Dietary vitamin E reduces labile iron in rat tissues

Previous studies have shown that dietary vitamin E reduced generation and/or levels of superoxide. As superoxide has potential to release iron from its transport and storage proteins, and labile or available form of iron is capable of catalyzing the formation of reactive hydroxyl radicals, the effect of dietary vitamin E on labile iron pool was studied in rats. One-month-old Sprague-Dawley male and female rats were fed a basal vitamin E-deficient diet supplemented with 0, 20, 200, or 2,000 IU vitamin E/kg diet for 90 days. The levels of labile iron were measured in the liver, kidney, spleen, heart and skeletal muscle. Additionally, the levels of lipid peroxidation products were measured. The results showed that, except for labile iron in the heart of male rats, dietary vitamin E dose dependently reduced the levels of labile iron and lipid peroxidation products in all tissues of male and female rats. The findings suggest that dietary vitamin E may protect against oxidative tissue damage by reducing the generation and/or level of superoxide, which in turn attenuates the release of iron from its protein complexes.     

Synergy in Free Radical Generation is Blunted by High-Fat Diet Induced Alterations in Skeletal Muscle Mitochondrial Metabolism

Due to their role in cellular energetics and metabolism, skeletal muscle mitochondria appear to play a key role in the development of insulin resistance and type II diabetes. High-fat diet can induce higher levels of reactive oxygen species (ROS), evidenced by hydrogen peroxide (H₂O₂) emission from mitochondria, which may be causal for insulin resistance in skeletal muscle. The underlying mechanisms are unclear. Recent published data on single substrate (pyruvate, succinate, fat) metabolism in both normal diet (CON) and high-fat diet (HFD) states of skeletal muscle allowed us to develop an integrated mathematical model of skeletal muscle mitochondrial metabolism. Model simulations suggested that long-term HFD may affect specific metabolic reaction/pathways by altering enzyme activities. Our model allows us to predict oxygen consumption and ROS generation for any combination of substrates. In particular, we predict a synergy between (iso-membrane potential) combinations of pyruvate and fat in ROS production compared to the sum of ROS production with each substrate singly in both CON and HFD states. This synergy is blunted in the HFD state.     

Preventive effect of dietary quercetin on disuse muscle atrophy by targeting mitochondria in denervated mice

Quercetin is a major dietary flavonoid in fruits and vegetables. We aimed to clarify the preventive effect of dietary quercetin on disuse muscle atrophy and the underlying mechanisms. We established a mouse denervation model by cutting the sciatic nerve in the right leg (SNX surgery) to lack of mobilization in hind-limb. Preintake of a quercetin-mixed diet for 14 days before SNX surgery prevented loss of muscle mass and atrophy of muscle fibers in the gastrocnemius muscle (GM). Phosphorylation of Akt, a key phosphorylation pathway of suppression of protein degradation, was activated in the quercetin-mixed diet group with and without SNX surgery. Intake of a quercetin-mixed diet suppressed the generation of hydrogen peroxide originating from mitochondria and elevated mitochondrial peroxisome proliferator-activated receptor-γ coactivator 1α mRNA expression as well as NADH dehydrogenase 4 expression in the GM with SNX surgery. Quercetin and its conjugated metabolites reduced hydrogen peroxide production in the mitochondrial fraction obtained from atrophied muscle. In C2C12 myotubes, quercetin reached the mitochondrial fraction. These findings suggest that dietary quercetin can prevent disuse muscle atrophy by targeting mitochondria in skeletal muscle tissue through protecting mitochondria from decreased biogenesis and reducing mitochondrial hydrogen peroxide release, which can be related to decreased hydrogen peroxide production and/or improvements on antioxidant capacity of mitochondria.     

Effect of methionine dietary supplementation on mitochondrial oxygen radical generation and oxidative DNA damage in rat liver and heart

Methionine restriction without energy restriction increases, like caloric restriction, maximum longevity in rodents. Previous studies have shown that methionine restriction strongly decreases mitochondrial reactive oxygen species (ROS) production and oxidative damage to mitochondrial DNA, lowers membrane unsaturation, and decreases five different markers of protein oxidation in rat heart and liver mitochondria. It is unknown whether methionine supplementation in the diet can induce opposite changes, which is also interesting because excessive dietary methionine is hepatotoxic and induces cardiovascular alterations. Because the detailed mechanisms of methionine-related hepatotoxicity and cardiovascular toxicity are poorly understood and today many Western human populations consume levels of dietary protein (and thus, methionine) 2–3.3 fold higher than the average adult requirement, in the present experiment we analyze the effect of a methionine supplemented diet on mitochondrial ROS production and oxidative damage in the rat liver and heart mitochondria. In this investigation male Wistar rats were fed either a L-methionine-supplemented (2.5 g/100 g) diet without changing any other dietary components or a control (0.86 g/100 g) diet for 7 weeks. It was found that methionine supplementation increased mitochondrial ROS generation and percent free radical leak in rat liver mitochondria but not in rat heart. In agreement with these data oxidative damage to mitochondrial DNA increased only in rat liver, but no changes were observed in five different markers of protein oxidation in both organs. The content of mitochondrial respiratory chain complexes and AIF (apoptosis inducing factor) did not change after the dietary supplementation while fatty acid unsaturation decreased. Methionine, S-AdenosylMethionine and S-AdenosylHomocysteine concentration increased in both organs in the supplemented group. These results show that methionine supplementation in the diet specifically increases mitochondrial ROS production and mitochondrial DNA oxidative damage in rat liver mitochondria offering a plausible mechanism for its hepatotoxicity.     

Mitochondrial catalase and oxidative injury

Mitochondria dysfunction induced by reactive oxygen species (ROS) is related to many human diseases and aging. In physiological conditions, the mitochondrial respiratory chain is the major source of ROS. ROS could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules like glutathione and vitamin E. However, in pathological conditions, these antioxidants are often unable to deal with the large amount of ROS produced. This inefficiency of antioxidants is even more serious in mitochondria, because mitochondria in most cells lack catalase. Therefore, the excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins and mDNA, which can then cause cells to die of necrosis or apoptosis. In order to study the important role of mitochondrial catalase in protecting cells from oxidative injury, a HepG2 cell line overexpressing catalase in mitochondria was developed by stable transfection of a plasmid containing catalase cDNA linked with a mitochondria leader sequence which would encode a signal peptide to lead catalase into the mitochondria. Mitochondria catalase was shown to protect cells from oxidative injury induced by hydrogen peroxide and antimycin A. However, it increased the sensitivity of cells to tumor necrosis factor-alpha-induced apoptosis by changing the redox-oxidative status in the mitochondria. Therefore, the antioxidative effectiveness of catalase when expressed in the mitochondrial compartment is dependent upon the oxidant and the locus of ROS production.     

Topology of superoxide production from different sites in the mitochondrial electron transport chain

We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15%) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria.     

The long life of birds: the rat-pigeon comparison revisited

The most studied comparison of aging and maximum lifespan potential (MLSP) among endotherms involves the 7-fold longevity difference between rats (MLSP 5y) and pigeons (MLSP 35y). A widely accepted theory explaining MLSP differences between species is the oxidative stress theory, which purports that reactive oxygen species (ROS) produced during mitochondrial respiration damage bio-molecules and eventually lead to the breakdown of regulatory systems and consequent death. Previous rat-pigeon studies compared only aspects of the oxidative stress theory and most concluded that the lower mitochondrial superoxide production of pigeons compared to rats was responsible for their much greater longevity. This conclusion is based mainly on data from one tissue (the heart) using one mitochondrial substrate (succinate). Studies on heart mitochondria using pyruvate as a mitochondrial substrate gave contradictory results. We believe the conclusion that birds produce less mitochondrial superoxide than mammals is unwarranted. We have revisited the rat-pigeon comparison in the most comprehensive manner to date. We have measured superoxide production (by heart, skeletal muscle and liver mitochondria), five different antioxidants in plasma, three tissues and mitochondria, membrane fatty acid composition (in seven tissues and three mitochondria), and biomarkers of oxidative damage. The only substantial and consistent difference that we have observed between rats and pigeons is their membrane fatty acid composition, with rats having membranes that are more susceptible to damage. This suggests that, although there was no difference in superoxide production, there is likely a much greater production of lipid-based ROS in the rat. We conclude that the differences in superoxide production reported previously were due to the arbitrary selection of heart muscle to source mitochondria and the provision of succinate. Had mitochondria been harvested from other tissues or other relevant mitochondrial metabolic substrates been used, then very different conclusions regarding differences in oxidative stress would have been reached.     

Fat intake reverses the beneficial effects of low caloric intake on skeletal muscle mitochondrial H(2)O(2) production

Food restriction is the most effective modulator of oxidative stress and it is believed that a reduction in caloric intake per se is responsible for the reduced generation of reactive oxygen species (ROS) by mitochondria. Hydrogen peroxide (H(2)O(2)) generation and oxygen consumption (O(2)) by skeletal muscle mitochondria were determined in a peculiar strain of rats (Lou/C) characterized by a self-low-caloric intake and a dietary preference for fat. These rats were fed either with a standard high-carbohydrate (HC) or a high-fat (HF) diet and the results were compared to those measured in Wistar rats fed a HC diet. H(2)O(2) production was significantly reduced in Lou/C rats fed a HC diet; this effect was not due to a lower O(2) consumption but rather to a decrease in rotenone-sensitive NADH-ubiquinone oxidoreductase activity and increased expression of uncoupling proteins 2 and 3. The reduced H(2)O(2) generation displayed by Lou/C rats was accompanied by a significant inhibition of permeability transition pore (PTP) opening. H(2)O(2) production was restored and PTP inhibition was relieved when Lou/C rats were allowed to eat a HF diet, suggesting that the reduced oxidative stress provided by low caloric intake is lost when fat proportion in the diet is increased.     

The exceptional longevity of the naked mole‐rat may be explained by mitochondrial antioxidant defenses

Naked mole‐rats (NMRs) are mouse‐sized mammals that exhibit an exceptionally long lifespan (>30 vs. <4 years for mice), and resist aging‐related pathologies such as cardiovascular and pulmonary diseases, cancer, and neurodegeneration. However, the mechanisms underlying this exceptional longevity and disease resistance remain poorly understood. The oxidative stress theory of aging posits that (a) senescence results from the accumulation of oxidative damage inflicted by reactive oxygen species (ROS) of mitochondrial origin, and (b) mitochondria of long‐lived species produce less ROS than do mitochondria of short‐lived species. However, comparative studies over the past 28 years have produced equivocal results supporting this latter prediction. We hypothesized that, rather than differences in ROS generation, the capacity of mitochondria to consume ROS might distinguish long‐lived species from short‐lived species. To test this hypothesis, we compared mitochondrial production and consumption of hydrogen peroxide (H₂O₂; as a proxy of overall ROS metabolism) between NMR and mouse skeletal muscle and heart. We found that the two species had comparable rates of mitochondrial H₂O₂ generation in both tissues; however, the capacity of mitochondria to consume ROS was markedly greater in NMRs. Specifically, maximal observed consumption rates were approximately two and fivefold greater in NMRs than in mice, for skeletal muscle and heart, respectively. Our results indicate that differences in matrix ROS detoxification capacity between species may contribute to their divergence in lifespan.     

Coordinate induction of peroxisomal acyl-CoA oxidase and UCP-3 by dietary fish oil: a mechanism for decreased body fat deposition

Rats fed dietary fats rich in 20- and 22-carbon polyenoic fatty acids deposit less fat and expend more energy at rest than rats fed other types of fats. We hypothesized that this decrease in energetic efficiency was the product of: (a) enhanced peroxisomal fatty acid oxidation and/or (b) the up-regulation of genes encoding proteins that were involved with enhanced heat production, i.e. mitochondrial uncoupling proteins (UCP-2, UCP-3) and peroxisomal fatty acid oxidation proteins. Two groups of male Fisher 344 rats 3-4 week old (n=5 per group) were pair fed for 6 weeks a diet containing 40% of its energy fat derived from either fish oil or corn oil. Epididymal fat pads from rats fed the fish oil diet weighed 25% (P < 0.05) less than those found in rats fed corn oil. The decrease in fat deposition associated with fish oil ingestion was accompanied by a significant increase in the abundance of skeletal muscle UCP-3 mRNA. The level of UCP-2 mRNA skeletal muscle was unaffected by the type of dietary oil, but the abundance of UCP-2 mRNA in the liver and heart were significantly lower (P < 0.05) in rats fed fish oil than in rats fed corn oil. In addition to inducing UCP-3 expression, dietary fish oil induced peroxisomal acyl-CoA oxidase gene expression 2-3 fold in liver, skeletal muscle and heart. These data support the hypothesis that dietary fish oil reduces fat deposition by increasing the expression of mitochondrial uncoupling proteins and increasing fatty acid oxidation by the less efficient peroxisomal pathway.     

Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity

Oxidative modification of cellular components may contribute to tissue dysfunction during aging. In skeletal muscle, contractile activity increases the generation of reactive oxygen and nitrogen species (ROS). The question of whether contraction-induced ROS generation is further increased in skeletal muscle of the elderly is important since this influences recommendations on their exercise participation. Three different approaches were used to examine whether aging influences contraction-induced ROS generation. Hind limb muscles of adult and old mice underwent a 15-min period of isometric contractions and we examined ROS generation by isolated skeletal muscle mitochondria, ROS release into the muscle extracellular fluid using microdialysis techniques, and the muscle glutathione and protein thiol contents. Resting skeletal muscle of old mice compared with adult mice showed increased ROS release from isolated mitochondria, but no changes in the extracellular levels of superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical activity or muscle glutathione and protein thiol contents. Skeletal muscle mitochondria isolated from both adult and old mice after contractile activity showed significant increases in hydrogen peroxide release compared with pre-contraction values. Contractions increased extracellular hydroxyl radical activity in adult and old mice, but had no significant effect on extracellular hydrogen peroxide or nitric oxide in either group. In adult mice only, contractile activity increased the skeletal muscle release of superoxide. A similar decrease in muscle glutathione and protein thiol contents was seen in adult and old mice following contractions. Thus, contractile activity increased skeletal muscle ROS generation in both adult and old mice with no evidence for an age-related exacerbation of ROS generation.     

Vitamin E regulation of mitochondrial superoxide generation

The mitochondrion is the greatest source, as well as the target, of reactive oxygen species (ROS). Increasing evidence indicates that vitamin E can act as a biological modifier independently of its antioxidant activity. Experimental evidence available shows that vitamin E is capable of dose-dependently regulating mitochondrial generation of superoxide and hydrogen peroxide. Vitamin E may modulate mitochondrial production and levels of superoxide by preventing electron leakage, by mediating the superoxide generation systems directly and/or by scavenging superoxide generated. By downregulating mitochondrial generation of superoxide and related ROS, vitamin E not only attenuates oxidative damage but also modulates the expression and activation of signal transduction pathways and other redox-sensitive biological modifiers.     

Mitochondrial function, content and ROS production in rat skeletal muscle: effect of high-fat feeding

A high intake of dietary fat has been suggested to diminish mitochondrial functioning in skeletal muscle, possibly attributing to muscular fat accumulation. Here we show however, that an 8-week high-fat dietary intervention did not affect intrinsic functioning of rat skeletal muscle mitochondria assessed by respirometry, neither on a carbohydrate- nor on a lipid-substrate. Interestingly, PPARGC1A protein increased by approximately 2-fold upon high-fat feeding and we observed inconsistent results on different markers of mitochondrial density. Mitochondrial ROS production, assessed by electron spin resonance spectroscopy remained unaffected. Intramyocellular lipid levels increased significantly illustrating that a reduced innate mitochondrial function is not a prerequisite for intra-muscular fat accumulation.     

一次性力竭运动对大鼠PHB1表达及线粒体功能的影响

目的：观察一次性力竭运动后大鼠脑、心、骨骼肌组织和线粒体中PHB1含量的变化及对大鼠线粒体功能的影响,探寻PHB1与线粒体功能和能量代谢的关系。方法：健康雄性SD大鼠40只,随机分为2组（n=20）：对照组和一次性力竭运动组,大鼠进行一次性急性跑台运动建立力竭运动模型。收集各组大鼠的心、脑和骨骼肌组织样品并提取线粒体,检测其呼吸功能和ROS的变化。用Western blot方法检测组织和线粒体中PHB1蛋白表达水平;用分光光度计检测各器官中ATP含量以及线粒体中复合体V活性（ATP合酶活性）。结果：①一次性力竭运动后脑、心肌、骨骼肌中ATP含量显著性降低;②一次性力竭运动后脑、心肌、骨骼肌线粒体中复合体V活性、RCR、ROS显著性降低,ST4均显著性升高,ST3无显著性差异。③一次性力竭运动后心、脑、骨骼肌线粒体中PHB1的表达显著性减少。④通过相关性分析得出：一次性力竭运动后心、脑、骨骼肌中ATP含量与心、脑、骨骼肌中复合体V活性呈正相关;心、脑、骨骼肌中ATP含量和心、脑骨骼肌中PHB1的表达呈正相关。结论：一次性力竭运动后,降低线粒体氧化磷酸化功能,使大鼠脑、骨骼肌线粒体内ROS生成增加,PHB1的表达、ATP含量和复合体V活性均下降。一次性力竭运动使得大鼠线粒体内PHB1表达降低,线粒体功能减弱,机体能量代谢降低。     

Changes in mitochondrial reactive oxygen species synthesis during differentiation of skeletal muscle cells

Myogenesis is accompanied by an intensive metabolic remodeling. We investigated the mitochondrial reactive oxygen species (ROS) generation at different levels of skeletal muscle differentiation: in C2C12 myoblasts, in C2C12 myotubes and in adult mouse skeletal muscle. Differentiation was accompanied by an increase in mitochondrial content and respiratory chain activity. The detected ROS production levels correlated with mitochondrial content, being the lowest in the myoblasts. Unlike the adult skeletal muscle, myoblast ROS production was significantly stimulated by the complex I inhibitor rotenone. Our results show that mitochondria are an important ROS source in skeletal muscle cells. The substantial changes in mitochondrial ROS synthesis during skeletal muscle differentiation can be explained by intensive bioenergetic remodeling.     

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single ∼33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 °C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 °C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.     

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.     

Mitochondrial-targeted antioxidants protect skeletal muscle against immobilization-induced muscle atrophy

Prolonged periods of muscular inactivity (e.g., limb immobilization) result in skeletal muscle atrophy. Although it is established that reactive oxygen species (ROS) play a role in inactivity-induced skeletal muscle atrophy, the cellular pathway(s) responsible for inactivity-induced ROS production remain(s) unclear. To investigate this important issue, we tested the hypothesis that elevated mitochondrial ROS production contributes to immobilization-induced increases in oxidative stress, protease activation, and myofiber atrophy in skeletal muscle. Cause-and-effect was determined by administration of a novel mitochondrial-targeted antioxidant (SS-31) to prevent immobilization-induced mitochondrial ROS production in skeletal muscle fibers. Compared with ambulatory controls, 14 days of muscle immobilization resulted in significant muscle atrophy, along with increased mitochondrial ROS production, muscle oxidative damage, and protease activation. Importantly, treatment with a mitochondrial-targeted antioxidant attenuated the inactivity-induced increase in mitochondrial ROS production and prevented oxidative stress, protease activation, and myofiber atrophy. These results support the hypothesis that redox disturbances contribute to immobilization-induced skeletal muscle atrophy and that mitochondria are an important source of ROS production in muscle fibers during prolonged periods of inactivity.     

Role of mitochondria in exercise-induced oxidative stress in skeletal muscle from hyperthyroid rats

Previous study showed that exercise induces higher oxidative damage and respiratory capacity reduction in hyperthyroid than in euthyroid skeletal muscle. Because impaired cell function can result from mitochondrial dysfunction, we evaluated the changes induced by exercise in oxygen consumption of skeletal muscle mitochondria from euthyroid and hyperthyroid rats. The mitochondrial function was related with indices of oxidative damage and nitric oxide production, scavenger levels and mitochondrial ROS production rates. Our results show that exercise increased state 4 and decreased state 3 respiration, and the highest changes happened in hyperthyroid preparations. This was consistent with the observation that oxidative damage and NO(*) derivative content were increased by T(3) administration and exercise, reaching the highest levels in hyperthyroid exercised rats. Our results also indicate that the high mitochondrial oxidative damage induced by T(3) and exercise is due to enhanced ROS production, which is dependent on increases in mitochondrial content and reduction degree, respectively, of autoxidizable electron carriers.