Induction Of Mitochondrial Permeability Transition Research Articles

Carvedilol: Just Another Beta-Blocker or a Powerful Cardioprotector?

Beta-blockers have been used to treat ischemic heart disease, due to negative chronotropic and inotropic properties, thus inducing a decrease in myocardial consumption of oxygen and nutrients, allowing a better balance between nutritional needs and the supply provided by the coronary blood flow. Recent developments in cell biology allowed us to understand that not all beta-blockers are equal, as their intracellular mechanisms of action can be very different. This paper will focus on carvedilol, a non-selective beta-blocker with alfa-blocker properties, currently used to treat hypertension, heart failure and coronary artery disease. Effects of carvedilol on cardiac mitochondria, their relation to its antioxidant properties, and how these can improve cardiomyocyte resistance to aggression and cardiac function will be discussed. We will begin by depicting the effect of carvedilol on mitochondrial parameters, namely oxidative phosphorylation, calcium homeostasis and energy production. Then we will focus on the mitochondrial permeability transition (MPT) and how the antioxidant properties of carvedilol can be used to minimize oxidative stress, a powerful inducer of MPT. Carvedilol will also be highlighted as an enzyme modulator, focusing on its importance to prevent doxorubicin (DOX) cardiotoxicity. The mitochondrial-related mechanism of cardioprotection involving carvedilol will also be addressed, as we will discuss some clinical pieces of evidence showing the importance of mechanisms previously depicted. In conclusion, based upon its molecular mechanisms of action, carvedilol seems to be a unique beta-blocker. These unique characteristics can help us understand the positive impact of carvedilol on the prognosis of patients with heart disease.

Fe(III) Shifts the Mitochondria Permeability Transition-Eliciting Capacity of Mangiferin to Protection of Organelle

Mangiferin acts as a strong antioxidant on mitochondria. However, when in the presence of Ca(2+), mangiferin elicits mitochondrial permeability transition (MPT), as evidenced by cyclosporin A-sensitive mitochondrial swelling. We now provide evidence, by means of electrochemical and UV-visible spectroscopical analysis, that Fe(III) coordinates with mangiferin. The resulting mangiferin-Fe(III) complex does not elicit MPT and prevents MPT by scavenging reactive oxygen species. Indeed, the complex protects mitochondrial membrane protein thiols and glutathione from oxidation. Fe(III) also significantly increases the ability of mangiferin to scavenge the 2,2-diphenyl-1-picrylhydrazyl radical, as well as to display antioxidant activity toward antimycin A-induced H(2)O(2) production and t-butyl hydroperoxide-promoted membrane lipid peroxidation in mitochondria. We postulate that coordination with Fe(III) constitutes a potential protective mechanism toward the prooxidant action of mangiferin and other catechol-containing antioxidants regarding MPT induction. Potential therapeutic relevance of this finding for conditions of pathological iron overload is discussed.

Mitochondrial permeability transition induced by novel pyridothiopyranopyrimidine derivatives: Potential new antimitochondrial antitumour agents

New pyridothiopyranopyrimidine derivatives (PTP1 and PTP2) were synthesised. Evaluation of the antiproliferative activity showed a significant capacity of the two compounds to inhibit cell growth. Investigation of the mechanism of action reveals that PTP1 interferes with the mitochondrial functions by inducing both swelling of the mitochondrial matrix and collapse of the electrical potential. These phenomena are fully prevented by typical inhibitors of the mitochondrial permeability transition, and are accompanied by the release of cytochrome c in the cytosol. The estimation of the redox state of thiol groups and glutathione suggests that the induction of permeability transition mediated by PTP1 is the result of an oxidative stress. The ability of cyclosporin A to prevent the antiproliferative effect of PTP1 indicates the induction of mitochondrial permeability transition as the molecular event responsible for the inhibition of cell growth. PTP1 also induces DNA fragmentation in intact cells. As regards PTP2, the presence of the p-toluensulphonamido group makes the lead chromophore unable to induce any effect on mitochondria.

Role of cyclophilin D in the resistance of brain mitochondria to the permeability transition

The mitochondrial permeability transition (MPT) is involved in both necrosis and apoptosis. Cyclophilin D (CypD) is an important component of the MPT. Brain mitochondria are more resistant to the MPT when compared to heart or liver mitochondria. We found that this increased resistance correlates with low expression of CypD in brain when compared to heart or liver. In newborn rats, sensitivity of brain mitochondria to the MPT and CypD expression are significantly higher than in mature animals. In an in vitro model of neuronal development, mitochondria in differentiated neuronal-like cells exert a higher calcium threshold toward MPT induction and express significantly less CypD when compared to undifferentiated precursor cells. Gain and loss of function experiments confirm the role of CypD in sensitivity to the MPT. Together our data indicate that the increased calcium threshold of brain mitochondria to the MPT correlates with low expression of CypD in brain; and that neuronal cells lose CypD during differentiation and become less sensitive to the MPT induction. This may be a protection mechanism that raises the threshold of brain tissue against injuries.

Overexpression of adenine nucleotide translocase reduces Ca 2+ signal transmission between the ER and mitochondria

The adenine nucleotide translocase (ANT), besides transferring ATP from the mitochondrial matrix to the rest of the cell, has also been proposed to be involved in mitochondrial permeability transition (MPT), and accordingly in mitochondrial Ca 2+ homeostasis. In order to assess the role of ANT in Ca 2+ signal transmission from the endoplasmic reticulum (ER) to mitochondria, we overexpressed the various ANT isoforms and measured the matrix [Ca 2+] ([Ca 2+] m) increases evoked by stimulation with IP 3-dependent agonists. ANT overexpression reduced the amplitude of the [Ca 2+] m peak following Ca 2+ release, an effect that was markedly greater for ANT-1 and ANT-3 isoforms than for ANT-2. Three further observations might explain these findings. First, the effect was partially reversed by treating the cells with cyclosporine A, suggesting the involvement of MPT. Second, the effect was paralleled by alterations of the 3D structure of the mitochondria. Finally, ANT-1 and ANT-3 overexpression also caused a reduction of ER Ca 2+ loading that caused a marginal decrease in the cytosolic Ca 2+ responses. Overall, these data provide evidence for the involvement of ANT-1 and ANT-3 in the induction of MPT and indicate the relevance of this phenomenon in ER–mitochondria Ca 2+ transfer.

Free radical scavenging action of the natural polyamine spermine in rat liver mitochondria

The isoflavonoid genistein, the cyclic triterpene glycyrrhetinic acid, and salicylate induce mitochondrial swelling and loss of membrane potential (ΔΨ) in rat liver mitochondria (RLM). These effects are Ca 2+-dependent and are prevented by cyclosporin A and bongkrekik acid, classic inhibitors of mitochondrial permeability transition (MPT). This membrane permeabilization is also inhibited by N-ethylmaleimide, butylhydroxytoluene, and mannitol. The above-mentioned pro-oxidants also induce an increase in O 2 consumption and H 2O 2 generation and the oxidation of sulfhydryl groups, glutathione, and pyridine nucleotides. All these observations are indicative of the induction of MPT mediated by oxidative stress. At concentrations similar to those present in the cell, spermine can prevent swelling and ΔΨ collapse, that is, MPT induction. Spermine, by acting as a free radical scavenger, in the absence of Ca 2+ inhibits H 2O 2 production and maintains glutathione and sulfhydryl groups at normal reduced level, so that the critical thiols responsible for pore opening are also consequently prevented from being oxidized. Spermine also protects RLM under conditions of accentuated thiol and glutathione oxidation, lipid peroxidation, and protein oxidation, suggesting that its action takes place by scavenging the hydroxyl radical.

Tetrandrine concentrations not affecting oxidative phosphorylation protect rat liver mitochondria from oxidative stress

The effects of tetrandrine (6,6′, 7,12-tetramethoxy-2, 2′-dimethyl-berbaman) on the mitochondrial function were assessed on oxidative stress, mitochondrial permeability transition (MPT), and bioenergetics of rat liver mitochondria. At concentrations lower than 100 nmol/mg protein, tetrandrine decreased the hydrogen peroxide formation, the extent of lipid peroxidation, the susceptibility to Ca 2+-induced opening of MPT pore, and inhibited the inner membrane anion channel activity, not significantly affecting the mitochondrial bioenergetics. High tetrandrine concentrations (100–300 nmol/mg protein) stimulated succinate-dependent state 4 respiration, while some inhibition was observed for state 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations. The respiratory control ratio and the transmembrane potential were depressed but the adenosine diphosphate to oxygen (ADP/O) ratio was less affected. A slight increase of the inner mitochondrial membrane permeability to H + and K + by tetrandrine was also observed. It was concluded that low concentrations of tetrandrine afford protection against liver mitochondria injury promoted by oxidative-stress events, such as hydrogen peroxide production, lipid peroxidation, and induction of MPT. Conversely, high tetrandrine concentrations revealed toxicological effects expressed by interference with mitochondrial bioenergetics, as a consequence of some inner membrane permeability to H + and K + and inhibition of the electron flux in the respiratory chain. The direct immediate protective role of tetrandrine against mitochondrial oxidative stress may be relevant to clarify the mechanisms responsible for its multiple pharmacological actions.

Protective effect of N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine (PF9601N) on mitochondrial permeability transition.

PF9601N, N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine, an monoamine oxidase (MAO) B inhibitor, has shown neuroprotective properties against dopaminergic toxins. To elucidate the mechanisms involved in this protection, the effect of PF9601N on mitochondria was assessed. PF9601N prevents mitochondrial swelling, drop in the electrical potential and oxidation of sulfhydryl groups, glutathione and pyridine nucleotides induced by Ca(2+). These observations demonstrate the protective effect of PF9601N on the induction of mitochondrial permeability transition. This protection is due to the interaction of the secondary protonated amino group in the molecule with pore-forming structures and to its antioxidant property, rather than to inhibition of MAO B activity. PF9601N also prevents the release of cytochrome c from mitochondria, suggesting its potential inhibitory effect on mitochondria-mediated apoptosis. The low IC(50) value for this inhibition, in comparison with deprenyl, make it a more efficient compound than propargylamines and other amines in protecting the bioenergetic functions of mitochondria.

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: Uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca 2+ at different conditions, a mitochondrial permeability transition (MPT) linked to protein thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8′-paepalantine dimer, and vioxanthin were assayed at 1–50 μM on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 μM Ca 2+; inhibition of these processes was assessed in non-energized organelles in the presence of 300 μM t-butyl hydroperoxide plus 500 μM Ca 2+. Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca 2+. Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin.

Doxorubicin-induced thiol-dependent alteration of cardiac mitochondrial permeability transition and respiration

Doxorubicin (DOX) is a highly effective treatment for several forms of cancer. However, clinical experience shows that DOX induces a cumulative and dose-dependent cardiomyopathy that has been ascribed to redox-cycling of the drug on the mitochondrial respiratory chain generating free radicals and oxidative stress in the process. Mitochondrial dysfunction including induction of the mitochondrial permeability transition (MPT) and inhibition of mitochondrial respiration have been implicated as major determinants in the pathogenesis of DOX cardiotoxicity. The present work was aimed at investigating whether the inhibition of mitochondrial respiration occurs secondarily to MPT induction in heart mitochondria isolated from DOX-treated rats and whether one or both consequences of DOX treatment are related with oxidation of protein thiol residues. DOX-induced oxidative stress was associated with the accumulation of products of lipid peroxidation and the depletion of alpha-tocopherol in cardiac mitochondrial membranes. No changes in mitochondrial coenzyme Q9 and Q10 concentrations were detected in hearts of DOX-treated rats. Cardiac mitochondria from DOX-treated rats were more susceptible to diamide-dependent induction of the MPT. Although DOX treatment did not affect state 4 respiration, state 3 respiration was decreased in heart mitochondria isolated from DOX-treated rats, which was reversed in part by adding either cyclosporin A or dithiothreitol, but not Trolox. The results suggest that in DOX-treated rats, (i) induction of the MPT is at least in part responsible for decreased mitochondrial respiration, (ii) heart mitochondria are more susceptible to diamide induced-MPT, (iii) thiol-dependent alteration of mitochondrial respiration is partially reversible ex vivo with dithiothreitol. Collectively, these data are consistent with the thesis that thiol-dependent alteration of MPT and respiration is an important factor in DOX-induced mitochondrial dysfunction.

Depletion of adenine nucleotide translocator protein in heart mitochondria from doxorubicin-treated rats—Relevance for mitochondrial dysfunction

Doxorubicin (DOX) is a highly effective treatment for several forms of cancer. However, DOX induces a cumulative and dose-dependent cardiomyopathy that has been ascribed to redox-cycling of the molecule on the mitochondrial complex I generating in the process increased oxidative stress. Mitochondrial dysfunction, including induction of the mitochondrial permeability transition (MPT) and inhibition of mitochondrial respiration have been implicated as major determinants in the pathogenesis of DOX cardiotoxicity. The adenine nucleotide translocator (ANT) has been suggested to be a principal component of the MPT pore and a possible target for DOX-induced cardiotoxicity. Nonetheless, no definitive evidence has been presented showing that altered ANT activity is due to decreased amount of the protein. By using carboxyatractyloside as a specific modulator of ANT activity and Western blotting, we observed that following DOX treatment in rats: (1) the amount of “functional ANT” that contributes to cardiac mitochondrial respiration with different substrates is reduced, (2) titrations with carboxyatracyloside revealed a lower threshold for MPT induction and most importantly, (3) a specific decrease in the amount of the ANT protein. This study identifies the ANT as one important target for DOX-induced cardiac toxicity and correlates the decrease in ANT protein concentration with inhibition of mitochondrial respiration and increased ability to form or at least regulate MPT pores.

Role of mitochondrial membrane permeability transition in N-nitrosofenfluramine-induced cell injury in rat hepatocytes

The role of mitochondrial membrane permeability transition in N-nitrosofenfluramine-induced cell injury was studied in mitochondria and hepatocytes isolated from rat liver. Mitochondrial permeability transition has been proposed as a common final pathway in acute cell death through mitochondrial dysfunction. In isolated mitochondria, N-nitrosofenfluramine (0.25 to 1.0 mM) in the presence of Ca 2+ (50 μM) elicited a concentration-dependent induction of mitochondrial swelling dependent on mitochondrial permeability transition and the release of cytochrome c, both of which were prevented by pretreatment with a specific inhibitor of mitochondrial permeability transition, cyclosporin A (0.2 μM). The effects of N-nitrosofenfluramine on mitochondria were more potent than those of fenfluramine, which is a sympathomimetic amine with anorectic action. The pretreatment of isolated hepatocytes with cyclosporin A (2 μM) partially but not completely prevented N-nitrosofenfluramine (0.6 mM; a low toxic dose)-induced cell death, loss of cellular ATP, formation of cell blebs and decrease in mitochondrial membrane potential. These results suggest that the onset of N-nitrosofenfluramine-induced cytotoxicity is linked to mitochondrial failure dependent upon induction of mitochondrial permeability transition accompanied by mitochondrial depolarization, the release of cytochrome c and depletion of intracellular ATP through uncoupling of oxidative phosphorylation.

Two Critical Factors Affecting the Release of Mitochondrial Cytochrome c as Revealed by Studies Using N,N′-Dicyclohexylcarbodiimide as an Atypical Inducer of Permeability Transition

N,N'-dicyclohexylcarbodiimide (DCCD) was earlier reported to have stimulatory effects on mitochondrial respiration and to induce mitochondrial swelling, when it was added to mitochondrial suspensions. These data seem to imply that DCCD caused the mitochondrial permeability transition (PT), but this possibility had never been investigated. In the present study, effects of DCCD on the mitochondrial structure and function were studied in detail. DCCD was found to induce mitochondrial PT in a cyclosporine A-insensitive manner. Electron microscopic analysis also supported the induction of the mitochondrial PT by DCCD. However, different from many other PT inducers, DCCD failed to cause massive release of mitochondrial cytochrome c. To understand the relationship between the induction of mitochondrial PT and the release of mitochondrial cytochrome c, we compared the actions of DCCD on mitochondrial structure and function with those of Ca2+, known as an ordinary PT inducer. As a result, two parameters considered to be critical for controlling the release of mitochondrial cytochrome c on the induction of PT were mitochondrial volume and the velocity of mitochondrial oxygen consumption.

Oxidative Stress Is Responsible for Mitochondrial Permeability Transition Induction by Salicylate in Liver Mitochondria

The interaction of salicylate with the respiratory chain of liver mitochondria generates hydrogen peroxide and, most probably, other reactive oxygen species, which in turn oxidize thiol groups and glutathione. This oxidative stress, confirmed by the prevention of action by antioxidant agents, leads to the induction of the mitochondrial permeability transition in the presence of Ca2+. This phenomenon induces further increase of oxidative damage resulting in impairment of oxidative phosphorylation and beta-oxidation, cardinal features of Reye's syndrome in the liver. Mitochondrial permeability transition induction also induces the release of cytochrome c and apoptotic inducing factor from mitochondria, suggesting that salicylate also behaves as a pro-apoptotic agent. The reactive group of salicylate for inducing oxidative stress is the hydroxyl group which, by interacting with a Fe-S cluster of mitochondrial Complex I, the so-called N-2(Fe-S) center, produces reactive oxygen species.

Dephosphorylation of the Rieske iron–sulfur protein after induction of the mitochondrial permeability transition

In the mitochondrial permeability transition (MPT), MPT pores open to cause the mitochondrial inner membrane to become non-selectively permeable to molecules of mass up to 1500 Da. In this study, we used proteomics to investigate protein changes after MPT induction. Isolated rat liver mitochondria were incubated with various MPT inducers, including CaCl 2, tert-butylhydroperoxide, and phenylarsine oxide, in the presence and absence of the MPT inhibitor, cyclosporin A. MPT induction was confirmed by an absorbance swelling assay. Mitochondrial membrane proteins prepared from control and treated mitochondria were separated by two-dimensional (2D) gel electrophoresis and stained with SyproRuby or Coomassie blue. Proteins of interest were further identified by mass spectrometry. 2D gel electrophoresis by isoelectric focusing and SDS–PAGE consistently showed a protein spot that shifted to a more basic isoelectric point after the MPT. This shift was prevented by CsA but did not occur after protonophoric uncoupling. Mass spectrometry identified this protein as the Rieske iron–sulfur protein (RISP) of ubiquinol–cytochrome c reductase (Complex III). Phosphatase treatment of sonicated mitochondria caused the same shift in RISP as occurred in MPT inducer-treated mitochondria. 2D gel electrophoresis by blue-native-PAGE and SDS–PAGE showed that RISP existed as an apparent monomer in mitochondrial membranes in addition to forming a complex with ubiquinol–cytochrome c reductase. These findings suggest that RISP may be part of MPT pores and that dephosphorylation of RISP may play a role in regulation of the MPT.

Agaric acid induces mitochondrial permeability transition through its interaction with the adenine nucleotide translocase. Its dependence on membrane fluidity

The effect of agaric acid as inducer of mitochondrial permeability transition was studied. It was found that: (i) agaric acid (AA) promoted efflux of accumulated Ca 2+, collapse of transmembrane potential, and mitochondrial swelling; (ii) these effects depend on membrane fluidity; (iii) ADP inhibited the effect of AA on Ca 2+ efflux, and (iv) AA blocked binding of the sulfhydryl reagent, eosin-5-maleimide, to the adenine nucleotide translocase. It is proposed that AA induces pore opening through binding of the citrate moiety to the ADP/ATP carrier; this interaction must be stabilized by insertion of the alkyl chain in the lipid milieu of the membrane.

Molecular basis of morphological changes in mitochondrial membrane accompanying induction of permeability transition, as revealed by immuno-electron microscopy

The mitochondrial inner membrane typically shows a condensed structure when examined by electron microscopy. However, this typical structure is known to disappear upon induction of the mitochondrial permeability transition (PT). This change in the appearance of the mitochondrial membrane structure that accompanies the induction of PT is thought to reflect changes in the permeability of inner mitochondrial membrane; however, its molecular basis has remained uncertain. In the present study, changes in membrane status were examined by immuno-electron microscopy using antibodies against the voltage-dependent anion channel (VDAC), β-subunit of F 1-ATPase (F 1β), and cytochrome c (cyt. c). In control mitochondria, antibody against VDAC was observed at the rim of the mitochondria, whereas antibodies against F 1β and cytochrome c bound these molecules inside of the mitochondria. However, in PT-induced mitochondria, all three antibodies were observed at the mitochondrial rim. These results strongly suggest that the inner mitochondrial membrane is shoved to the rim region of mitochondria upon induction of mitochondrial PT.

Carbenoxolone Induces Oxidative Stress in Liver Mitochondria, Which Is Responsible for Transition Pore Opening

Carbenoxolone (Cbx), a derivative of glycyrrhetinic acid, which has been found to affect mineralocorticoid and glucocorticoid receptors, induces swelling and membrane potential collapse when added to Ca(2+)-loaded liver mitochondria at 10 microM concentrations. These effects are strictly correlated with hydrogen peroxide generation, increase in oxygen uptake, and sulfhydryl and pyridine nucleotide oxidation. Cyclosporin A, bongkrekic acid, and N-ethylmaleimide completely abolish all the above-described effects, suggesting that Cbx can be considered an inducer of mitochondrial permeability transition by means of oxidative stress. Cbx can also trigger the apoptotic pathway because the above events are also correlated with the loss of cytochrome c. These effects are probably related to the conjugated carbonyl oxygen in C-11, which produces reactive oxygen species by interacting with the mitochondrial respiratory chain, mainly at the level of complex I but, most likely, also with complex III. The oxidative stress induced by Cbx, which is responsible for pore opening, excludes that this is related to a genomic effect of the compound.

Inhibition Mechanisms of Mitochondrial Permeability Transition by 4-Hydroxytamoxifen: Protection of NAD(P)H and Thiol Group Oxidation

The effects of 4-hydroxytamoxifen (OHTAM) on the mitochondrial permeability transition (MPT) induced by Ca2+ plus peroxynitrite (ONOO−) or phenylarsine oxide (PhAsO) were studied to clarify its mechanisms of MPT inhibition. Ca2+ plus ONOO− induced mitochondrial swelling, membrane potential (Δ Ψ) depolarization, and Ca2+ release. OHTAM, when preincubated with mitochondria, prevents those events, and if added after their induction this drug promotes a time-dependent reversal of Δ Ψ depolarization and Ca2+ release associated with MPT induction, because these events are also inhibited by cyclosporine A (CyA). Preincubation with OHTAM also inhibits thiol group oxidation associated with the MPT promoted by ONOO− and allows the NAD(P)+ to recover their reduced state faster and in a higher extension. The mitochondrial swelling and Ca2+ release after MPT induction with Ca2+ plus PhAsO are inhibited by OHTAM; similarly to CyA, the oxidation of NAD(P)H induced by this combination is also inhibited. According to these data, the MPT inhibition by OHTAM may be related to its antioxidant capacity and the binding to target protein components of the MPT, preventing the oxidation of NAD(P)H and thiol groups, an event that increases the sensitivity to MPT induction.

Reactive oxygen species and the mitochondrial signaling pathway of cell death.

Reactive oxygen species (ROS) are produced as a by-product of cellular metabolic pathways and function as a critical second messenger in a variety of intracellular signaling pathways. Thus, a defect or deficiency in the anti-oxidant defense system on the one hand and/or the excessive intracellular generation of ROS on the other renders a cell oxidatively stressed. As a consequence, direct or indirect involvement of ROS in numerous diseases has been documented. In most of these cases, the deleterious effect of ROS is a function of activation of intracellular cell-death circuitry. To that end, involvement of ROS at different phases of the apoptotic pathway, such as induction of mitochondrial permeability transition and release of mitochondrial death amplification factors, activation of intracellular caspases and DNA damage, has been clearly established. For instance, the ROS-induced alteration of constitutive mitochondrial proteins, such as the voltage-dependent anion channel (VDAC) and/or the adenine nucleotide translocase (ANT) can induce the pro-apoptotic mitochondrial membrane permabilization. Not only do these observations provide insight into the intricate mechanisms underlying a variety of disease states, but they also present novel opportunities for the design and development of more effective therapeutic strategies.