Butyrate increases catalase activity and protects rat pulmonary artery smooth muscle cells against hyperoxia

A protective effect of butyrate against hyperoxia was found with adult rat pulmonary artery smooth muscle cells. Butyrate (5mM) when added just prior to the hyperoxic exposure (95%) markedly decreased lactate dehydrogenase release from cells during 68 hours of exposure (22% release with butyrate versus 98% without). The uptake and reduction of a tetrazolium compound as another index of cell viability also showed similar improvement with butyrate. Butyrate was associated with a striking increase of catalase to three times the control in the air exposed group while GSH content and the activities of superoxide dismutase and glutathione peroxidase were not significantly changed. In the groups exposed to hyperoxia alone, both enzyme activities were decreased compared to the air exposed controls. When butyrate was present with hyperoxia, the superoxide dismutase was maintained closer to the air exposed control values and the catalase activity remained nearly twice as high as the air exposed control cells. These results suggest that butyrate protects rat pulmonary artery smooth muscle cells from hyperoxia by increasing catalase activity which may help to preserve superoxide dismutase activity. This may be a good model to determine the biological significance of catalase and its interrelationships with other antioxidant systems within the cell.     

Superoxide, hydrogen peroxide, and oxygen toxicity in two free-living nematode species

Two species of free-living nematodes, Turbatrix aceti and Caenorhabditis elegans, exhibited a marked sensitivity to 3 atm of 100% O2. Environmental changes in pH and temperature, which altered nematode respiration, resulted in alterations in the survival of these organisms under high pO2. Levels of defensive enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and dianisidine peroxidase were measured in the two species. No changes in the level of superoxide dismutase or catalase activity were induced by exposure of the nematodes to high pO2. Manipulation of these two enzymes was however achieved using the inhibitors 3-amino-1,2,4-triazole and diethyldithiocarbamate. 3-Amino-1,2,4-triazole (20 mM) eliminated greater than or equal to 80% of the catalase activity in vivo and diethyldithiocarbamate (5 mM) decreased the level of CuZn superoxide dismutase by greater than or equal to 70%. Both of these compounds increased the sensitivity of C. elegans to high pO2 toxicity. Compounds capable of intracellular redox-cycling with O2- -production, such as plumbagin, increased CN- -resistant respiration in the nematodes and imposed an O2-dependent toxicity. These experiments demonstrate the toxicity of intracellular O2- and H2O2 in nematodes and the importance of superoxide dismutase and catalase in providing a defense against these toxic molecules in vivo.     

Oxygen toxicity in control and H2O2-resistant Chinese hamster fibroblast cell lines

Following exposure to 95% oxygen, clonogenic cell survival was assayed and qualitative morphologic changes were observed in a Chinese hamster fibroblast cell line (HA-1). The time in 95% O2 necessary to clonogenically inactivate 90% of the cells was inversely related to the cell density of the cultures at the beginning of hyperoxic exposure (from 1 to 6 X 10(4) cells/cm2). The O2-induced loss in clonogenicity and evidence of morphologic injury were shown to be significantly delayed (17-22 h) in an H2O2-resistant variant of the parental HA-1 cell line. After the delay in onset of clonogenic cell killing or morphologic injury, the process of injury proceeded in a similar fashion in both cell lines. The H2O2-resistant cell line demonstrated significantly greater catalase activity (20-fold), CuZn superoxide dismutase activity (2-fold), and Se-dependent glutathione peroxidase activity (1.5-fold). The greater activities of CuZn superoxide dismutase and catalase were accompanied by similarly greater quantities of immunoreactive protein as determined by immunoblotting. These data demonstrate that the cells adapted and/or selected for growth in a highly peroxidative environment also became refractory to O2-induced toxicity, which may be related to increased expression of antioxidant enzymes. However, the magnitude of this cross-resistance to O2 toxicity was less than the magnitude of the cellular resistance to the toxicity of exogenous H2O2, suggesting that in this system the toxicity of 95% oxygen is not identical to H2O2-mediated cytotoxicity.     

Effect of normobaric hyperoxia on antioxidant defenses of HeLa and CHO cells

The effect of increased intracellular oxygen activation on cellular antioxidant defenses in CHO and HeLa cells was studied. In both cell types, hyperoxic exposure (up to 4 days, 600-700 mm Hg O2) and in CHO cells menadione (up to 3 days, 15 microM) failed to affect the enzymatic antioxidant defenses Mn-containing superoxide dismutase (Mn-SOD), CuZn-SOD, catalase and glutathione peroxidase. The markedly increased antioxidant enzyme activities observed in a recently obtained oxygen-tolerant CHO variant persisted under normoxia. These data suggest that the synthesis of antioxidant enzymes is constitutive. Glutathione levels of HeLa cells did not respond to hyperoxia whereas in CHO cells hyperoxia and menadione exposure resulted in a 2- and 7-fold increase in glutathione contents, respectively. However, considering the large variations in glutathione contents observed under normal culture conditions, it is uncertain whether this increase is to be considered as a true adaptive response.     

Induction of chromosomal aberrations in active oxygen-generating systems. I. Effects of paraquat in Chinese hamster cells in culture

A possible role for the superoxide anion radical (O2-) in the clastogenicity of paraquat (PQ) was investigated in cultured Chinese hamster cells. When cells were treated with 0.8 mg/ml of PQ for 3 h followed by 21 h of recovery time, structural chromosome aberrations were induced in about 50% of the metaphases examined. Almost all aberrations were of the chromatid-type and involved exclusively gaps and breaks. The induction of chromosomal aberrations by PQ was enhanced by a 1-h pretreatment with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Diethyl maleate, a glutathione scavenger, also enhanced the induction of chromosomal aberrations, but 3-aminotriazole, an inhibitor of catalase, showed no such effects. Enhanced induction of chromosomal aberrations was also observed when PQ-treated cells were cultured at a high oxygen concentration (80%). The present results suggest that the production of chromosomal aberrations by PQ may be directly or indirectly related to the generation of O2-, but not to the formation of hydrogen peroxide by the dismutation reaction of O2- or of other active oxygen species including the hydroxyl radical and singlet oxygen.     

Hyperoxia results in transient oxidative stress and an adaptive response by antioxidant enzymes in goldfish tissues

The effects of hyperoxia on the status of antioxidant defenses and markers of oxidative damage were evaluated in goldfish tissues. The levels of lipid peroxides, thiobarbituric acid reactive substances, carbonyl proteins and the activities of some antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of goldfish, Carassius auratus L., over a time course of 3-12 h of hyperoxia exposure followed by 12 or 36 h of normoxic recovery. Exposure to high oxygen resulted in an accumulation of protein carbonyls in tissues throughout hyperoxia and recovery whereas lipid peroxides and thiobarbituric acid reactive substances accumulated transiently under short-term hyperoxia stress (3-6 h) but were then strongly reduced. This suggests that hyperoxia stimulated an enhancement of defenses against lipid peroxidation or mechanisms for enhancing the catabolism of peroxidation products. The activities of principal antioxidant enzymes, superoxide dismutase and catalase, were not altered under hyperoxia but catalase increased during normoxic recovery; activities may rise in anticipation of further hyperoxic excursions. In most tissues, the activities of glutathione-utilizing enzymes (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) as well as glucose-6-phosphate dehydrogenase, were not affected under hyperoxia but increased sharply during normoxic recovery. Correlations between some enzyme activities and oxidative stress markers were found, for example, an inverse correlation was seen between levels of thiobarbituric acid reactive substances and glutathione-S-transferase activity in liver and catalase and glucose-6-phosphate dehydrogenase in kidney. The results suggest that liver glutathione-S-transferase plays an important role in detoxifying end products of lipid peroxidation accumulated under hyperoxia stress.     

Amelioration of glucose induced hemolysis of human erythrocytes by vitamin E

Cells under aerobic condition are always threatened with the insult of reactive oxygen species, which are efficiently taken care of by the highly powerful antioxidant systems of the cell. The erythrocytes (RBCs) are constantly exposed to oxygen and oxidative stress but their metabolic activity is capable of reversing the injury under normal conditions. In vitro hemolysis of RBCs induced by 5, 10 and 20 mM glucose was used as a model to study the free radical induced damage of biological membranes in hyperglycemic conditions and the protection rendered by vitamin E on the same. RBCs are susceptible to oxidative damage, peroxidation of the membrane lipids, release of hemoglobin (hemolysis) and alteration in activity of antioxidant enzymes catalase and superoxide dismutase. The glucose induced oxidative stress and the protective effect of vitamin E on cellular membrane of human RBCs manifested as inhibition of membrane peroxidation and protein oxidation and restoration of activities of superoxide dismutase and catalase, was investigated.Thiobarbituric acid reactive substances are generated from decomposition of lipid peroxides and their determination gives a reliable estimate of the amount of lipid peroxides present in the membrane. Vitamin E at 18 μg/ml (normal serum level) strongly enhanced the RBC resistance to oxidative lysis leading to only 50–55% hemolysis in 24 h, whereas RBCs treated with 10 and 20 mM glucose without vitamin E leads to 70–80% hemolysis in 24 h. Levels of enzymic antioxidants catalase, superoxide dismutase and nonenzymic antioxidants glutathione showed restoration to normal levels in presence of vitamin E. The study shows that vitamin E can protect the erythrocyte membrane exposed to hyperglycemic conditions and so a superior antioxidant status of a diabetic patient may be helpful in retarding the progressive tissue damage seen in chronic diabetic patients.     

Superoxide dismutase-rich bacteria. Paradoxical increase in oxidant toxicity

Superoxide dismutase is considered important in protection of aerobes against oxidant damage, and increased tolerance to oxidant stress is associated with induction of this enzyme. However, the importance of superoxide dismutase in this tolerance is not clear because conditions which promote the synthesis of superoxide dismutase likewise affect other antioxidant enzymes and substances. To clarify the role of superoxide dismutase per se in organismal defense against oxidant-generating drugs, we employed Escherichia coli transformed with multiple copies of the gene for bacterial iron superoxide dismutase. These bacteria have greater than ten times the superoxide dismutase activity of wild-type E. coli but, importantly, are normal in other oxidant defense parameters including catalase, peroxidases, glutathione, and glutathione reductase. High superoxide dismutase and control bacteria were exposed to the O2- -generating drug paraquat and to elevated pO2. We find; high superoxide dismutase E. coli are more readily killed by paraquat under aerobic, but not anaerobic, conditions. During exposure to paraquat, high superoxide dismutase E. coli accumulate more H2O2. Coincidentally, the reduced glutathione content of high superoxide dismutase E. coli declines more than in control E. coli. E. coli with high superoxide dismutase activity are also more readily killed by hyperoxia. Interestingly, the susceptibility of the parental and high superoxide dismutase E. coli to killing by exogenous H2O2 is not significantly different. Thus, under these experimental conditions, greatly enhanced superoxide dismutase activity accelerates H2O2 formation. The increased H2O2 probably accounts for the exaggerated sensitivity of high superoxide dismutase bacteria to oxidant-generating drugs. These results support the concept that the product of superoxide dismutase, H2O2, is at least as hazardous as the substrate, O2-. We conclude that effective organismal defense against reactive oxygen species may require balanced increments in antioxidant enzymes and cannot necessarily be improved by increases in the activity of single enzymes.     

Magnitude of Hyperoxic Stress and Degree of Lung Maturity Determine the Nature of Pulmonary Antioxidant Response in the Guinea Pig

The ability of the immature lung to induce antioxidant defences in response to hyperoxic stress was examined. Preterm guinea pigs (65 days gestation, term = 68 d) were exposed to either 21+ O2, 85+ O2 or 95+ O2 for 72 hours. Exposure to 85+ O2 increased lung catalase, glutathione peroxidase and manganese superoxide dismutase activities in comparison to air controls. Exposure to 95+ O2 resulted only in an increase in glutathione peroxidase activity. Bronchoalveolar lavage fluid GSH concentration was increased by a similar amount by both exposure regimes, while lung copper/zinc superoxide dismutase activity was unchanged by either treatment. Comparison of the antioxidant response of term and preterm animals exposed to 85+ O2 for 72 hours indicated a greater response in the lung of the preterm animals. Manganese superoxide dismutase activity was elevated in both term and preterm animals, while catalase and glutathione peroxidase activities were elevated only in preterm animals. The extent of microvascular permeability as indicated by bronchoalveolar lavage fluid protein concentration, was lower in preterm animals than in term animals. We conclude that the immature lung can respond to hyperoxic stress by antioxidant induction and that the nature of the response is dependent, in part, both on the severity of the stress and on the maturity of the lung.     

Magnitude of Hyperoxic Stress and Degree of Lung Maturity Determine the Nature of Pulmonary Antioxidant Response in the Guinea Pig

The ability of the immature lung to induce antioxidant defences in response to hyperoxic stress was examined. Preterm guinea pigs (65 days gestation, term = 68 d) were exposed to either 21+ O2, 85+ O2 or 95+ O2 for 72 hours. Exposure to 85+ O2 increased lung catalase, glutathione peroxidase and manganese superoxide dismutase activities in comparison to air controls. Exposure to 95+ O2 resulted only in an increase in glutathione peroxidase activity. Bronchoalveolar lavage fluid GSH concentration was increased by a similar amount by both exposure regimes, while lung copper/zinc superoxide dismutase activity was unchanged by either treatment. Comparison of the antioxidant response of term and preterm animals exposed to 85+ O2 for 72 hours indicated a greater response in the lung of the preterm animals. Manganese superoxide dismutase activity was elevated in both term and preterm animals, while catalase and glutathione peroxidase activities were elevated only in preterm animals. The extent of microvascular permeability as indicated by bronchoalveolar lavage fluid protein concentration, was lower in preterm animals than in term animals. We conclude that the immature lung can respond to hyperoxic stress by antioxidant induction and that the nature of the response is dependent, in part, both on the severity of the stress and on the maturity of the lung.     

Effects of culture conditions an hyperoxia on antioxidant enzymes in pig pulmonary artery and aortic endothelium

Because hyperoxia induces early injury to lung endothelial cells and since tolerance to hyperoxia is correlated with increased lung antioxidant enzyme activity, we measured superoxide dismutase, catalase and glutathione peroxidase in both fresh isolates and primary cultures of endothelial cells from pig pulmonary artery and aorta. Cultured endothelial cells were studied at confluency and up to 5 days thereafter under control or hyperoxic conditions. In both types of confluent cell, total and cyanide-insensitive superoxide dismutase increased when compared to fresh cells. The most conspicuous postconfluency change in both types of endothelial cell was a marked decrease in gluthathione peroxidase, which could be prevented by the addition of selenomethionine to culture media. A 5-day exposure to hyperoxia resulted in a 2-fold increase in cyanide-insensitive superoxide dismutase in both aortic and pulmonary artery endothelial cells. In view of a similar decrease in DNA in both types of cells despite some differences in enzyme levels, oxygen cytotoxicity could not be related to a particular antioxidant enzyme profile.     

Hyperoxia causes oxygen free radical-mediated membrane injury and alters myocardial function and hemodynamics in the newborn

Newborn children can be exposed to high oxygen levels (hyperoxia) for hours to days during their medical and/or surgical management, and they also can have poor myocardial function and hemodynamics. Whether hyperoxia alone can compromise myocardial function and hemodynamics in the newborn and whether this is associated with oxygen free radical release that overwhelms naturally occurring antioxidant enzymes leading to myocardial membrane injury was the focus of this study. Yorkshire piglets were anesthetized with pentobarbital sodium (65 mg/kg), intubated, and ventilated to normoxia. Once normal blood gases were confirmed, animals were randomly allocated to either 5 h of normoxia [arterial Po(2) (Pa(O(2))) = 83 +/- 5 mmHg, n = 4] or hyperoxia (Pa(O(2)) = 422 +/- 33 mmHg, n = 6), and myocardial functional and hemodynamic assessments were made hourly. Left ventricular (LV) biopsies were taken for measurements of antioxidant enzyme activities [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)] and malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) as an indicator of oxygen free radical-mediated membrane injury. Hyperoxic piglets suffered significant reductions in contractility (P < 0.05), systolic blood pressure (P < 0.03), and mean arterial blood pressure (P < 0.05). Significant increases were seen in heart rate (P < 0.05), whereas a significant 11% (P < 0.05) and 61% (P < 0.001) reduction was seen in LV SOD and GPx activities, respectively, after 5 h of hyperoxia. Finally, MDA and 4-HNE levels were significantly elevated by 45% and 38% (P < 0.001 and P = 0.02), respectively, in piglets exposed to hyperoxia. Thus, in the newborn, hyperoxia triggers oxygen free radical-mediated membrane injury together with an inability of the newborn heart to upregulate its antioxidant enzyme defenses while impairing myocardial function and hemodynamics.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

Effects of experimentally altered glutathione levels on life span, metabolic rate, superoxide dismutase, catalase and inorganic peroxides in the adult housefly, Musca domestica

Effects of varied levels of glutathione, an intracellular redox buffer, were examined in the adult male housefly in order to study the inter-relationship between enzymic and non-enzymic antioxidant defenses. An increase of over 100% in the concentrations of glutathione was induced by the administration of 3 mM L-2-oxothiazolidine-4-carboxylate (LOC), which increases the intracellular level of cysteine. A decrease in glutathione concentration of up to 85% was achieved by the administration of L-buthionine-SR-sulfoximine (BUS), which irreversibly inhibits glutamylcysteine synthetase. Life spans of houseflies were shortened by a decrease in the glutathione concentration, but were not prolonged by augmentation of glutathione. Metabolic rate and superoxide dismutase activity were independent of glutathione concentration. H2O2 was increased by both experimental regimes, whereas catalase activity was decreased by BUS. Results suggest that catalase activity is influenced by glutathione concentration.     

Characteristics of mutagenesis by glyoxal in Salmonella typhimurium: contribution of singlet oxygen

The characteristics of mutagenesis by glyoxal in Salmonella tester strains TA100 and TA104, and particularly a possible role of active oxygen species, were investigated. Glyoxal was converted into a non-mutagenic chemical with glutathione (GSH) by glyoxalase I, and the mutagenic activity was enhanced by the depletion of intracellular GSH. Glyoxal caused the reduction of nitro blue tetrazolium, which was suppressed by the addition of 2,5-diphenylfuran, superoxide dismutase (SOD) and catalase (CAT), scavengers of singlet oxygen (1O2), superoxide radical (O2-) and hydrogen peroxide (H2O2), respectively. However, only the 1O2 scavenger almost completely suppressed the mutagenic activity of glyoxal. Mutagenicity assays using strains pretreated with N,N-diethyldithiocarbamate of a SOD inhibitor and strains with low levels of SOD and CAT indicated that the mutagenesis by glyoxal was independent of intracellular levels of SOD and CAT, though glyoxal itself repressed them. Therefore, all the results suggest that 1O2 formed from glyoxal is related to its mutagenesis, but that neither O2- nor H2O2 is intracellularly predominantly related to it. The action of glyoxal against SOD and CAT, and the formation of glyoxal adducts with amino acids as their components are also discussed.     

Effect of free-radical scavengers on enumeration of thermally stressed cells of Staphylococcus aureus MF-31.

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

Oxidative stress decreases antioxidant enzyme activities in reaggregation cultures of rat brain cells

The brain has been suggested to be especially sensitive to damage by reactive oxygen species. In this study, we examined the effects of hyperoxic conditions on the activities and mRNA levels of antioxidant enzymes in reaggregation cultures of rat forebrain cells. Cultures were exposed to 80% oxygen for 12–60 h starting on Days 17 and 33 in culture. Superoxide dismutase activities and mRNA levels were not affected by hyperoxia, whereas catalase activity was slightly decreased after 24 h in 80% oxygen at Day 17. Glutathione peroxidase activity was markedly decreased already after 12 h of hyperoxia, and decreased activities of glutathione reductase and glucose-6-phosphate dehydrogenase were also noted. The glutathione peroxidase mRNA levels were increased in hyperoxic cultures at Day 17 but not at Day 33. These results suggest that the enzymatic defense mechanisms against reactive oxygen species in the brain are rather weak and deteriorate during oxidative stress but that a potential for compensatory upregulation exists at least during the first postnatal weeks.     

Sustained Photoproduction of Ammonia from Nitrate by Anacystis nidulans

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells

Rat heart ornithine decarboxylate activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase has a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O2- on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect upon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of Km and a reduction of Vmax with respect to control activity. The exposure of rats to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.