Mitochondrial Peroxide Generation Research Articles

Elevated phospholipid hydroperoxide glutathione peroxidase (GPX4) expression modulates oxylipin formation and inhibits age-related skeletal muscle atrophy and weakness

Our previous studies support a key role for mitochondrial lipid hydroperoxides as important contributors to denervation-related muscle atrophy, including muscle atrophy associated with aging. Phospholipid hydroperoxide glutathione peroxidase 4 (GPX4) is an essential antioxidant enzyme that directly reduces phospholipid hydroperoxides and we previously reported that denervation-induced muscle atrophy is blunted in a mouse model of GPX4 overexpression. Therefore, the goal of the present study was to determine whether GPX4 overexpression can reduce the age-related increase in mitochondrial hydroperoxides in skeletal muscle and ameliorate age-related muscle atrophy and weakness (sarcopenia). Male C57Bl6 WT and GPX4 transgenic (GPX4Tg) mice were studied at 3 to 5 and 23–29 months of age. Basal mitochondrial peroxide generation was reduced by 34% in muscle fibers from aged GPX4Tg compared to old WT mice. GPX4 overexpression also reduced levels of lipid peroxidation products: 4-HNE, MDA, and LOOHs by 38%, 32%, and 84% respectively in aged GPX4Tg mice compared to aged WT mice. Muscle mass was preserved in old GPX4 Tg mice by 11% and specific force generation was 21% higher in old GPX4Tg versus age matched male WT mice. Oxylipins from lipoxygenases (LOX) and cyclooxygenase (COX), as well as less abundant non-enzymatically generated isomers, were significantly reduced by GPX4 overexpression. The expression of cPLA2, 12/15-LOX and COX-2 were 1.9-, 10.5- and 3.4-fold greater in old versus young WT muscle respectively, and 12/15-LOX and COX-2 levels were reduced by 37% and 35%, respectively in muscle from old GPX4Tg mice. Our study suggests that lipid peroxidation products may play an important role in the development of sarcopenia, and their detoxification might be an effective intervention in preventing muscle atrophy.

Muscle mitochondrial catalase expression prevents neuromuscular junction disruption, atrophy, and weakness in a mouse model of accelerated sarcopenia.

BackgroundOxidative stress and damage are associated with a number of ageing phenotypes, including age‐related loss of muscle mass and reduced contractile function (sarcopenia). Our group and others have reported loss of neuromuscular junction (NMJ) integrity and increased denervation as initiating factors in sarcopenia, leading to mitochondrial dysfunction, generation of reactive oxygen species and peroxides, and loss of muscle mass and weakness. Previous studies from our laboratory show that denervation‐induced skeletal muscle mitochondrial peroxide generation is highly correlated to muscle atrophy. Here, we directly test the impact of scavenging muscle mitochondrial hydrogen peroxide on the structure and function of the NMJ and muscle mass and function in a mouse model of denervation‐induced muscle atrophy CuZnSOD (Sod1 −/− mice, Sod1KO).MethodsWhole‐body Sod1KO mice were crossed to mice with increased expression of human catalase (MCAT) targeted specifically to mitochondria in skeletal muscle (mMCAT mice) to determine the impact of reduced hydrogen peroxide levels on key targets of sarcopenia, including mitochondrial function, NMJ structure and function, and indices of muscle mass and function.ResultsFemale adult (~12‐month‐old) Sod1KO mice show a number of sarcopenia‐related phenotypes in skeletal muscle including reduced mitochondrial oxygen consumption and elevated reactive oxygen species generation, fragmentation, and loss of innervated NMJs (P < 0.05), a 30% reduction in muscle mass (P < 0.05), a 36% loss of force generation (P < 0.05), and a loss of exercise capacity (305 vs. 709 m in wild‐type mice, P < 0.05). Muscle from Sod1KO mice also shows a 35% reduction in sarco(endo)plasmic reticulum ATPase activity (P < 0.05), changes in the amount of calcium‐regulating proteins, and altered fibre‐type composition. In contrast, increased catalase expression in the mMCAT × Sod1KO mice completely prevents the mitochondrial and NMJ‐related phenotypes and maintains muscle mass and force generation. The reduction in exercise capacity is also partially inhibited (~35%, P < 0.05), and the loss of fibre cross‐sectional area is inhibited by ~50% (P < 0.05).ConclusionsTogether, these striking findings suggest that scavenging of mitochondrial peroxide generation by mMCAT expression efficiently prevents mitochondrial dysfunction and NMJ disruption associated with denervation‐induced atrophy and weakness, supporting mitochondrial H2O2 as an important effector of NMJ alterations that lead to phenotypes associated with sarcopenia.

HyPer2 imaging reveals temporal and heterogeneous hydrogen peroxide changes in denervated and aged skeletal muscle fibers in vivo

To determine the role of denervation and motor unit turnover in the age-related increase in skeletal muscle oxidative stress, the hydrogen peroxide (H2O2) specific, genetically-encoded, fluorescent cyto-HyPer2 probe was expressed in mouse anterior tibialis (AT) muscle and compared with ex vivo measurements of mitochondrial oxidant generation. Crush of the peroneal nerve induced increased mitochondrial peroxide generation, measured in permeabilised AT fibers ex vivo and intra vital confocal microscopy of cyto-HyPer2 fluorescence showed increased cytosolic H2O2 in a sub-set (~24%) of individual fibers associated with onset of fiber atrophy. In comparison, mitochondrial peroxide generation was also increased in resting muscle from old (26 month) mice compared with adult (6–8 month) mice, but no age effect on fiber cytosolic H2O2in vivo was seen. Thus ageing is associated with an increased ability of muscle fibers to maintain cytosolic redox homeostasis in the presence of denervation-induced increase in mitochondrial peroxide generation.

Redox responses in skeletal muscle following denervation

Previous studies have shown a significant increase in the mitochondrial generation of hydrogen peroxide (H2O2) and other peroxides in recently denervated muscle fibers. The mechanisms for generation of these peroxides and how the muscle responds to these peroxides are not fully established. The aim of this work was to determine the effect of denervation on the muscle content of proteins that may contribute to mitochondrial peroxide release and the muscle responses to this generation. Denervation of the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles in mice was achieved by surgical removal of a small section of the peroneal nerve prior to its entry into the muscle. An increase in mitochondrial peroxide generation has been observed from 7 days and sustained up to 21 days following denervation in the TA muscle fibers. This increased peroxide generation was reduced by incubation of skinned fibers with inhibitors of monoamine oxidases, NADPH oxidases or phospholipase A2 enzymes and the muscle content of these enzymes together with peroxiredoxin 6 were increased following denervation. Denervated muscle also showed significant adaptations in the content of several enzymes involved in the protection of cells against oxidative damage. Morphological analyses indicated a progressive significant loss of muscle mass in the TA muscle from 7 days up to 21 days following denervation due to fiber atrophy but without fiber loss. These results support the possibility that, at least initially, the increase in peroxide production may stimulate adaptations in an attempt to protect the muscle fibers, but that these processes are insufficient and the increased peroxide generation over the longer term may activate degenerative and atrophic processes in the denervated muscle fibers.

EFFECT OF DENERVATION ON REDOX‐REGULATION IN SKELETAL MUSCLE

Changes in skeletal muscle mass and function occur during aging and are accompanied by loss of integrity and stability of the neuromuscular junction. Previous studies from our group have shown a significant increase in the mitochondrial generation of hydrogen peroxide (H202) and other peroxides in denervated muscle fibers. The mechanisms of generation of these peroxides and the effect on muscle responses are unknown. Our aim was to determine the effect of denervation on proteins that may contribute to peroxide release and proteins that regulate redox responses.The Anterior Tibialis (AT) and Extensor Digitorum Longus (EDL) muscles of wild type mice were denervated under anaesthesia by surgical removal of a small section of the peroneal nerve. Western blotting and immunohistochemical techniques were used to examine protein contents. AT and EDL muscles of thy‐1YFP mice, in which YFP expression in neuronal cells allow visualisation of the muscle innervation, were also denervated and used for morphological examination and analysis of muscle mitochondrial peroxide release.Previous work from our group has shown that, following denervation, hydrogen peroxide can be generated by several sources within mitochondria (Pollock et al, Free Rad. Biol. Med 2017) and the current data show that the increased peroxide generation was maintained to 21 days and was associated with a significant increase in Monoamine Oxidase A and NADPH Oxidase components. An increased content of proteins involved in the activation of the NADPH oxidase complex such as Peroxiredoxin 6 and Phospholipase A2 was also observed together with a significant increase in the content of several enzymes involved in the protection of cells against oxidative damage. Oxyblot analysis to assess the levels of carbonylation, showed that by 7 days post‐denervation there was a significant increase in protein carbonylation of low molecular weight (25–35 kDa) proteins. Morphological analysis indicated a progressive significant loss of muscle mass in the AT muscle from 7 days up to 21 days following denervation due to fiber atrophy without fiber loss. These results support the possibility that (at least initially) the increase in peroxide production may stimulate adaptations to protect the muscle fibers but, together with data on structural changes indicating significant fiber atrophy from 7 days after denervation, increased peroxide generation over the longer term may activate degenerative processes, such as apoptosis of the denervated muscle fibers.Support or Funding InformationThis project was funded by the US National Institutes of Health (NIA) and the Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. According to the results, it may be proposed that hypoxia can decrease S-nitrosylation of soluble-H-Ras in D PC12 cells and abolish the inhibitory effect of NO-HRas in mitochondrial oxidative metabolism.

Denervated muscle fibers induce mitochondrial peroxide generation in neighboring innervated fibers: Role in muscle aging

Disruption of neuromuscular junctions and denervation of some muscle fibers occurs in ageing skeletal muscle and contribute to loss of muscle mass and function. Aging is associated with mitochondrial dysfunction and loss of redox homeostasis potentially occurs through increased mitochondrial generation of reactive oxygen species (ROS). No specific link between increased mitochondrial ROS generation and denervation has been defined in muscle ageing. To address this, we have examined the effect of experimental denervation of all fibers, or only a proportion of the fibers, in the mouse tibialis anterior (TA) muscle on muscle mitochondrial peroxide generation. Transection of the peroneal nerve of mice caused loss of pre-synaptic axons within 1–3 days with no significant morphological changes in post-synaptic structures up to 10 days post-surgery when decreased TA mass and fiber size were apparent. Mitochondria in the denervated muscle showed increased peroxide generation by 3 days post-transection. Use of electron transport chain (ETC) substrates and inhibitors of specific pathways indicated that the ETC was unlikely to contribute to increased ROS generation, but monoamine oxidase B, NADPH oxidase and phospholipase enzymes were implicated. Transection of one of the 3 branches of the peroneal nerve caused denervation of some TA muscle fibers while others retained innervation, but increased mitochondrial peroxide generation occurred in both denervated and innervated fibers. Thus the presence of recently denervated fibers leads to increased ROS generation by mitochondria in neighboring innervated fibers providing a novel explanation for the increased mitochondrial oxidative stress and damage seen with aging in skeletal muscles.

175 - Effect of Partial Denervation on Mitochondrial ROS Generation in Skeletal Muscle; a Role in Sarcopenia?

With advancing age there is an increased occurrence of denervated skeletal muscle fibres, which is associated with morphological breakdown of neuromuscular junctions (NMJs). Dramatic changes in mitochondrial generation of hydrogen peroxide (H 2 O 2 ) have been reported in skeletal muscle of rodents following experimental nerve transection 1 . We hypothesised that denervation of even a limited number of fibers, such as that observed during ageing, could influence H 2 O 2 generation by both the denervated and neighbouring innervated fibers within a muscle. Thy1-YFP mice (expressing YFP in neuronal cells and so allowing visualisation of these cells only) underwent surgical transection of 1 of the 3 nerve branches entering the anterior tibialis (AT) muscle. Following recovery for up to 7 days, the AT muscles were quickly excised and samples taken for morphological assessment or for assessment of mitochondrial H 2 O 2 generation. Confocal imaging of skeletal muscle preparations allowed visualisation and mapping of NMJ integrity. Analysis revealed that at 7 days post-surgery the innervation status of individual NMJs varied across the muscle and four regions were identified in which the muscle fibres were either fully innervated, fully denervated or had a mix of innervated and denervated fibres. From each region, small bundles of fibres were removed, saponin permeablised and mitochondrial peroxide generation assessed using the Amplex Red assay. Peroxide release was assessed in state 1 respiration and in the presence of some electron transport chain substrates. Following partial denervation, oxidation of Amplex red was increased by 40-70 fold compared to sham in the bundles of fibers from all regions of the AT muscle. An increase comparable to those observed in fully denervated muscles in our previous experiments. These data show that innervated muscle fibers respond to denervation of adjacent fibers by increasing mitochondrial peroxide generation. Thus data support our hypothesis that loss of innervation in a proportion of muscle fibres is sufficient to disrupt mitochondrial H 2 O 2 generation with consequences for the entire muscle and we propose that this process is relevant to understanding muscle peroxide changes in sarcopenia. Reference Muller FL et al 2007. AM J. Physiol. Supported by the UK BBSRC and NIA (AG-20591).

181 - The Role of Denervation in Cytokine-Mediated Muscle Dysfunction in Old Mice

Age-related loss of muscle mass and function greatly influences the quality of life of older people. Denervation and increases in pro-inflammatory cytokines are proposed to play a substantial role in this muscle dysfunction. Full denervation of muscle of adult mice causes an increase in the rate of mitochondrial peroxide production1 and data from our laboratory have further demonstrated that this peroxide generation by muscle remained significantly elevated for up to 10 days after denervation, compared with sham controls. Further data suggest that increased mitochondrial peroxide generation may mediate cytokine production by muscle cells2. We hypothesise that the denervation evident in some muscle fibres of old mice results in increased mitochondrial peroxide generation which in turn results in increased production of cytokines by muscle, further contributing to muscle dysfunction. The aim of this study was (1) to use intra-vital imaging in Thy1-CFP mice to examine the structure of peripheral axons and neuromuscular junctions in single muscle fibers in vivo and to monitor H2O2 generation by denervated muscle fibers using AAV6-Hyper2-Cyto injected mice and (2) to examine the effects of increased H2O2 on NF-κB activation and the production of pro-inflammatory cytokines in these fibers. Data demonstrated that the increased mitochondrial peroxide generation in fully denervated muscle of adult mice was associated with activation of NF-κB and increased production of pro-inflammatory cytokines including IL-6, MCP1 and CXCL1. These data suggest that denervation of individual muscle fibers in old mice may result in elevations in H2O2 production in both denervated and innervated fibers and that this may play a role in the increased activation of NF-κB and production of pro-inflammatory cytokines seen in muscles of old mice. Current work focuses on assessment of neuromuscular transmission using electrophysiology in single fibers to determine which age-related changes in neuromuscular junction structure are associated with a lack of neuromuscular activity and cytokine production. References Muller FL, et al 2007. Am J. Physiol. (Reg) 293:R1159-1168. Lightfoot AP, et al 2015. Redox biology. 6:253-259, 2015. Supported by the UK MRC and NIA (AG-20591).

Effect of Muscle Fiber Denervation on Mitochondrial Hydrogen Peroxide Generation in Adjacent Innervated Muscle Fibres: Role in Sarcopenia?

Nerve transection causes dramatic increases in mitochondrial generation of hydrogen peroxide (H2O2) in the denervated skeletal muscle of rodents. We hypothesized that denervation of a limited number of fibers, such as that observed in muscles during aging, could influence H2O2 generation by both denervated and innervated fibers throughout the muscle. Thy1‐YFP mice (expressing YFP in neurons) underwent full transection of the peroneal nerve or transection of only one of the 3 branches entering the anterior tibialis (AT) muscle. Following up to 10days recovery, generation of H2O2 by mitochondria in permeablised fibers with intact mitochondra from different portions of the AT was assessed using Amplex Red. Results demonstrated increased H2O2 generation by 3days after full transection compared with sham control, which remained elevated at 10days. In situ imaging showed loss of innervation of endplates with no evidence of post‐synaptic changes. Immuno‐fluorescent staining for markers of denervation showed neuronal cell adhesion molecule positive muscle fibers by 7days post‐surgery. Transection of the single branch of the peroneal nerve caused denervation of a portion of the AT and mitochondrial H2O2 generation in the denervated portion was elevated by 3days post‐transection in a similar manner to the fully denervated muscle. Denervation of some just fibers was associated with increased mitochondrial H2O2 generation in all areas of the muscle. Thus data support the hypothesis that the effect of partial denervation on muscle H2O2 generation is mirrored in fully innervated fibers of the same muscle, an effect which may be mirrored in sarcopenia. Supported by the UK Biotechnology and Biological Sciences Research Council.

Ginsenoside Rd attenuates mitochondrial permeability transition and cytochrome C release in isolated spinal cord mitochondria: involvement of kinase-mediated pathways.

Ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. However, the effects of Rd on spinal cord mitochondrial dysfunction and underlying mechanisms are still obscure. In this study, we sought to investigate the in vitro effects of Rd on mitochondrial integrity and redox balance in isolated spinal cord mitochondria. We verified that Ca2+ dissipated the membrane potential, provoked mitochondrial swelling and decreased NAD(P)H matrix content, which were all attenuated by Rd pretreatment in a dose-dependent manner. In contrast, Rd was not able to inhibit Ca2+ induced mitochondrial hydrogen peroxide generation. The results of Western blot showed that Rd significantly increased the expression of p-Akt and p-ERK, but had no effects on phosphorylation of PKC and p38. In addition, Rd treatment significantly attenuated Ca2+ induced cytochrome c release, which was partly reversed by antagonists of Akt and ERK, but not p-38 inhibitor. The effects of bisindolylmaleimide, a PKC inhibitor, on Rd-induced inhibition of cytochrome c release seem to be at the level of its own detrimental activity on mitochondrial function. Furthermore, we also found that pretreatment with Rd in vivo (10 and 50 mg/kg) protected spinal cord mitochondria against Ca2+ induced mitochondrial membrane potential dissipation and cytochrome c release. It is concluded that Rd regulate mitochondrial permeability transition pore formation and cytochrome c release through protein kinases dependent mechanism involving activation of intramitochondrial Akt and ERK pathways.

Ethylmalonic Acid Induces Permeability Transition in Isolated Brain Mitochondria

Predominant accumulation of ethylmalonic acid (EMA) in tissues and biological fluids is a characteristic of patients affected by short chain acyl-CoA dehydrogenase deficiency and ethylmalonic encephalopathy. Neurological abnormalities are frequently found in these disorders, but the mechanisms underlying the brain injury are still obscure. Since hyperlacticacidemia is also found in many affected patients indicating a mitochondrial dysfunction; in the present work, we evaluated the in vitro and ex vivo effects of EMA plus Ca(2+) on mitochondrial integrity and redox balance in succinate-supported brain organelles. We verified that the evaluated parameters were disturbed only when EMA was associated with exogenous micromolar Ca(2+) concentrations. Thus, we found that this short chain organic acid plus Ca(2+) dissipated the membrane potential and provoked mitochondrial swelling, as well as impaired the mitochondrial Ca(2+) retention capacity, resulting in a rapid Ca(2+) release and decreased NAD(P)H matrix content. In contrast, EMA was not able to stimulate mitochondrial hydrogen peroxide generation. We also observed that all these effects were prevented by the mitochondrial Ca(2+) uptake inhibitor ruthenium red and the mitochondrial permeability transition (MPT) inhibitors cyclosporin A (CsA) and ADP. Furthermore, mitochondria isolated from rat brains after in vivo intrastriatal administration of EMA was more susceptible to Ca(2+)-induced swelling, which was fully prevented by CsA and ADP. Finally, EMA significantly decreased striatal slice viability, which was attenuated by CsA. The data strongly indicate that EMA reduced the mitochondrial threshold for Ca(2+)-induced MPT reinforcing the role of this cation in EMA-induced disruption of mitochondrial bioenergetics. It is, therefore, presumed that EMA acting synergistically with Ca(2+) compromises mitochondrial energy homeostasis in the central nervous system that may explain at least in part the neurologic alterations presented by patients with abnormal levels of this organic acid.

Inorganic Polyphosphates Regulate Hexokinase Activity and Reactive Oxygen Species Generation in Mitochondria of Rhipicephalus (Boophilus) microplus Embryo

The physiological roles of polyphosphates (poly P) recently found in arthropod mitochondria remain obscure. Here, the possible involvement of poly P with reactive oxygen species generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondrial hexokinase and scavenger antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase were assayed during embryogenesis of R. microplus. The influence of poly P3 and poly P15 were analyzed during the period of higher enzymatic activity during embryogenesis. Both poly Ps inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exopolyphosphatase activity was stimulated by the hexokinase reaction product, glucose-6-phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondrial hexokinase is also active. The superoxide dismutase, catalase and glutathione reductase activities were higher during embryo cellularization, at the end of embryogenesis and during embryo segmentation, respectively. All of the enzymes were stimulated by poly P3. However, superoxide dismutase was not affected by poly P15, catalase activity was stimulated only at high concentrations and glutathione reductase was the only enzyme that was stimulated in the same way by both poly Ps. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

Effect of KCl-medium on succinate oxidation and hydrogen peroxide generation in mitochondria of sugar beet taproot

The effect of substitution of KCl for sucrose in the reaction medium on succinate oxidation and hydrogen peroxide generation was investigated in the mitochondria isolated from stored taproots of sugar beet (Beta vulgaris L.). In a sucrose-containing medium, oxidation of succinate was inhibited by oxaloacetate; this inhibition was especially pronounced upon a decrease in substrate concentration and eliminated in the presence of glutamate, which removed oxaloacetate in the course of transamination. Irrespective of succinate concentration, substitution of KCl for sucrose in the medium considerably enhanced suppression of succinate oxidation apparently as a result of slow activation of succinate dehydrogenase (SDH) by its substrate. In this case, mitochondria showed the symptoms of uncoupling, lower values of membrane potential (ΔΨ), respiratory control (RC), and ADP/O induced by electrophoretic transport of potassium via K+ channel of mitochondria. KCl-dependent suppression of succinate oxidation by taproot mitochondria was accompanied by a considerable inhibition of H2O2 production as compared with the sucrose-containing medium. These results indicate that in the presence of potassium ions, ΔΨ dissipates, suppression of succinate oxidation by oxaloacetate increases, and succinate-dependent generation of ROS in sugar beet mitochondria is inhibited. A possible physiological role of oxaloacetate-restricted SDH activity in the suppression of respiration of storage organs protecting mitochondria from oxidative stress is discussed.

Reduced antioxidant capacity and diet-induced atherosclerosis in uncoupling protein-2-deficient mice

Vascular dysfunction in response to reactive oxygen species (ROS) plays an important role in the development and progression of atherosclerotic lesions. In most cells, mitochondria are the major source of cellular ROS during aerobic respiration. Under most conditions the rates of ROS formation and elimination are balanced through mechanisms that sense relative ROS levels. However, a chronic imbalance in redox homeostasis is believed to contribute to various chronic diseases, including atherosclerosis. Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane protein shown to be a negative regulator of macrophage ROS production. In response to a cholesterol-containing atherogenic diet, C57BL/6J mice significantly increased expression of UCP2 in the aorta, while mice lacking UCP2, in the absence of any other genetic modification, displayed significant endothelial dysfunction following the atherogenic diet. Compared with wild-type mice, Ucp2(-/-) mice had decreased endothelial nitric oxide synthase, an increase in vascular cell adhesion molecule-1 expression, increased ROS production, and an impaired ability to increase total antioxidant capacity. These changes in Ucp2(-/-) mice were associated with increased aortic macrophage infiltration and more numerous and larger atherosclerotic lesions. These data establish that in the vasculature UCP2 functions as an adaptive antioxidant defense to protect against the development of atherosclerosis in response to a fat and cholesterol diet.

Inhibition by methylated organoarsenicals of the respiratory 2-oxo-acid dehydrogenases

Inorganic arsenic that is ingested through drinking water or inhalation is metabolized by biological methylation pathways into organoarsenical metabolites. It is now becoming understood that this metabolism that was formerly considered to be detoxification may contribute as much or more to increasing the toxicity of arsenic. One proposed mode of the toxic action of arsenic and its organoarsenic metabolites is through its binding to proteins and inactivating their enzymatic activity. The classic case has been considered the affinity of the proximal 1,3 sulfhydryl groups of the lipoic acid cofactor of the pyruvate dehydrogenase complex for arsenic. A 2:1 stoichiometry of sulfhydryl to arsenic groups has been measured in proteins and arsenical complexes can be synthesized using free d, l-lipoic acid. The relative importance of this site for arsenic binding has come in to question through the use of methylating bifunctional arsenic complexes, and the suggestion that arsenic inhibits the pyruvate dehydrogenase complex indirectly by elevating mitochondrial hydrogen peroxide generation. In order to separate the effects of direct trivalent arsenite toxicity from that of hydrogen peroxide and activated oxygen, we studied the inhibition of the PDH complex under conditions that did not generate hydrogen peroxide but did expose the lipoic acid group in its reduced state to arsenicals. We also studied the effects of arsenicals in the inhibition of the α-ketoglutarate dehydrogenase complex. We found that only trivalent arsenical compounds inhibited the activity of both dehydrogenase complexes and only when the lipoic acid was in its reduced form. Arsenite inhibited both enzyme complexes approximately equivalently while monomethylarsenite inhibited the PDH complex to a greater extent than the KGDH complex – although both complexes were very sensitive to inhibition by this complex. Dimethylarsenite inhibition of both complexes was only observed with longer pre-incubation periods. Cumulative inhibition by the reduced arsenical was observed for all complexes indicating a binding mode of inhibition that is dependent upon lipoic acid being in its reduced state.

Hearts in adult offspring of copper-deficient dams exhibit decreased cytochrome c oxidase activity, increased mitochondrial hydrogen peroxide generation and enhanced formation of intracellular residual bodies

The long-term effects of low dietary copper (Cu) intake during pregnancy and lactation on cardiac mitochondria in first-generation adult rats was examined. Rat dams were fed diets containing either low (1 mg/kg Cu) or adequate (6 mg/kg Cu) levels of dietary Cu beginning 3 weeks before conception and ending 3 weeks after birth. Cytochrome c oxidase (CCO) activity was 51% lower in isolated cardiac mitochondria from 21-day-old offspring of Cu-deficient dams than in the offspring of Cu-adequate dams. CCO activities in the cardiac mitochondria of 63- and 290-day-old offspring were 22% lower and 14% lower, respectively, in the offspring of Cu-deficient dams after they had been repleted with adequate dietary Cu from the time they were 21 days old. Electron micrographs showed that the size of residual bodies and the cellular volume they occupied in cardiomyocytes rose significantly between 63 and 290 days in the Cu-repleted offspring of Cu-deficient dams, but not in the offspring of Cu-adequate dams. The rate of hydrogen peroxide generation by cardiac mitochondria also was 24% higher in the 290-day-old repleted offspring of Cu-deficient dams than in the offspring of Cu-adequate dams. The increase in hydrogen peroxide production by cardiac mitochondria and in the relative volume and size of dense deposits in cardiomyocytes is consistent with increased oxidative stress and damage resulting from prolonged reduction of CCO activity in the offspring of Cu-deficient dams.

Oxidative stress is involved in the permeabilization of the inner membrane of brain mitochondria exposed to hypoxia/reoxygenation and low micromolar Ca2+

From in vivo models of stroke it is known that ischemia/reperfusion induces oxidative stress that is accompanied by deterioration of brain mitochondria. Previously, we reported that the increase in Ca2+ induces functional breakdown and morphological disintegration in brain mitochondria subjected to hypoxia/reoxygenation (H/R). Protection by ADP indicated the involvement of the mitochondrial permeability transition pore in the mechanism of membrane permeabilization. Until now it has been unclear how reactive oxygen species (ROS) contribute to this process. We now report that brain mitochondria which had been subjected to H/R in the presence of low micromolar Ca2+ display low state 3 respiration (20% of control), loss of cytochrome c, and reduced glutathione levels (75% of control). During reoxygenation, significant mitochondrial generation of hydrogen peroxide (H2O2) was detected. The addition of the membrane permeant superoxide anion scavenger TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) suppressed the production of H2O2 by brain mitochondria metabolizing glutamate plus malate by 80% under normoxic conditions. TEMPOL partially protected brain mitochondria exposed to H/R and low micromolar Ca2+ from decrease in state 3 respiration (from 25% of control to 60% of control with TEMPOL) and permeabilization of the inner membrane. Membrane permeabilization was obvious, because state 3 respiration could be stimulated by extramitochondrial NADH. Our data suggest that ROS and Ca2+ synergistically induce permeabilization of the inner membrane of brain mitochondria exposed to H/R. However, permeabilization can only partially be prevented by suppressing mitochondrial generation of ROS. We conclude that transient deprivation of oxygen and glucose during temporary ischemia coupled with elevation in cytosolic Ca2+ concentration triggers ROS generation and mitochondrial permeabilization, resulting in neural cell death.

Effect of Auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.