Oxygen effects on maize leaf superoxide dismutase and glutathione reductase

Exposure of maize seedlings to an atmosphere containing 75% O₂ and 350 ppm CO₂ resulted in a two- to three-fold increase in glutathione reductase activity in leaf tissue within 48 hr after initiation of the O₂ treatment. This elevated level of glutathione reductase was still evident in plants maintained in the hyperopic environment for 7 days. Superoxide dismutase activity was not altered from its level in control tissue during the 7-day experimental period. These results suggest a key role for glutathione reductase in the protection of photosynthetic tissue against detrimental effects of intermediate reduction products of O₂.     

Temporal and regional changes of superoxide dismutase and glutathione peroxidase activities in rats exposed to focal cerebral ischemia

Reactive oxygen species are important cause of tissue injury during cerebral ischemia and reperfusion (I/R). Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are intracellular enzymes responsible for endogenous antioxidant defense of tissues affected by I/R. The aim of this study was to examine temporal and regional changes of SOD and GSH-Px activities in animals exposed to transient focal cerebral ischemia. Male Wistar Hannover rats were subjected to the right middle cerebral artery occlusion for 2?h. The animals were sacrificed immediately, 0·5, 1, 2, 3, 6, 24, 48, 72 or 168?h after ischemic procedure. SOD and GSH-Px activities were determined spectrophotometrically in the hippocampus and parietal cortex, both unilaterally and contralaterally to the occlusion. Sham-operated animals were used as the control group. Our results indicated that transient focal cerebral ischemia causes significant changes in SOD activities in the hippocampus and parietal cortex such as in GSH-Px activities in the parietal cortex, unilaterally and contralaterally to the lesion in rats during different reperfusion periods. Statistically significant activation of GSH-Px was registered neither in the right nor in the left hippocampus of ischemic animals. Copyright ? 2012 John Wiley & Sons, Ltd.     

Subcellular distribution and activities of superoxide dismutase,catalase, glutathione peroxidase,and glutathione reductase in the southern armyworm,Spodoptera eridania

In mid-fifth-instar larvae of the southern armyworm, Spodoptera eridania, the subcellular distribution of four antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR)—were examined. Two-thirds (4.26 units ·mg protein^?1) of the SOD activity was found in the cytosol, and one-thirds (2.13 units ·mg protein^?1) in the mitochondria. CAT activity was unusually high and not restricted to the microsomal fraction where peroxisomes are usually isolated. The activity was distributed as follows: cytosol (163 units) mitochondria (125 units) and microsomes (119 units). Similar to CAT, the subcellular compartmentalization of both GPOX and GR was unusual. No activity was detected in the cytosol, but in mitochondria and microsomes, GR levels were 5.49 and 3.09 units. Although GPOX activity exhibited 14–16-fold enrichment in mitochondria and microsomes, respectively, over the 850g crude homogenate, the level was negligible (mitochondria = 1.4 × 10^?3 units; microsomes = 1.6 × 10^?3 units), indicating that this enzyme is absent. The unusual distribution of CAT has apparently evolved as an evolutionary answer to the absence of GR from the cytosol, and the lack of GPOX activity.     

Overexpression of Mn-superoxide dismutase in maize leaves leads to increased monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities

The effect of increased Mn-superoxide dismutase (SOD) on antioxidant enzymes and metabolites was studied using transformed maize, TG1+ and TG2+. The progeny of the backcross of each of the primary transformants with the parental line generated two populations denoted M6884 and M6885. These were grown at optimal (25 degrees C) and sub-optimal (18, 14 and 10 degrees C) temperatures to assess the impact of elevated SOD activity on cold tolerance and the antioxidant defences in maize. The plants of the M6885 population had similar foliar SOD activities to the untransformed maize plants. Within the segregating M6884 population 50% of the plants had elevated SOD activity (up to four times the activity of the untransformed controls) and 50% of the plants contained the product of the transgene. In untransformed plants grown at 25 degrees C and 18 degrees C, SOD activity was not detectable in mesophyll extracts. Similarly, increased foliar SOD activity in the M6884 transformed maize did not lead to detectable mesophyll SOD activity. Increased foliar KCN-insensitive SOD activities were accompanied by enhancement of monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities; enzymes which are localized exclusively in the leaf mesophyll tissues. Increased foliar SOD activity had no effect on the hydrogen peroxide, glutathione or ascorbate contents of the leaves. This suggests that increased recycling of reduced ascorbate was required to compensate for enhanced hydrogen peroxide production in transformed plants.     

Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity

Automated assays for catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase are presented. The assay for catalase is based on the peroxidatic activity of the enzyme. The glutathione peroxidase and reductase assays measure the consumption of NADPH following the reduction of t-butyl hydroperoxide and oxidized glutathione, respectively. The assay for superoxide dismutase is based on the reduction of cytochrome c. All assays utilize the Cobas FARA clinical automated analyzer and provide considerable time savings over the manual assays.     

A supramolecular bifunctional artificial enzyme with superoxide dismutase and glutathione peroxidase activities

For constructing a bifunctional antioxidative enzyme with both superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, a supramolecular artificial enzyme was successfully constructed by the self-assembly of the Mn(III)meso-tetra[1-(1-adamantyl methyl ketone)-4-pyridyl] porphyrin (MnTPyP-M-Ad) and cyclodextrin-based telluronic acid (2-CD-TeO₃H) through host-guest interaction in aqueous solution. The self-assembly of the adamantyl moieties of Mn(III) porphyrin and the β-CD cavities of 2-CD-TeO₃H was demonstrated by the NMR spectra. In this supramolecular enzyme model, the Mn(III) porphyrin center acted as an efficient active site of SOD and tellurol moiety endowed GPx activity. The SOD-like activity (IC₅₀) of the new catalyst was found to be 0.116 μM and equals to 2.56% of the activity of the native SOD. Besides this, supramolecular enzyme model also showed a high GPx activity, and a remarkable rate enhancement of 27-fold compared to the well-known GPx mimic ebselen was observed. More importantly, the supramolecular artificial enzyme showed good thermal stability.     

Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves

The influence of varied Mg supply (10-1000 micromolar) and light intensity (100-580 microeinsteins per square meter per second) on the concentrations of ascorbate (AsA) and nonprotein SH-compounds and the activities of superoxide dismutase (SOD; EC 1.15.11) and the H₂O₂ scavenging enzymes, AsA peroxidase (EC 1.11.1.7), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were studied in bean (Phaseolus vulgaris L.) leaves over a 13-day period. The concentrations of AsA and SH-compounds and the activities of SOD and H₂O₂ scavenging enzymes increased with light intensity, in particular in Mg-deficient leaves. Over the 12-day period of growth for a given light intensity, the concentrations of AsA and SH-compounds and the activities of these enzymes remained more or less constant in Mg-sufficient leaves. In contrast, in Mg-deficient leaves, a progressive increase was recorded, particularly in concentrations of AsA and activities of AsA peroxidase and glutathione reductase, whereas the activities of guaiacol peroxidase and catalase were only slightly enhanced. Partial shading of Mg-deficient leaf blades for 4 days prevented chlorosis, and the activities of the O₂^.− and H₂O₂ scavenging enzymes remained at a low level. The results demonstrate the role of both light intensity and Mg nutritional status on the regulation of O₂^.− and H₂O₂ scavenging enzymes in chloroplasts.     

Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging

Sunflower ( Helianthus annuus L.) seeds progressively lost their ability to germinate at 25°C, the optimal temperature for germination, after accelerated aging was carried out at 45°C (a temperature too high to permit germination) in water or at 76 or 100% relative humidity (RH). The deleterious effects of the high-temperature treatment increased with increasing seed moisture content. Incubation of seeds at 45°C in water resulted in electrolyte leakage, which indicated a loss of membrane integrity. A relationship between leakage and loss of seed viability could not be assumed, since no increase in electrolyte efflux occurred after aging al 100% RH. Accelerated aging induced accumulation of malondialdehyde, suggesting that seed deterioration was associated with lipid peroxidation. However, there was no direct relationship between lipid peroxidation and deterioration in membrane integrity. Loss of seed viability was also associated with a decrease in superoxide dismutase, catalase and glutathione reductase activities. Finally, the results obtained suggest that sunflower seed deterioration during accelerated aging is closely related to a decrease in the activities of detoxifying enzymes and to lipid peroxidation.     

Carbaryl-induced effects on glutathione content, glutathione reductase and superoxide dismutase activity of the cyanobacterium Nostoc muscorum

The carbamate insecticide carbaryl, at concentrations of 10 mg/l and above, significantly stimulated glutathione reductase (GR) and superoxide dismutase (SOD) activity in the cyanobacterium Nostoc muscorum. A low content of total glutathione (GSH + GSSG), decreased photosynthetic activity, and an increased level of H₂O₂ was observed in pesticide treated cyanobacteria. As no glutathione peroxidase was observed in this species, stimulation of GR and SOD activity, higher production of H₂O₂, and low glutathione level was attributed to the utilization of GSH to remove H₂O₂ spontaneously and nonenzymatically under conditions of pesticide toxicity.     

Ferric reductase, superoxide dismutase and alkaline phosphatase activities in siderophore producing fungi

Enzymes associated with release of iron from internalized ferrated siderophore (ferrisiderophore reductase), with damage to the cell at high iron concentration (superoxide dismutase) and siderophore synthesis (alkaline phosphatase), were examined in 3 test fungi viz., Aspergillus sp. ABp4, Aureobasidium pullulans and Rhizopus sp. Extracellular ferrisiderophore reductase activity was present in all the three fungi, but Aureobasidium pullulans, that showed the highest activity (84.3 microM min(-1)), was the only one to produce intra-cellular ferric reductase (147.9 microM min(-1)). Superoxide dismutase was produced by Aureobasidium pullulans and Rhizopus sp., but not by Aspergillus sp. ABp4, that showed intra-cellular enzyme activity in case of ferric reductase and alkaline phosphatase. Maximum SOD activity was seen in Aureobasidium pullulans both extra-cellularly (93.83 ng ml(-1)) and intra-cellularly (57.14 ng ml(-1)). All the test fungi examined, produced intra-cellular alkaline phosphatase. There was no extracellular alkaline phosphatase. Among the three fungi, Aureobasidium pullulans showed highest alkaline phosphatase activity (129.9 microM min(-1)) and Aspergillus sp. ABp4 the least (76.4 microM min(-1)).     

Photoinhibition of photosystem II in tobacco plants overexpressing glutathione reductase and poplars overexpressing superoxide dismutase

We studied photoinhibition in two cultivars of tobacco ( Nicotiana tabacum L.) expressing the bacterial gor gene in the cytosol and in four lines of poplar ( Populus tremula × P. alba ) expressing the FeSOD gene of Arabidopsis thaliana in the chloroplast. The respective total activities of glutathione reductase (EC 1.6.4.2) in leaves of gor tobaccos and superoxide dismutase (EC 1.15.1.1) in the FeSOD poplars were 5–8 times higher than in the respective untransformed control plants. Leaves of control and transformed plants were subjected to high-light stress at 20°C, and photoinhibition of photosystem II (PSII) was measured by oxygen evolution and chlorophyll fluorescence. The leaves were illuminated both in the presence and absence of lincomycin, which inhibits chloroplast protein synthesis. In both cases, the time course of loss of PSII activity was identical in plants overproducing superoxide dismutase (SOD) and in the untransformed controls, suggesting that the ability to convert superoxide to hydrogen peroxide is not a limiting factor in protection against photoinhibition, or in the repair of photoinhibitory damage or that the site of O₂⁻ production is not accessible to the transgene product. The rate constant of photoinhibition, measured in lincomycin-treated leaves, was smaller in glutathione reductase (GR) overproducing tobacco cv. Samsun than in the respective wild-type, but this difference was not seen in cv. Bel W3. The steady-state level of PSII activity measured when the PSII repair cycle was allowed to equilibrate with photoinhibitory damage under high light was not higher in the GR overproducing cv. Samsun, suggesting that the repair of photoinhibitory damage was not enhanced in plants overproducing GR in the cytosol.     

Correlative links between superoxide dismutase, catalase and glutathione peroxidase activities in mouse liver

Qualitative and quantitative differences in correlative and regressive links between superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase were assessed in the mice liver by two- and three-dimensional statistical methods. Paired linear correlation analysis indicated SOD-CAT tandem as the correlatively acting enzymatic pair. Three-dimensional analysis revealed uniform response surfaces which exhibited higher activities at disproportional values of the other two and lower activities at proportional activities of the other two enzymes. The direct effect of the enzymes on each other was positive [table: see text] while the effect of their product was always negative.     

Radiation resistance and the CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase activities of seven human cell lines

CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase form the primary enzymic defense against toxic oxygen reduction metabolites in cells. To test the importance of these protective enzymes in the cellular radiation response, the enzymic activities of seven different human cell lines were determined in parallel with their clonogenic survival characteristics. A positive correlation between the content of glutathione peroxidase in cell lines and their extrapolation numbers (n) and quasithreshold doses (Dq) was detected. Between the cellular contents of the other enzymes and D0, n, and Dq no positive correlations could be established. An interesting finding was a very high Mn superoxide dismutase content in a malignant mesothelioma cell line P7, which had an extremely high D0, 5.0 Gy.     

Effects of dietary selenium and tocopherol on glutathione peroxidase and superoxide dismutase activities in rat phagocytes

Glutathione peroxidase activities (GSH-Px) of peritoneal exudate polymorphonuclear neutrophils, pulmonary alveolar macrophages, and peritoneal exudate macrophages of rats depleted of dietary selenium for four to six weeks were markedly lower than the corresponding activities in rats fed the same diet supplemented with 0.5 ppm selenium as sodium selenite. GSH-Px in phagocytes from selenium-supplemented rats adequate or deficient in tocopherol status did not differ significantly. In selenium deficient animals, the residual GSH-Px of polymorphonuclear neutrophils and peritoneal macrophages, but not of alveolar macrophages were slightly higher in tocopherol-deficient rats than in tocopherol-supplemented animals. Superoxide dismutase activities of each cell type were comparable and were not significantly affected by dietary selenium or tocopherol.     

CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in glutathione-deficient human fibroblasts

The effect of genetically determined glutathione deficiency on the fibroblast content of CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase was investigated. No significant differences between glutathione-deficient and -proficient human fibroblasts were revealed. There was a large variation in the content of the investigated enzymes in fibroblasts grown and analysed on different occasions. Whereas the contents of CuZn superoxide dismutase, catalase and glutathione peroxidase did not deviate much from what has been found in other human cell-lines and tissues, the fibroblasts were found to contain exceptional amounts of Mn superoxide dismutase.     

A spectrophotometric assay for superoxide dismutase activities in crude tissue fractions.

A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.     

Biological variability of superoxide dismutase and glutathione peroxidase in blood

The enzymatic antioxidant defences of mammalian cells include copper-zinc superoxide dismutase (SOD)(Cu Zn-SOD; EC 1.15.1.1) which catalyses the dismutation of superoxide anions (O₂^.-) to hydrogen peroxide(H², O²)and a seleno-dependent glutathione peroxidase (GSH-px) (GSH-px; EC 1.11.1.9) which catalyses the degradation of H²O² to H²O and O². The measurement of these enzyme activities is often used as a possible biological index of oxidative stress in various clinical conditions. Complete understanding of such information requires knowledge of the random biological fluctuation of the enzyme activity which occurs in each individual. In the present investigation we examined this normal variability in 12 healthy volunteers (four women and eight men) aged 23–45 years, over 6 months. The intra-individual coefficients of variation (estimated using analysis of variance techniques) were 15% (SOD) and 13% (GSH-px). The analytical goal for imprecision was achieved for both enzymes, i.e. it was less than one half of the measured intra-individual variation. Both enzymes showed marked individuality, indicating that an individual's reference values are more useful than population-based data. The critical difference required for significant changes in serial results is 45% for SOD and 40% for GSH-px.     

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.     

Microinjection of antibodies against superoxide dismutase and glutathione peroxidase

Antibodies were prepared against glutathione peroxidase, superoxide dismutase, and catalase. Inhibition of the enzyme activity was obtained with anti-Gpx and anti-SOD antibodies but not with anti-CAT antibodies. The antibodies were then injected into human fibroblasts and bovine chondrocytes in culture either under normal conditions or under 1 atm of oxygen. The injected anti-Gpx and anti-SOD antibodies increased the mortality rate of the fibroblasts incubated under 1 atm of oxygen. However, when cells were incubated under normal atmosphere, anti-Gpx antibodies inhibited the division while anti-SOD antibodies increased this capacity. Anti-Gpx antibodies injected into chondrocytes decreased their viability. Injection of control antiserum had no effect. These data stress the primary importance of Gpx as antioxidant under all conditions and the relative efficiency of SOD according to the balance between the radical production and the activity of the other antioxidant systems.     

Effects of paraquat administration on longevity, oxygen consumption, lipid peroxidation, superoxide dismutase, catalase, glutathione reductase, inorganic peroxides and glutathione in the adult housefly

The effects of oxidative stress in the adult male housefly were examined by the administration of 1 mM paraquat. Houseflies exhibit NADH and NADPH-diaphorase activity. Paraquat caused a significant decrease in life span, metabolic rate and the concentration of thiobarbituric acid-reactants. Concentrations of reduced glutathione and inorganic peroxides were increased by paraquat. Paraquat stimulated the activity of catalase but did not affect activities of superoxide dismutase and glutathione reductase. The levels of oxidized glutathione and the rate of fluorescent age pigment accumulation were unaffected by paraquat. Results indicate that paraquat toxicity does not result from lipid peroxidation.