The consequences of increased oxidative stress, measured as the level of malondialdehyde (MDA) during ischemia/reperfusion, were studied in 48 patients in the acute phase of myocardial infarction (AMI) and a control group (21 blood donors). The serum levels of a-tocopherol and b-carotene were followed. Immediately after the treatment onset the level of a-tocopherol started to decrease, reaching a plateau after 24 h. The consumption of b-carotene was delayed by 90 min. Steady decline was detected during the whole time interval studied (48 h). Glutathione peroxidase (GPx) activity, as a representative of antioxidant enzymes, was estimated in whole blood. The influx of oxygenated blood was accompanied by a stimulation of GPx activity, which reached its maximum at the time of completed reperfusion. When comparing the AMI patients with the control group, the levels of MDA were found significantly increased, which indicates that oxidative stress is already increased during ischemia. Lower antioxidant levels found in the patients might either already be the result of vitamin consumption during ischemia or be a manifestation of their susceptibility to AMI. Monitored consumption of a-tocopherol and b-carotene during reperfusion indicated that in the case of patients, whose level of antioxidant vitamins is below the threshold limit, a further substantial decrease of antioxidant vitamins during reperfusion could enhance the oxidative damage of the myocardium., V. Mužáková, R. Kanďár, P. Vojtíšek, J. Skalický, R. Vaňková, A. Čegan, Z. Červinková., and Obsahuje bibliografii
Opening of the mitochondrial membrane permeability transition pore (MPTP) is an important factor in the activation of apoptotic and necrotic processes in mammalian cells. In a previous paper we have shown that cardiac mitochondria from neonatal rats are more resistant to calcium load than mitochondria from adult animals. In this study we have analyzed the ontogenetic development of this parameter both in heart and in liver mitochondria. We found that the high resistance of heart mitochondria decreases from day 14 to adulthood. On the other hand, we did not observe a similar age-dependent sensitivity in liver mitochondria, particularly in the neonatal period. Some significant but relatively smaller increase could be observed only after day 30. When compared with liver mitochondria cardiac mitochondria were more resistant also to the peroxide activating effect on calcium-induced mitochondrial swelling. These data thus indicate that the MPTP of heart mitochondria is better protected against damaging effects of the calcium load and oxidative stress. We can only speculate that the lower sensitivity to calcium-induced swelling may be related to the higher ischemic tolerance of the neonatal heart., Z. Drahota, ... [et al.]., and Obsahuje seznam literatury
The present study describes the estimation of acetaminophen (AAP) toxicity in cultured rat hepatocytes. We used different concentrations of AAP - 1, 2. 5, 5, 10 and 20 mM, to test influence of AAP on cellular viability, functional capacity and oxidative status at given time intervals. WST-1 test showed decrease of dehydrogenase activity in 5, 10 and 20 mM AAP to 75 % of control values after 1 hour of incubation. At 12 h of treatment, all AAP concentrations decreased WST-1 signal; no enzyme activity was found since 18 h in cells treated with 20 mM AAP according to LDH leakage test performed at 24 h of incubation. Functional capacity was tested by albumin assay where the decrease was strictly related to AAP dose. Intracellular oxidative status was assessed by analysis of GSH/GSSG levels and time course of ROS production and glutathione reductase (GR) activity. Increased ROS prod uction was found already after 3 h of incubation in 2.5, 5, 10 and 20 mM AAP, respectively. The highest ROS production was measured after 12 h treatment. GR activity was decreased already after 3 h of incubation and remained also decreased in cells treated with 2.5, 5, 10 and 20 mM AAP during further incubation., Tomáš Roušar ... [et al.]., and Obsahuje seznam literatury
The aim of the present work was to investigate a new mechanism likely contributing to the toxic action of acetaminophen, especially to explore the possible inhibition of glutathione reductase through an acetaminophen-glutathione conjugate (APAP-SG). APAP-SG conjugate was synthesized by organic synthesis and purified by column chromatography. The inhibitory effect of the conjugate on two types of glutathione reductase (from yeasts and rat hepatocytes) was tested spectrophotometrically. We found that the enzyme activity was reduced similarly after the treatment with 2.96 mM acetaminophenglutathione conjugate in both yeast and hepatocyte glutathione reductases (GR); the enzyme activity was inhibited to 52.7±1.5 % (2.4±0.3 mU/ml) in yeast GR (control activity was 5.6±0.3 mU/ml) and to 48.1±8.8 % (2.2±0.2 mU/ml) in rat hepatocytes lysate GR (control activity was 5.2±0.2 mU/ml). In addition, the enzyme activity (from hepatocytes lysate) was decreased to 79±7 %, 67±2 % and 39±7 %, in 0.37, 1.48 and 3.7 mM concentration of the conjugate, respectively. We found that glutathione reductase, the essential enzyme of the antioxidant system, was dose-dependently inhibited by the product of acetaminophen metabolism - the conjugate of acetaminophen and glutathione., T. Roušar ... [et al.]., and Obsahuje bibliografii a bibliografické odkazy
D-galactosamine is a hepatotoxic agent, which induces diffuse injury of liver tissue followed by the regeneration process. Our data showed a high increase of serum aminotransferases after D-galactosamine administration, which indicates a high extent of liver injury. When lipid emulsion was applied immediately after D-galactosamine, the increase of serum aminotransferases was greatly reduced. In addition, the decrease of the cytochrome c oxidase activity induced by D-galactosamine was not observed after lipid emulsion administration and the increase of total liver oxidative capacity in the regeneration period due to activated mitochondrial biogenesis was accelerated. All these findings indicate a protective effect of lipid emulsion administration against D-galactosamine toxicity., R. Ferenčíková, Z. Červinková, Z. Drahota., and Obsahuje bibliografii
Mitochondria as an energy generating cell device are very sensitive to oxidative damage. Our previous findings obtained in hepatocytes demonstrated that Complex I of the respiratory chain is more sensitive to oxidative damage than other respiratory chain complexes. We present additional data on isolated mitochondria showing that palmityl carnitine oxidation is strongly depressed at a low (200 μM) tert-butyl hydroperoxide (tBHP) concentration, while oxidation of the flavoprotein-dependent substrate - succinate is not affected and neither is ATP synthesis inhibited by tBHP. In the presence of tBHP, the respiratory control index for palmityl carnitine oxidation is strongly depressed, but when succinate is oxidized the respiratory control index remains unaffected. Our findings thus indicate that flavoprotein-dependent substrates could be an important nutritional factor for the regeneration process in the necrotic liver damaged by oxidative stress., Z. Červinková, H. Rauchová, P. Křiváková, Z. Drahota., and Obsahuje bibliografii a bibliografické odkazy
Using high-resolution oxygraphy, we tested the changes of various parameters characterizing the mitochondrial energy provision system that were induced by peroxidative damage. In the presence of succinate as respiratory substrate, 3 mM t-butyl hydroperoxide increased respiration in the absence of ADP, which indicated partial uncoupling of oxidative phosphorylation. Low activity of coupled respiration was still maintained as indicated by the ADP-activated and oligomycin-inhibited respiration. However, during the incubation the phosphorylative capacity decreased as indicated by the continuous decrease of the mitochondrial membrane potential. Under these experimental conditions the maximum capacity of the succinate oxidase system was inhibited by 50 % in comparison with values obtained in the absence of t-butyl hydroperoxide. Our data thus indicate that the oxygraphic evaluation of mitochondrial function represents a useful tool for evaluation of changes participating in peroxidative damage of cell energy metabolism., P. Křiváková, A. Lábajová, Z. Červinková, Z. Drahota., and Obsahuje bibliografii a bibliografické odkazy
The concentration-dependence of tert-butyl hydroperoxide (BHP) inhibitory effect on oxygen consumption in isolated rat liver mitochondria was measured in the presence of various respiratory substrates. Strong inhibitory effect at low concentrations of BHP (15-30 μM) was found for oxoglutarate and palmitoyl carnitine oxidation. Pyruvate and glutamate oxidation was inhibited at higher concentrations of BHP (100-200 μM). Succinate oxidation was not affected even at 3.3 mM BHP. Determination of mitochondrial membrane potential has shown that in the presence of NADH-dependent substrates the membrane potential was dissipated by BHP but was completely restored after addition of succinate. Our data thus indicate that beside peroxidative damage of complex I also various mitochondrial NADH-dependent dehydrogenases are inhibited, but to a different extent and with different kinetics. Our data also show that succinate could be an important nutritional substrate protecting hepatocytes during peroxidative damage., R. Endlicher ... [et al.]., and Obsahuje seznam literatury
Acetaminophen overdose is the most often cause of acute liver injury. The toxic mechanism is linked to formation of an active metabolite that reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). This compound has been recognized to be non-toxic generally. Our preliminary results showed, however, that APAP-SG could possess a toxic effect too. Therefore, the aim of our study was to prepare, purify and to test possible toxicity of APAP-SG. We prepared APAP-SG using organic synthesis. The conjugate was purified by preparative HPLC and its structure was confirmed using mass spectrometry. Final purity of APAP-SG was >98 %. We estimated a toxic effect of APAP-SG in isolated rat liver mitochondria using a fluorescent ROS probe. We assessed ROS production in presence of complex I or complex II substrates. The increase of ROS-dependent fluorescence in presence of glutamate/malate was 104±13 % and 130±10 % in 1 mM and 5 mM APAP-SG, respectively, in comparison with controls. ROS production related to presence of complex II substrate was enhanced 4-times in APAP-SG (5 mM) treated mitochondria (compared to controls). We conclude, we proved our hypothesis that APAP-SG conjugate is able to induce a mitochondrial impairment leading to enhanced ROS production., T. Roušar, ... [et al.]., and Obsahuje seznam literatury