Sergio Di Meo

Mitochondria in Exercise-Induced Oxidative Stress

In recent years it has been suggested that reactive oxygen species (ROS) are involved in the damage to muscle and other tissues induced by acute exercise. Despite the small availability of direct evidence for ROS production during exercise, there is an abundance of literature providing indirect support that oxidative stress occurs during exercise. The electron transport associated with the mitochondrial respiratory chain is considered the major process leading to ROS production at rest and during exercise. It is widely assumed that during exercise the increased electron flow through the mitochondrial electron transport chain leads to an increased rate of ROS production. On the other hand, results obtained by in vitro experiments indicate that mitochondrial ROS production is lower in state 3 (ADP-stimulated) than in state 4 (basal) respiration. It is possible, however, that factors, such as temperature, that are modified in vivo during intense physical activity induce changes (uncoupling associated with loss of cytochrome oxidase activity) leading to increased ROS production. The mitochondrial respiratory chain could also be a potential source of ROS in tissues, such as liver, kidney and nonworking muscles, that during exercise undergo partial ischemia because of reduced blood supply. Sufficient oxygen is available to interact with the increasingly reduced respiratory chain and enhance the ROS generation. At the cessation of exercise, blood flow to hypoxic tissues resumes leading to their reoxygenation. This mimics the ischemia-reperfusion phenomenon, which is known to cause excessive production of free radicals. Apart from a theoretical rise in ROS, there is little evidence that exercise-induced oxidative stress is due to its increased mitochondrial generation. On the other hand, if mitochondrial production of ROS supplies a remarkable contribution to exercise-induced oxidative stress, mitochondria should be a primary target of oxidative damage. Unfortunately, there are controversial reports concerning the exercise effects on structural and functional characteristics of mitochondria. However, the isolation of mitochondrial fractions by differential centrifugation has shown that the amount of damaged mitochondria, recovered in the lightest fraction, is remarkably increased by long-lasting exercise.

Mitochondrial metabolism of reactive oxygen species.

For a long time mitochondria have mainly been considered for their role in the aerobic energy production in eukaryotic cells, being the sites of the oxidative phosphorylation, which couples the electron transfer from respiratory substrates to oxygen with the ATP synthesis. Subsequently, it was showed that electron transfer along mitochondrial respiratory chain also leads to the formation of radicals and other reactive oxygen species, commonly indicated as ROS. The finding that such species are able to damage cellular components, suggested mitochondrial involvement in degenerative processes underlying several diseases and aging. More recently, a new role for mitochondria, as a system able to supply protection against cellular oxidative damage, is emerging. Experimental evidence indicates that the systems, evolved to protect mitochondria against endogenously produced ROS, can also scavenge ROS produced by other cellular sources. It is possible that this action, particularly relevant in physio-pathological conditions leading to increased cellular ROS production, is more effective in tissues provided with abundant mitochondrial population. Moreover, the mitochondrial dysfunction, resulting from ROS-induced inactivation of important mitochondrial components, can be attenuated by the cell purification from old ROS-overproducing mitochondria, which are characterized by high susceptibility to oxidative damage. Such an elimination is likely due to two sequential processes, named mitoptosis and mitophagy, which are usually believed to be induced by enhanced mitochondrial ROS generation. However, they could also be elicited by great amounts of ROS produced by other cellular sources and diffusing into mitochondria, leading to the elimination of the old dysfunctional mitochondrial subpopulation.

Effect of thyroid state on H2O2 production by rat liver mitochondria

It has been suggested that activation of mitochondrial respiration by thyroid hormone results in oxidative tissue injury secondary to increased reactive oxygen species production. In order to throw light on this subject, the effects of thyroid state on O2 consumption and H2O2 release by rat liver mitochondria were investigated. Hypothyroidism decreased the rates of O2 consumption and H2O2 release by succinate or pyruvate/malate-supplemented mitochondria during both State 4 and State 3 respiration, whereas hyperthyroidism increased such rates. Conversely, with both substrates and during either respiration phase, the percentage of O2 released as H2O2 was not significantly affected by thyroid state. On the other hand, the capacity of mitochondria to remove H2O2 increased by about 17% in hyperthyroid rats and decreased by about 35% in hypothyroid ones. This result indicates that the ratio between H2O2 production and release and so the percentage of O2 turned into H2O2 instead of being reduced to water increase in the transition from hypothyroid to hyperthyroid state. In light of previous observations that mitochondrial content of cytochromes and ubiquinone also increases in such a transition, the modifications of H2O2 production appear to be due to a modulation by thyroid hormone of the mitochondrial content of the autoxidisable electron carriers. This view is supported by measurements of H2O2 release in the presence of respiratory inhibitors, which show that the thyroid state-linked changes in H2O2 production occur at H2O2 generator sites of both Complex I and Complex III.

Role of ROS and RNS Sources in Physiological and Pathological Conditions

There is significant evidence that, in living systems, free radicals and other reactive oxygen and nitrogen species play a double role, because they can cause oxidative damage and tissue dysfunction and serve as molecular signals activating stress responses that are beneficial to the organism. Mitochondria have been thought to both play a major role in tissue oxidative damage and dysfunction and provide protection against excessive tissue dysfunction through several mechanisms, including stimulation of opening of permeability transition pores. Until recently, the functional significance of ROS sources different from mitochondria has received lesser attention. However, the most recent data, besides confirming the mitochondrial role in tissue oxidative stress and protection, show interplay between mitochondria and other ROS cellular sources, so that activation of one can lead to activation of other sources. Thus, it is currently accepted that in various conditions all cellular sources of ROS provide significant contribution to processes that oxidatively damage tissues and assure their survival, through mechanisms such as autophagy and apoptosis.

Effect of thyroid state on susceptibility to oxidants and swelling of mitochondria from rat tissues.

The effects of the thyroid state on oxidative damage, antioxidant capacity, susceptibility to in vitro oxidative stress and Ca(2+)-induced permeabilization of mitochondria from rat tissues (liver, heart, and gastrocnemious muscle) were examined. Hypothyroidism was induced by administering methimazole in drinking water for 15 d. Hyperthyroidism was elicited by a 10 d treatment of hypothyroid rats with triiodothyronine (10 micro g/100 g body weight). Mitochondrial levels of hydroperoxides and protein-bound carbonyls significantly decreased in hypothyroid tissues and were reported above euthroid values in hypothyroid rats after T(3) treatment. Mitochondrial vitamin E levels were not affected by changes of animal thyroid state. Mitochondrial Coenzyme Q9 levels decreased in liver and heart from hypothyroid rats and increased in all hyperthyroid tissues, while Coenzyme Q10 levels decreased in hypothyroid liver and increased in all hyperthyroid tissues. The antioxidant capacity of mitochondria was not significantly different in hypothyroid and euthyroid tissues, whereas it decreased in the hyperthyroid ones. Susceptibility to in vitro oxidative challenge decreased in mitochondria from hypothyroid tissues and increased in mitochondria from hyperthyroid tissues, while susceptibility to Ca(2+)-induced swelling decreased only in hypothyroid liver mitochondria and increased in mitochondria from all hyperthyroid tissues. The tissue-dependence of the mitochondrial susceptibility to stressful conditions in altered thyroid states can be explained by different thyroid hormone-induced changes in mitochondrial ROS production and relative amounts of mitochondrial hemoproteins and antioxidants. We suggest that susceptibilities to oxidants and Ca(2+)-induced swelling may have important implications for the thyroid hormone regulation of the turnover of proteins and whole mitochondria, respectively.

Effect of ischemia–reperfusion on heart mitochondria from hyperthyroid rats

OBJECTIVE We investigated the effect of hyperthyroidism on the functional response of mitochondria to ischemia-reperfusion and its relationship with changes in mitochondrial susceptibility to stress conditions. METHODS Hyperthyroidism was elicited by ten daily intraperitoneal injections of T3 (10 microg/100 g body weight). Mitochondria were isolated at 3000xg (M3) from homogenates of hearts perfused by the Langendorff technique after either 25 min reperfusion following 20 min ischemia or 45 min perfusion (controls). Rates of O2 consumption and H2O2 release with complex II-linked substrate, capacity to remove H2O2, extent of oxidative damage, levels of liposoluble antioxidants, such as ubiquinols and vitamin E, and susceptibility to Ca2+ -induced swelling were determined. RESULTS During reperfusion, hyperthyroid hearts displayed a significant tachycardia together with a low functional recovery. In comparison to the respective controls, mitochondria from both euthyroid and hyperthyroid hearts subjected to ischemia-reperfusion protocol exhibited decreases in the rate of O2 consumption, capacity to remove H2O2, and concentration of antioxidants, and increases in the rate of H2O2 release, concentration of hydroperoxides and protein-bound carbonyls, and susceptibility to Ca2+ -induced swelling. Such changes were higher in mitochondria from hyperthyroid hearts. The increase in the protein percent content and cytochrome oxidase activity of a mitochondrial fraction isolated at 8000xg (M8) from hyperthyroid hearts after reperfusion, suggests that the decline of mitochondrial respiration of M3 fraction could be due to the degradation of the oldest, mature mitochondria endowed of high oxidative capacity, but low antioxidant capacity, which would be lost by heavy mitochondrial fraction and recovered in the light fraction. CONCLUSIONS The higher susceptibility to ischemia-reperfusion of the heart from hyperthyroid animals is associated with a significant increase in mitochondrial dysfunction.

Effect of vitamin E on the response to ischemia-reperfusion of langendorff heart preparations from hyperthyroid rats

Hyperthyroidism has been reported to decrease heart antioxidant capacity and increase its susceptibility to in vitro oxidative stress. This may affect the heart response to ischemia-reperfusion, a condition that increases free radical production. We compared the functional recovery from in vitro ischemia-reperfusion (Langendorff) of hearts from euthyroid (E), hyperthyroid (H, ten daily intraperitoneal injections of T3, 10 microg/100g body weight), vitamin E-treated (VE, ten daily intramuscular injections, 20 mg/100g body weight) and hyperthyroid vitamin E-treated (HVE) rats. We also determined lipid peroxidation, tissue antioxidant capacity and the tissue capability to face an oxidative stress in vitro. A significant tachycardia was displayed during reperfusion following 20 min ischemia by the hyperthyroid hearts, together with a low recovery of left ventricular developed pressure (LVDP) and left ventricular dP/dt(max). When H hearts were paced at 300 beats/min, the functional recovery (LVDP and dP/dt(max)) was close to 100% and significantly higher than in E paced hearts. At the end of the ischemia-reperfusion protocol, myocardium antioxidant capacity was significantly lower, whereas lipid peroxidation and the susceptibility to in vitro oxidative stress were higher in the T3 treated (H) than in euthyroid rats. The in vitro tachycardic response, the reduction in the antioxidant capacity and the increase in lipid peroxidation were prevented by treatment of hyperthyroid rats with vitamin E (HVE). These results suggest that the tachycardic response to reperfusion following chronic T3 pretreatment was associated with the reduced capability of the heart to face oxidative stresses in hyperthyroidism.

Role of nitric oxide in the reperfusion induced injury in hyperthyroid rat hearts.

We recently reported that hyperthyroidism affects the heart response to ischemia/reperfusion. A significant tachycardia during reperfusion was associated with an increase in the oxidative stress of hearts from T3-treated animals. In the present study we checked the possible role of nitric oxide (NO) in this major stress induced by the hyperthyroid state. We compared the functional recovery from ischemia/reperfusion of Langendorff preparations from euthyroid (E) and hyperthyroid (H, ten daily intraperitoneal injections of T3, 10 μg/100 g body weight) rats, in the presence and in the absence of 0.2 mM Nω-nitro-L-arginine (L-NNA). At the end of the ischemia/reperfusion protocol (10 min preischemic perfusion, 20 min global ischemia, 30 min reperfusion) lipid peroxidation, antioxidant capacity (CA) and susceptibility to in vitro oxidative stress were determined on heart homogenates. The main effect of hyperthyroidism on the reperfusion functional response was confirmed to be a strong tachycardic response (154% recovery at 25 min reperfusion) accompanied by a low recovery in both left ventricular diastolic pressure (LVDP) and left ventricular dP/dtmax. This functional response was associated with a reduction in CA and an increase in both lipid peroxidation and susceptibility to oxidative stress. Perfusion of hearts with L-NNA per se had small but significant negative chronotropic and positive inotropic effects on preischemic performance of euthyroid rat hearts only. More importantly, L-NNA perfusion completely blocked the reperfusion tachycardic response in the hyperthyroid rats. Concomitantly, myocardium oxidative state (lipid peroxidation, CA and in vitro susceptibility to oxidative stress) of L-NNA perfused hearts was similar to that of E animals. These results suggest that the higher reperfusion-induced injury occurring in hyperthyroid animals is associated with overproduction of nitric oxide.

Effect of thyroid state on rate and sites of H2O2 production in rat skeletal muscle mitochondria.

The purpose of this study was to investigate the effects of thyroid state on rates and sites of H(2)O(2) production in rat muscle mitochondria. With Complex I- and Complex II-linked substrates, hypothyroidism decreased and hyperthyroidism increased the rates of O(2) consumption during State 4 and State 3 respiration and the rates of H(2)O(2) release during State 4 respiration. During State 3, the rates of H(2)O(2) release were not affected by thyroid state. However, the mitochondrial capacity to remove H(2)O(2) increased in the transition from hypothyroid to hyperthyroid state, thus suggesting that an increase in H(2)O(2) production rate also occurred in such a transition during State 3 respiration. The observation that mitochondrial coenzyme Q levels and cytochrome oxidase activities are higher in the hyperthyroid and lower in the hypothyroid groups suggests that the modifications of H(2)O(2) production are due to a modulation by thyroid hormone of the mitochondrial content of autoxidizable electron carriers. This idea is supported by measurements of H(2)O(2) release in the presence of respiratory inhibitors. In fact, such measurements indicate that the thyroid state-linked changes in H(2)O(2) production occur at both generator sites of the respiratory chain.

Poly(ADP-ribosyl)ation of proteins and germ cell development in hyperthyroid rat testes

The effect of increased serum levels of thyroid hormone (triiodothyronine, T3) on young rat testis spermatogenesis was studied by analysing molecular and morphological parameters. Hyperthyroidism was induced by either T3-treatment or 2- and 10-day cold exposure. The poly(ADP-ribosyl)ation of proteins catalysed by poly(ADP-ribose) polymerase, which is particularly active at specific stages of rat spermatogenesis, was analysed as molecular index of DNA damage and cell stress. Poly(ADP-ribose) polymerase activity rose after both T3-treatment and 2- and 10-day cold exposure, with a trend of 10-day cold-exposed rats towards control values. In all hyperthyroid rats poly(ADP-ribose) turnover, as a contribution of both poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase), was enhanced with respect to euthyroid animals. Poly(ADP-ribosyl)ation of proteins occurred with long and branched polymers suggesting an increased involvement of the modification system in DNA repair. Morphological changes of germ tissue were observed in hyperthyroid rats, mainly a high reduction of mature cells in the seminiferous tubule, and evidence of germ cell apoptosis was obtained by TUNEL method. In control animals germ cell apoptosis was within physiological levels. Conversely, in hyperthyroid rats a dramatic increase in the number of TUNEL-positive cells (some spermatogonia and numerous primary spermatocytes) was found, even though the increase was lower in 10-day than in 2-day cold-exposed animals.