Mitochondrial Reactive Oxygen Species Formation Research Articles

Manganese superoxide dismutase and aldehyde dehydrogenase deficiency increase mitochondrial oxidative stress and aggravate age-dependent vascular dysfunction

Imbalance between pro- and antioxidant species (e.g. during aging) plays a crucial role for vascular function and is associated with oxidative gene regulation and modification. Vascular aging is associated with progressive deterioration of vascular homeostasis leading to reduced relaxation, hypertrophy, and a higher risk of thrombotic events. These effects can be explained by a reduction in free bioavailable nitric oxide that is inactivated by an age-dependent increase in superoxide formation. In the present study, mitochondria as a source of reactive oxygen species (ROS) and the contribution of manganese superoxide dismutase (MnSOD, SOD-2) and aldehyde dehydrogenase (ALDH-2) were investigated. Age-dependent effects on vascular function were determined in aortas of C57/Bl6 wild-type (WT), ALDH-2(-/-), MnSOD(+/+), and MnSOD(+/-) mice by isometric tension measurements in organ chambers. Mitochondrial ROS formation was measured by luminol (L-012)-enhanced chemiluminescence and 2-hydroxyethidium formation with an HPLC-based assay in isolated heart mitochondria. ROS-mediated mitochondrial DNA (mtDNA) damage was detected by a novel and modified version of the fluorescent-detection alkaline DNA unwinding (FADU) assay. Endothelial dysfunction was observed in aged C57/Bl6 WT mice in parallel to increased mitochondrial ROS formation and oxidative mtDNA damage. In contrast, middle-aged ALDH-2(-/-) mice showed a marked vascular dysfunction that was similar in old ALDH-2(-/-) mice suggesting that ALDH-2 exerts age-dependent vasoprotective effects. Aged MnSOD(+/-) mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage. The correlation between mtROS formation and acetylcholine-dependent relaxation revealed that mitochondrial radical formation significantly contributes to age-dependent endothelial dysfunction.

The positive inotropic effect of endothelin-1 is mediated by mitochondrial reactive oxygen species

We have previously demonstrated the participation of reactive oxygen species (ROS) in the positive inotropic effect of a physiological concentration of Angiotensin II (Ang II, 1 nM). The objective of the present work was to evaluate the role and source of ROS generation in the positive inotropic effect produced by an equipotent concentration of endothelin-1 (ET-1, 0.4 nM). Isolated cat ventricular myocytes were used to measure sarcomere shortening with a video-camera, superoxide anion ( O 2 −) with chemiluminescence, and ROS production and intracellular pH (pH i) with epifluorescence. The ET-1-induced positive inotropic effect (40.4 ± 3.1%, n = 10, p < 0.05) was associated to an increase in ROS production (105 ± 29 fluorescence units above control, n = 6, p < 0.05). ET-1 also induced an increase in O 2 − production that was inhibited by the NADPH oxidase blocker, apocynin, and by the blockers of mitochondrial ATP-sensitive K + channels (mK ATP), glibenclamide and 5 hydroxydecanoic acid. The ET-1-induced positive inotropic effect was inhibited by apocynin (0.3 mM; 6.3 ± 6.6%, n = 13), glibenclamide (50 μM; 8.8 ± 3.5%, n = 6), 5 hydroxydecanoic acid (500 μM; 14.1 ± 8.1, n = 9), and by scavenging ROS with MPG (2 mM; 0.92 ± 5.6%, n = 8). ET-1 enhanced proton efflux (J H ) carried by the Na +/H + exchanger (NHE) after an acid load, effect that was blocked by MPG. Consistently, the ET-induced positive inotropic effect was also inhibited by the NHE selective blocker HOE642 (5 μM; 9.37 ± 6.07%, n = 7). The data show that the effect of a concentration of ET-1 that induces an increase in contractility of about 40% is totally mediated by an intracellular pathway triggered by mitochondrial ROS formation and stimulation of the NHE.

MITOCHONDRIAL COMPLEX III IS INVOLVED IN PROAPOPTOTIC BAK-INDUCED MICROVASCULAR ENDOTHELIAL CELL HYPERPERMEABILITY

It has been shown that the intrinsic mitochondrial apoptotic cascade is activated in vascular hyperpermeability after conditions such as hemorrhagic shock. Studies from our laboratory demonstrated mitochondrial reactive oxygen species (ROS) formation in endothelial cells during vascular hyperpermeability. We hypothesized that the participation of mitochondrial ROS in the intrinsic apoptotic cascade results in microvascular endothelial cell hyperpermeability. The purpose of this study was to identify the site(s) of ROS formation in the mitochondrial complex(es) that leads to hyperpermeability. Rat lung microvascular endothelial cell monolayers were pretreated with inhibitors of the complex(es) (I-V) before the activation of the mitochondrial apoptotic cascade using the proapoptotic peptide BAK (BH3). Inhibitors of the xanthine oxidase, nicotinamide adenine dinucleotide phosphate (reduced form) oxidase, NOS, and cytochrome P-450 monooxygenase were also studied. The hyperpermeability was determined by the fluorescence of fluorescein isothiocyanate-albumin that leaked across endothelial cells and ROS production by 2',7& rime;-dichlorofluorescein diacetate. Cytochrome c levels were also measured. BAK (BH3)-transfected cells showed increased ROS, cytosolic cytochrome c, and hyperpermeability (P<0.05). Complex III inhibitors antimycin A (10 microM) and stigmatellin (10 microM) attenuated BAK (BH3)-mediated ROS formation and hyperpermeability (P<0.05). The complex III inhibition decreased BAK (BH3)-mediated cytochrome c release. The results suggest that mitochondrial ROS formation, particularly at respiratory chain complex III, is involved in BAK-induced monolayer hyperpermeability.

Interrelation between the inhibition of glycolytic flux by silibinin and the lowering of mitochondrial ROS production in perifused rat hepatocytes

Silibinin, the most biologically active component of the polyphenolic extract from milk thistle seeds, is widely used to prevent many types of hepatobiliary disorders. Recent evidence suggests new applications for this ancient medication, notably for the treatment of type 2 diabetes owing to its antihyperglycemic properties. As we have lately demonstrated that silibinin lowered glucose production from various gluconeogenic substrates in perifused rat hepatocytes, the aim of this study was to examine the effect of silibinin on both oxidative glucose utilization and reactive oxygen species (ROS) generation since the release of ROS secondary to an increased mitochondrial metabolism may contribute to diabetic damage. We found that silibinin dose-dependently reduced glycolysis from carbohydrates in a cell perifusion system via an inhibitory effect targeted on pyruvate kinase activity. Furthermore, a dramatic effect upon oxidative phosphorylation was shown, as evidenced by a fall in ATP-to-ADP ratio, together with an increase in lactate-to-pyruvate ratio. The most attractive finding was that silibinin, at a concentration as low as 10 µM, fully mitigated the rise in metabolic flow-driven ROS formation. In addition, studies on isolated liver mitochondria revealed that this low dose of silibinin depressed ROS production linked to the electron transfer chain activity. From these results, one may tentatively suggest that interesting activities for silibinin, beyond its general antioxidant status, could be expected from its potential clinical use, especially in pathological conditions when mitochondrial ROS formation is severely enhanced.

MnSOD and ALDH‐2 deficiency potentiate aging‐associated vascular dysfunction, mitochondrial oxidative stress and mtDNA strand breaks

Vascular aging is associated with progressive deterioration of vascular homeostasis and reduced relaxation. These effects can be explained by a reduction in free bioavailable NO that is inactivated by an age‐dependent increase in superoxide formation. In the present study mitochondria as a source of reactive oxygen species (ROS) and the contribution of manganese superoxide dismutase (MnSOD) and aldehyde dehydrogenase (ALDH‐2) were investigated. Age‐dependent effects on vascular function were determined in aortae of these mice by isometric tension measurements. Mitochondrial ROS formation was measured by enhanced chemiluminescence in isolated heart mitochondria. ROS‐mediated mitochondrial DNA (mtDNA) damages were detected by the fluorescent‐detection alkaline DNA unwinding (FADU)‐assay. Expression of antioxidant proteins were assayed by quantitative RT‐PCR. Endothelial dysfunction was observed in aged C57/Bl6 WT mice in parallel to increased mitochondrial ROS formation and mtDNA damage. In contrast, middle‐aged ALDH‐2−/− mice showed a marked vascular dysfunction that was similar in old ALDH‐2−/− mice. Aged MnSOD+/− mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage. Mitochondrial radical formation significantly contributes to age‐dependent endothelial‐dysfunction.

New insights into bioactivation of organic nitrates, nitrate tolerance and cross-tolerance

Organic nitrates still represent a group of very effective anti-ischemic drugs used for the treatment of patients with stable angina, acute myocardial infarction and chronic congestive heart failure. Long-term therapy with organic nitrates, however, results in a rapid development of nitrate tolerance blunting their hemodynamic and antiischemic efficacy. Recent studies revealed that mitochondrial reactive oxygen species (ROS) formation and a subsequent oxidative inactivation of nitrate reductase, the mitochondrial aldehyde dehydrogenase (ALDH-2), play an important role for the development of nitrate and crosstolerance. The present review focuses firstly on the role of ALDH-2 for organic nitrate bioactivation and secondly on the role of oxidative stress in the development of tolerance and cross-tolerance (endothelial dysfunction) in response to various organic nitrates. Finally, we would like to draw the reader's attention to the protective properties of the organic nitrate pentaerithrityl tetranitrate (PETN), which, in contrast to all other organic nitrates, is able to upregulate enzymes with a strong antioxidative capacity thereby preventing tolerance and the development of endothelial dysfunction.

ROS production by adrenodoxin does not cause apoptosis in fission yeast

We previously showed that production of reactive oxygen species (ROS) caused by overexpression of the mitochondrial electron transfer protein adrenodoxin (Adx) induces apoptosis in mammalian cells. In the fission yeast Schizosaccharomyces pombe, ROS are also produced in cells that undergo an apoptotic-like cell death, but it is not yet clear whether they are actually causative for this phenomenon or whether they are merely produced as a by-product. Therefore, the purpose of this study was to trigger mitochondrial ROS production in fission yeast by overexpression of either wildtype Adx (Adx-WT) or of several activated Adx mutants and to investigate its consequences. It was found that strong expression of either Adx-WT or Adx-S112W did not produce any ROS, while Adx-D113Y caused a twofold and Adx1-108 a threefold increase in ROS formation as compared to basal levels. However, no typical apoptotic markers or decreased viability could be observed in these strains. Since we previously observed that an increase in mitochondrial ROS formation of about 60% above basal levels is sufficient to strongly induce apoptosis in mammalian cells, we conclude that S. pombe is either very robust to mitochondrial ROS production or does not undergo apoptotic cell death in response to mitochondrial ROS at all.

Role of mitochondria in non‐alcoholic fatty liver disease

Mitochondrial dysfunction is involved in the three stages of the transition from lack of exercise and excessive food intake to insulin resistance, diabetes and non-alcoholic steatohepatitis (NASH). In muscle, lack of exercise, a fat-rich diet, a polymorphism in peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1), and possibly age-related mitochondrial DNA (mtDNA) mutations may variously combine their effects to decrease PGC-1 expression, mitochondrial biogenesis and fat oxidation. Together with excessive food intake, insufficient fat oxidation causes fat accumulation and cellular stress in myocytes. The activation of Jun N-terminal kinase and protein kinase C-theta triggers the serine phosphorylation and inactivation of the insulin receptor substrate, and hampers the insulin-mediated translocation of glucose transporter-4 to the plasma membrane. Initially, the trend for increased blood glucose increases insulin secretion by pancreatic beta-cells. High plasma insulin levels compensate for insulin resistance in muscle and maintain normal blood glucose levels. Eventually, however, increased uncoupling protein-2 expression and possibly acquired mtDNA mutations in pancreatic beta-cells can blunt glucose-mediated adenosine triphosphate (ATP) formation and insulin secretion, to cause diabetes in some patients. High plasma glucose and/or insulin levels induce hepatic lipogenesis and cause hepatic steatosis. In fat-engorged hepatocytes, several vicious cycles involving tumor necrosis factor-alpha, reactive oxygen species (ROS), peroxynitrite, and lipid peroxidation products alter respiratory chain polypeptides and mtDNA, thus partially blocking the flow of electrons in the respiratory chain. The overreduction of upstream respiratory chain complexes increases mitochondrial ROS and peroxynitrite formation. Oxidative stress increases the release of lipid peroxidation products and cytokines, which together trigger the liver lesions of NASH.

Gap‐junction blocker carbenoxolone differentially enhances NMDA‐induced cell death in hippocampal neurons and astrocytes in co‐culture

The beneficial or detrimental role of gap junction communication in the pathophysiology of brain injury is still controversial. We used co-cultures of hippocampal astrocytes and neurons, where we identified homocellular astrocyte-astrocyte and heterocellular astrocyte-neuron coupling by fluorescence recovery after photobleaching, which was decreased by the gap junction blocker carbenoxolone (CBX). In these cultures, we determined the cell type-specific effects of CBX on the excitotoxic damage caused by N-methyl-D-aspartate (NMDA). We determined in both astrocytes and neurons the influence of CBX, alone or together with NMDA challenge, on cytotoxicity using propidium iodide labeling. CBX alone was not cytotoxic, but CBX treatment differentially accelerated the NMDA-induced cell death in both astrocytes and neurons. In addition, we measured mitochondrial potential using rhodamine 123, membrane potential using the oxonol dye bis(1,3-diethylthiobarbituric acid)trimethine oxonol, cytosolic Ca(2+) level using fura-2, and formation of reactive oxygen species (ROS) using dihydroethidium. CBX alone induced neither an intracellular Ca(2+) rise nor a membrane depolarization. However, CBX elicited a mitochondrial depolarization in both astrocytes and neurons and increased the ROS formation in neurons. In contrast, NMDA caused a membrane depolarization in neurons, coinciding with intracellular Ca(2+) rise, but neither mitochondrial depolarization nor ROS production seem to be involved in NMDA-mediated cytotoxicity. Pre-treatment with CBX accelerated the NMDA-induced membrane depolarization and prevented the repolarization of neurons after the NMDA challenge. We hypothesize that these effects are possibly mediated via blockage of gap junctions, and might be involved in the mechanism of CBX-induced acceleration of excitotoxic cell death, whereas the CBX-induced mitochondrial depolarization and ROS formation are not responsible for the increase in cytotoxicity. We conclude that both in astrocytes and neurons gap junctions provide protection against NMDA-induced cytotoxicity.

Mitochondrial oxidative stress and nitrate tolerance – comparison of nitroglycerin and pentaerithrityl tetranitrate in Mn-SOD+/-mice

BackgroundChronic therapy with nitroglycerin (GTN) results in a rapid development of nitrate tolerance which is associated with an increased production of reactive oxygen species (ROS). According to recent studies, mitochondrial ROS formation and oxidative inactivation of the organic nitrate bioactivating enzyme mitochondrial aldehyde dehydrogenase (ALDH-2) play an important role for the development of nitrate and cross-tolerance.MethodsTolerance was induced by infusion of wild type (WT) and heterozygous manganese superoxide dismutase mice (Mn-SOD+/-) with ethanolic solution of GTN (12.5 μg/min/kg for 4 d). For comparison, the tolerance-free pentaerithrityl tetranitrate (PETN, 17.5 μg/min/kg for 4 d) was infused in DMSO. Vascular reactivity was measured by isometric tension studies of isolated aortic rings. ROS formation and aldehyde dehydrogenase (ALDH-2) activity was measured in isolated heart mitochondria.ResultsChronic GTN infusion lead to impaired vascular responses to GTN and acetylcholine (ACh), increased the ROS formation in mitochondria and decreased ALDH-2 activity in Mn-SOD+/- mice. In contrast, PETN infusion did not increase mitochondrial ROS formation, did not decrease ALDH-2 activity and accordingly did not lead to tolerance and cross-tolerance in Mn-SOD+/- mice. PETN but not GTN increased heme oxygenase-1 mRNA in EA.hy 926 cells and bilirubin efficiently scavenged GTN-derived ROS.ConclusionChronic GTN infusion stimulates mitochondrial ROS production which is an important mechanism leading to tolerance and cross-tolerance. The tetranitrate PETN is devoid of mitochondrial oxidative stress induction and according to the present animal study as well as numerous previous clinical studies can be used without limitations due to tolerance and cross-tolerance.

Polyethylene Glycol–Superoxide Dismutase Prevents Endotoxin-induced Cardiac Dysfunction

Sepsis produces significant mitochondrial and contractile dysfunction in the heart, but the role of superoxide-derived free radicals in the genesis of these abnormalities is not completely understood. The study was designed to test the hypothesis that superoxide scavenger administration prevents endotoxin-induced cardiac mitochondrial and contractile dysfunction. Four groups of rats were studied, and animals were injected with either saline, endotoxin, endotoxin plus polyethylene glycol-adsorbed-superoxide dismutase (PEG-SOD; a free-radical scavenger), or PEG-SOD alone. Animals were killed 48 h after injections. We then measured cardiac mitochondrial generation of reactive oxygen species (ROS), formation of free-radical reaction products (protein carbonyls, lipid aldehydes, nitrotyrosine), mitochondrial function, and cardiac contractile function. Endotoxin elicited increases in cardiac mitochondrial ROS formation (p < 0.001), increases in cardiac levels of free-radical reaction products, reductions in mitochondrial ATP generation (p < 0.001), and decrements in cardiac pressure-generating capacity (p < 0.01). Administration of PEG-SOD blocked formation of free-radical reaction products, prevented mitochondrial dysfunction, and preserved cardiac contractility. For example, mitochondrial ATP generation was 923 +/- 50, 392 +/- 32, 753 +/- 25, and 763 +/- 36 nmol/min/mg, respectively, for control, endotoxin, endotoxin + PEG-SOD, and PEG-SOD groups (p < 0.001). In addition, cardiac systolic pressure generation at a diastolic pressure of 15 mm Hg averaged 110 +/- 11, 66 +/- 7, 129 +/- 10 and 124 +/- 5 mm Hg, respectively, for control, endotoxin, endotoxin + PEG-SOD, and PEG-SOD groups (p < 0.01). These data indicate that superoxide-derived oxidants play a critical role in the development of cardiac mitochondrial and contractile dysfunction in endotoxin-induced sepsis.

Reactive oxygen species, but not Ca2+overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion

We investigated the role of pH, reactive oxygen species (ROS), Ca2+, and the mitochondrial permeability transition (MPT) in pH-dependent ischemia-reperfusion injury to adult rat myocytes. Myocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 3 h to simulate ischemia. To simulate reperfusion, myocytes were reoxygenated at pH 6.2 or 7.4 for 2 h. Some myocytes were treated with MPT blockers (cyclosporin A and N-methyl-4-isoleucine cyclosporin) and antioxidants (desferal, diphenylphenylene diamine, and 2-mercaptopropionyl glycine). Mitochondrial membrane potential, inner membrane permeabilization, and ROS formation were imaged with tetramethylrhodamine methyl ester, calcein, and chloromethyldichlorofluorescein diacetate, respectively. For Ca2+ imaging, myocytes were coloaded with rhod-2 and fluo-4 to evaluate mitochondrial and cytosolic Ca2+, respectively. After 10 min of reperfusion at pH 7.4, calcein redistributed across the mitochondrial inner membrane, an event preceded by mitochondrial ROS formation and accompanied by hypercontracture, mitochondrial depolarization, and then cell death. Acidotic reperfusion, antioxidants, and MPT blockers each prevented the MPT, depolarization, hypercontraction, and cell killing. Antioxidants, but neither MPT blockers nor acidotic reperfusion, inhibited ROS formation after reperfusion. Furthermore, anoxic reperfusion at pH 7.4 prevented cell death. Both mitochondrial and cytosolic Ca2+ increased during ischemia but recovered in the first minutes of reperfusion. Mitochondrial and cytosolic Ca2+ overloading again occurred late after reperfusion. This late Ca2+ overloading was blocked by MPT inhibition. Intramitochondrial Ca2+ chelation by cold loading/warm incubation of BAPTA did not prevent cell death after reperfusion. In conclusion, mitochondrial ROS, together with normalization of pH, promote MPT onset and subsequent myocyte death after reperfusion. In contrast, Ca2+ overloading appears to be the consequence of bioenergetic failure after the MPT and is not a factor promoting MPT onset.

Metformin delays the manifestation of diabetes and vascular dysfunction in Goto–Kakizaki rats by reduction of mitochondrial oxidative stress

This study was undertaken to test the hypothesis that hyperglycaemia induces the generation of reactive oxygen species (ROS) by mitochondria and that the oxidative stress thereby exerted is diminished by treatment with metformin. As a parameter of mitochondrial ROS formation, the activity of mitochondrial aconitase activity was determined using Goto-Kakizaki (GK) rats as model of type 2 diabetes. In parallel with the development of diabetes (glucose, insulin), the generation of oxidative stress was determined in aortic tissue, heart and kidney of GK rats by measurement of lipid peroxides, oxidized proteins (carbonyl activity) and mitochondrial aconitase activity. Vascular activity was determined in aortae by measuring the endothelium-dependent vasodilatation in response to acetylcholine, and vasoconstriction in response to phenylephrine. At the age of 12-14 weeks, blood glucose levels rose dramatically from 7.5 up to 16.2 mM, indicating the manifestation of an overt diabetes. In addition, the glucose tolerance was impaired. The increase in blood glucose was not accompanied by changes in plasma insulin. Whereas the lipid peroxides in plasma only showed a tendency to increase, the amount of oxidized proteins (carbonyl moieties) increased from 4.6 to 10.9 micromol/mg protein (2.4 fold). In addition, the lipid peroxides in tissue were increased. Mitochondrial aconitase activity was reduced in the aorta and kidney, but not in the heart of diabetic animals. Treatment with metformin nearly normalized the hyperglycaemia and prevented the rise in carbonyl, tissue lipid peroxides and the fall in aconitase activity. Whereas the endothelium-dependent vasodilatation was not affected by the diabetes, the reaction of aortae in response to phenylephrine was strongly enhanced, changes which were prevented by treatment with metformin. These observations provide in vivo evidence that the generation of ROS plays an important role in the onset of diabetes and the development of vascular dysfunction in GK rats with type 2 diabetes.

Aging sensitizes toward ROS formation and lipid peroxidation in PS1M146L transgenic mice

Mutations in the presenilins (PS) account for the majority of familial Alzheimer disease (FAD) cases. To test the hypothesis that oxidative stress can underlie the deleterious effects of presenilin mutations, we analyzed lipid peroxidation products (4-hydroxynonenal (HNE) and malondialdehyde) and antioxidant defenses in brain tissue and levels of reactive oxygen species (ROS) in splenic lymphocytes from transgenic mice bearing human PS1 with the M146L mutation (PS1M146L) compared to those from mice transgenic for wild-type human PS1 (PS1wt) and nontransgenic littermate control mice. In brain tissue, HNE levels were increased only in aged (19–22 months) PS1M146L transgenic animals compared to PS1wt mice and not in young (3–4 months) or middle-aged mice (13–15 months). Similarly, in splenic lymphocytes expressing the transgenic PS1 proteins, mitochondrial and cytosolic ROS levels were elevated to 142.1 and 120.5% relative to controls only in cells from aged PS1M146L animals. Additionally, brain tissue HNE levels were positively correlated with mitochondrial ROS levels in splenic lymphocytes, indicating that oxidative stress can be detected in different tissues of PS1 transgenic mice. Antioxidant defenses (activities of antioxidant enzymes Cu/Zn-SOD, GPx, or GR) or susceptibility to in vitro oxidative stimulation was unaltered. In summary, these results demonstrate that the PS1M146L mutation increases mitochondrial ROS formation and oxidative damage in aged mice. Hence, oxidative stress caused by the combined effects of aging and PS1 mutations may be causative for triggering neurodegenerative events in FAD patients.

The mitochondrial uncoupler 2,4‐dinitrophenol attenuates tissue damage and improves mitochondrial homeostasis following transient focal cerebral ischemia

Ischemic stroke is caused by acute neuronal degeneration provoked by interruption of cerebral blood flow. Although the mechanisms contributing to ischemic neuronal degeneration are myriad, mitochondrial dysfunction is now recognized as a pivotal event that can lead to either necrotic or apoptotic neuronal death. Lack of suitable 'upstream' targets to prevent loss of mitochondrial homeostasis has, so far, restricted the development of mechanistically based interventions to promote neuronal survival. Here, we show that the uncoupling agent 2,4 dinitrophenol (DNP) reduces infarct volume approximately 40% in a model of focal ischemia-reperfusion injury in the rat brain. The mechanism of protection involves an early decrease in mitochondrial reactive oxygen species formation and calcium uptake leading to improved mitochondrial function and a reduction in the release of cytochrome c into the cytoplasm. The observed effects of DNP were not associated with enhanced cerebral perfusion. These findings indicate that compounds with uncoupling properties may confer neuroprotection through a mechanism involving stabilization of mitochondrial function.

The Effects of Methylmercury on Mitochondrial Function and Reactive Oxygen Species Formation in Rat Striatal Synaptosomes Are Age-Dependent

Methylmercury (MeHg) is especially toxic to the developing central nervous system. In order to understand the reasons for this age-dependent vulnerability, we compared the effects of MeHg on formation of reactive oxygen species (ROS) and mitochondrial function in striatal synaptosomes obtained from rats of various ages. Basal ROS levels were greater, and basal mitochondrial function was lower, in synaptosomes from younger animals, compared to adult animals. MeHg induced ROS formation in synaptosomes from rats of all ages, although the increases were greatest in synaptosomes from the younger animals. MeHg also reduced mitochondrial metabolic function, as assessed by MTT reduction, as well as mitochondrial membrane potential; again, the greatest changes were seen in synaptosomes from early postnatal animals. These age-dependent differences in susceptibility to MeHg are most likely due to a less efficient ROS detoxifying system and lower activity of mitochondrial enzymes in tissue from young animals.

Heterozygous Deficiency of Manganese Superoxide Dismutase in Mice (Mn-SOD+/-): A Novel Approach to Assess the Role of Oxidative Stress for the Development of Nitrate Tolerance

Nitroglycerin (GTN)-induced tolerance was reported to be associated with increased levels of reactive oxygen species (ROS) in mitochondria. In the present study, we further investigated the role of ROS for the development of nitrate tolerance by using heterozygous manganese superoxide dismutase knock-out mice (Mn-SOD+/-). Mn-SOD is acknowledged as a major sink for mitochondrial superoxide. Vasodilator potency of mouse aorta in response to acetylcholine and GTN was assessed by isometric tension studies. Mitochondrial ROS formation was detected by 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-enhanced chemiluminescence and mitochondrial aldehyde dehydrogenase (ALDH-2) activity was determined by a high-performance liquid chromatography-based assay. Aortic rings from Mn-SOD+/- mice showed normal endothelial function and vasodilator responses to GTN. In contrast, preincubation of aorta with GTN or long-term GTN infusion caused a marked higher degree of tolerance as well as endothelial dysfunction in Mn-SOD+/- compared with wild type. Basal as well as GTN-stimulated ROS formation was significantly increased in isolated heart mitochondria from Mn-SOD+/- mice, correlating well with a marked decrease in ALDH-2 activity in response to in vitro and in vivo GTN treatment. The data presented indicate that deficiency in Mn-SOD leads to a higher degree of tolerance and endothelial dysfunction associated with increased mitochondrial ROS production in response to in vitro and in vivo GTN challenges. These data further point to a crucial role of ALDH-2 in mediating GTN bioactivation as well as development of GTN tolerance and underline the important contribution of ROS to these processes.

Synergistic effects of hydrogen peroxide and ethanol on cell viability loss in PC12 cells by increase in mitochondrial permeability transition

The promoting effect of ethanol against the cytotoxicity of hydrogen peroxide (H 2O 2) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with H 2O 2 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. In PC12 cells and dopaminergic neuroblastoma SH-SY5Y cells, the promoting effect of ethanol on the H 2O 2-induced cell death was increased with exposure time. Ethanol promoted the nuclear damage, change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to H 2O 2 in PC12 cells. Catalase, carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the H 2O 2 and ethanol-induced mitochondrial dysfunction and cell injury. The results show that the ethanol treatment promotes the cytotoxicity of H 2O 2 against PC12 cells. Ethanol may enhance the H 2O 2-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that ethanol as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by oxidants.

Mitochondrion‐Targeted Photosensitizer Enhances the Photodynamic Effect‐Induced Mitochondrial Dysfunction and Apoptosis

Recently, the mitochondrion has been considered as a novel pharmacological target for anticancer therapy due to its crucial role involved in arbitrating cell apoptosis. We have previously demonstrated that 488-nm laser irradiation induced a specific mitochondrial reactive oxygen species (mROS) formation and apoptotic death. In this study, we used a second generation of photosensitizers, the benzoporphyrin-derivative monoacid ring A (BPD-MA). We investigated specifically mechanisms at the mitochondrial level for BPD-MA coupled with 690-nm laser irradiation, the photodynamic effect (PDE) of BPD-MA, using conventional and laser scanning imaging microscopy in intact C6 glioma cells. We demonstrated BPD-MA localized mainly in the mitochondrial area. The phototoxicity induced by 1-10 J 690-nm laser irradiation was minor as compared to that induced by 488-nm laser irradiation. Unlike other mitochondrion-targeted photosensitizers, the dark toxicity induced by BPD-MA (0.05-5 mg/mL, effective doses used for the PDE) was relatively low. Nevertheless, the PDE of BPD-MA using 0.5 mg/mL coupled with 5J 690-nm irradiation induced profound and rapid (< 1 min) mitochondrial swelling, mROS formation, and severe plasma membrane blebbing as compared to that induced by 488-nm laser irradiation (< 10 min). Later, the PDE of BPD-MA resulted in positive propidium iodide cell-death stain and positive TUNEL apoptotic nuclear stain and DNA laddering. Finally, the PDT of BPD-MA also instantaneously promoted the mitochondrion to diminish its covalent binding with a mitochondrial marker, MitoTracker Green. We conclude that the PDT of BPD-MA targeted primarily and compellingly the mitochondrion to induce effective mitochondria-mediated apoptosis and thus may serve as a powerful photosensitizer for clinical cancer therapy.

Inhibition of MG132-induced mitochondrial dysfunction and cell death in PC12 cells by 3-morpholinosydnonimine

The effect of 3-morpholinosydnonimine (SIN-1) against the cytotoxicity of MG132, a proteasome inhibitor, in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with MG132 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS), and depletion of GSH. Addition of SIN-1, a producer of nitric oxide (NO) and superoxide, differentially reduced the MG132-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 150 μM. Carboxy-PTIO, superoxide dismutase, Mn-TBAP, and ascorbate prevented the inhibitory effect of SIN-1 on the cytotoxicity of MG132. SIN-1 inhibited the MG132-induced change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents in PC12 cells. S-nitroso- N-acetyl- dl-penicillamine reduced the MG132-induced cell death in PC12 cells, whereas peroxynitrite and H 2O 2 did not affect the cytotoxicity of MG132. The results suggest that NO and superoxide liberated from SIN-1 exert an inhibitory effect against the cytotoxicity of MG132. SIN-1 may inhibit the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability that is associated with oxidative damage.