The effect of melaxen and valdoxan on the activity of the glutathione antioxidant system in rats with experimental hyperthyroidism

The activity of the glutathione system and conjugated diene content (CD) have been investigated in the liver and blood serum of rats with experimental hyperthyroidism treated with melaxen and valdoxan. The study of glutathione reductase (GR), glutathione peroxidase (GP) and glutathione transferase (GST) activities increased under this pathology has shown that administration of these compounds decreased these activities towards control levels. Melaxen and valdoxan administration increased reduced glutathione (GSH) content as compared with untreated hyperthyroid rats. This increase may be associated with its decreased utilization for detoxification of toxic products of free radical oxidation (FRO). Administration of the melatonin correcting drugs also tended to normalize the CD level increased in the liver and blood serum of hyperthyroid rats. Results of this study indicate that melaxen and valdoxan exhibit positive effect on free radical homeostasis. This appears to be accompanied by a decrease in the load of the glutathione antioxidant system in comparison with the examined pathology.     

The dose-dependent effect of 3,5-dicarbomethoxyphenylbiguanide on activity of the glutathione antioxidant system in heart and blood serum of rats with experimental myocardial infarction

Administration of a synthetic compound with predicted anti-ischemic and cardioprotective activity, 3,5-dicarbomethoxyphenylbiguanide (3,5-DCMPBG) to rats with experimental myocardial infarction led to a decrease in the lipid peroxidation level, glutathione peroxidase activity, the level of reduced glutathione, activity of NADP-isocitrate dehydrogenase in the heart and blood serum, and activity of glucoso-6-phosphate dehydrogenase in heart in comparison with their levels in untreated animals with myocardial infarction. This may be attributed to a decrease of free radical processes and reduction of antioxidant system loading induced by the protective effect of the administered compound. At the same time the increase glutathione reductase activity observed under these conditions in the heart and blood serum is probably associated with specific influence of 3,5-DCMPBG on this enzyme.     

Effect of cisplatin on lipid peroxidation in pig blood platelets.

The effect of cisplatin (cis-diamminedichloroplatinum II) on the activities of the blood platelet antioxidative enzymes (glutathione peroxidase, superoxide dismutase) as well as on the peroxidation of blood platelet lipids was investigated. Cisplatin was found to cause a significant inhibition of platelet free radical scavering enzyme activity and an increase of malonyldialdehyde levels in blood platelets incubated with cisplatin in vitro.     

Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits.

The present study investigated changes in rate of free radical production, antioxidant enzyme activity, and glutathione status immediately after and 24 h after acute muscle stretch injury in 18 male New Zealand White rabbits. There was no change in free radical production in injured muscles, compared with noninjured controls, immediately after injury (time 0; P = 0.782). However, at 24 h postinjury, there was a 25% increase in free radical production in the injured muscles. Overall, there was an interaction (time and treatment) effect (P = 0.005) for free radical production. Antioxidant enzyme activity demonstrated a treatment (injured vs. control) and interaction effect for both glutathione peroxidase (P = 0.015) and glutathione reductase (P = 0.041). There was no evidence of lipid peroxidation damage, as measured by muscle malondialdehyde content. An interaction effect occurred for both reduced glutathione (P = 0.008) and total glutathione (P = 0.015). Morphological analysis (hematoxylin and eosin staining) showed significant polymorphonuclear cell infiltration of the damaged region at 24 h postinjury. We conclude that acute mechanical muscle stretch injury results in increased free radical production within 24 h after injury. Antioxidant enzyme and glutathione systems also appear to be affected during this early postinjury period.     

Electron paramagnetic resonance decay constant and oxidative stresses in liver microsomes of the selenium-deficient rat

The free radical-reducing activity and the membrane fluidity of liver microsomes from selenium-deficient (SeD) rats were examined by means of electron paramagnetic resonance (EPR) spin label method using nitroxyl-labeled stearic acids. Our findings show that the membrane fluidity and lipid peroxidation levels in SeD rat liver microsome were relatively unchanged compared with normal rat. In contrast, SeD caused the induction of liver microsomal cytochrome P-450 activity. The nitroxyl spin probes are substrates for reduction-relating cytochrome P-450. Previous in vivo studies suggested that the total liver free radical reduction activity in SeD rat was decreased. In contrast, SeD caused the induction of liver microsomal cytochrome P-450 activity, and the reduction rate of nitroxyl radical existing at shallow depth in membrane was increased. Selenium-deficient rats experienced an increase in hydrogen peroxide (H2O2) due to a pronounced loss of glutathione peroxidase (GSH-Px) activity. This masked the overall reduction rate of the nitroxyl spin probe by reoxidation of the hydroxylamine form. Although the SeD condition caused induction of liver cytochrome P-450 and chronic increased H2O2, this did not result in oxidative liver damage. An increased level of glutathione in SeD liver was also evident, likely due to the absence of GSH-Px activity. Using the EPR spin label method, we have shown that SeD causes complicated redox changes in the liver, notably, alterations in the levels of cytochrome P-450 and GSH-Px systems.     

One- and two-electron oxidation of reduced glutathione by peroxidases

The oxidation of glutathione by horseradish peroxidase forms a thiyl free radical as demonstrated with the spin trapping ESR technique. Reactions of this thiyl free radical result in oxygen consumption, which is inhibited by the radical trap 5,5-dimethyl-1-pyrroline-N-oxide. In contrast to L-cysteine oxidation, glutathione oxidation is highly hydrogen peroxide-dependent. The oxidation of glutathione by glutathione peroxidase forms glutathione disulfide without forming a thiyl radical intermediate, except in the presence of the thiyl radical-generating horseradish peroxidase.     

Actions of melatonin in the reduction of oxidative stress

Melatonin was discovered to be a direct free radical scavenger less than 10 years ago. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant. In terms of direct free radical scavenging, melatonin interacts with the highly toxic hydroxyl radical with a rate constant equivalent to that of other highly efficient hydroxyl radical scavengers. Additionally, melatonin reportedly neutralizes hydrogen peroxide, singlet oxygen, peroxynitrite anion, nitric oxide and hypochlorous acid. The following antioxidative enzymes are also stimulated by melatonin: superoxide dismutase, glutathione peroxidase and glutathione reductase. Melatonin has been widely used as a protective agent against a wide variety of processes and agents that damage tissues via free radical mechanisms.     

Glutathione and ascorbate reduction of the acetaminophen radical formed by peroxidase. Detection of the glutathione disulfide radical anion and the ascorbyl radical

The acetaminophen phenoxyl radical was generated by the oxidation of acetaminophen by horseradish peroxidase in a fast-flow ESR experiment, and its reaction with glutathione and ascorbate was studied. Glutathione reduces the phenoxyl radical of acetaminophen to regenerate acetaminophen and form the thiyl radical of glutathione. This thiyl radical reacts with the thiolate anion of glutathione to form the disulfide radical anion, which was detected and characterized by ESR spectroscopy. In the presence of ascorbate, the ascorbyl radical was produced by the reduction of the acetaminophen phenoxyl radical by ascorbate. This reaction results in the complete reduction of the free radical of acetaminophen, whereas the glutathione reduction of the phenoxyl radical of acetaminophen was not complete on the fast-flow ESR time scale of milliseconds. This suggests that ascorbate rather than glutathione is more likely to react with the acetaminophen phenoxyl free radical in vivo. In the presence of both ascorbate and higher concentrations of glutathione, the reaction with ascorbate is dominant. When cysteine was used in the place of reduced glutathione in the above assay system, the disulfide radical anion of cystine was observed in a manner similar to glutathione. These reactions may have significance in the detoxification of acetaminophen and the free radical metabolites of xenobiotics in general. Only in cells containing low levels of ascorbate can glutathione play a direct role in the detoxification of the acetaminophen phenoxyl radical.     

Evaluation of the fluidity and functionality of the renal cortical brush border membrane in experimental diabetes in rats

The present study was aimed at addressing the effect of hyperglycemia on the renal cortical brush border membrane. The fluidity and the functionality of the renal cortical brush border membrane have been evaluated after 6 weeks of streptozotocin-induced diabetes in rats. Lipid peroxidation and protein oxidation were first performed to confirm a state of oxidative stress. The fluidity of the brush border membrane of diabetic rats decreased significantly by 15.76%. There was an increase in the amount of early (19.39%) and advanced (42.23%) glycation end-products suggesting the accumulation of significant amount of non-enzymic glycation products at 6 weeks of diabetes. Although, the activities of both gamma-glutamyl transpeptidase and alkaline phosphatase of the brush border membrane decreased, that of the latter decreased to a significant extent with an increase in K(m) (81%) and no change in the V(max). A study of the activities of glutathione-dependent antioxidant enzymes in the renal cortical homogenates showed that the activities of glutathione peroxidase and glyoxalase II were altered significantly. Our study seems to suggest that increased free radical generation accompanied by non-enzymic glycation may be responsible for oxidative stress and an increased rigidity of the diabetic brush border membrane. Alkaline phosphatase may thus serve as a potentially useful marker of free radical induced damage to the renal cortical brush border membrane. The results also suggest that enhanced susceptibility to oxidative stress during early stages may be an important factor in the development of secondary complications of diabetes.     

Vitamin E in young and old human red blood cells

Young and old human red blood cells contain about the same amount of alpha-tocopherol, a compound which has previously been shown to be the major lipid-soluble, chain-breaking antioxidant present in such cells. Since red blood cells lose up to ca. 20% of lipid material from their membrane as they age, the alpha-tocopherol/membrane-lipid ratio actually rises with age rather than declining as might have been expected on the basis of the free radical theory of aging. The alpha-tocopherol/arachidonic acid moiety ratios increase in the order: young red blood cells less than old red blood cells less than plasma, which argues against the suggested membrane stabilizing effect of alpha-tocopherol/arachidonic acid moiety complexes.     

Red cell peroxide metabolism in diabetes mellitus

In a group of normal controls and in a group of diabetics subdivided for type we evaluated the following red blood cell parameters: superoxide dismutase (SOD), glutathione peroxidase (GSH.Px), catalase (C-ase), glutathione content (GSH) and membrane-protein-sulphydryl groups (P-SH). There was no difference in the enzymatic activities of superoxide dismutase, glutathione peroxidase and catalase in normals and in the diabetics. However, the erythrocyte GSH content as well as membrane P-SH groups discriminate the normals from diabetics and also the diabetics subdivided for type. None of these parameters was related to the erythrocyte filterability considered as a reflection of the red blood cell deformability. These findings reveal that the diabetic red blood cell is less protected from oxidant agents.     

Zinc-induced damage to carp (Cyprinus carpio L.) erythrocytes in vitro

Fish erythrocytes were used to elucidate the effect of zinc ions on the cell antioxidant defence system. It was detected that an increase of the Zn2+ concentration (0.01-1 mM) leads to a marked decrease (p < 0.05) in the catalase and the glutathione peroxidase activities. We observed a loss of 14-39% activity of glutathione peroxidase, and 16-20% diminution for catalase. No significant changes were found in case of the superoxide dismutase. Incubation of red blood cells with zinc brought about a decrease of the erythrocyte thiol group content. Treatment of carp erythrocytes with zinc ions also resulted in enhanced hemolysis and in the induction of significant (p < 0.001) changes in the intracellular glucose level. The increase of glucose concentration in the erythrocytes was correlated with increased concentration of metal in the incubation medium. It was proposed that Zn could affect transport systems across the red blood cells and therefore increased the permeability of the membranes to small molecules (e.g. hexose), and led to hemolysis. Zinc ions could act as a potential cell toxicant, leading to disturbances in functions of the antioxidant defence system and to alterations in the erythrocyte membrane properties.     

State of the antioxidant system during induction in rats of primary generalized epileptic activity

Antioxidation system in the brain and blood of rats with generalized bemegride-induced epileptic activity was studied. Antioxidation enzyme activity (superoxide dismutase, glutathione peroxidase and glutathione reductase) and alpha-tocopherol content were determined at an early convulsive stage, immediately after generalized seizures and 10-15 min after seizure. Antioxidation enzyme activity and alpha-tocopherol level in the brain homogenate and blood remained unchanged at any stages of investigation. It is suggested that the increased level of lipid peroxidation products in the brain and blood of rats upon the development of bemegride-induced epileptic activity is not related to the decrease in antioxidation system activity. The effect is mediated by the activation of the reaction initiating free radical brain lipid transformations.     

Inhibition of selenium dependent glutathione peroxidase and superoxide dismutase in rats by diethyldithiocarbamate: effect of pre-administration of alpha-tocopherol

Rats pre-administered with alpha-tocopherol (10 mgs/day) for 7 days afforded a significant protection at the tissue level against the lowering of superoxide dismutase and glutathione peroxidase, especially the selenium-dependent glutathione peroxidase. The protective action of alpha-tocopherol in the diethyldithiocarbamate treated rats may be attributed to its antioxidant/free radical scavenging action. It is concluded that selenium-dependent glutathione peroxidase and alpha-tocopherol act in a complementary fashion to block free radical formation.     

Molecular bases of the treatment of Alzheimer's disease with antioxidants: prevention of oxidative stress

Alzheimer's disease is associated with a systemic oxidative stress situation which can be followed in vivo by determining biomarkers such as plasma lipoperoxides and TBARS levels and the oxidation degree of glutathione in red blood cells. It has been observed that Alzheimer's patients show an increased level of plasma TBARS, which indicates a higher free radical oxidation of plasma unsaturated phospholipids, and an increased oxidation of red blood cells glutathione, which indicates oxidative stress in peripheral cells. This latter, glutathione oxidation, was found to correlate statistically with the cognitive status of the patients. Treatment with vitamin E resulted in an improved cognitive performance only of those patients in which the tocopherol acted as an antioxidant, according to blood indicative markers of oxidative stress. Indeed, the effect of vitamin E on Alzheimer's disease patients showed considerable variations both in its antioxidant function and in its capacity to improve cognitive functions. An important conclusion from the reported results is that epidemiological or clinical studies that aim to test the effect of antioxidant supplementation on given functions should include the determination of the antioxidant status of the patients by the measurement of blood markers of oxidative stress.     

The oxidant/antioxidant network: role of melatonin

Melatonin is now known to be a multifaceted free radical scavenger and antioxidant. It detoxifies a variety of free radicals and reactive oxygen intermediates including the hydroxyl radical, peroxynitrite anion, singlet oxygen and nitric oxide. Additionally, it reportedly stimulates several antioxidative enzymes including glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and superoxide dismutase; conversely, it inhibits a prooxidative enzyme, nitric oxide synthase. Melatonin also crosses all morphophysiological barriers, e.g., the blood-brain barrier, placenta, and distributes throughout the cell; these features increase the efficacy of melatonin as an antioxidant. Melatonin has been shown to markedly protect both membrane lipids and nuclear DNA from oxidative damage. In every experimental model in which melatonin has been tested, it has been found to resist macromolecular damage and the associated dysfunction associated with free radicals.     

Cytotoxic aldehyde generation in heart following acute iron-loading

Although the mechanism of myocardial failure following acute iron poisoning is not known, excess iron-catalyzed free radical generation is conjectured to play a role. The effects of time (0 to 360 minutes) on total iron concentrations, glutathione peroxidase activity, and cytotoxic aldehyde production in heart of mice (B6D2F1, n = 65) were first investigated following acute iron-loading (20 mg iron dextran i.p./mouse). In a subsequent experiment, the effects of dose (0 to 80 mg iron dextran i.p./mouse, n = 75) on the aforementioned parameters were investigated. Our results show that the concentrations of cytotoxic aldehydes: (1) significantly differ over-time, with corresponding increases in total concentrations of iron (r = 0.93, p < 0.001); and (2) increase parallel to the total dose of iron administered (r = 0.95, p < 0.001). Furthermore, dose-and time-dependent alterations to glutathione peroxidase activity are observed, which is most likely due to an acute up-regulation of the enzyme as an endogenous protective response to increased free radical activity in the heart subsequent to iron-loading. While no single mechanism is likely to account for the complex pathophysiology of acute iron-induced heart failure, our results shown that iron-loading can result in significant free radical generation, as quantified by cytotoxic aldehydes, in heart tissue of mice. This is the first report on the effects of time and dose on cytotoxic aldehyde generation and glutathione peroxidase activity in heart of mice following acute iron-loading.     

Effect of quercetin on daunorubicin-induced heart mitochondria changes in rats

Cancer therapy with daunorubicin is limited by its cardiotoxicity. It has been suggested that daunorubicin-induced free radical generation can be involved. The precise molecular mechanism of daunorubicin-induced cardiotoxicity is still not well understood but it is believed that mitochondria play an important role in this process. It has been reported that flavonoids with antioxidant properties may prevent anthracycline-induced cardiotoxicity. In this work, we investigated the effects of daunorubicin and quercetin on mitochondrial enzyme activities such as ATPase, glutathione peroxidase (GPx) and glutathione reductase (GR). Moreover, we also studied the changes of outer mitochondrial membrane using synchronous fluorescence spectra. The activity of ATPase and GR were significantly increased after daunorubicin application. Pretreatment with quercetin significantly alleviated this increase. On the other hand, GPx activity was significantly decreased and quercetin prevented this decrease. Treatment with quercetin alone had no significant effect on the enzyme activity studied. Quercetin also completely prevented daunorubicin-induced changes in fluorescence of the outer mitochondrial membrane. In conclusion, our data indicate that quercetin may be useful in mitigating daunorubicin-induced cardiotoxicity.     

Environmental induction models for the investigation of activity: changes in glutathione peroxidase, a crucial factor of the antioxidant defence

Glutathione peroxidase enzyme superfamily plays significant role in the elimination of reactive oxygen free radicals in the animals. Many characteristics of these proteins have been revealed already, but their regulation is still not known. Several data suggest that some environmental factors have certain regulatory effect, while others propose strict genetic regulation. In this report we present four different environmental induction models in which New Zealand white rabbits were used as experimental animals. In three models, free radical load of different origin, lipidperoxide load, application of a glutathione depletor or a prooxidant agent, was introduced. Beside these negative models a positive model was also constructed in which additive selenium was supplied. Glutathione peroxidase activity was measured in blood serum, erythrocyte haemolysate and liver. Reduced glutathione, and malondialdehyde concentration in the liver were also determined. According to the results, the established models are capable for analysing the enzyme activity x environmental interactions.     

The role of selenium peroxidases in the protection against oxidative damage of membranes

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.