene deletions. Digitonin-solubilized mitochondrial extracts prepared from yeast coq4 point mutants show that Coq3p and Coq4 polypeptides no longer co-migrate as high molecular mass complexes by one- and two-dimensional Blue Native-PAGE. Similarly, gel filtration chromatography confirms that O-methyltransferase activity, Coq3p, Coq4p, and Coq7p migration are disorganized in the coq4-1 mutant mitochondria. The data suggest that Coq4p plays an essential role in organizing a Coq enzyme complex required for Q biosynthesis.     

Modes of coenzyme Q function in electron transfer

Summary In the mitochondrial respiratory chain, coenzyme Q acts in different ways. A diffusable coenzyme Q pool as a common substrate-like intermediate links the low-potential complexes with complex III. Its diffusion in the lipids is not rate-limiting for electron transfer, but its content is not saturating for maximal rate of NADH oxidation. Protein-bound coenzyme Q is involved in energy conservation, and may be part of enzyme supercomplexes, as in succinate cytochromec reductase. The reason for lack of kinetic saturation of the respiratory chain by quinone concentration is in the low extent of solubility of monomeric coenzyme Q in the membrane lipids. Assays of respiratory enzymes are performed using water soluble coenzyme Q homologs and analogs; several problems exist in using oxidized quinones as acceptors of coenzyme Q reductases. In particular, for complex I no acceptor appears to favorably substitute the endogenous quinone. In addition, quinone reduction sites in complex III compete with the sites in the dehydrogenases, particularly when using duroquinone. The different extent by which these sites operate when different donor substrates (NADH, succinate, glycerol-3-phosphate) are used is best explained by different exposure of the quinone acceptor sites in the dehydrogenases.     

Inhibition by coenzyme Q of ethanol- and carbon tetrachloride-stimulated lipid peroxidation in vivo and catalyzed by microsomal and mitochondrial systems

The ability of coenzyme Q to inhibit lipid peroxidation in intact animals as well as in mitochondrial, submitochondrial, and microsomal systems has been tested. Rats fed coenzyme Q prior to being treated with carbon tetrachloride or while being treated with ethanol excrete less thiobarbituric acid-reacting material in the urine than such rats not fed coenzyme Q. Liver homogenates, mitochondria, and microsomes isolated from rats treated with carbon tetrachloride and ethanol catalyze lipid peroxidation at rates which exceed those from animals also fed coenzyme Q. The rate of lipid peroxidation catalyzed by submitochondrial particles isolated from hearts of young, old, and endurance trained elderly rats was inversely proportional to the coenzyme Q content of the submitochondrial preparation in assays in which succinate was employed to reduce the endogenous coenzyme Q. Reduced, but not oxidized, coenzyme Q inhibited lipid peroxidation catalyzed by rat liver microsomal preparations. These results provide additional evidence in support of an antioxidant role for coenzyme Q.     

N-acetylcysteine, coenzyme Q10 and superoxide dismutase mimetic prevent mitochondrial cell dysfunction and cell death induced by d-galactosamine in primary culture of human hepatocytes

d-Galactosamine (d-GalN) induces reactive oxygen species (ROS) generation and cell death in cultured hepatocytes. The aim of the study was to evaluate the cytoprotective properties of N-acetylcysteine (NAC), coenzyme Q₁₀ (Q₁₀) and the superoxide dismutase (SOD) mimetic against the mitochondrial dysfunction and cell death in d-GalN-treated hepatocytes. Hepatocytes were isolated from liver resections. NAC (0.5 mM), Q₁₀ (30 μM) or MnTBAP (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (1 mg/mL) were co-administered with d-GalN (40 mM) in hepatocytes. Cell death, oxidative stress, mitochondrial transmembrane potential (MTP), ATP, mitochondrial oxidized/reduced glutathione (GSH) and Q₁₀ ratios, electronic transport chain (ETC) activity, and nuclear- and mitochondria-encoded expression of complex I subunits were determined in hepatocytes. d-GalN induced a transient increase of mitochondrial hyperpolarization and oxidative stress, followed by an increase of oxidized/reduced GSH and Q₁₀ ratios, mitochondrial dysfunction and cell death in hepatocytes. The cytoprotective properties of NAC supplementation were related to a reduction of ROS generation and oxidized/reduced GSH and Q₁₀ ratios, and a recovery of mitochondrial complexes I + III and II + III activities and cellular ATP content. The co-administration of Q₁₀ or MnTBAP recovered oxidized/reduced GSH ratio, and reduced ROS generation, ETC dysfunction and cell death induced by d-GalN. The cytoprotective properties of studied antioxidants were related to an increase of the protein expression of nuclear- and mitochondrial-encoded subunits of complex I. In conclusion, the co-administration of NAC, Q₁₀ and MnTBAP enhanced the expression of complex I subunits, and reduced ROS production, oxidized/reduced GSH ratio, mitochondrial dysfunction and cell death induced by d-GalN in cultured hepatocytes.     

Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC–EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.     

Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes

Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ₁₀), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 μM H₂O₂)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ₁₀ can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ₁₀ not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ₁₀ prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ₁₀ preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ₁₀ may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies.     

Inhibitors of catalase-amyloid interactions protect cells from beta-amyloid-induced oxidative stress and toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.     

Saccharomyces cerevisiae Coq9 polypeptide is a subunit of the mitochondrial coenzyme Q biosynthetic complex

Coenzyme Q (Q) is a redox active lipid that is an essential component of the electron transport chain. Here, we show that steady state levels of Coq3, Coq4, Coq6, Coq7 and Coq9 polypeptides in yeast mitochondria are dependent on the expression of each of the other COQ genes. Submitochondrial localization studies indicate Coq9p is a peripheral membrane protein on the matrix side of the mitochondrial inner membrane. To investigate whether Coq9p is a component of a complex of Q-biosynthetic proteins, the native molecular mass of Coq9p was determined by Blue Native-PAGE. Coq9p was found to co-migrate with Coq3p and Coq4p at a molecular mass of approximately 1 MDa. A direct physical interaction was shown by the immunoprecipitation of HA-tagged Coq9 polypeptide with Coq4p, Coq5p, Coq6p and Coq7p. These findings, together with other work identifying Coq3p and Coq4p interactions, identify at least six Coq polypeptides in a multi-subunit Q biosynthetic complex.     

Protective effect of ursodeoxycholic acid on liver mitochondrial function in rats with alloxan-induced diabetes: link with oxidative stress

We investigated the effects of ursodeoxycholic acid (UDCA) on mitochondrial functions and oxidative stress and evaluated their relationships in the livers of rats with alloxan-induced diabetes. Diabetes was induced in male Wistar rats by a single alloxan injection (150 mg kg^− 1 b.w., i.p.). UDCA (40 mg kg^− 1 b.w., i.g., 30 days) was administered from the 5th day after the alloxan treatment. Mitochondrial functions were evaluated by oxygen consumption with Clark oxygen electrode using succinate, pyruvate + malate or palmitoyl carnitine as substrates and by determination of succinate dehydrogenase and NADH dehydrogenase activities. Liver mitochondria were used to measure chemiluminiscence enhanced by luminol and lucigenin, reduced liver glutathione and the end-products of lipid peroxidation. The activities of both NADH dehydrogenase and succinate dehydrogenase as well as the respiratory control (RC) value with all the substrates and the ADP/O ratio with pyruvate + malate and succinate as substrates were significantly decreased in diabetic rats. UDCA developed the beneficial effect on the mitochondrial respiration and oxidative phosphorylation parameters in alloxan-treated rats, whereas the activities of mitochondrial enzymes were increased insignificantly after the administration of UDCA. The contents of polar carbonyls and MDA as well as the chemiluminescence with luminol were elevated in liver mitochondria of diabetic rats. The treatment with UDCA normalized all the above parameters measured except the MDA content. UDCA administration prevents mitochondrial dysfunction in rats treated with alloxan and this process is closely connected with inhibition of oxidative stress by this compound.     

Alpha-synuclein overexpression and aggregation exacerbates impairment of mitochondrial functions by augmenting oxidative stress in human neuroblastoma cells

Overexpression of alpha-synuclein and oxidative stress has been implicated in the neuronal cell death in Parkinson's disease. Alpha-synuclein associates with mitochondria and excessive accumulation of alpha-synuclein causes impairment of mitochondrial functions. However, the mechanism of mitochondrial impairment caused by alpha-synuclein is not fully understood. We recently reported that alpha-synuclein associates with mitochondria and that overexpression of alpha-synuclein causes nitration of mitochondrial proteins and release of cytochrome c from the mitochondria [Parihar M.S., Parihar A., Fujita M., Hashimoto M., Ghafourifar P. Mitochondrial association of alpha-synuclein causes oxidative stress. Cell Mol Life Sci. 2008a;65:1272–1284]. The present study shows that overexpression of alpha-synuclein A53T or A30P mutants or wild-type in human neuroblastoma cells augmented aggregation of alpha-synuclein. Immunoblotting and immuno-gold electron transmission microscopy show localization of alpha-synuclein aggregates within the mitochondria of overexpressing cells. Overexpressing cells show increased mitochondrial reactive oxygen species, increased protein tyrosine nitration, decreased mitochondrial transmembrane potential, and hampered cellular respiration. These findings suggest an important role for mitochondria in cellular responses to alpha-synuclein.     

Inhibition of specific electron transport pathways leads to oxidative stress and decreased Candida albicans proliferation

Candida parapsilosis mitochondria contain three respiratory chains: the classical respiratory chain (CRC), a secondary parallel chain (PAR) and an “alternative” oxidative pathway (AOX). We report here the existence of similar pathways in C. albicans. To observe the capacity of each pathway to sustain yeast growth, C. albicans cells were cultured in the presence of inhibitors of these pathways. Antimycin A and KCN totally abrogated yeast growth, while rotenone did not prevent proliferation. Furthermore, rotenone promoted only partial respiratory inhibition. Lower concentrations of KCN that promote partial inhibition of respiration did not inhibit yeast growth, while partial inhibition of respiration with antimycin A did. Similarly, AOX inhibitor BHAM decreased O₂ consumption slightly but completely stunted cell growth. Reactive oxygen species production and oxidized glutathione levels were enhanced in cells treated with antimycin A or BHAM, but not rotenone or KCN. These findings suggest that oxidative stress prevents C. albicans growth.     

Closure of VDAC causes oxidative stress and accelerates the Ca-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca²⁺-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O₂⁻) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O₂⁻ from the IMS and sensitizing to the Ca²⁺-induced MPT. Treatment of isolated rat liver mitochondria with 5 μM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8 ± 1.4 min within a range of 100-250 μM Ca²⁺. G3139-mediated acceleration of the MPT was reversed by 20 μM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O₂⁻ in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O₂⁻ accumulation. Mathematical modeling of generation and turnover of O₂⁻ within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O₂⁻. In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O₂⁻, which causes an internal oxidative stress and sensitizes mitochondria toward the Ca²⁺-induced MPT.