Citrate carrier activity and cardiolipin level in eel (Anguilla anguilla) liver mitochondria

The activity of the tricarboxylate (citrate) carrier has been assayed in intact liver mitochondria from yellow eel (Anguilla anguilla) and compared to that from rat. The eel-citrate carrier specific activity was approximately 1.7-fold higher than that assayed in rat-liver mitochondria. The content of the main mitochondrial phospholipids, phosphatidylethanolamine and phosphatidylcholine, did not show a significant difference between the two species, while in eel a higher cardiolipin level was observed. Fatty acid composition of eel-liver mitochondrial phospholipids was characterised by a large amount of unsaturated fatty acids, dominated by octadecaenoic acid (C(18:1) (n-9)) and docosahexaenoic acid (C(22:6) (n-3)). The cardiolipin fatty acid pattern of eel-liver mitochondria showed, with respect to the rat, a higher C(20:5) (n-3) and C(22:6) (n-3) content and a lower amount of C(18:2) (n-6) and C(20:4) (n-6). A noticeable activity of lipogenic enzymes was also detected in eel liver cytosol. The results of this study suggest that the remarkable activity of the citrate carrier in eel-liver mitochondria can most likely be ascribed to a considerable cardiolipin level. A covariance of citrate carrier and lipogenic enzyme activities was observed.     

Isoalantolactone induces reactive oxygen species mediated apoptosis in pancreatic carcinoma PANC-1 cells

Isoalantolactone, a sesquiterpene lactone compound possesses antifungal, antibacteria, antihelminthic and antiproliferative activities. In the present study, we found that isoalantolactone inhibits growth and induces apoptosis in pancreatic cancer cells. Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of reactive oxygen species, cardiolipin oxidation, reduced mitochondrial membrane potential, release of cytochrome c and cell cycle arrest at S phase. N-Acetyl Cysteine (NAC), a specific ROS inhibitor restored cell viability and completely blocked isoalantolactone-mediated apoptosis in PANC-1 cells indicating that ROS are involved in isoalantolactone-mediated apoptosis. Western blot study showed that isoalantolactone increased the expression of phosphorylated p38 MAPK, Bax, and cleaved caspase-3 and decreased the expression of Bcl-2 in a dose-dependent manner. No change in expression of phosphorylated p38 MAPK and Bax was found when cells were treated with isoalantolactone in the presence of NAC, indicating that activation of these proteins is directly dependent on ROS generation. The present study provides evidence for the first time that isoalantolactone induces ROS-dependent apoptosis through intrinsic pathway. Furthermore, our in vivo toxicity study demonstrated that isoalantolactone did not induce any acute or chronic toxicity in liver and kidneys of CD1 mice at dose of 100 mg/kg body weight. Therefore, isoalantolactone may be a safe chemotherapeutic candidate for the treatment of human pancreatic carcinoma.     

Mitochondrial complex I dysfunction in rat heart with aging: critical role of reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered a key factor in the heart aging process. Mitochondrial respiration is an important site of ROS generation and a potential contributor to heart functional changes with aging. We have examined the effects of aging on various parameters related to mitochondrial bioenergetics in rat heart, such as complex I activity, oxygen consumption, membrane potential, ROS production, and cardiolipin content and oxidation. A loss in complex I activity, state 3 respiration, and membrane potential was found in mitochondria with aging. The capacity of mitochondria to produce H(2)O(2) was significantly increased in aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, significantly decreased as a function of aging, whereas there was a significant increase in the level of oxidized cardiolipin. The lower complex I activity in mitochondria from aged rats could be almost completely restored to the level of young heart by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that aging causes heart mitochondrial complex I deficiency, which can be attributed to ROS-induced cardiolipin peroxidation. These results may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with heart aging.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin

Reactive oxygen species (ROS) are considered a key factor in mitochondrial dysfunction associated with brain aging process. Mitochondrial respiration is an important source of ROS and hence a potential contributor to brain functional changes with aging. In this study, we examined the effect of aging on cytochrome c oxidase activity and other bioenergetic processes such as oxygen consumption, membrane potential and ROS production in rat brain mitochondria. We found a significant age-dependent decline in the cytochrome c oxidase activity which was associated with parallel changes in state 3 respiration, membrane potential and with an increase in H₂O₂ generation. The cytochrome aa₃ content was practically unchanged in mitochondria from young and aged animals. The age-dependent decline of cytochrome c oxidase activity could be restored, in situ, to the level of young animals, by exogenously added cardiolipin. In addition, exposure of brain mitochondria to peroxidized cardiolipin resulted in an inactivation of this enzyme complex. It is suggested that oxidation/depletion of cardiolipin could be responsible, at least in part, for the decline of cytochrome c oxidase and mitochondrial dysfunction in brain aging. Melatonin treatment of old animals largely prevented the age-associated alterations of mitochondrial bioenergetic parameters. These results may prove useful in elucidating the molecular mechanisms underlying mitochondrial dysfunction associated with brain aging process, and may have implications in etiopathology of age-associated neurodegenerative disorders and in the development of potential treatment strategies.     

Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage

The aim of this study was to investigate the influence of reactive oxygen species (ROS) on the activity of complex I and on the cardiolipin content in bovine heart submitochondrial particles (SMP). ROS were generated through the use of xanthine/xanthine oxidase (X/XO) system. Treatment of SMP with X/XO resulted in a large production of superoxide anion, detected by acetylated cytochrome c method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to ROS generation resulted in a marked loss of complex I activity and to parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD+catalase. Exogenous added cardiolipin was able to almost completely restore the ROS-induced loss of complex I activity. No restoration was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, nor with peroxidized cardiolipin. These results demonstrate that ROS affect the mitochondrial complex I activity via oxidative damage of cardiolipin which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those pathophysiological conditions characterized by an increase in the basal production of reactive oxygen species such as aging, ischemia/reperfusion and chronic degenerative diseases.     

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.     

Susceptibility of mitochondrial electron-transport complexes to oxidative damage. Focus on cytochrome c oxidase

Abstract

Reactive oxygen species (ROS) are associated with a number of mitochondrial disorders. These include: ischemia/reperfusion injury, Parkinson's disease, Alzheimer's disease, neurodegenerative diseases, and other age-related degenerative changes. ROS can be generated at numerous sites within the cell, but the mitochondrial electron transport chain is recognized as the major source of intracellular ROS. Two mitochondrial electron-transfer complexes are major sources of ROS: complex I and complex III. Oxidative damage to either of these complexes, or to electron transport complexes that are in close proximity to these ROS sources, e.g., cytochrome c oxidase, would be expected to inhibit electron transport. Such inhibition would lead to increased electron leakage and more ROS production, much like the well-known effect of adding electron transport inhibitors. Recent studies reveal that ROS and lipid peroxidation products are effective inhibitors of the electron-transport complexes. In some cases, inactivation of enzymes correlates with chemical modification of only a small number of unusually reactive amino acids. In this article, we review current knowledge of ROS-induced alterations within three complexes: (1) complex IV; (2) complex III; and (3) complex I. Our goal is to identify “hot spots” within each complex that are easily chemically modified and could be responsible for ROS-induced inhibition of the individual complexes. Special attention has been placed on ROS-induced damage to cardiolipin that is tightly bound to each of the inner membrane protein complexes. Peroxidation of the bound cardiolipin is thought to be particularly important since its close proximity and long residence time on the protein make it an especially effective reagent for subsequent ROS-induced damage to these proteins.     

The stability and activity of respiratory Complex II is cardiolipin-dependent

Respiratory Complex II of the mitochondrial inner membrane serves as a link between the tricarboxylic acid cycle and the electron transport chain. Complex II dysfunction has been implicated in a wide range of heritable mitochondrial diseases, including cancer, by a mechanism that likely involves the production of reactive oxygen species (ROS). Using Complex II enzymes reconstituted into nanoscale lipid bilayers (nanodiscs) with varying lipid composition, we demonstrate for the first time that the phospholipid environment, specifically the presence of cardiolipin, is critical for the assembly and enzymatic activity of the complex, as well as in the curtailment of ROS production.     

Mitochondrial dysfunction associated with cardiac ischemia/reperfusion can be attenuated by oxygen tension control. Role of oxygen-free radicals and cardiolipin

Reactive oxygen species (ROS) are considered an important factor in ischemia/reperfusion injury to cardiac myocites. Mitochondrial respiration is an important source of ROS generation and hence a potential contributor to cardiac reperfusion injury. Appropriate treatment strategy could be particularly useful to limit this ROS generation and associated mitochondrial dysfunction. In the present study, we examined the effect of lowering the oxygen tension, at the onset of the reperfusion, on various parameters of mitochondrial bioenergetics in rat heart tissue. After isolation of mitochondria from control, ischemic, normoxic and hypoxic reperfused rat heart, various bioenergetic parameters were evaluated such as rates of mitochondrial oxygen consumption, complex I and complex III activity, H2O2 production and in addition, the degree of lipid peroxidation, cardiolipin content and cardiolipin oxidation. We found that normoxic reperfusion significantly altered all these mitochondrial parameters, while hypoxic reperfusion had a protective effect attenuating these alterations. This effect appears to be due, at least in part, to a reduction of mitochondrial ROS generation with subsequent preservation of cardiolipin integrity, protection of mitochondrial function and improvement of post-ischemic hemodynamic function of the heart.     

Oxidation-induced changes in human lens epithelial cells 2. Mitochondria and the generation of reactive oxygen species

The relationships among reactive oxygen species (ROS) generation, lipid compositional changes, antioxidant power, and mitochondrial membrane potential were determined in a human lens epithelial cell line, HLE-B3. Cells grown in a hyperoxic atmosphere grew linearly for about 3 days, and then progressively died. Total antioxidant power and ROS generation increased by 50 and 43%, respectively, in cells grown in a hyperoxic atmosphere compared to those cultured in a normoxic atmosphere. By specifically uncoupling the mitochondrial proton gradient, we determined that the mitochondria are most likely the major source of ROS generation. ROS generation correlated inversely with mitochondrial membrane potential and the amount of cardiolipin, factors likely to contribute to loss of cell viability. Our results support the idea that hyperoxic damage to HLE-B3 cells derives from enhanced generation of ROS from the mitochondrial electron transport chain resulting in the oxidation of cardiolipin. With extended hyperoxic insult, the oxidants overwhelm the antioxidant defense system and eventually cell death ensues.     

Mitochondria,oxidative stress and cell death

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of “free” cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.     

Molecular mechanism of 'mitocan'-induced apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and Bcl-2 family proteins

Mitochondria have emerged recently as effective targets for novel anti-cancer drugs referred to as 'mitocans'. We propose that the molecular mechanism of induction of apoptosis by mitocans, as exemplified by the drug alpha-tocopheryl succinate, involves generation of reactive oxygen species (ROS). ROS then mediate the formation of disufide bridges between cytosolic Bax monomers, resulting in the formation of mitochondrial outer membrane channels. ROS also cause oxidation of cardiolipin, triggering the release of cytochrome c and its translocation via the activated Bax channels. This model may provide a general mechanism for the action of inducers of apoptosis and anticancer drugs, mitocans, targeting mitochondria via ROS production.     

A mitochondrial thioredoxin-sensitive mechanism regulates TGF-β-mediated gene expression associated with epithelial–mesenchymal transition

Transforming growth factor (TGF)-β is a pro-oncogenic cytokine that induces the epithelial–mesenchymal transition (EMT), a crucial event in tumor progression. During TGF-β-mediated EMT in NMuMG mouse mammary epithelial cells, we observed sustained increases in reactive oxygen species (ROS) levels in the cytoplasm and mitochondria with a concomitant decrease in mitochondrial membrane potential and intracellular glutathione levels. In pseudo ρ0 cells, whose respiratory chain function was impaired, the increase in intracellular ROS levels was abrogated, suggesting an important role of mitochondrial activity as a trigger for TGF-β-stimulated ROS generation. In line with this, TGF-β-mediated expression of the EMT marker fibronectin was inhibited not only by chemicals that interfere with ROS signaling but also by exogenously expressed mitochondrial thioredoxin (TXN2) independent of Smad signaling. Of note, TGF-β-mediated induction of HMGA2, a central mediator of EMT and metastatic progression, was similarly impaired by TXN2 expression, revealing a novel mechanism involving a thiol oxidation reaction in mitochondria, which regulates TGF-β-mediated gene expression associated with EMT.     

Identification of dicarboxylate carrier Slc25a10 as malate transporter in de novo fatty acid synthesis

Mitochondrial solute carrier family 25 member 10 (Slc25a10) transports dicarboxylates such as malate or succinate across the mitochondrial inner membrane. Although fatty acid synthesis in adipose tissue or the liver is initiated by citrate transport in exchange for malate across the mitochondrial membrane, the transporter responsible for supplying malate during citrate transport has not been identified. In the present study, we clarified the role of Slc25a10 in supplying malate for citrate transport and examined the effect of Slc25a10 suppression on the lipogenic pathway and lipid accumulation. We have reported an Slc25a10 increase in white adipose tissue in obese mouse models and a decrease in a fasted mouse model using expression profiles. Next, we examined the effect of Slc25a10 suppression by small interfering RNA on citrate transport in the lipogenic cell lines HepG2 and 3T3-L1. We observed that inhibition of malate transport by Slc25a10 suppression significantly reduced the citrate transport from the mitochondria to the cytosol. We also found that suppression of Slc25a10 down-regulated the lipogenic pathway, indicated by decreases in ACC1 expression and malonyl-CoA level. Furthermore, suppression of Slc25a10 decreased triglyceride lipid accumulation in adipose-differentiated 3T3-L1 cells. These results suggested that Slc25a10 plays an important role in supplying malate for citrate transport required for fatty acid synthesis and indicated that inhibition of Slc25a10 might effectively reduce lipid accumulation in adipose tissues.     

The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis

Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.     

Adenosine induces apoptosis in human liver cancer cells through ROS production and mitochondrial dysfunction

Mitochondria are the most important sensor for apoptosis. Extracellular adenosine is well reported to induce apoptosis of tumor cells. Here we found that extracellular adenosine suppresses the cell growth by induction of apoptosis in BEL-7404 liver cancer cells, and identified a novel mechanism that extracellular adenosine triggers apoptosis by increasing Reactive Oxygen Species (ROS) production and mitochondrial membrane dysfunction in the cells. We observed that adenosine increases ROS production, activates c-Caspase-8 and -9 and Caspase effectors, c-Caspase-3 and c-PARP, induces accumulation of apoptosis regulator Bak, decreases Bcl-xL and Mcl-1, and causes the mitochondrial membrane dysfunction and the release of DIABLO, Cytochrome C, and AIF from mitochondria to cytoplasm in the cells; ROS inhibitor, NAC significantly reduces adenosine-induced ROS production; it also shows the same degree of blocking adenosine-induced loss of mitochondrial membrane potential (MMP) and apoptosis. Our study first observed that adenosine increases ROS production in tumor cells and identified the positive feedback loop for ROS-mediated mitochondrial membrane dysfunction which amplifies the death signals in the cells. Our findings indicated ROS production and mitochondrial dysfunction play a key role in adenosine-induced apoptosis of 7404 cells.     

Sirtuin-3 (SIRT3) Protein Attenuates Doxorubicin-induced Oxidative Stress and Improves Mitochondrial Respiration in H9c2 Cardiomyocytes

Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction.     

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

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

Mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress and improve insulin secretion in glucotoxicity and glucolipotoxicity

Mitochondrial oxidative damage is thought to play a key role in pancreatic β-cell failure in the pathogenesis of type 2 diabetes. Despite this, the potential of mitochondria-targeted antioxidants to protect pancreatic β-cells against oxidative stress has not yet been studied. Therefore, we investigated if mitochondria-targeted antioxidants protect pancreatic β-cells such as RINm5F and HIT-T15 cells against oxidative stress under glucotoxic and glucolipotoxic conditions. When β-cells were incubated under these conditions, the expression levels of mitochondrial electron transport chain complex subunits, mitochondrial antioxidant enzymes (such as MnSOD and Prx3), β-cell apoptosis, lipogenic enzymes (such as ACC, FAS and ABCA1), intracellular lipid accumulation, oxidative stress, ER stress, mitochondrial membrane depolarization, nuclear NF- κB and sterol regulatory element binding protein 1c (SREBP1c) were all increased, in parallel with decreases in intracellular ATP content, citrate synthase enzymatic activity and glucose-stimulated insulin secretion. These changes were consistent with elevated mitochondrial oxidative stress, and incubation with the mitochondria-targeted antioxidants, MitoTempol or Mitoquinone (MitoQ), prevented these effects. In conclusion, mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress, promote their survival, and increase insulin secretion in cell models of the glucotoxicity and glucolipotoxicity associated with Type 2 diabetes.