C60 Fullerene Reduces 3-Nitropropionic Acid-Induced Oxidative Stress Disorders and Mitochondrial Dysfunction in Rats by Modulation of p53, Bcl-2 and Nrf2 Targeted Proteins.

C60 fullerene as a potent free radical scavenger and antioxidant could be a beneficial means for neurodegenerative disease prevention or cure. The aim of the study was to define the effects of C60 administration on mitochondrial dysfunction and oxidative stress disorders in a 3-nitropropionic acid (3-NPA)-induced rat model of Huntington's disease. Animals received 3-NPA (30 mg/kg i.p.) once a day for 3 consecutive days. C60 was applied at a dose of 0.5 mg/kg of body weight, i.p. daily over 5 days before (C60 pre-treatment) and after 3-NPA exposure (C60 post-treatment). Oxidative stress biomarkers, the activity of respiratory chain enzymes, the level of antioxidant defense, and pro- and antiapoptotic markers were analyzed in the brain and skeletal muscle mitochondria. The nuclear and cytosol Nrf2 protein expression, protein level of MnSOD, γ-glutamate-cysteine ligase (γ-GCLC), and glutathione-S-transferase (GSTP) as Nrf2 targets were evaluated. Our results indicated that C60 can prevent 3-NPA-induced mitochondrial dysfunction through the restoring of mitochondrial complexes' enzyme activity, ROS scavenging, modulating of pro/antioxidant balance and GSH/GSSG ratio, as well as inhibition of mitochondria-dependent apoptosis through the limitation of p53 mitochondrial translocation and increase in Bcl-2 protein expression. C60 improved mitochondrial protection by strengthening the endogenous glutathione system via glutathione biosynthesis by up-regulating Nrf2 nuclear accumulation as well as GCLC and GSTP protein level.

The Effect Of NO Donor on Calcium Uptake and Reactive Nitrogen Species Production in Mitochondria.

BACKGROUND/AIMS: NO and reactive nitrogen species (RNS) are thought to be physiologically important effectors of mitochondrial calcium transport, but this issue was not studied in a living organism. According to literature, the modulation of Ca2+ uptake could influence RNS production via the action on mitochondrial NO synthase (mtNOS). The aim of this work was to study the effect of in vivo administration of NO donor nitroglycerine (NG) on matrix Ca2+ accumulation, RNS production and mtNOS activity. METHODS: Ca2+ uptake was studied spectrophotometrically with arsenazo-III. The amounts of stable RNS (nitrite, nitrate and nitrosothiols) and L-citrulline, the product of enzymatic NOS activity, were determined analytically. RESULTS: NG administration resulted in dose-dependent short-term increase in Ca2+-uptake accompanied by essential rise in L-citrulline and RNS content in mitochondria. In parallel, dose-dependent elevation of hydroperoxide production was detected. Ca2+-uniporter activity was not affected, but mitochondrial permeability transition pore (MPTP) was effectively blocked by NO. CONCLUSION: Our results indicate that MPTP blockage by NO was the primary cause for the increase in calcium uptake which eventually resulted in the activation of mtNOS and RNS production. Improved Ca2+ accumulation in mitochondria, together with MPTP blockage, may contribute to well-known cardioprotective effects of pharmacological donors of nitric oxide.

The effect of atp-dependent potassium uptake on mitochondrial functions under acute hypoxia.

The opening of mitochondrial K(+) АТР-channel (mtK(+) АТР-channel) is supposed to be important in the modulation of mitochondrial functions under hypoxia, but the underlying mechanisms have not been clarified yet. The aim of this work was to study the effect of acute hypoxia on mtK(+) АТР-channel activity and to estimate the contribution of the channel in the modulation of mitochondrial functions. MtK(+) АТР-channel activity was assessed polarographically from the rate of State 4 respiration and by potentiometric monitoring of potassium efflux from deenergized mitochondria. It was shown that hypoxia reliably increased mtK(+) АТР-channel activity, which resulted in the changes of respiration rates (increase of State 4 and suppression of State 3 respiration), uncoupling (the decrease of respiratory control ratio) and suppression of phosphorylation. These effects were well mimicked by mtK(+) АТР-channel opener diazoxide (DZ) in isolated rat liver mitochondria. MtK(+) АТР-channel opening in vitro suppressed phosphorylation too, but increased phosphorylation efficiency, while mtK(+) АТР-channel blockers reduced it dramatically. The correlation was established between mtK(+) АТР-channel activity and the endurance of the rats to physical training under hypoxia. Hypoxia improved physical endurance, but treatment by mtK(+) АТР-channel blockers glibenklamide and 5-hydroxydecanoate (5-HD) prior to hypoxia strongly reduced both the channel activity and the endurance limits. This was in accord with the observation that under glibenklamide and 5-HD administration hypoxia failed to restore mtK(+) АТР-channel activity. Based on the experiments, we came to the conclusion that mtK(+) АТР-channel opening played a decisive role in the regulation of energy metabolism under acute hypoxia via the modulation of phosphorylation system in mitochondria.

The effect of intermittent hypoxic training on lung and heart tissues of healthy rats.

INTRODUCTION: Recently, particular attention has been focused on the problem of the beneficial influence of intermittent hypoxia (IH) on the human organism. However, knowledge regarding the negative effects of intermittent hypoxic training (IHT) on cellular adaptive mechanisms remains limited. The aim of the present study was to investigate: 1) lung and heart ultrastructural changes under IHT; and 2) the adequateness of morphological and morphometric methods to determine the constructive and destructive displays of hypoxia. MATERIAL AND METHODS: Adult male Wistar rats underwent IHT every day for 7-28 days. Lung and heart tissues were assessed by morphological and cellular morphometric methods. RESULTS: We observed evident ultra structural changes of the lung air-blood barrier (LABB) by the 7-10(th) day of training. Structural damage of LABB was most considerable after 2 weeks of IHT exposure, its ultrastructure partially normalized by the end of the IHT 4-weeks course: there was diminishing of LABB hydration and disappearance of areas of its destruction. The structural changes in the heart blood-tissue barrier (HBTB) were considerably less marked compared with those in LABB during the 1(st) and 2(nd) weeks of training. Heart tissue structural changes increased by the end of the fourth week of IHT. Both tissue cells revealed no significant necrotic damage of mitochondria after IHT, while changes relating to the energy-directed restructuring of mitochondria were observed. We hypothesized that acute moderate hypoxia promotes a specific type of mitosis in lung and heart tissues. CONCLUSIONS: Ultrastructural changes in the rat lung and heart tissues depend on IHT duration. The phenomenon of "micromitochondria within mitochondria" is an additional adaptive mechanism for IH exposure.

Diazoxide affects mitochondrial bioenergetics by the opening of mKATP channel on submicromolar scale.

BACKGROUND: Cytoprotection afforded by mitochondrial ATP-sensitive K+-channel (mKATP-channel) opener diazoxide (DZ) largely depends on the activation of potassium cycle with eventual modulation of mitochondrial functions and ROS production. However, generally these effects were studied in the presence of Mg∙ATP known to block K+ transport. Thus, the purpose of our work was the estimation of DZ effects on K+ transport, K+ cycle and ROS production in rat liver mitochondria in the absence of Mg∙ATP. RESULTS: Without Mg·ATP, full activation of native mKATP-channel, accompanied by the increase in ATP-insensitive K+ uptake, activation of K+-cycle and respiratory uncoupling, was reached at ≤0.5 µM of DZ,. Higher diazoxide concentrations augmented ATP-insensitive K+ uptake, but not mKATP-channel activity. mKATP-channel was blocked by Mg·ATP, reactivated by DZ, and repeatedly blocked by mKATP-channel blockers glibenclamide and 5-hydroxydecanoate, whereas ATP-insensitive potassium transport was blocked by Mg2+ and was not restored by DZ. High sensitivity of potassium transport to DZ in native mitochondria resulted in suppression of mitochondrial ROS production caused by the activation of K+-cycle on sub-micromolar scale. Based on the oxygen consumption study, the share of mKATP-channel in respiratory uncoupling by DZ was found. CONCLUSIONS: The study of mKATP-channel activation by diazoxide in the absence of MgATP discloses novel, not described earlier, aspects of mKATP-channel interaction with this drug. High sensitivity of mKATP-channel to DZ results in the modulation of mitochondrial functions and ROS production by DZ on sub-micromolar concentration scale. Our experiments led us to the hypothesis that under the conditions marked by ATP deficiency affinity of mKATP-channel to DZ can increase, which might contribute to the high effectiveness of this drug in cardio- and neuroprotection.

C60 Fullerene Prevents Restraint Stress-Induced Oxidative Disorders in Rat Tissues: Possible Involvement of the Nrf2/ARE-Antioxidant Pathway.

The effects of C60FAS (50 and 500 µg/kg) supplementation, in a normal physiological state and after restraint stress exposure, on prooxidant/antioxidant balance in rat tissues were explored and compared with the effects of the known exogenous antioxidant N-acetylcysteine. Oxidative stress biomarkers (ROS, O2·-, H2O2, and lipid peroxidation) and indices of antioxidant status (MnSOD, catalase, GPx, GST, γ-GCL, GR activities, and GSH level) were measured in the brain and the heart. In addition, protein expression of Nrf2 in the nuclear and cytosol fractions as well as the protein level of antiradical enzyme MnSOD and GSH-related enzymes γ-GCLC, GPx, and GSTP as downstream targets of Nrf2 was evaluated by western blot analysis. Under a stress condition, C60FAS attenuates ROS generation and O2·- and H2O2 releases and thus decreases lipid peroxidation as well as increases rat tissue antioxidant capacity. We have shown that C60FAS supplementation has dose-dependent and tissue-specific effects. C60FAS strengthened the antiradical defense through the upregulation of MnSOD in brain cells and maintained MnSOD protein content at the control level in the myocardium. Moreover, C60FAS enhanced the GSH level and the activity/protein expression of GSH-related enzymes. Correlation of these changes with Nrf2 protein content suggests that under stress exposure, along with other mechanisms, the Nrf2/ARE-antioxidant pathway may be involved in regulation of glutathione homeostasis. In our study, in an in vivo model, when C60FAS (50 and 500 µg/kg) was applied alone, no significant changes in Nrf2 protein expression as well as in activity/protein levels of MnSOD and GSH-related enzymes in both tissues types were observed. All these facts allow us to assume that in the in vivo model, C60FAS affects on the brain and heart endogenous antioxidative statuses only during the oxidative stress condition.

Antarctica challenges the new horizons in predictive, preventive, personalized medicine: preliminary results and attractive hypotheses for multi-disciplinary prospective studies in the Ukrainian "Akademik Vernadsky" station.

BACKGROUND: Antarctica is a unique place to study the health condition under the influence of environmental factors on the organism in pure form. Since the very beginning of the scientific presence of Ukraine in the Antarctic, biomedical research has been developed for the monitoring of individual biomarkers of winterers and medical accompaniment in Antarctic expeditions. The aim of the study was to analyze and discuss the retrospective data of long-term monitoring and observations in Ukrainian Antarctica station "Akademik Vernadsky," providing multi-scale biomedical information with regard to conditions of a perfect isolation from technological and social influences and under extreme environmental factors. METHODS: Medical and biological studies have been performed with the participation of all 20 Ukrainian wintering expeditions. We surveyed 200 males aged 20-60 years (mean age 37 years). Extensive medical examinations were carried out before the expedition, during the selection of candidates, and after returning, and particular functions were monitored during the entire stay in Antarctica. The medical records were analyzed to study the reaction of the human organism on phenomena like "Antarctic syndrome," dysadaptation, anxiety, desynchronosis, photoperiodism, influence of climatic and meteofactors like "Schumann resonance," infrasound, "ozone hole," and "sterile" environment; important aspects of its role on human health were precisely studied and discussed. RESULTS: The examinations showed the multi-level symptoms of the processes of dysregulation and dysadaptation, as functional tension in the sympathetic-adrenal system rights, especially during urgent adaptation to the Antarctic (1-month stay at the station) and, to a lesser extent, after returning from an expedition to Kyiv. At the initial, adaptation to the conditions of the Antarctic levels of urinary catecholamines (epinephrine, norepinephrine, dopamine, DOPA) increased compared with the start of the expedition (23.2 ± 4.3 and 53.3 ± 5 2 mmol/l, p < 0.001; 67.1 ± 12.3 and 138.3 ± 16.9 mmol/l, p < 0.01; 1749.6 ± 476.5 vs 7094.6 ± 918.3 mmol/l, p < 0.001; 129.6 ± 12.3 and 349.9 ± 40.6 mmol/l, p < 0.001, respectively). In the blood serum of 100 % of the expedition, we found an increase of oxidative stress markers-the level of TBARS increased by 41.2 %, i.e., the activation of free radical peroxidation. Thus, in 80 % of the participants, we observed a reduction in the activity of the SOD antiradical enzyme vs 58 % in the controls. Changes in brain electrical activity after a long stay at the Antarctic stations showed increasing delta rhythms, signs of CNS protective inhibition, likely due to hypoxia. We found changes in the concentrations of microelements (iron, copper, zinc, etc.) in the blood of winterers after the expedition. The polychrome-adaptive method of correcting the changes of the psycho-emotional state in a monochrome Antarctic environment was successfully applied. CONCLUSIONS: The preliminary results of the retrospective study and our own observations of the fundamental physiological mechanisms of the negative influence of extreme environmental factors on an organism in the absence of man-made origin factors allow the determination of many mechanisms of "pre-pathology" processes which promise to develop the pathogenetically based pro-active prevention methods for a number of common diseases to set prospective interdisciplinary research in predictive, preventive, and personalized medicine.

Erratum to: Antarctica challenges the new horizons in predictive, preventive, personalized medicine: preliminary results and attractive hypotheses for multi-disciplinary prospective studies in the Ukrainian "Akademik Vernadsky" station.

[This corrects the article DOI: 10.1186/s13167-016-0060-8.].

Tissue oxygenation and mitochondrial respiration under different modes of intermittent hypoxia.

We compared the results of five modes of intermittent hypoxia training (IHT) on gastrocnemius muscle Po2 and heart and liver mitochondrial respiration in rats. Minutes of hypoxia, %O2, and recovery minutes on air in each mode were: 1) 5, 12%, 5; 2) 15, 12%, 15; 3) 5, 12%, 15; 4) 5, 7%, 5; and 5) 5, 7%, 15. Mode 1 proved best in that Pmo2 dropped minimally at the end of every hypoxic bout and recovered quickly after each bout. One, 2, and 3 week IHT in mode 1 each increased tissue PO2 in both normoxic and 30 min severe hypoxic (7% O2) tests. Adaptation to IHT in Mode 1 caused the substrate-dependent reorganization of liver and heart mitochondrial energy metabolism favoring NADH-dependent oxidation and improving the efficiency of oxidative phosphorylation. Mitochondrial adaptation occurred after 14 days of IHT in liver tissue, but after 21 days in myocardium, and was preserved during the 3 months following IHT termination. When using Mode 2, positive changes were also registered, but were less pronounced. Other IHT modes provoked negative effects on Pmo2 levels, both during hypoxic periods and reoxygenation. In conclusion, the most effective IHT regimen is 5 min 12% O2 with 5 min breaks, five cycles per day during 2 or 3 weeks depending on the task of IHT.