Mitochondria-specific Antioxidant Research Articles

Local Redox Modifications in Skeletal Muscle Differentially Affect Sarcoplasmic Reticulum Calcium Release and Muscle Force Generation

Aims: Reactive oxygen species are implicated in reducing muscle force production through oxidative cellular damage in skeletal muscle following repetitive fatiguing muscle contractions. We investigated whether shifting cellular redox status from an oxidized to reduced state could improve recovery of force production in skeletal muscle following fatigue. Methods: Mechanically-dissected intact single fibers from flexor digitorum brevis muscles of C57/BL6 mice were fatigued at physiological temperature (32 °C) with brief 150 ms duration 70 Hz tetani every 1 s for a total of 60 contractions. Fibers were superfused with the reducing agent dithiothretrol (DTT 1 mM, n = 10) for 20 min after fatigue-induced force loss was established. Results: The reducing agent, DTT, temporarily improved low-frequency (30 Hz) contractile force by 65% by increasing myofibrillar Ca2+ sensitivity without affecting sarcoplasmic reticulum (SR) Ca2+ release during recovery from fatigue. Intriguingly, addition of the oxidizing agent tert-butyl hydroperoxide (T-BOOH 10 μM, n=7) also temporarily improved 30 Hz force by 47% without affecting SR Ca2+ release. We then determined whether antioxidants and inhibitors of free radical production could prevent in the first place the oxidative damage associated with fatigue. We found that a mitochondria specific antioxidant (SS-31) and nitric oxide synthase inhibitor (L-NAME) recovered fatigue-induced impairments in SR Ca2+ release without affecting 30 Hz force. Conclusion: Antioxidants and inhibition of free radicals improves Ca2+ release during recovery, but has no effect on improving low-frequency force production. On the other hand, acute change in intracellular milieu using redox agents improves force development but does not affect Ca2+ release. Thus, proteins involved in SR Ca2+ release and myofibrillar contractile proteins show different dependency on the local redox micro-environment.

The role of the mitochondrial oxidative stress in the cytotoxic effects of the green tea catechin, (–)‐epigallocatechin‐3‐gallate, in oral cells

The tea catechin, (-)-epigallocatechin-3-gallate (EGCG), has potential cancer preventive effects. The prooxidant activity of EGCG may play a role in these effects. Here, we report that EGCG exerted cytotoxic effects against oral cancer cell lines (IC50 = 83-95μM). EGCG treatment resulted in formation of extracellular reactive oxygen species (ROS), however, these ROS were rapidly cleared (half-life = 1.7 h). EGCG treatment increased the production of mitochondrial H2 O2 in SCC-25 cells (0-6 h) before the induction of apoptosis. Subsequently, an opening of the mitochondrial transition pore and a decrease in mitochondrial membrane potential were observed. The mitochondria-specific antioxidant, MitoTEMPO, reduced these effects. HGF-1 human gingival fibrobasts were resistant to EGCG (IC50 > 200μM) and EGCG-induced ROS. EGCG induced differential expression of genes related to antioxidant defense in oral cancer cells and gingival fibroblasts: metallothionein 3, superoxide dismutase 2/3, and thioredoxin reductase 2 were downregulated in SCC-25 cells, but upregulated in HGF-1 cells. We conclude that induction of mitochondrial ROS and mitochondrial dysfunction by EGCG play a role in the inhibition of oral cancer, and that gingival fibroblasts are spared from these effects in part because of a selective induction of antioxidant responsive genes.

Role of mitochondria-specific antioxidants and their derivatives in drug discovery and development for Alzheimer's disease

Role of mitochondria-specific antioxidants and their derivatives in drug discovery and development for Alzheimer's disease

Selective deposition of dietary α-Lipoic acid in mitochondrial fraction and its synergistic effect with α-Tocoperhol acetate on broiler meat oxidative stability

The use of bioactive antioxidants in feed of broiler to mitigate reactive oxygen species (ROS) in biological systems is one of promising nutritional strategies. The aim of present study was to alleviate ROS production in mitochondrial fraction (MF) of meat by supplemented dietary antioxidant in feed of broiler. For this purpose, mitochondria specific antioxidant: α-lipoic acid (25 mg, 75 mg and 150 mg) with or without combination of α-tocopherol acetate (200 mg) used in normal and palm olein oxidized oil (4%) supplemented feed. One hundred and eighty one day old broiler birds were randomly divided into six treatments and provided the mentioned feed from third week. Feed intake, feed conversion ratio (FCR) remained statistically same in all groups while body weight decreased in supplemented groups accordingly at the end of study. The broiler meat MF antioxidant potential was significantly improved by feeding supplemented feed estimated as 1,1-di phenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, 2,2-azinobis-(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS+) and thiobarbituric acid reactive substances (TBARS). The maximum antioxidant activity was depicted in group fed on 150 mg/kg α-lipoic acid (ALA) and 200 mg/kg α-tocopherol acetate (ATA) (T4) in both breast and leg MF. Moreover, TBARS were higher in leg as compared to breast MF. Although, oxidized oil containing feed reduced the growth, lipid stability and antioxidant potential of MF whilst these traits were improved by receiving feed containing ALA and ATA. ALA and ATA showed higher deposition in T4 group while least in group received oxidized oil containing feed (T5). Positive correlation exists between DPPH free radical scavenging activity and the ABTS + reducing activity. In conclusion, ALA and ATA supplementation in feed had positive effect on antioxidant status of MF that consequently diminished the oxidative stress in polyunsaturated fatty acid enriched meat.

Age‐associated attenuation of autophagy underlies ryanodine receptor hyperactivity

Aging is associated with reduced autophagy and altered mitochondrial function; yet, the link between these processes and altered calcium (Ca) homeostasis remains unknown. We used ventricular myocytes from young (5–9 months) and old (4–5 years) rabbit hearts to test the hypothesis that reduced autophagy leads to dysfunctional mitochondria that produces ROS, leading to oxidation of ryanodine receptors (RyRs) and changes in Ca homeostasis. Markers of autophagy were measured via western blotting and β‐galactosidase staining. Age‐associated changes in ROS production, mitochondrial membrane potential, and Ca handling were determined in myocytes using confocal microscopy. Specific markers confirmed reduced autophagy. In aged myocytes, ROS production was increased and mitochondrial membrane was depolarized; both parameters were exacerbated with beta‐adrenergic agonist isoproterenol. Also with isoproterenol, aged myocytes showed decreased Ca transient amplitude, SR Ca load, and latency of spontaneous Ca waves, which were reversed with the mitochondria‐specific antioxidant mito‐TEMPO. These data suggest that age‐related attenuation of autophagy may lead to mitochondrial dysfunction that underlies increased thiol‐oxidation of RyRs. This process could explain the reduced RyR refractoriness leading to spontaneous Ca release in senescent cardiomyocytes making aged hearts more prone to arrhythmia.

Targeting of mitochondrial reactive oxygen species production does not avert lipid-induced insulin resistance in muscle tissue from mice

High-fat, high-sucrose diet (HF)-induced reactive oxygen species (ROS) levels are implicated in skeletal muscle insulin resistance and mitochondrial dysfunction. Here we investigated whether mitochondrial ROS sequestering can circumvent HF-induced oxidative stress; we also determined the impact of any reduced oxidative stress on muscle insulin sensitivity and mitochondrial function. The Skulachev ion (plastoquinonyl decyltriphenylphosphonium) (SkQ), a mitochondria-specific antioxidant, was used to target ROS production in C2C12 muscle cells as well as in HF-fed (16 weeks old) male C57Bl/6 mice, compared with mice on low-fat chow diet (LF) or HF alone. Oxidative stress was measured as protein carbonylation levels. Glucose tolerance tests, glucose uptake assays and insulin-stimulated signalling were determined to assess muscle insulin sensitivity. Mitochondrial function was determined by high-resolution respirometry. SkQ treatment reduced oxidative stress in muscle cells (-23% p < 0.05), but did not improve insulin sensitivity and glucose uptake under insulin-resistant conditions. In HF mice, oxidative stress was elevated (56% vs LF p < 0.05), an effect completely blunted by SkQ. However, HF and HF+SkQ mice displayed impaired glucose tolerance (AUC HF up 33%, p < 0.001; HF+SkQ up 22%; p < 0.01 vs LF) and disrupted skeletal muscle insulin signalling. ROS sequestering did not improve mitochondrial function. SkQ treatment reduced muscle mitochondrial ROS production and prevented HF-induced oxidative stress. Nonetheless, whole-body glucose tolerance, insulin-stimulated glucose uptake, muscle insulin signalling and mitochondrial function were not improved. These results suggest that HF-induced oxidative stress is not a prerequisite for the development of muscle insulin resistance.

Reactive oxygen species contribute to the development of arrhythmogenic Ca2+ waves during β‐adrenergic receptor stimulation in rabbit cardiomyocytes

While β-adrenergic receptor (β-AR) stimulation leads to positive inotropic effects, it can also induce arrhythmogenic Ca2+ waves. β-AR stimulation increases mitochondrial oxygen consumption and, thereby, the production of reactive oxygen species (ROS). We therefore investigated the role of ROS in the generation of Ca2+ waves during β-AR stimulation in rabbit ventricular myocytes. Isoproterenol (ISO) increased Ca2+ transient amplitude during systole, sarcoplasmic reticulum (SR) Ca2+ load and the occurrence of Ca2+ waves during diastole. These effects, however, developed at different time points during ISO application.While SR Ca2+ release and load reached a maximum level after 3 min, Ca2+ waves occurred at the highest frequency only after 6 min of ISO application.Measurement of intra-SR-free Ca2+ concentration ([Ca2+]SR) showed an initial increase of SR Ca2+ load followed by a gradual decline over time during ISO application. This decline of [Ca2+]SR was not due to decreased SR Ca2+ uptake, but instead was the result of increased SRCa2+ leak mainly in the form of Ca2+ waves. ISO application led to significant RyR phosphorylation at the protein kinase A (PKA)-specific site, which remained relatively stable throughout β-AR activation.Moreover, β-AR stimulation significantly increased ROS production after 4–6 min of ISO application. The ROS scavenger Tiron and the superoxide dismutase mimetic MnTBPA abolished the ISO-mediated ROS production. The mitochondria-specific antioxidant Mito-Tempo and an inhibitor of the electron transport chain, rotenone, also effectively prevented the ISO-mediated ROS production. Scavenging ROS during ISO application decreased the occurrence of Ca2+ waves and partially prevented augmentation of SRCa2+ leak, but did not affect the increase of Ca2+ transient amplitude. Treatment of myocytes with ISO for 15 min significantly reduced the free thiol content in RyRs. These data suggest that increased mitochondrial ROS production during β-AR stimulation causes RyR oxidation. Together with RyR phosphorylation, oxidation of RyRs increases diastolic SR Ca2+ leak to a critical level leading to the generation of arrhythmogenic Ca2+ waves.

Mitochondria Specific Antioxidants and their Derivatives in the Context of the Drug Development for Neurodegeneration and Cancer

Neurodegeneration and cancer are fast becoming the leading causes of age-associated disability, dementia and ultimately death worldwide. Although oxidative stress has been intensely studied, little analysis has been done in chronic oxidative stress-induced mitochondrial models. In this regard, DNA-overproliferation and/or deletion initiate mitochondrial deregulation causing energy failure, which has been implicated in the pathogenesis of Alzheimer’s disease (AD), tumor growth, and metastasis. In this regard, decline in mitochondrial normal homeostasis during the development and maturation of the neurodegeneration, tumor growth, and/or metastases is characterized by tissue and cellular oxygen deficiency, which leads to subcellular energy defects. In addition, the overexpression of the cascades initiates the formation and release of large amounts of reactive free radicals [mainly nitric oxide (NO) via the overexpression of NO synthases], which cause the oxidative stress, cellular alterations, and concomitant mitochondrial lesions and decline in normal organ function. The present study explores the intimate, i.e. direct relationship between chronic oxidative stress and mitochondrial damage as a vital life-supporter for cells and/or the microcirculatory systems whose damage occurs before the development of human AD. Our study highlights the effects of chronic oxidative stress-induced mitochondrial DNA over-proliferation and/or deletion and mitochondrial enzyme activities during the development of human AD. Mitochondrial DNA damage also leads to other pathologies, including colorectal cancer in liver metastasis, and malignant brain cancers. We hypothesize that mitochondrial lesions, especially mitochondrial DNA abnormalities, are detrimental to cell viability and thus mitochondrial DNA damage could be used as a new diagnostic tool and/or criterion for the early detection of AD and other diseases. Further extension of this approach will enable us not only for the better understanding of the blood brain barrier (BBB) homeostasis, which most likely plays a key role in the development of AD and some of forms of the cancer, but also for the development of new and more specific treatment strategies.

Mitochondrion-Specific Antioxidants as Drug Treatments for Alzheimer Disease

Age-related dementias such as Alzheimer disease (AD) have been linked to vascular disorders like hypertension, diabetes and atherosclerosis. These risk factors cause ischemia, inflammation, oxidative damage and consequently reperfusion, which is largely due to reactive oxygen species (ROS) that are believed to induce mitochondrial damage. At higher concentrations, ROS can cause cell injury and death which occurs during the aging process, where oxidative stress is incremented due to an accelerated generation of ROS and a gradual decline in cellular antioxidant defense mechanisms. Neuronal mitochondria are especially vulnerable to oxidative stress due to their role in energy supply and use, causing a cascade of debilitating factors such as the production of giant and/or vulnerable young mitochondrion who's DNA has been compromised. Therefore, mitochondria specific antioxidants such as acetyl-L-carnitine and R-alphalipoic acid seem to be potential treatments for AD. They target the factors that damage mitochondria and reverse its effect, thus eliminating the imbalance seen in energy production and amyloid beta oxidation and making these antioxidants very powerful alternate strategies for the treatment of AD.

Reactive Oxygen Species, NADPH Oxidases, and Hypertension

Reactive oxygen species (ROS) produced in the neuronal, renal, and vascular systems not only influence cardiovascular physiology but are also strongly implicated in pathological signaling leading to hypertension. Different sources of ROS have been identified, ranging from xanthine-xanthine oxidase and mitochondria to NADPH oxidase (Nox) enzymes. Of 7 Nox family members, Nox1, Nox2, and Nox4 (and Nox5 in humans) influence the cardiovascular system. Their activation processes and cell and tissue distribution vary widely, adding complexity to understanding their functional roles. Whether these systems act collectively or independently in disease conditions is unclear, but recently feed forward mechanisms have been established between ROS sources. Studies published in Hypertension over the last few years are the focus of this review, and they provide a framework with which to consider the roles of Nox enzymes in neuronal, renal, and vascular hypertensive mechanisms, as well as cardiac remodeling, and their relationships with other ROS-generating systems. ### Neuronal ROS in Hypertension Redox signaling in the central nervous system is well recognized in neuronal control of blood pressure (BP), as well as in response to angiotensin II (Ang II) and aldosterone, which are linked to ROS-dependent hypertension. Recently, new roles for ROS have been described in the hypothalamus and brain stem, nucleus tractus solitarius (NTS), subfornical organ (SFO), rostral ventrolateral medulla, and area postrema (Figure 1). Figure 1. Neuronal NADPH oxidase–dependent ROS involved in central regulation of hypertension. NADPH oxidase homologues, mainly Nox2 and Nox4, are found in different regions of the neuronal system and are reported to have a role in the neuropathogenesis of hypertension by enhancing the sympathetic nerve activity. Nox-induced ROS initiate a forward loop in cross-activation of different receptors and between Nox and mitochondrial ROS. OVLT indicates organum vasculosum of the lamina terminalis; PVN, paraventricular nucleus; PP, posterior pituitary; AP, area postrema; RVLM, rostral ventrolateral medulla. Several …

Role of mitochondrial reactive oxygen species in osteoclast differentiation

Previously we showed that hypoxia-induced mitochondrial respiratory stress in RAW 264.7 macrophages and other cells caused activation of retrograde signaling (also known as mitochondrial respiratory stress signaling) and the appearance of tartrate-resistant acid phosphatase (TRAP)-positive cells. In the present study, we used N-acetyl cysteine and ascorbate (general antioxidants) and MitoQ, a mitochondria-specific antioxidant, to investigate the role of intracellular reactive oxygen species (ROS) in osteoclast differentiation. Our results show that hypoxia-mediated mitochondrial dysfunction, as tested by disruption of mitochondrial transmembrane potential, was suppressed by MitoQ as well as by the other antioxidants. These agents also suppressed the activation of mitochondrial retrograde signaling. Interestingly, in terms of molar concentrations, MitoQ was more than 1000-fold more effective than general antioxidants in suppressing the receptor activator of nuclear factor-B ligand-induced differentiation of RAW 264.7 cells into multinucleated and TRAP-positive osteoclasts. We propose that mitochondrial function and intramitochondrial ROS play important roles in osteoclastogenesis.

Changes in the expression of mitochondrial peroxiredoxin and thioredoxin in neurons and glia and their protective effects in experimental cerebral ischemic damage

We observed chronological changes in the mitochondrial-specific antioxidant enzymes peroxiredoxin 3 (Prx3) and thioredoxin 2 (Trx2) and their neuroprotective effects in the hippocampal CA1 region after 5 min of transient cerebral ischemia in gerbils. In the sham-operated group, weak Prx3 and Trx2 immunoreactivity was detected in the stratum pyramidale. Prx3 immunoreactivity was increased in pyramidal neurons and expressed in microglia 1 and 3 days, respectively, after ischemia/reperfusion (I/R). Trx2 immunoreactivity in pyramidal neurons increased 30 min and 1 day after I/R and decreased 6 h after I/R. Trx2 immunoreaction was expressed in astrocytes at 3 days postischemia. The intraventricular administration of Prx3 or Prx3/Trx2 (16 μg/20 μl, icv) using an osmotic pump significantly reduced ischemia-induced hyperactivity in a spontaneous motor test and protected CA1 pyramidal neurons from the ischemic damage. In addition, the activation of astrocytes and microglia was decreased in the ischemic CA1 region after Prx3/Trx2 treatment. In addition, treatment with Prx3 or Prx3/Trx2 significantly reduced lipid peroxidation and the release of cytochrome c from mitochondria and cytoplasm in the ischemic CA1 region. These results suggest that changes in the expression of Prx3 and Trx2 in the hippocampal CA1 region after I/R may be associated with the delayed neuronal death of CA1 pyramidal cells induced by transient cerebral ischemia, and that treatment with Prx3 or Prx3/Trx2 in ischemic brains shows a potent neuroprotective effect against ischemic damage by reducing lipid peroxidation and mitochondrial-mediated apoptosis by I/R.

Attenuation of Angiotensin II–Induced Vascular Dysfunction and Hypertension by Overexpression of Thioredoxin 2

Reactive oxygen species increase in the cardiovascular system during hypertension and in response to angiotensin II. Because mitochondria contribute to reactive oxygen species generation, we sought to investigate the role of thioredoxin 2, a mitochondria-specific antioxidant enzyme. Mice were created with overexpression of human thioredoxin 2 (Tg(hTrx2) mice) and backcrossed to C57BL/6J mice for > or =6 generations. Twelve-week-old male Tg(hTrx2) or littermate wild-type mice were made hypertensive by infusion of angiotensin II (400 ng/kg per minute) for 14 days using osmotic minipumps. Systolic arterial blood pressure was not different between Tg(hTrx2) and wild-type animals under baseline conditions (101+/-1 respective 102+/-1 mm Hg). The angiotensin II-induced hypertension in wild-type mice (145+/-2 mm Hg) was significantly attenuated in Tg(hTrx2) mice (124+/-1 mm Hg; P<0.001). Aortic endothelium-dependent relaxation was significantly reduced in wild-type mice after angiotensin II infusion but nearly unchanged in transgenic mice. Elevated vascular superoxide and hydrogen peroxide levels, as well as expression of NADPH oxidase subunits in response to angiotensin II infusion, were significantly attenuated in Tg(hTrx2) mice. Mitochondrial superoxide anion levels were augmented after angiotensin II infusion in wild-type mice, and this was blunted in Tg(hTrx2) mice. Angiotensin II infusion significantly increased myocardial superoxide formation, heart weight, and cardiomyocyte size in wild-type but not in Tg(hTrx2) mice. These data indicate a major role for mitochondrial thioredoxin 2 in the development of cardiovascular alterations and hypertension during chronic angiotensin II infusion. Thioredoxin 2 may represent an important therapeutic target for the prevention and treatment of hypertension and oxidative stress.

Mitochondrial abnormalities in spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is a motor neuron disease caused by polyglutamine expansion mutation in the androgen receptor (AR). We investigated whether the mutant protein alters mitochondrial function. We found that constitutive and doxycycline-induced expression of the mutant AR in MN-1 and PC12 cells, respectively, are associated with depolarization of the mitochondrial membrane. This was mitigated by cyclosporine A, which inhibits opening of the mitochondrial permeability transition pore. We also found that the expression of the mutant protein in the presence of ligand results in an elevated level of reactive oxygen species, which is blocked by the treatment with the antioxidants co-enzyme Q10 and idebenone. The mutant protein in MN-1 cells also resulted in increased Bax, caspase 9 and caspase 3. We assessed the effects of mutant AR on the transcription of mitochondrial proteins and found altered expression of the peroxisome proliferator-activated receptor γ coactivator 1 and the mitochondrial specific antioxidant superoxide dismutase-2 in affected tissues of SBMA knock-in mice. In addition, we found that the AR associates with mitochondria in cultured cells. This study thus provides evidence for mitochondrial dysfunction in SBMA cell and animal models, either through indirect effects on the transcription of nuclear-encoded mitochondrial genes or through direct effects of the mutant protein on mitochondria or both. These findings indicate possible benefit from mitochondrial therapy for SBMA.

Thioredoxin 1 and Thioredoxin 2 Have Opposed Regulatory Functions on Hypoxia-inducible Factor-1α

Hypoxia inducible factor 1 (HIF-1), a key regulator for adaptation to hypoxia, is composed of HIF-1alpha and HIF-1beta. In this study, we present evidence that overexpression of mitochondria-located thioredoxin 2 (Trx2) attenuated hypoxia-evoked HIF-1alpha accumulation, whereas cytosolic thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount. Transactivation of HIF-1 is decreased by overexpression of Trx2 but stimulated by Trx1. Inhibition of proteasomal degradation of HIF-1alpha in Trx2-overexpressing cells did not fully restore HIF-1alpha protein levels, while HIF-1alpha accumulation was enhanced in Trx1-overexpressing cells. Reporter assays showed that cap-dependent translation is increased by Trx1 and decreased by Trx2, whereas HIF-1alpha mRNA levels remained unaltered. These data suggest that thioredoxins affect the synthesis of HIF-1alpha. Trx1 facilitated synthesis of HIF-1alpha by activating Akt, p70S6K, and eIF-4E, known to control cap-dependent translation. In contrast, Trx2 attenuated activities of Akt, p70S6K, and eIF-4E and provoked an increase in mitochondrial reactive oxygen species production. MitoQ, a mitochondria specific antioxidant, reversed HIF-1alpha accumulation as well as Akt activation under hypoxia in Trx2 cells, supporting the notion of translation control mechanisms in affecting HIF-1alpha protein accumulation.

The role of docosahexaenoic acid in mediating mitochondrial membrane lipid oxidation and apoptosis in colonocytes

Docosahexaenoic acid (DHA, 22:6 n-3) from fish oil, and butyrate, a fiber fermentation product, work coordinately to protect against colon tumorigenesis in part by inducing apoptosis. We have recently demonstrated that dietary DHA is incorporated into mitochondrial membrane phospholipids, thereby enhancing oxidative stress induced by butyrate metabolism. In order to elucidate the subcellular origin of oxidation induced by DHA and butyrate, immortalized young adult mouse colonocytes were treated with 0-200 microM DHA or linoleic acid (LA, 18:2 n-6; control) for 72 h with or without 5 mM butyrate for the final 24 h. Cytosolic reactive oxygen species, membrane lipid oxidation, and mitochondrial membrane potential (MP), were measured by live-cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells exhibited a 151% increase in lipid oxidation (P < 0.01), compared with no butyrate treatment, which could be blocked by a mitochondria-specific antioxidant, 10-(6'-ubiquinoyl) decyltriphenylphosphonium bromide (MitoQ) (P < 0.05). Butyrate treatment of LA pretreated cells did not show any significant effect. In the absence of butyrate, DHA treatment, compared with LA, increased resting MP by 120% (P < 0.01). In addition, butyrate-induced mitochondrial membrane potential (MP), dissipation was 21% greater in DHA primed cells as compared with LA at 6 h. This effect was reversed by preincubation with inhibitors of the mitochondrial permeability transition pore, cyclosporin A or bongkrekic acid (1 microM). The functional importance of these events is supported by the demonstration that DHA and butyrate-induced apoptosis is blocked by MitoQ. These data indicate that DHA and butyrate potentiate mitochondrial lipid oxidation and the dissipation of MP which contribute to the induction of apoptosis.

Mitochondrial redox state regulates transcription of the nuclear-encoded mitochondrial protein manganese superoxide dismutase: a proposed adaptive response to mitochondrial redox imbalance

Overexpression of human manganese superoxide dismutase (MnSOD) in mouse NIH/3T3 cells using an inducible retroviral system led to alterations in the mitochondrial redox state since levels of reactive oxygen species rapidly increased after induction of human MnSOD ( Antioxid. Redox Signal. 6:489–500; 2004). Alterations in exogenous human MnSOD led to large increases in levels of endogenous mouse MnSOD ( sod2) and thioredoxin 2 ( txn2) mRNAs, but smaller increases in MnSOD and thioredoxin 2 protein expression. Tight regulation of mitochondrial protein levels seems to be necessary for optimal cellular function, since mitochondrial antioxidant protein levels did not increase to the same extent as antioxidant protein mRNA levels. We hypothesize that these changes in antioxidant proteins are adaptations to the altered mitochondrial redox state elicited by MnSOD overexpression. The mitochondrial-specific antioxidant MitoQ reversed cell growth inhibition, and greatly decreased levels of endogenous sod2 and txn2 transcripts following induction of exogenous MnSOD. Elevated levels of mouse sod2 transcripts resulted from transcriptional activation of the endogenous sod2 gene since actinomycin D prevented transcription of this gene. Therefore, the mitochondrial redox state appears to modulate a nuclear-driven biochemical event, i.e., transcriptional activation of a nuclear gene encoding a protein targeted to mitochondria.

Manganese potentiates lipopolysaccharide-induced expression of NOS2 in C6 glioma cells through mitochondrial-dependent activation of nuclear factor kappaB

Neuronal injury in manganese neurotoxicity (manganism) is thought to involve activation of astroglial cells and subsequent overproduction of nitric oxide (NO) by inducible nitric oxide synthase (NOS2). Manganese (Mn) enhances the effects of proinflammatory cytokines on expression of NOS2 but the molecular basis for this effect has not been established. It was postulated in the present studies that Mn enhances expression of NOS2 through the cis-acting factor, nuclear factor kappaB (NF-κB). Exposure of C6 glioma cells to lipopopolysaccharide (LPS) resulted in increased expression of NOS2 and production of NO that was dramatically potentiated by Mn and was blocked through overexpression of mutant IκBα (S32/36A). LPS-induced DNA binding of p65/p50 was similarly enhanced by Mn and was decreased by mutant IκBα. Phosphorylation of IκBα was potentiated by Mn and LPS and was not blocked by U0126, a selective inhibitor of ERK1/2. Mn decreased mitochondrial membrane potential and increased matrix calcium, associated with a rise in intracellular reactive oxygen species (ROS) that was attenuated by the mitochondrial-specific antioxidant, MitoQ. Blocking mitochondrial ROS also attenuated the enhancing effect of Mn on LPS-induced phosphorylation of IκBα and expression of NOS2, suggesting a link between Mn-induced mitochondrial dysfunction and activation of NF-κB. Overexpression of a dominant-negative mutant of the NF-κB-interacting kinase (Nik) prevented enhancement of LPS-induced phosphorylation of IκBα by Mn. These data indicate that Mn augments LPS-induced expression of NOS2 in C6 cells by increasing mitochondrial ROS and activation of NF-κB.

Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells.

Throughout the lifetime of an individual, light is focused onto the retina. The resulting photooxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration (AMD), the leading cause of legal blindness in the developed world, involves oxidative stress and death of the retinal pigment epithelium (RPE) followed by death of the overlying photoreceptors. Evidence suggests that damage due to exposure to light plays a role in AMD and other age-related eye diseases. In this work a system for light-induced damage and death of the RPE, based on the human ARPE-19 cell line, was used. Induction of mitochondria-derived reactive oxygen species (ROS) is shown to play a critical role in the death of cells exposed to short-wavelength blue light (425 +/- 20 nm). ROS and cell death are blocked either by inhibiting the mitochondrial electron transport chain or by mitochondria-specific antioxidants. These results show that mitochondria are an important source of toxic oxygen radicals in blue light-exposed RPE cells and may indicate new approaches for treating AMD using mitochondria-targeted antioxidants.

Endotoxin Pretreatmentin VivoIncreases the Mitochondrial Respiratory Capacity in Rat Hepatocytes

Administration of sublethal doses of endotoxin produces tolerance to subsequent oxidative stress in diverse animal models. Although endotoxin induces antioxidant enzymes, particularly manganous superoxide dismutase (Mn-SOD), the phenomenon of tolerance remains incompletely understood. Previously I determined that endotoxin treatment in rats increased lung mitochondrial respiration-dependent (i.e., independent of Mn-SOD) scavenging of superoxide anion. Because nonenzymatic scavenging of superoxide anion correlates with the mitochondrial membrane energy gradient, I hypothesized that endotoxin increases the mitochondrial transmembrane potential. Endotoxin treatment (500 μg/kg intraperitoneally 48 h earlier) increased the hepatocyte mitochondrial transmembrane potential as determined by two separate methods: the intramitochondrial sequestration of triphenylmethylphosphonium (electrical potential or ΔΨ) and the fluorescence intensity of the hepatocyte mitochondria when stained with rhodamine-123 andexamined by confocal microscopy. These findings suggest that endotoxin treatment increased the total mitochondrial membrane potential per hepatocyte. In parallel, endotoxin treatment increased the fluorescence intensity of hepatocyte mitochondria after staining with 10-N-nonyl-acridine orange, a dye that binds to the mitochondrial inner membrane independently of the transmembrane potential. This suggests that an increase in mitochondrial inner membrane mass is responsible for the net increase in inner membrane potential per cell following endotoxin pretreatment. These findings complement previous studies in which endotoxin treatment increased the mitochondrial-specific antioxidant Mn-SOD and support the more recent finding that endotoxin treatment also increased nonenzymatic scavenging of superoxide by lung mitochondria. Taken together, these observations suggest that mitochondrial biogenesis, and the subsequent increase in both enzymatic and nonenzymatic scavenging of superoxide anion, is a central feature of endotoxin-mediated tolerance to oxidative stress.