Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.     

Superoxide dismutase 1 protects retinal cells from oxidative damage

Bolstering the endogenous oxidative damage defense system is a good strategy for development of treatments to combat neurodegenerative diseases in which oxidative damage plays a role. A first step in such treatment development is to determine the role of various components of the defense system in cells that degenerate. In this study, we sought to determine the role of superoxide dismutase 1 (SOD1) in two models of oxidative damage-induced retinal degeneration. In one model, paraquat is injected into the vitreous cavity and then enters retinal cells and generates reactive oxygen species (ROS) that cause progressive retinal damage. Assessment of retinal function with serial electroretinograms (ERGs) showed that sod1 -/- mice were much more sensitive than sod1 +/+ mice to the damaging effects of paraquat, while sod1 +/- mice showed intermediate sensitivity. Compared to sod1 +/+ mice, sod1 -/- mice showed greater paraquat-induced oxidative damage and apoptosis. In the second model, mice were exposed to hyperoxia for several weeks, and sod1 -/- mice showed significantly greater reductions in ERG amplitudes than sod1 +/+ mice. In both of these models, transgenic mice carrying a sod1 transgene driven by a beta-actin promoter showed less oxidative stress-induced reduction in ERG amplitudes. These data demonstrate that SOD1 protects retinal cells against paraquat- and hyperoxia-induced oxidative damage and suggest that overexpression of SOD1 should be considered as one component of ocular gene therapy to prevent oxidative damage-induced retinal degeneration.     

Superoxide anion production and superoxide dismutase and catalase activities in Coxiella burnetii.

Coxiella burnetii was examined for superoxide anion (O2-) production and superoxide dismutase and catalase activities. The organism generated O2- at pH 4.5 but not at pH 7.4. The rickettsia displayed superoxide dismutase activity distinguishable from that of the host cell (L-929 mouse fibroblast). Catalase activity was maximal at pH 7.0 and diminished at pH 4.5. These enzymes may account, in part, for the ability of this obligate intracellular parasite to survive within phagocytes.     

Superoxide dismutase: the balance between prevention and induction of oxidative damage

Cu,Zn-superoxide dismutase (SOD1) has been shown to be effective in several free radical mediated diseases, although some studies have pointed toward SOD1 toxicity at a high concentrations. In the present study, the balance between prevention and induction of damage by SOD1 has been investigated both in vitro and in vivo. In vitro superoxide was generated using xanthine/xanthine oxidase. In vivo superoxide was generated using the redox cycling compound doxorubicin. Furthermore, we determined the pharmacokinetics of lecithinized SOD1 (PC-SOD) in order to compare the results obtained in vivo with those obtained in vitro. It was found that in vitro high concentrations of SOD1 induce hydroxylation of coumarin 3-carboxylic acid (3-CCA). This could be caused by a peroxidative action of SOD1 or formation of the highly reactive hydroxyl radicals. Any signs of toxicity are absent in vivo because these concentrations are not reached. It can be concluded that SOD1 possesses a large therapeutic window and application of SOD1 or its derivatives for strengthening the body's defenses against oxidative stress in a variety of pathologies seems safe.     

Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress

Bifidobacteria are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). To improve oxidative-stress tolerance, the superoxide dismutase (SOD) gene from Streptococcus thermophilus (StSodA) and the heme-dependent catalase (KAT) gene from Lactobacillus plantarum (LpKatL) were heterologously expressed in Bifidobacterium longum strain NCC2705. Three types of strain NCC2705 transformants were obtained: with transgenic SOD expression, with transgenic KAT expression, and with coexpression of the two genes. Intracellular expression of the genes and their functional role in oxidative-stress resistance were evaluated. In response to oxidative stress, B. longum NCC2705/pDP401-LpKatL (expressing LpKatL) and NCC2705/pDP-Kat-Sod (coexpressing LpKatL and StSodA) rapidly degraded exogenous H₂O₂ and the peroxides generated as a byproduct of aerobic cultivation, preventing oxidative damage to DNA and RNA. Individual expression of StSodA or LpKatL both improved B. longum NCC2705 cell viability. Survival rate of strain NCC2705 was further improved by combining SOD and KAT expression. The two enzymes played complementary roles in ROS-scavenging pathways, and coexpression led to a synergistic beneficial effect under conditions of intensified oxidative stress. Our results illustrate that heterogeneous expression of heme-dependent KAT and Mn²⁺-dependent SOD is functional in the B. longum oxidative-stress response, and synergistic protection is achieved when their expressions are combined.     

Superoxide dismutase and catalase protect cultured hepatocytes from the cytotoxicity of acetaminophen

Superoxide dismutase, catalase and mannitol prevent the killing of cultured hepatocytes by acetaminophen in the presence of an inhibitor of glutathione reductase, BCNU. Under these conditions, the cytotoxicity of acetaminophen depends upon its metabolism, since beta-naphthoflavone, an inhibitor of mixed function oxidation, prevents the cell killing. In hepatocytes made resistant to acetaminophen by pretreatment with the ferric iron chelator, deferoxamine, addition of ferric or ferrous iron restores the sensitivity to acetaminophen. In such a situation, both superoxide dismutase and catalase prevent the killing by acetaminophen in the presence of ferric iron. By contrast, catalase, but not superoxide dismutase, prevents the cell killing dependent upon addition of ferrous iron. These results document the participation of both superoxide anion and hydrogen peroxide in the killing of cultured hepatocytes by acetaminophen and suggest that hydroxyl radicals generated by an iron catalyzed Haber-Weiss reaction mediate the cell injury.     

Superoxide dismutase and catalase activities in purified Frankia vesicles

Superoxide dismutase (EC 1.15.1.1.) and catalase (EC 1.11.1.6.) activities of Frankia cells grown in the presence of ammonium were very high in comparison with those of other prokaryaotes and particularly Rhizobium . Furthermore, these activities were significantly enhanced under nitrogen-fixing conditions where vesicles were produced. By using a single-step sucrose gradient, Frankia vesicles were isolated and appeared intact and free of hyphal contamination. The contents of superoxide dismutase and catalase in the purified vesicles were similar to those in preparations containing both vesicles and hyphae. These results suggest an important role of superoxide dismutase and catalase in the protection of the overall nitrogen-fixation process against O₂ in Frankia vesicles. Beside the protective role played by the thick walls of the vesicles, the presence of specialized enzymes is emphasized.     

Superoxide dismutase and catalase levels in halophilic vibrios.

Superoxide dismutase (SOD) and catalase (CAT) levels were determined for several aerobically grown halophilic vibrios and compared with those found in aerobically grown Escherichia coli K-12. The SOD levels ranged from 25 to 103.6 U/mg of protein for the vibrios compared with 44.6 U/mg of protein for E. coli. The CAT levels ranged from 2.1 to 32.1 U/mg of protein. Electrophoretic analysis of cell extracts revealed that the halophilic vibrios tested possessed only one detectable SOD enzyme, except one strain which possessed two distinct enzymes, as compared with the three SOD enzymes in aerobically grown E. coli K-12. A comparison of anaerobically and aerobically grown vibrios revealed a three- to fourfold increase in SOD activity in the aerobic cells, suggesting that oxygen acts as an inducer for SOD in the vibrios as has been reported for E. coli. In one strain, Vibrio parahaemolyticus 27519, both SOD enzymes were observed in low levels in anaerobic and at higher levels in aerobically grown cells as compared with only one SOD enzyme in anaerobically grown E. coli. This suggests that differences in SOD regulation occur between the two genera. Our results indicate that halophilic vibrios possess SOD, which could enhance viruulence by allowing the organisms to survive in oxygenated environments.     

Superoxide dismutase and catalase activities in carotenoid-synthesizing fungi Blakeslea trispora and Neurospora crassa under the oxidative stress

The addition of menadione into the medium during cultivation of Neurospora crassa in the dark activated its constitutive superoxide dismutase. Exposure to light not only activated superoxide dismutase and catalase, but also increased the content of neurosporaxanthin. Superoxide dismutase activity in the mixed (+/-) mycelium of Blakeslea trispora synthesizing beta-carotene in the dark was much lower than that in Neurospora crassa. The superoxide dismutase activity further decreased in oxidative stress. The catalase activity decreased with an increase in the content of beta-carotene. Our results indicate that neurosporaxanthin possesses photoprotective properties in Neurospora crassa. In Blakeslea trispora (+/-) fungi, this compound acts as a major antioxidant during inactivation of enzymes that detoxify reactive oxygen species.     

Coimobilization of superoxide dismutase, catalase and peroxidase

For preparationing the polyenzyme antioxidant complex, containing superoxide dismutase (SOD), catalase and horseradish peroxidase (HRP), the different successivities of those enzymes co-immobilization were compared. The optimum successivity is provided by simultaneous co-immobilization of covalently bound HRP with the SOD and catalase. The catalytic enzyme activity and the catalase operational stability was kinetically characterized in various samples. For one sample, the influence of ascorbate, glutathione and ethanol on the catalase kinetic parameters was studied. A possible scheme of different processes at the H2O2 decomposition in the presence of co-immobilized SOD, catalase, HRP and the substrates-reductans was discussed.     

Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis: role of mitochondrial superoxide

The purpose of this study was to test the hypothesis whether Mito-carboxy proxyl (Mito-CP), a mitochondria-targeted nitroxide, inhibits peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC). Glucose/glucose oxidase (Glu/GO)-induced oxidative stress was monitored by dichlorodihydrofluorescein oxidation catalyzed by intracellular H(2)O(2) and transferrin receptor-mediated iron transported into cells. Pretreatment of BAECs with Mito-CP significantly diminished H(2)O(2)- and lipid peroxide-induced intracellular formation of dichlorofluorescene and protein oxidation. Electron paramagnetic resonance (EPR) studies confirmed the selective accumulation of Mito-CP into the mitochondria. Mito-CP inhibited the cytochrome c release and caspase-3 activation in cells treated with peroxides. Mito-CP inhibited both H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, overexpression of transferrin receptor (TfR), and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. In contrast, the "untargeted" carboxy proxyl (CP) nitroxide probe did not protect the cells from peroxide-induced oxidative stress and apoptosis. However, both CP and Mito-CP inhibited superoxide-induced cytochrome c reduction to the same extent in a xanthine/xanthine oxidase system. We conclude that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function.     

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.     

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.     

Stabilization of L-ascorbic acid by superoxide dismutase and catalase

The effects of superoxide dismutase (SOD) and catalase on the autoxidation rate of L-ascorbic acid (ASA) in the absence of metal ion catalysts were examined. The stabilization of ASA by SOD was confirmed, and the enzyme activity of SOD, which scavenges the superoxide anion formed during the autoxidation of ASA, contributed strongly to this stabilization. The stabilization of ASA by catalase was observed for the first time; however, the specific enzyme ability of catalase would not have been involved in the stabilization of ASA. Such proteins as bovine serum albumin (BSA) and ovalbumin also inhibited the autoxidation of ASA, therefore it seems that non-specific interaction between ASA and such proteins as catalase and BSA might stabilize ASA and that the non-enzymatic superoxide anion scavenging ability of proteins might be involved.     

Inactivation of catalase and superoxide dismutase by singlet oxygen derived from photoactivated dye

Both superoxide dismutase (SOD) and catalase are key enzymes in the antioxidant system of the cells that work to maintain low steady-state concentrations of the reactive oxygen species. When exposed to a singlet oxygen-producing system composed of dye, such as methylene blue or rose bengal, and visible light both SOD and catalase were susceptible to oxidative modification and damage as indicated by the loss of activity, fragmentation and aggregation of peptide as well as by the formation of carbonyl groups. Histidine, a powerful quenching agent for singlet oxygen, and the polyamines, such as spermine and spermidine, were effective at protecting the activity loss mediated by illuminated dye, whereas spin traps were only mildly effective. The structural alterations of modified enzymes were indicated by the increase in susceptibility to proteases, the change in absorption spectra and in fluorescence spectra. The singlet oxygen-mediated damage to SOD and catalase may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.