Variable alpha-tocopherol stimulation and protection of glutathione peroxidase activity in established and malignant fibroblasts.

Studies on glutathione (GSH) metabolism in an established baby hamster kidney fibroblast cell line (BHK-21/C13) and in its polyoma virus-transformed counterpart (BHK-21/PyY) have revealed a significant stimulation of intracellular GSH peroxidase (GSHpx) activity (selenium-independent plus selenium-dependent) by alpha-tocopherol supplementation (14 microM). This stimulation was found to be much greater in the transformed cells. Other GSH-requiring enzyme activities (i.e. GSH reductase and GSH S-transferase) were unaltered by alpha-tocopherol treatment, suggesting a degree of specificity in its action on GSHpx. In unsupplemented growth media, the GSHpx activity in both cell lines was significantly decreased by oxidative stress. However, the same stress applied to the alpha-tocopherol-supplemented cells had no effect on the stimulated GSHpx activity, suggesting that some protection was afforded by the alpha-tocopherol.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

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.     

In vitro stimulatory effect of anti-apoptotic seminal vesicle protein 4 on purified peroxidase enzymes

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.     

Lipid and steroid hydroperoxides as substrates for the non-selenium-dependent glutathione peroxidase.

The reduction of linoleic acid hydroperoxide catalysed by rat liver cytosol was previously shown to be catalysed by a selenium-dependent glutathione peroxidase. In contrast, the enzyme responsible in guinea-pig liver cytosol is not selenium-dependent and appears to be a glutathione transferase.     

Myocardial Antioxidant Defense Mechanisms: Time Related Changes After Reperfusion of the Ischemic Rat Heart

It is well known that reperfusion damage of ischemic myocardium may be attributed to alterations in the antioxidant defense system against free radical aggression. In addition, the degree of myocardial damage may depend on the duration and severity of ischemia that precedes reperfusion. We carried out serial ischemic experiments (10, 30, 60 and 120 min) in ex-vivo rat hearts followed by 30 min reperfusion and we assayed the glutathione-dependent enzymatic activities (selenium-dependent glutathione-peroxidase: GSH-Px; selenium-independent glutathione peroxidase: GST-Px; glutathione-transferase: GST and glutathione-reductase: GS-SG-Red), Catalase activity (CAT) and non-proteic thiol compounds (NP-SH) at the end of reperfusion. We found a significant reduction of NP-SH, GSH-Px and CAT in ischemic/ reperfused hearts from 30 min on, while GST activity was increased. In addition, we observed the appearance of a selenium-independent glutathione peroxidase activity (GST-Px) belonging to the GST system. In conclusion, we found the longer the duration of ischemia the greater the inbalance between the myocardial antioxidant system especially the GST activation, suggesting in particular for GST-Px, a role in the control of the damage against oxygen toxicity during ischemia/reperfusion.     

Effect of alpha-tocopherol on superoxide dismutase and glutathione lipoperoxidase activities of mouse liver cytosol and mitochondria

The maximal radioactivity of mouse liver cytosol was observed 18 hours after intraperitoneal injection of 5-CH3-[3H] alpha-tocopherol emulsion with Tween 80. Intraperitoneal injections of Tween 80 (500 mg/kg) without alpha-tocopherol significantly decreased the glutathione lipoperoxidase and superoxide dismutase activities of liver cytosol. Vitamin E effect on the glutathione lipoperoxidase activity was weakly pronounced, while the superoxide dismutase activities of cytosol and liver mitochondria were markedly increased thereby. It was assumed that natural free radical scavengers can induce enzymatic utilization of superoxide radical anions, while synthetic free radical scavengers can trigger on enzymatic systems of lipoperoxide utilization.     

Glutathione and glutathione metabolizing enzymes in yeasts

Total glutathione content, glutathione peroxidase, glutathione transferase and glutathione reductase activities have been measured in 12 species of yeasts. All the strains tested contained glutathione, though in different amounts, as well as the above mentioned enzymes. To discriminate between the selenium-dependent and the selenium-independent form, glutathione peroxidase activity has been measured with both H₂O₂ and cumene hydroperoxide. Rhodotorula glutinis appeared to be the only strain in which the selenium-dependent form was not found, but this yeast exhibited the highest level of selenium-independent glutathione peroxidase activity as compared to the other strains.     

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.     

In vivo inhibition of selenium dependent glutathione peroxidase and superoxide dismutase in rats by diethyldithiocarbamate

Intraperitoneal injection of rats with diethyldithiocarbamate (1.2 g/kg body wt) led to maximum diminution of superoxide dismutase activity at 1 hr by 86 and 84% in liver and red blood cell respectively with a gradual return to the normal level at 48 hr after administration of injection. Significant inhibition of selenium-dependent glutathione peroxidase was also observed, which returned to normal at 48 hr after administration of injection. However, maximum decline in its activity was at 12 hr by 52 and 73% in liver and red blood cells respectively. No significant difference in tissue level of selenium-independent glutathione peroxidase was observed during time course study after diethyldithiocarbamate administration. It is possible that inhibition of superoxide dismutase by diethyldithiocarbamate leads to accumulation of superoxide anion which in turn inactivates selenium-dependent glutathione peroxidase by its reaction with selenium at the active site of the enzyme.     

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.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

Pro-oxidant and antioxidant processes in gas gland and other tissues of cod (Gadus morhua)

Partial reduction of molecular oxygen produces reactive oxyradicals, including the superoxide anion radical (O _- ² ) and hydroxyl radical (·OH). The gas gland functions under hyperoxic and acidic conditions and therefore is likely to be subjected to enhanced oxidative stress. Aspects of pro- and antioxidant processes in gas gland were compared with other tissues likely to be subject to differing degrees of oxyradical production, viz. liver (site of chemically-mediated oxyradical production), gills and skeletal muscle. Antioxidant enzyme activities (superoxide dismutase, catalase, selenium-dependent and total glutathione peroxidase) per g wet weight were highest in liver and lowest in muscle. Catalase and glutathione peroxidase activies per g wet weight were higher in gills than in gas gland, whereas the reverse was seen for superoxide dismutase. Cytosolic superoxide dismutase activities per mg protein were two- and nine-fold higher in gas gland than in liver and gills. The pH characteristics of the antioxidant enzymes were generally similar in all the tissues. Glutathione, vitamin E and unsaturated (peroxidizable) lipid levels were generally highest in liver followed by gas gland. Lipid peroxidation (malonaldehyde equivalents) was evident in all tissues except gas gland. Hydrogen peroxide and O _- ² were involved in the NAD(P)H-dependent ferric/EDTA-mediated formation of ·OH (as measured by 2-keto-4-methiolbutyrate oxidation) by mitochondrial and postmitochondrial fractions of gas gland. Tissue maximal potentials for ·OH production paralled superoxide dismutase but not catalase or glutathione peroxidase activities. Overall, the results confirm the presence of effective antioxidant defences in gas gland and support previous workers' contentions of a central role for superoxide dismutase in this process.Abbreviations EDTA di-sodium ethylenediaminetetra-acetic acid - G-6-P glucose-6-phosphate - GPX total glutathione peroxidase - GSH reduced glutathione - GSSG oxidised glutathione - GST glutathion-S-transferase - HPLC high performance liquid chromatography - KMBA 2-keto-4-methiolbutyric acid - MOPS 3-[N-morpholino] propane-sulphonic acid - PMS postmitochondrial supernatant - Se-GPX selenium-dependent glutathion peroxidase - SOD superoxide dismutase - TCA trichloroacetic acid     

Free radicals, lipid peroxidation and muscular ischemia]

The role of oxygen free radicals in ischemia and reperfusion injury of skeletal muscle has not been well defined, partly because of the relative resistance of this tissue to normothermic ischemia. Under normal conditions small quantities of oxygen free radicals are produced but they are quenched by intracellular free radical scavenging enzymes (superoxide dismutase, catalase and glutathione peroxidase) or alpha-tocopherol. The increase in malondialdehyde suggests increased lipid peroxidation initiated by free radical reactions. Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. The results of recent studies indicate that: a) oxygen free-radicals mediates, at least in part, the increased microvascular permeability produced by reoxygenation, b) free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged ischemia. Additional evidence support the hypothesis of the interrelationship between ischemic tissue and inflammatory cells. So capillary plugging by granulocytes and oxygen free radical formation may contribute to the ischemic injury.     

Quantitative study of natural antioxidant systems for cellular nitrofurantoin toxicity

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5.10(-5) M in the culture medium. The protection afforded by antioxidants was then tested: alpha-tocopherol gave at 10(-4) M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.     

Alterations of the plasma selenium concentrations and the activities of tissue peroxide metabolism enzymes in streptozotocin-induced diabetic rats

The activity of aortic glutathione peroxidase, a selenium-dependent enzyme, significantly decreased in rats 4 and 8 months after the injection of streptozotocin (STZ). Catalase activity was shown to occur at low levels in rat aorta and was not influenced by the diabetic state. Superoxide dismutase activity was less than detectable. The activity of selenium-dependent glutathione peroxidase in kidney, but not in lung and liver, increased in diabetic rats. Catalase and superoxide dismutase activities in the kidney were not altered. The plasma lipid peroxide value increased in diabetic rats. The selenium content in plasma of diabetic rats increased markedly while the increase in plasma glutathione peroxidase activities was insignificant. The observed abnormalities in plasma of STZ rats were improved by insulin treatment. The defects in glutathione peroxidase in the diabetic rat aorta were restored by insulin treatment. These results may suggest that the capacity of the antioxidative defense system in the aorta decreased in the diabetic state, and this may help clarify the mechanism of the pathogenesis of endothelial dysfunction associated with diabetes.     

The content of lipid peroxidation products and the activity of antioxidant enzymes in the myocardium and liver of rats with various vitamin E allowances

After 2 month of feeding vitamin E-supplemented diet (100.6 and 0 mg/kg; group I-control, II and III, respectively) the concentration of lipid peroxidation products (diene conjugates, malondialdehyde, Schiff's bases) and activity of antioxidant enzymes (superoxide dismutase, glutathione peroxidase) was estimated in rat heart and liver. Although the content of alpha-tocopherol in organs of group II was significantly decreased, the concentration of peroxidation products and enzyme activities was unchanged. Moreover, these parameters were constant in rat liver of group III. The heart was more sensitive because in group III to vitamin E deficiency (the alpha-tocopherol level was dropped fourfold) the concentration of diene conjugates and malondialdehyde was increased and superoxide dismutase activity was decreased. Thus insufficiency of vitamin E may result in selective alterations of myocardial functions. In addition, vitamin E may be useful instrument for correction of free radical oxidation and antioxidant system activity in the heart.     

Selenite-induced variation in glutathione peroxidase activity of three mammalian cell lines: no effect on radiation-induced cell killing or DNA strand breakage

The selenium-dependent glutathione peroxidase activities of three mammalian cell lines, HT29, P31, and N-18, cultured in medium with low serum content, increased about 2-, 5-, and 40-fold, respectively, after supplementation with 100 nM selenite. Catalase, CuZn superoxide dismutase, and Mn superoxide dismutase activities were not generally influenced by selenite supplementation, and there was only a minor nonselenium-dependent glutathione peroxidase activity in the investigated cell lines. Gamma-irradiated control and selenite-supplemented cells showed no changes in the surviving fractions, as estimated by clonogenic survival or [3H]-thymidine uptake, nor were there any significant differences between the two groups in the induction of DNA strand breaks after gamma irradiation under repairing (37 degrees C) or nonrepairing (0 degrees C) conditions. The results suggest that selenium-dependent glutathione peroxidase does not contribute significantly to the radiation resistance of cultured mammalian cells.     

Regulation by catecholamines and cAMP of enzymes of thiol and disulfide metabolism

In vivo exo- and endogenous catecholamines have no influence on the activities of thioredoxin reductase, glutathione reductase, thiol transferase and nonselenium-dependent glutathione peroxidase. At the same time catecholamines activate via beta-adrenoceptors glutathione S-transferase and selenium-dependent glutathione peroxidase from many tissues and inhibit gamma-glutamyl transferase from kidney. In vitro cAMP has identical effects on the activities of the above enzymes. The possible significance of regulation of glutathione metabolism enzymes is discussed.     

Effect of the herbicide 4-CPA on human erythrocyte antioxidant enzymes in vitro

To investigate the possible role of oxygen free radicals and oxidant stress in the toxic effects of phenoxyherbicides, we studied the in vitro effect of 4-chlorophenoxyacetic acid (4-CPA) on various human erythrocyte antioxidant enzymes, namely glucose-6-phosphate dehydrogenase, catalase, selenium-dependent glutathione peroxidase, glutathione reductase and Cu/Zn-superoxide dismutase. 4-CPA added in a dose of 1 ppm to human erythrocytes for 1 h caused a significant reduction in glucose-6-phosphate dehydrogenase (P <0.001) and catalase (P <0.001) activities, but did not significantly affect the activities of other enzymes. Such selective inactivation of specific erythrocyte antioxidant enzymes may play a role in the toxic effects of phenoxyherbicides.