Mitochondrial involvement in non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is an increasing recognized condition that may progress to end-stage liver disease. There are consistent evidences that mitochondrial dysfunction plays a central role in NASH whatever its origin. Mitochondria are the key controller of fatty acids removal and this is part of an intensive gene program that modifies hepatocytes to counteract the excessive fat storage. Mitochondrial dysfunction participates at different levels in NASH pathogenesis since it impairs fatty liver homeostasis and induces overproduction of ROS that in turn trigger lipid peroxidation, cytokines release and cell death. In this review we briefly recall the role of mitochondria in fat metabolism and energy homeostasis and focus on the role of mitochondrial impairment and uncoupling proteins in the pathophysiology of NASH progression. We suggest that mitochondrial respiratory chain, UCP2 and redox balance cooperate in a common pathway that permits to set down the mitochondrial redox pressure, limits the risk of oxidative damage, and allows the maximal rate of fat removal. When the environmental conditions change and high energy supply occurs, hepatocytes are unable to replace their ATP store and steatosis progress to NASH and cirrhosis. The beneficial effects of some drugs on mitochondrial function are also discussed.     

NEFA‐induced ROS impaired insulin signalling through the JNK and p38MAPK pathways in non‐alcoholic steatohepatitis

The aim of this study was to investigate the changes in hepatic oxidative phosphorylation (OXPHOS) complexes (COs) in patients and cows with non‐alcoholic steatohepatitis (NASH) and to investigate the mechanism that links mitochondrial dysfunction and hepatic insulin resistance induced by non‐esterified fatty acids (NEFAs). Patients and cows with NASH displayed high blood NEFAs, TNF‐α and IL‐6 concentrations, mitochondrial dysfunction and insulin resistance. The protein levels of peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α), mitofusin‐2 (Mfn‐2) and OXPHOS complexes (human: COI and COIII; cow: COI‐IV) were significantly decreased in patients and cows with NASH. NEFA treatment significantly impaired mitochondrial function and, increased reactive oxygen species (ROS) production, and excessive ROS overactivated the JNK and p38MAPK pathways and induced insulin resistance in cow hepatocytes. PGC‐1α and Mfn‐2 overexpression significantly decreased the NEFA‐induced ROS production and TNF‐α and IL‐6 mRNA expressions, reversed the inhibitory effect of NEFAs on mitochondrial function and attenuated the overactivation of the ROS‐JNK/p38MAPK pathway, alleviated insulin resistance induced by NEFAs in cow hepatocytes and HepG2 cells. These findings indicate that NEFAs induce mitochondrial dysfunction and insulin resistance mediated by the ROS‐JNK/p38MAPK pathway. PGC‐1α or Mfn‐2 overexpression reversed the lipotoxicity of NEFAs on mitochondrial dysfunction and insulin resistance. Our study clarified the mechanism that links hepatic mitochondrial dysfunction and insulin resistance in NASH.     

Hepatotoxicity due to mitochondrial dysfunction

Mitochondria are involved in fatty acid β-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation, which provide most of the cell energy. Mitochondria are also the main source of reactive oxygen species in the cell and are involved in cell demise through opening of the mitochondrial permeability transition pore. It was therefore to be expected that mitochondrial dysfunction could be a major mechanism of drug-induced liver disease. Microvesicular steatosis (which may cause liver failure, coma, and death) is the consequence of severe impairment of mitochondrial β-oxidation. Endogenous compounds (such as cytokines or female sex hormones) or xenobiotics (including toxins such as ethanol and drugs such as aspirin, valproic acid, ibuprofen, or zidovudine) can inhibit β-oxidation directly or through a primary effect on the mitochondrial genome or the respiratory chain itself. In some patients, infections and cytokines, or inborn errors of β-oxidation enzymes or the mitochondrial genome, may favor the appearance of drug-induced microvesicular steatosis. Nonalcoholic steatohepatitis may develop under conditions causing prolonged, microvesicular, and/or macrovacuolar steatosis. In this condition, chronic impairment of mitochondrial β-oxidation (causing steatosis) and the respiratory chain (increasing the production of ROS) lead to lipid peroxidation, which, in turn, may cause the diverse lesions of steatohepatitis, namely, necrosis, inflammation, Mallory's bodies, and fibrosis. Finally, mitochondria are involved in several forms of drug-induced cytolytic hepatitis, through inhibition or uncoupling of respiration or through a drug-induced or reactive metabolite-induced mitochondrial permeability transition. The latter effect commits hepatocytes to either apoptosis or necrosis, depending on the number of organelles that have undergone the permeability transition. This revised version was published online in July 2006 with corrections to the Cover Date.     

Apolipoprotein A-I expression suppresses COX-2 expression by reducing reactive oxygen species in hepatocytes

Abnormal lipid metabolism may contribute to the increase of reactive oxygen species (ROS) and inflammation in the pathogenesis of non-alcoholic steatohepatitis (NASH). Apolipoprotein A-I (apoA-I) accepts cellular cholesterol and phospholipids transported by ATP-binding cassette transporter A1 to generate nascent high density lipoprotein particles. Previous studies revealed that the overexpression of ABCA1 or apoA-I alleviated hepatic lipid levels by modifying lipid transport. Here, we examined the effect of apoA-I overexpression on ROS and genes involved in inflammation in both BEL-7402 hepatocytes and mice. Human apoA-I was overexpressed by transfection in BEL-7402 hepatocytes and by an adenoviral vector in C57BL/6J mice fed a methionine choline-deficient diet. The overexpression of apoA-I in both models resulted in decreased ROS and lipid peroxidation levels, as well as a reduced MAPK phosphorylation and decreased expression levels of c-Fos and COX-2. These results suggest that apoA-I overexpression can reduce steatosis by decreasing ROS levels and suppressing COX-2-induced inflammation in hepatocytes. MAPK and c-Fos are involved in this regulatory process.     

Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis

The worldwide rising prevalence of obesity and insulin resistance is associated with a parallel increase in nonalcoholic fatty liver disease (NAFLD). NAFLD is characterized by excess accumulation of triglyceride in the hepatocyte due to increased inflow of free fatty acids and/or de novo lipogenesis caused by various drugs and multiple defects in energy metabolism. Accumulation of lipids in the hepatocyte impairs the oxidative capacity of the mitochondria, increasing the reduced state of the electron transport chain (ETC) complexes and stimulating peroxisomal and microsomal pathways of fat oxidation. The consequent increased generation of reactive oxygen species (ROS) and reactive aldehydic derivatives causes oxidative stress and cell death, via ATP, NAD, and glutathione depletion and DNA, lipid, and protein damage. Oxidative stress also triggers production of inflammatory cytokines, causing inflammation and a fibrogenic response. This ultimately results in the development of nonalcoholic steatohepatitis (NASH), which can result in end-stage liver disease. The current therapeutic strategies for NASH treatment are mostly directed toward correction of the risk factors. Stimulation of mitochondrial function may also prevent NASH development, protecting the cell against the increased flux of reduced substrates to the ETC and ROS generation.     

Oxidatively damaged proteins of heart mitochondrial electron transport complexes

Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I-V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.     

Glutathione depletion and the production of reactive oxygen species in isolated hepatocyte suspensions

Diethyl maleate (DEM) (5 mM) and ethyl methanesulfonate (EMS) (35 mM) treatments rapidly depleted cellular reduced glutathione (GSH) below detectable levels (1 nmol/10(6) cells), and induced lipid peroxidation and necrotic cell death in freshly isolated rat hepatocytes. In hepatocytes incubated with 2.5 mM DEM and 10 mM EMS, however, the complete depletion of cellular GSH observed was not sufficient to induce lipid peroxidation or cell death. Instead, DEM- and EMS-induced lipid peroxidation and cell death were dependent on increased reactive oxygen species (ROS) production as measured by increases in dichlorofluorescein fluorescence. The addition of antioxidants (vitamin E succinate and deferoxamine) prevented lipid peroxidation and cell death, suggesting that lipid peroxidation is involved in the sequence of events leading to necrotic cell death induced by DEM and EMS. To investigate the subcellular site of ROS generation, the cytochrome P450 inhibitor, SKF525A, was found to reduce EMS-induced lipid peroxidation but did not protect against the loss of cell viability, suggesting a mitochondrial origin for the toxic lipid peroxidation event. In agreement with this conclusion, mitochondrial electron transport inhibitors (rotenone, thenoyltrifluoroacetone and antimycin A) increased EMS-induced lipid peroxidation and cell death, while the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, blocked EMS- and DEM-mediated ROS production and lipid peroxidation. Furthermore, EMS treatment resulted in the significant loss of mitochondrial alpha-tocopherol shortly after its addition, and this loss preceded losses in cellular alpha-tocopherol levels. Treatment of hepatocytes with cyclosporin A, a mitochondrial permeability transition inhibitor, oxypurinol, a xanthine oxidase inhibitor, or BAPTA-AM, a calcium chelator, provided no protection against EMS-induced cell death or lipid peroxidation. Our results indicate that DEM and EMS induce cell death by a similar mechanism, which is dependent on the induction of ROS production and lipid peroxidation, and mitochondria are the major source for this toxic ROS generation. Cellular GSH depletion in itself does not appear to be responsible for the large increases in ROS production and lipid peroxidation observed.     

In Vitro Effect of Copper Chloride Exposure on Reactive Oxygen Species Generation and Respiratory Chain Complex Activities of Mitochondria Isolated from Broiler Liver

This study is to examine if Cu(2+) can act directly on mitochondria or indirectly by producing reactive oxygen species (ROS), isolated broiler hepatic mitochondria were exposed to different concentrations of Cu(2+) (10, 30, 50?μM). Respiratory chain complex activities, ROS generation, respiratory control ratio (RCR) and mitochondrial membrane potential were investigated. Dose-dependent inhibition of respiratory chain complexes and induction of ROS were observed, which coincided with decreasing RCR both with glutamate?+?malate or succinate. Further investigation indicated that the membrane potential determined by rhodamine 123 release decreased after CuCl(2) exposure at 30 and 50?μM. In addition, the effects of the antioxidants NAC (200?μM) and GSH (200?μM) were studied at 50?μM Cu(2+). The results indicate that Cu can induce mitochondrial dysfunction in excessive dose and the effect of Cu(2+) exposure on respiratory chain is not site-specific, and antioxidants can protect the mitochondrial function by reducing the formation of free radicals.     

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.     

Tetramethylphenylenediamine-induced hepatocyte cytotoxicity caused by lysosomal labilisation and redox cycling with oxygen activation

It has already been reported that in vivo muscle necrosis induced by various phenylenediamine derivatives correlated with their in vitro autoxidation rate [9]. Now in a more detailed investigation of the cytotoxic mechanism of a ring-methylated phenylenediamine known as tetramethylphenylenediamine or durenediamine (DD) towards isolated rat hepatocytes has been carried out. Cytotoxicity was preceded by ROS formation which was markedly increased by inactivating DT-diaphorase or catalase but were prevented by a subtoxic concentration of the mitochondrial respiratory inhibitor cyanide. This suggests that ROS generation could be attributed to a futile two-electron redox cycle involving oxidation of phenylenediamine to the corresponding diimine by the mitochondrial electron transfer chain and re-reduction by the DT-diaphorase. Endocytosis inhibitors, lysosomotropic agents or lysosomal protease inhibitors also prevented DD-induced cytotoxicity suggesting that DD-induced ROS caused lysosomal damage and protease activation in hepatocytes. Furthermore preincubation with deferoxamine (a ferric iron chelator) or addition of antioxidants, catalase or ROS scavengers (mannitol, tempol or dimethylsulfoxide) prevented DD cytotoxicity. These results suggest that H(2)O(2) reacts with lysosomal Fe(2+) to form "ROS" which causes lysosomal lipid peroxidation, membrane disruption, protease release and cell death.     

Alteration of mitochondrial supercomplexes assembly in metabolic diseases

Deregulation of nutrient, hormonal, or neuronal signaling produces metabolic alterations that result in increased mitochondrial reactive oxygen species (ROS) production. The associations of the mitochondrial respiratory chain components into supercomplexes could have pathophysiological relevance in metabolic diseases, as supramolecular arrangements, by sustaining a high electron transport rate, might prevent ROS generation. In this review, the relationship between mitochondrial dysfunction and supercomplex arrangement of the mitochondrial respiratory chain components in obesity, insulin resistance, hepatic steatosis and diabetes mellitus is summarized and discussed.     

Involvement of free radicals and oxidative stress in NAFLD/NASH

Abstract

Non-alcoholic fatty liver disease (NAFLD) is now the most common liver disease affecting high proportion of the population worldwide. NAFLD encompasses a large spectrum of conditions ranging from fatty liver to non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and cancer. NAFLD is considered as a multifactorial disease in relation to the pathogenic mechanisms. Oxidative stress has been implicated in the pathogenesis of NAFLD and NASH and the involvement of reactive oxygen species (ROS) has been suggested. Many studies show the association between the levels of lipid oxidation products and disease state. However, often neither oxidative stress nor ROS has been characterized, despite oxidative stress is mediated by multiple active species by different mechanisms and the same lipid oxidation products are produced by different active species. Further, the effects of various antioxidants have been assessed in human and animal studies, but the effects of drugs are determined by the type of active species, suggesting the importance of characterizing the active species involved. This review article is focused on the role of free radicals and free radical-mediated lipid peroxidation in the pathogenesis of NAFLD and NASH, taking characteristic features of free radical-mediated oxidation into consideration. The detailed analysis of lipid oxidation products shows the involvement of free radicals in the pathogenesis of NAFLD and NASH. Potential beneficial effects of antioxidants such as vitamin E are discussed.     

Mechanical ventilation induces diaphragmatic mitochondrial dysfunction and increased oxidant production

Mechanical ventilation (MV) is a life-saving intervention used in patients with acute respiratory failure. Unfortunately, prolonged MV results in diaphragmatic weakness, which is an important contributor to the failure to wean patients from MV. Our laboratory has previously shown that reactive oxygen species (ROS) play a critical role in mediating diaphragmatic weakness after MV. However, the pathways responsible for MV-induced diaphragmatic ROS production remain unknown. These experiments tested the hypothesis that prolonged MV results in an increase in mitochondrial ROS release, mitochondrial oxidative damage, and mitochondrial dysfunction. To test this hypothesis, adult (3–4 months of age) female Sprague–Dawley rats were assigned to either a control or a 12-h MV group. After treatment, diaphragms were removed and mitochondria were isolated for subsequent respiratory and biochemical measurements. Compared to control, prolonged MV resulted in a lower respiratory control ratio in diaphragmatic mitochondria. Furthermore, diaphragmatic mitochondria from MV animals released higher rates of ROS in both State 3 and State 4 respiration. Prolonged MV was also associated with diaphragmatic mitochondrial oxidative damage as indicated by increased lipid peroxidation and protein oxidation. Finally, our data also reveal that the activities of the electron transport chain complexes II, III, and IV are depressed in mitochondria isolated from diaphragms of MV animals. In conclusion, these results are consistent with the concept that diaphragmatic inactivity promotes an increase in mitochondrial ROS emission, mitochondrial oxidative damage, and mitochondrial respiratory dysfunction.     

Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells

The mechanism of tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity in metabolically inhibited cells is unclear, although some studies have suggested that mitochondrial dysfunction and generation of reactive oxygen species may be involved. Here we studied the effect of TNFalpha on the redox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatment with TNFalpha and cycloheximide (TNFalpha/CHX) induced mitochondrial cytochrome c release, increased the steady-state reduction of cytochrome b, and decreased the steady-state reduction of cytochromes cc(1) and aa(3). TNFalpha/CHX treatment also induced lipid peroxidation, intracellular generation of reactive oxygen species, and cell death. Furthermore, as the cells died mitochondrial morphology changed from an orthodox to a hyperdense and condensed and finally to a swollen conformation. Antimycin A, a mitochondrial respiratory chain complex III inhibitor that binds to cytochrome b, blocked the formation of reactive oxygen species, suggesting that the free radicals are generated at the level of cytochrome b. Moreover, antimycin A, when added after 3 h of TNFalpha/CHX treatment, arrested the further release of cytochrome c and the cytotoxic response. We propose that the reduced cytochrome b promotes the formation of reactive oxygen species, lipid peroxidation of the cell membrane, and cell death.     

Mechanisms of the Statins Cytotoxicity in Freshly Isolated Rat Hepatocytes

Statins are potent drugs, used as lipid‐lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin‐treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin‐induced toxicity. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:287‐294, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21485     

Curcumin ameliorates rabbits's steatohepatitis via respiratory chain, oxidative stress, and TNF-α

The pathophysiology of nonalcoholic steatohepatitis (NASH) is still not fully understood, and available treatments are not entirely satisfactory. Steatosis progression to NASH is associated with deleterious action of reactive oxygen species, mitochondrial dysfunction, and inflammatory cytokines. We investigated the use of curcumin (compared to vitamin E) in the treatment of NASH. Experimental NASH was induced in rabbits by the intake of a high-fat diet. Oxidative stress status, histology, lipid metabolism, and TNF-α protein levels were assessed in liver. The high-fat diet induced pathologically assessed NASH, and compared to healthy controls, raised the levels of aminotransferases, reduced mitochondrial antioxidants, increased mitochondrial reactive oxygen species, and led to poor mitochondrial function as well as to higher TNF-α protein levels. Curcumin administration together with the high-fat diet led to rabbits with a lower NASH grade and lower levels of aminotransferases, higher values for mitochondrial antioxidants, lower mitochondrial reactive oxygen species, an improved mitochondrial function, and lower levels of TNF-α protein levels. Vitamin E treatment was unable to reduce NASH. In conclusion, curcumin might be useful in the management of NASH through a mechanism involving the antioxidant, anti-inflammatory, and mitochondrial-protective potential of curcumin.     

Role of Mitochondria in Human Aging

Mitochondria are the major intracellular source and target sites of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in animal and human cells. It has been demonstrated that mitochondrial respiratory function declines with age in various human tissues and that a defective respiratory chain results in enhanced production of ROS and free radicals in mitochondria. On the other hand, accumulating evidence now indicates that lipid peroxidation, protein modification and mitochondrial DNA (mtDNA) muutation are concurrently increased during aging. On the basis of these observations and the fact that the rate of cellular production of superoxide anions and hydrogen peroxide increases with age, it has recently been postulated that oxidative stress is a major contributory factor in the aging process. A causal relationship between oxidative modification and mutation of mtDNA, mitochondrial dysfunction and aging has emerged, although some details have remained unsolved. In this article, the role of mitochondria in the human aging process is reviewed on the basis of recent findings gathered from our and other laboratories.     

Mitochondrial response to oxidative and nitrosative stress in early stages of diabetes

Diabetes mellitus (DM) is associated with increased production of reactive oxygen and nitrogen species; consequently, an increase in lipid peroxidation and a decrease in antioxidants resulting in mitochondrial dysfunction. Using a rat model of DM induced by streptozotocin, we show the opposite: an increase in NO levels, S-nitrosylation, aconitase activity, and total glutathione and a decrease in lipid peroxidation at early stages of diabetes. These data imply that the decrease in lipid peroxidation is a vital early response to hyperglycemia to prevent escalation of ROS generation in mitochondria. These results also suggest a need for novel therapeutic targets to prevent the neurological consequences of diabetes.     

Antioxidant and prooxidant properties of mitochondrial Coenzyme Q

Coenzyme Q is both an essential electron carrier and an important antioxidant in the mitochondrial inner membrane. The reduced form, ubiquinol, decreases lipid peroxidation directly by acting as a chain breaking antioxidant and indirectly by recycling Vitamin E. The ubiquinone formed in preventing oxidative damage is reduced back to ubiquinol by the respiratory chain. As well as preventing lipid peroxidation, Coenzyme Q reacts with other reactive oxygen species, contributing to its effectiveness as an antioxidant. There is growing interest in using Coenzyme Q and related compounds therapeutically because mitochondrial oxidative damage contributes to degenerative diseases. Paradoxically, Coenzyme Q is also involved in superoxide production by the respiratory chain. To help understand how Coenzyme Q contributes to both mitochondrial oxidative damage and antioxidant defences, we have reviewed its antioxidant and prooxidant properties.     

High doses of sodium tungstate can promote mitochondrial dysfunction and oxidative stress in isolated mitochondria

Tungstate (W) is recognized as an agent of environmental pollution and a substitute to depleted uranium. According to some preliminary studies, tungstate toxicity is related to the formation of reactive oxygen species (ROS) under abnormal pathological conditions. The kidneys and liver are the main tungstate accumulation sites and important targets of tungstate toxicity. Since the mitochondrion is the main ROS production site, we evaluated the mechanistic toxicity of tungstate in isolated mitochondria for the first time, following a two‐step ultracentrifugation method. Our findings demonstrated that tungstate‐induced mitochondrial dysfunction is related to the increased formation of ROS, lipid peroxidation, and potential membrane collapse, correlated with the amelioration of adenosine triphosphate and glutathione contents. The present study indicated that mitochondrial dysfunction was associated with disruptive effects on the mitochondrial respiratory chain and opening of mitochondrial permeability transition (MPT) pores, which is correlated with cytochrome c release. Our findings suggest that high concentrations of tungstate (2 mM)‐favored MPT pore opening in the inner membranes of liver and kidney mitochondria of rats. Besides, the results indicated higher tungstate susceptibility in the kidneys, compared with the liver.