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.     

The interrelationship of superoxide dismutase and peroxidatic enzymes in the red cell.

Activities of superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) and catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) were determined during the course of incubation of red cell suspensions with 1,4-naphthoquinone-2-sulfonic acid. In the absence of glucose, incubation with napthoquinone sulfonate resulted in an inhibition of catalase and superoxide dismutase. The catalase inhibitor, 3-amino-1,2,4-triazole enhanced inactivation of catalase in the presence of naphthoquinone sulfonate and this in turn led to augmented inhibition of superoxide dismutase. The presence of glucose in the incubation medium prevented napthoquinone sulfonate-induced enzyme inhibition in the absence of aminotriazole, but had little effect in the presence of aminotriazole. The relevance of these findings to the cellular interrelationship of peroxidatic enzymes and superoxide dismutase is discussed.     

Up-regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild salt-tolerant tomato species Lycopersicon pennellii

The response of the antioxidative systems of leaf cell mitochondria and peroxisomes of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species Lycopersicon pennellii (Lpa) to NaCl 100 mM stress was investigated. Salt-dependent oxidative stress was evident in Lem mitochondria as indicated by their raised levels of lipid peroxidation and H2O2 content whereas their reduced ascorbate and reduced glutathione contents decreased. Concomitantly, SOD activity decreased whereas APX and GPX activities remained at control level. In contrast, the mitochondria of salt-treated Lpa did not exhibit salt-induced oxidative stress. In their case salinity induced an increase in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione-dependent peroxidase (GPX). Lpa peroxisomes exhibited increased SOD, APX, MDHAR and catalase activity and their lipid peroxidation and H2O2 levels were not affected by the salt treatment. The activities of all these enzymes remained at control level in peroxisomes of salt-treated Lem plants. The salt-induced increase in the antioxidant enzyme activities in the Lpa plants conferred cross-tolerance towards enhanced mitochondrial and peroxisomal reactive oxygen species production imposed by salicylhydroxamic acid (SHAM) and 3-amino-1,2,4-triazole (3-AT), respectively.     

The isolation and partial characterization of catalase and a peroxidase active fraction from human white adipose tissue.

1. A procedure is described for the purification of catalase and a peroxidase active fraction from human white adipose tissue. 2. Gel electrophoresis on SDS-PAGE revealed relative molecular masses of 202,900 and 208,600 for the active catalase and peroxidase molecules respectively (nonreducing conditions), as compared to 56,800 and 49,800 for the monomers under reducing conditions, thus indicating the likelihood of tetramers in the intact state. 3. The two purified enzymes differ with regard to pH optima (5-9 for catalase and 3 for peroxidase), temperature stability (up to 50 degrees C for catalase and 70 degrees C for peroxidase) and Km values towards H2O2 (38.9 mM for catalase and 7.69 mM for peroxidase, which was also active in oxidizing a number of o-dihydricphenols as second substrates). 4. The catalase enzyme showed uncompetitive inhibition by the irreversible inhibitor 3-amino-1,2,4-triazole (AT), Ki = 5.4 mM.     

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.     

Use of the inhibition of enzymatic antioxidant systems in order to evaluate their physiological importance

Chemical inhibitors of the different antioxidant enzymes were systematically testet either on purified enzymes of after incubation with human fibroblasts in culture. Inhibition values were obtained for catalase with aminotriazole, for superoxide dismutase with diethyldithiocarbamate, for glutathione peroxidase with mercaptosuccinate, for glutathione reductase with bischloroethylnitrosourea and for glutathione synthesis with buthionine sulfoximine. Viability of cells incubated with these inhibitors was then tested under normal conditions and under high oxygen pressure; the data were correlated with the above-mentioned inhibitory values. Cell viability was particularly affected when the glutathione-related enzymes, especially glutathione peroxidase, were inhibited.     

Leishmanial superoxide dismutase: A possible target for chemotherapy

Leishmania tropica promastigotes stimulate macrophages to produce activated oxygen as measured by luminol-enhanced chemiluminescence. Exogenous superoxide dismutase and catalase inhibit this by 95%, implying that both superoxide and hydrogen peroxide are generated. Whereas leishmania have undetectable levels of catalase, and very little glutathione peroxidase, they have relatively high amcunts of superoxide dismutase (23 units/mg protein). The leishmanial superoxide dismutase is cyanide-insensitive but azide- and peroxide-sensitive, suggesting that the enzyme may be iron-containing. Furthermore, the leishmanial superoxide dismutase is insensitive to diethyldithiocarbamate, which inhibits vertebrate enzymes. Thus, leishmania may contain a superoxide dismutase which is different from its host's enzyme. A specific inhibitor of this enzyme might serve as an antileishmanial agent.     

Antioxidant responses of the Mediterranean mussel, Mytilus galloprovincialis, to environmental variability of dissolved oxygen

Physiological responses of Mytilus galloprovincialis against environmental dissolved oxygen partial pressure (pO(2)) variation were studied in terms of the modulated induction of the main antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT) and selenium-dependent glutathione peroxidase (GPX). Field in vivo studies were performed at two sites of the Lagoon of Venice, characterized by different aquatic environmental conditions implying different pO(2). SOD and GPX are more active in gills, and their complementary role is discussed. CAT is more active in the digestive gland, where the enzyme dismutates H(2)O(2) derived from divalent reduction of O(2) performed by various oxidases in peroxisomes. Antioxidant enzyme activities are correlated with water dissolved oxygen (DO), especially in the gills. This tissue, because of its anatomical localization and its physiological role, responds to DO variations modulating the induction of the antioxidant enzymes as a protection mechanism against potential toxicity due to increases in ROS formation.     

Effect of chronic ethanol treatment under partial catalase inhibition on the activity of enzymes related to peroxide metabolism in rat liver and heart

1. In order to test the hypothesis that the alcoholic cardiomyopathy under partial catalase inhibition is associated with the activation of lipid peroxidation in cardiomyocytes (Panchenko et al., Experientia 43, 580-581, 1987), the effects of ethanol and catalase inhibitor 3-amino-1,2,4-triazole (aminotriazole) on rat heart and liver content of reduced glutathione and on the activity of enzymes related to peroxide metabolism: catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase were investigated. 2. In accordance with the data obtained by Kino (J. molec, cell. Cardiol. 13, 5-12, 1981), when ethanol (36% of dietary calories) and aminotriazole were simultaneously administered an alcoholic cardiomyopathy developed while in the liver moderate fatty degeneration was revealed. 3. Chronic combined or separate administration of ethanol and aminotriazole was shown to increase glutathione concentration and glutathione-S-transferase activity in rat liver. In the groups of animals which received isocaloric carbohydrates in the diet instead of ethanol the liver glucose-6-phosphate dehydrogenase was increased. 4. Acute and chronic aminotriazole injections led to catalase inactivation and in the latter case also to inhibition of the liver superoxide dismutase and glutathione peroxidase activities. 5. Ethanol and aminotriazole treatment did not alter the glutathione level and the activity of all enzymes tested (except catalase) in rat myocardium.     

DNA breakage induced by 1,2,4-benzenetriol: relative contributions of oxygen-derived active species and transition metal ions

We report here the relative roles of metals and selected reactive oxygen species in DNA damage by the genotoxic benzene metabolite 1,2,4-benzenetriol, and the interactions of antioxidants in affording protection. 1,2,4-Benzenetriol induces scission in supercoiled phage DNA in neutral aqueous solution with an effective dose (ED(50)) of 6.7 microM for 50% cleavage of 2.05 microg/ml supercoiled PM2 DNA. In decreasing order of effectiveness: catalase (20 U/ml), formate (25 mM), superoxide dismutase (20 U/ml), and mannitol (50 mM) protected, from 85 to 28%. Evidently, H(2)O(2) is the dominant active species, with O(2)(*)(-) and *OH playing subordinate roles. Desferrioxamine or EDTA inhibited DNA breakage by 81-85%, despite accelerating 1,2,4-benzenetriol autoxidation. Consistent with this suggestion of a crucial role for metals, addition of cupric, cuprous, ferric, or ferrous ions enhanced DNA breakage, with copper being more active than iron. Combinations of scavengers protected more effectively than any single scavenger alone, with implications for antioxidants acting in concert in living cells. Synergistic combinations were superoxide dismutase with *OH scavengers, superoxide dismutase with desferrioxamine, and catalase with desferrioxamine. Antagonistic (preemptive) combinations were catalase with superoxide dismutase, desferrioxamine with *OH scavengers, and catalase with *OH scavengers. The most striking aspect of synergism was the extent to which metal chelation (desferrioxamine) acted synergistically with either catalase or superoxide dismutase to provide virtually complete protection. Concluding, 1,2,4-benzenetriol-induced DNA damage occurs mainly by site-specific, Fenton-type mechanisms, involving synergism between several reactive intermediates. Multiple antioxidant actions are needed for effective protection.     

Xanthine oxidase, superoxide dismutase, catalase and lipid peroxidation in Mastomys natalensis: effect of Dipetalonema viteae infection

Status of xanthine oxidase, superoxide dismutase, catalase and lipid peroxidation, the enzymes metabolizing reactive oxygen intermediates in liver, lungs and spleen of M. natalensis during D. viteae infection was investigated. Xanthine oxidase and lipid peroxidation exhibited stimulation, while superoxide dismutase and catalase showed depression in liver and spleen of the infected animals. The filarial infection therefore appears to create O2 toxicity in these tissues. Lungs, on the other hand was found safe as it possessed elevated xanthine oxidase, superoxide dismutase and catalase. Lipid peroxidation in lungs operated below the control level. The impact of these changes in the establishment and development of the infection has been discussed.     

Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans

A number of facultatively anaerobic members of the genus Bacillus were screened for their catalase, diaminobenzidine peroxidase, and superoxide dismutase activities. A strain of Bacillus coagulans (7050) lacking peroxidatic activity and containing single catalatic and superoxide dismutase activities was selected. Responses of the superoxide dismutase activity and catalase level to the partial pressure of oxygen, and Fe and Mn levels, as well as to aerobic and fermentative metabolism, were determined. There appeared to be a relationship between high endogenous catalase levels and the high H2O2 evolution and KCN insensitivity of B. coagulans respiration. Bacillus coagulans 7050 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and screened for the expression of oxygen intolerance. All of the 38 stable oxygen sensitive mutants obtained had very low or completely absent catalatic activity and catalase protein. No mutant lacked superoxide dismutase, although five showed significantly lowered levels of the enzyme. Exogenous bovine liver catalase restored aerotolerance and reduced cell pleomorphism in the mutants.     

Induction and inactivation of catalase and superoxide dismutase of Escherichia coli by ozone

Oxyradicals have been implicated in ozone (O3) toxicity and in other oxidant stress. In this study, we investigated the effects of O3 on the biosynthesis of the antioxidant enzymes catalase and superoxide dismutase in Escherichia coli to determine their role in the defense against ozone toxicity. Inhibition of growth and loss of viability were observed in cultures exposed to ozone. Results also showed an increase in the activities of catalase and superoxide dismutase in cultures exposed to ozone, which was shown to be due to true induction rather than activation of preexisting apoproteins. Cessation of O3 exposure resulted in 30 min of continual high rate of catalase biosynthesis followed by a gradual decrease in the level of the enzyme approaching that of control cultures. This decrease was attributed to a concomitant cessation of de novo enzyme synthesis and dilution of preexisting enzyme by cellular growth. Ozonation of cell-free extracts showed that superoxide dismutase and catalase are subject to oxidative inactivation by ozone. In vivo induction of these enzymes may represent an adaptive response evolved to protect cells against ozone toxicity.     

Induction of 6-thioguanine-resistant mutants by hyperoxia and gamma-irradiation: effect of compromising cellular antioxidant systems

Hyperoxia and gamma-irradiation were found to be mutagenic in a transformed Syrian hamster cell line in a dose-dependent manner. The frequency of resistance to 6-thioguanine increased from 10 per 10(6) survivors after 48 h of growth in 70% O2 to 32.6 (highly significant) after 75 h. Increasing the oxygen tension to 95% resulted in a significant mutagenic response in only 44 h. At equitoxic doses, gamma-irradiation was 4 times more mutagenic than 70% O2. After growth in hyperoxia, the cells showed an enhancement of catalase activity, glutathione peroxidase activity and glutathione levels but there was little effect on superoxide dismutase activity. Diethyldithiocarbamate (3 mM, 1.5 h) was mutagenic in normoxia and potentiated the mutagenic activity of both gamma-irradiation and hyperoxia. Cells thus treated showed an 855 reduction in superoxide dismutase activity. When diethyldithiocarbamate was used in conjunction with a direct-acting alkylating agent, the mutagenic response was only additive. Depletion of cellular glutathione with buthionine sulfoximine (0.2 mM) or inhibition of catalase activity with aminotriazole (100 mM) was also effective in potentiating the mutagenic response of gamma-irradiation and hyperoxia. The data demonstrates that endogenously produced activated oxygen species are mutagenic to hamster cells in culture and suggest that aerobic organisms are subject to an unavoidable background risk due to living in an oxygen atmosphere.     

Association of Antioxidant Systems in the Protection of Human Fibroblasts Against Oxygen Derived Free Radicals

The protection of human diploid fibroblasts against high oxygen tension was investigated using various combinations of the three major antioxidant enzymes: superoxide dismutase, catalase and gluthathione peroxidase. α-Tocopherol, a well-known hydrophobic antioxidant, was also tested in combination with the different enzymes. Microinjection of solutions containing different combinations of the three enzymes was compared with the injection of each single enzyme. We observed that the protections given by catalase or superoxide dismutase on the one hand, and by glutathione peroxidase on the other hand, were additive. Surprisingly, the combinations of catalase and superoxide dismutase were less effective than catalase alone and was even toxic at low SOD concentrations. Addition of α-tocopherol following the injection of any of the three enzymes was highly beneficial, but the strongest synergistic effect was obtained with glutathione peroxidase. These results stress the importance of membrane protection by α-tocopherol and indirectly by glutathione peroxidase. They also showed that any injection leading to the decrease in the O₂^?. or H₂ O ₂ concentration combined with one of these two protectors is very beneficial for the cells probably by decreasing the OH concentration. This is also proven by the very good protective effect obtained with desferrioxamine.     

Exogenous spermidine differentially alters activities of some scavenging system enzymes, H(2)O(2) and superoxide radical levels in water-stressed cucumber leaves

In order to examine whether polyamines (PAs) modify the functioning of the scavenging system and oxidative stress levels in water-stressed plants, cucumber (Cucumis sativus L.) seedlings were treated with spermidine (Spd) prior to dehydration, and stress-evoked changes in superoxide dismutase (SOD) (EC 1.15.1.1), catalase (EC 1.11.1.6), guaiacol peroxidase (EC 1.11.1.7) activities, H(2)O(2) and superoxide radical levels were determined. Free PA content during Spd treatment and during the stress period were also determined. Exogenous application of Spd differentially influenced enzymes of the antioxidative system under stress conditions; we observed an increase of guaiacol peroxidase activity, and, to a lesser degree, a reduction of SOD and catalase activities in Spd-treated plants in comparison to untreated stressed plants. Hydrogen peroxide and superoxide radical contents were also reduced in stressed plants after Spd pretreatment. These positive effects were observed in the case of 1mM Spd concentration. A higher concentration (3mM) influenced negative, more significant stress-induced changes, but a lower concentration (0.1mM) had a very limited effect. In summary, PAs are able to moderate the activities of scavenging system enzymes and to influence oxidative stress intensity.     

Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus

Here we investigated H2O2 production and detoxification in the hematophagous hemiptera, Rhodnius prolixus. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radical (O2-). This reaction produces hydrogen peroxide, which is scavenged by antioxidant enzymes such as catalase (CAT). SOD and CAT activities were found in all tissues studied, being highest in the midgut. CAT was dose-dependently inhibited in vivo by injections of 3-amino-1,2,4-triazole (AT). Insects treated with AT showed a twofold increase in H2O2 levels. Injection of DL-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, also resulted in a fourfold increase in H2O2, together with stimulation of CAT activity. Simultaneous administration of both AT and BSO had a synergistic effect on midgut H2O2 content. Taken all together, our results suggest that CAT and glutathione-dependent mechanisms cooperate to control H2O2 concentration in the midgut cell and prevent hydroxyl radical generation by Fenton reaction in this tissue.     

Significance of alterations in hepatic antioxidant enzymes. Primacy of glutathione peroxidase.

The relative contributions of catalase and the selenoenzyme glutathione peroxidase (GSH-Px) were elucidated in the rat liver by selectively modulating the activities of these enzymes using dietary selenium (Se) and the catalase inhibitor 3-amino-1,2,4-triazole (3-AT). Increased peroxidation occurred only in Se-deficient rats with markedly reduced cytosolic and mitochondrial GSH-Px activities. Although 3-AT treatment resulted in a 75% reduction of hepatic catalase activity and also a 20% reduction of both cytosolic and mitochondrial superoxide dismutase (SOD) activity, no incremental increase in peroxidation was observed over that associated with Se deficiency. In Se-deficient animals, treatment with 3-AT resulted in a doubling of cytosolic GSH-Px. This was associated with a 49% elevation in hepatic Se suggesting that increased Se may have contributed to the enhanced GSH-Px activity. These results suggest that GSH-Px plays the pivotal role in preventing hepatic peroxidation. Furthermore, the effects of 3-AT in vivo are not restricted to inhibition of catalase activity insofar as it also affects cytosolic GSH-Px activity and cytosolic and mitochondrial SOD activities.     

Mechanism of the antibiotic action pyocyanine.

Exposure of Escherichia coli growing in a rich medium to pyocyanine resulted in increased intracellular levels of superoxide dismutase and of catalase. When these adaptive enzyme syntheses were prevented by nutritional paucity, the toxic action of pyocyanine was augmented. The antibiotic action of pyocyanine was dependent upon oxygen and was diminished by superoxide dismutase and by catalase, added to the suspending medium. Pyocyanine slightly augmented the respiration of E. coli suspended in a rich medium, but greatly increased the cyanide-resistant respiration. Pyocyanine was able to cause the oxidation of reduced nicotinamide adenine dinucleotide, with O2- production, in the absence of enzymatic catalysis. It is concluded that pyocyanine diverts electron flow and thus increases the production of O2- and H2O2 and that the antibiotic action of this pigment is largely a reflection of the toxicity of these products of oxygen reduction.     

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.