Tag Archives: reactive oxygen species

The effect of permeability transition pore opening on reactive oxygen species production in rat brain mitochondria

O. V. Akopova, L. I. Kolchinskaya, V. I. Nosar, A. N. Smirnov, M. K. Malysheva, I. N. Mankovska, V. F. Sagach

Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: luko@biph.kiev.ua

The influence of mitochondrial permeability transition pore (MPTP) opening on reactive oxygen species (ROS) production in the rat brain mitochondria was studied. It was shown that ROS production is regulated differently by the rate of oxygen consumption and membrane potential, dependent on steady-state or non-equilibrium conditions. Under steady-state conditions, at constant rate of Ca2+-cycling and oxygen consumption, ROS production is potential-dependent and decreases with the inhibition of respiration and mitochondrial depolarization. The constant rate of ROS release is in accord with proportional dependence of the rate of ROS formation on that of oxygen consumption. On the contrary, transition to non-equilibrium state, due to the release of cytochrome c from mitochondria and progressive respiration inhibition, results in the loss of proportionality in the rate of ROS production on the rate of respiration and an exponential rise of ROS production with time, independent of membrane potential. Independent of steady-state or non-equilibrium conditions, the rate of ROS formation is controlled by the rate of potential-dependent uptake of Ca2+ which is the rate-limiting step in ROS production. It was shown that MPTP opening differently regulates ROS production, dependent on Ca2+ concentration. At low calcium MPTP opening results in the decrease in ROS production because of partial mitochondrial depolarization, in spite of sustained increase in oxygen consumption rate by a cyclosporine A-sensitive component due to simultaneous work of Ca2+-uniporter and MPTP as Ca2+-influx and efflux pathways. The effect of MPTP opening at low Ca2+ concentrations is similar to that of Ca2+-ionophore, A-23187. At high calcium MPTP opening results in the increase of ROS release due to the rapid transition to non-equilibrium state because of cytochrome c loss and progressive gating of electron flow in respiratory chain. Thus, under physiological conditions MPTP opening at low intracellular calcium could attenuate oxidative damage and the impairment of neuronal functions by diminishing ROS formation in mitochondria.

Indicators of oxidative and nitrosative stress and activity of enzymes of nitric oxide metabolism in rats treated with 4-thiazolidinone derivatives possessing antineoplastic activity

L. I. Kоbylinska1, R. R. Panchuk2, R. B. Lesyk1,
B. S. Zіmenkovsky1, R. S. Stoika2

1Danylo Halytsky Lviv National Medical University, Ukraine;
2Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv;
e-mail: lesya8@gmail.com

Principal ways of generation and function of free oxygen and nitrogen radical metabolites, as well as the ways of their bio-neutralization in rats treated with potential anticancer drugs have been discussed. Three isatin-pyrazoline 4-thiazolidinone conjugates – Les-3288, Les-3833 and Les-3882 – were selected as the most perspective antineoplastic agents. Since the reactive oxygen species (ROS) and reactive nitrogen species are responsible for negative side effects of many anticancer drugs, we measured the indicators of the oxidative and nitrosative stress and the activity of enzymes of the nitric oxide metabolism in blood of rats treated with such compounds. It was found that both Les-3833 and doxorubicin used as a positive control increased the level of specific indicators of the oxidative and nitrosative stress, while Les-3288 and Les-3882 did not cause a significant elevation in ROS content. There were no big changes in the activity of either iNO-synthase or NO-reductase under the action of doxorubicin, while Les-3288 and Les-3882 induced a decrease in the activi­ty of iNO-synthase, and Les-3288 induced a decrease in the activity of NO-reductase. Thus, the content of low molecular weight indicators of the oxidative and nitrosative stress in blood of rats is of higher informative value than the level of activity of enzymes of the nitric oxide metabolism at the action of such toxic agents as anticancer drugs. These indicators should be used for assessment of toxic damage of tissues and organs by the anticancer drugs.

Modulation of LPS-induced ROS production and NF-κB nuclear translocation by N-stearoylethanolamine in macrophages

A. G. Berdyshev, H. V. Kosiakova, N. M. Hula

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kievlipids@gmail.com

N-Stearoylethanolamine (NSE) is a minor lipid that belongs to the N-acylethanolamines family that mediates a wide range of biological processes. The effect of the NSE on reactive oxygen species (ROS) production and NF-κB activation stimulated by lipopolysaccharide (LPS) in rat peritoneal macrophages (PM) was evaluated. PM were obtained from the rat peritoneal cavity. ROS were detected following DCFDA and DHE fluorescence. Nuclear translocation of p65 NF-κB was examined by immunofluorescent method using confocal microscopy. It was shown that NSE exposure to peritoneal macrophages (10-7 M) prior to 30 min LPS stimulation inhibited super oxide and hydrogen peroxide production and NF-κB translocation into nuclei. Thus, NSE exhibits therapeutic potential to treat inflammatory diseases associated with increased activation of macrophages.

Induction of plant cells heat resistance by hydrogen sulfide donor is mediated by H(2)O(2) generation with participation of NADPH oxidase and superoxide dismutase

Yu. E. Kolupaev1,2, E. N. Firsova1, Т. О. Yastreb1

1V.V. Dokuchaev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@ukr.net;
2V.N. Karazin Kharkiv National University, Ukraine

The participation of enzymatic systems carrying out generation and conversion of reactive oxygen species (ROS), in realization of the stress-protective effect of hydrogen sulfide (H2S) on wheat coleoptile cells was investigated. It has been shown that the treatment of isolated coleoptiles with a 100 μM hydrogen sulfide donor sodium hydrosulfide (NaHS) caused a transient enhancement of the generation of superoxide anion radical (O2•–), an increase of hydrogen peroxide content and superoxide dismutase (SOD) activity in them. The increase in ROS generation was eliminated by the inhibitor of NADPH oxidase imidazole, but not by the peroxidase inhibitor sodium azide. Treatment of coleoptiles with SOD inhibitor sodium diethyldithiocarbamate (DDC) enhanced the generation of O2•– and neutralized the effect of increasing H2O2 content induced by NaHS. One day after treatment with the H2S donor, the generation of ROS decreased to a control level, while the activity of antioxidant enzymes increased markedly and the resistance of coleoptiles to damaging heating­ was increased. These effects of the hydrogen sulfide donor were eliminated by coleoptiles’ treatment with inhibitors of NADPH oxidase (imidazole) and SOD (DDC). It was concluded that both NADPH oxidase, genera­ting O2•– , and SOD, which turns it into H2O2 performing signaling functions, are involved in the formation of a signal that induces protective systems and causes an increase in heat resistance of plant cells.

Regulation of the mitochondrial ATP-sensitive potassium channel in rat uterus cells by ROS

O. B. Vadzyuk, Yu. Yu. Mazur, S. O. Kosterin

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: vadzyuk@biochem.kiev.ua

In previous study we demonstrated the presence of ATP-sensitive potassium current in the inner mitochondrial membrane, which was sensitive to diazoxide and glybenclamide, in mitochondria isolated from the rat uterus. This current was supposed to be operated by mitochondrial ATP-sensitive potassium channel (mitoKATP). Regulation of the mitoKATP in uterus cells is not studied well enough yet.
It is well known that the reactive oxygen species (ROS) can play a dual role. They can dama­ge cells in high concentrations, but they can also act as messengers in cellular signaling, mediating survival of cells under stress conditions. ROS are known to activate mitoKATP during the oxidative stress in the brain and heart, conferring the protection of cells. The present study examined whether ROS mediate the mitoKATP activation in myometrium cells.
Oxidative stress was induced by rotenone. ROS generation was measured by 2′,7′-dichlorofluorescin diacetate. The massive induction of ROS production was demonstrated in the presence of rotenone. Hyperpolarization of the mitochondrial membrane was also detected with the use of the potential-sensitive dye DiOC6 (3,3′-dihexyloxacarbocyanine iodide). Diazoxide, a selective activator of mitoKATP, depolarized mitochondrial membrane either under oxidative stress or under normal conditions, while mitoKATP blocker glybenclamide effectively restored mitochondrial potential in rat myocytes. Estimated <K1/2> value for diazoxide to mitoKATP under normoxia was four times higher than under oxidative stress conditions: 5.01 ± 1.47∙10-6 М and 1.24 ± 0.21∙10-6 М respectively.
The ROS scavenger N-acetylcysteine (NAC) successfully eliminates depolarization of mitochondrial membrane by diazoxide under oxidative stress. These results suggest that elimination of ROS by NAC prevents the activation of mitoKATP under oxidative stress. Taking into account the higher affinity of diazoxide to mitoKATP under stress conditions than under normoxia, we conclude that the oxidative stress conditions are more favourable than normoxia for the activation of mitoKATP­. Thus we hypothesize that the ROS regulate the activity of the mitoKATP in myocytes.

Metabolic changes in living cells under electromagnetic radiation of mobile communication systems

I. L. Yakymenko1,2, E. P. Sidorik1, O. S. Tsybulin2

1R.E.Kavetsty Institute of Experimental Pathology, Oncology
and Radiobiology, National Academy of Sciences of Ukraine, Kyiv;
2Bila Tserkva National Agrarian University, Ukraine;
e-mail: iyakymen@gmail.com

Review is devoted to the analysis of biological effects of microwaves. The results of last years’ researches indicated the potential risks of long-term low-level microwaves exposure for human health. The analysis of metabolic changes in living cells under the exposure of microwaves from mobile communication systems indicates that this factor is stressful for cells. Among the reproducible effects of low-level microwave radiation are overexpression of heat shock proteins, an increase of reactive oxygen species level, an increase of intracellular Ca2+, damage of DNA, inhibition of DNA reparation, and induction of apoptosis. Extracellular-signal-regulated kinases ERK and stress-related kinases p38MAPK are involved in metabolic changes. Analysis of current data suggests that the concept of exceptionally thermal mechanism of biological effects of microwaves is not correct. In turn, this raises the question of the need to revaluation of modern electromagnetic standards based on thermal effects of non-ionizing radiation on biological systems.

Participation of the active oxygen forms in the induction of ascorbate peroxidase and guaiacol peroxidase under heat hardening of wheat seedlings

Yu. E. Kolupaev, O. I. Oboznyi

V. V. Dokuchayev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru

The influence of one-minute hardening heating at 42 °C on the dynamics of hydrogen peroxide generation and activity of antioxidant enzymes in roots of winter wheat seedlings has been investigated. It was shown that the content of hydrogen peroxide increased within the first 30 minutes after heat influence, whereupon it approached the level of control variant. The activity of superoxide dismutase (SOD) increased significantly within 10 min after heating and was maintained at a high level during 24 hours of observation. The activi­ty of ascorbate peroxidase and guaiacol peroxidase increased after 3-6 hours after the hardening and reached its maximum after 24 hours, when there was the most significant increase in heat resistance of seedlings. The short-term increase in hydrogen peroxide content caused by hardening heating was suppressed by treatment of seedlings with H2O2 scavenger dimethylthiourea, inhibitors of NADPH-oxidase (imidazole) and SOD (sodium diethyldithiocarbamate). All these effectors levelled the increase of activity of ascorbate peroxidase and guaiacol peroxidase and significantly inhibited the development of heat resistance of seedlings. The conclusion was made about the role of hydrogen peroxide produced with the participation of NADPH­-oxidase and SOD in the induction of antioxidant system by heat harde­ning of wheat seedlings.

Peculiarities of the free radical processes in rat liver mitochondria under toxic hepatitis on the background of alimentary protein deficiency

G. P. Kopylchuk, O. M. Voloshchuk

Yuriy Fedkovych Chernivtsi National University
Institute of Biology, Chemistry and Natural Resources, Ukraine;
e-mail: kopilchuk@gmail.com

The rate of superoxide anion radical, hydroxyl radical and hydrogen peroxide generation, the level of oxidative modification of mitochondrial proteins in the liver of rats with toxic hepatitis was investigated on the background of alimentary protein deficiency. We did not find significant increases of the intensity of free radical processes in liver mitochondria of rats maintained on the protein-deficient ration. The most significant intensification of free radical processes in liver mitochondria is observed under the conditions of toxic hepatitis, induced on the background of alimentary protein deprivation. Under these conditions the aggravation of all studied forms of reactive oxygen species generation was observed in liver mitochondria. The generation rates were increased as follows: O2 – by 1.7 times, Н2О2 – by 1.5 times, ОН – practically double on the background of accumulation of oxidized mitochondria-derived proteins. The established changes in thiol groups’ redox status of respiratory chain proteins insoluble in 0.05 M sodium-phosphate buffer (pH 11.5), and changes of their carbonyl derivatives content may be considered as one of the regulatory factors of mitochondrial energy-generating function.

Remodulating effect of doxorubicin on the state of iron-containing proteins, and redox characteristics of tumor with allowance for its sensitivity to cytostatic agents

V. F. Chekhun, Yu. V. Lozovska, A. P. Burlaka,
I. I. Ganusevich, Yu. V. Shvets, N. Yu. Lukyanova,
I. M. Todor, N. A. Tregubova, L. A. Naleskina

R. E. Kavetsky Institute of Experimental Pathology, Oncology
and Radiobiology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: Lozovskaya.2012@mail.ru

The study was aimed at determining the changes of metal-containing proteins in blood serum and tumor tissue of animals with parental and doxorubicin-resistant strains of Walker-256 carcinosarcoma before and after the cytostatic administration. It has been shown that upon doxorubicin action the levels of total iron and transferrin in the tissues from the both groups of animals decreased while that of ferritine simultaneously increased with more pronounced pattern in the group of animals with resistant tumor strain. It has been shown that upon the action of doxorubicin in tumor tissue of animals with different sensitivity to the cytostatic there could be observed oppositely directed changes in the redox state of these cells that in turn determined the content of “free iron” complexes, ROS generation and concentration of active forms of matrix metaloproteinase-2 and matrix metaloproteinase-9, namely, the increase of these indexes in animals with parental strain and their decrease in animals with the resistant one. So, our study has demonstrated the remodulating effect of doxorubicin on the state of metal-containing proteins and redox characteristics of tumor dependent on its sensitivity to cytostatic, at the levels of the tumor and an organism. These data may serve as a criterion for the development of programs for the correction of malfunction of iron metabolism aimed at elevating tumor sensitivity to cytostatic agents.

Signal mediators at induction of heat resistance of wheat plantlets by short-term heating

Yu. V. Karpets, Yu. E. Kolupaev, T. O. Yastreb

V. V. Dokuchaev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru

The effects of functional interplay of calcium ions, reactive oxygen species (ROS) and nitric oxide (NO) in the cells of wheat plantlets roots (Triticum aestivum L.) at the induction of their heat resistance by a short-term influence of hyperthermia (heating at the temperature of 42 °С during 1 minute) have been investigated. The transitional increase of NO and H2O2 content, invoked by heating, was suppressed by the treatment of plantlets with the antagonists of calcium EGTA (chelator of exocellular calcium), lanthanum chloride (blocker of calcium channels of various types) and neomycin (inhibitor of phosphatidylinositol-dependent phospholipase C). The rise of hydrogen peroxide content, caused by hardening, was partially suppressed by the action of inhibitors of nitrate reductase (sodium wolframate) and NO-synthase (NG-nitro-L-arginine methyl ester – L-NAME), and the increasing of nitric oxide content was suppressed by the treatment of plants with the antioxidant ionol and with the scavenger of hydrogen peroxide (dimethylthiourea). These compounds and antagonists of calcium also partially removed the effect of the rise of plantlets’ heat resistance, invoked by hardening heating. The conclusion on calcium’s role in the activation of enzymatic systems, generating reactive oxygen species and nitric oxide, and on the functional interplay of these signal mediators at the induction of heat resistance of plantlets by hardening heating is made.