Effect of ethanol on superoxide dismutase activity in cultured neural cells

Superoxide dismutase (EC 1.15.1.1) activity was investigated in several types of neural cells cultivated in the presence of 100 mM ethanol. Superoxide dismutase was inhibited by acute treatment with ethanol. Chronic treatment with ethanol specifically inhibited superoxide dismutase in glial cells. In all instances withdrawal of ethanol produced a quick return to control values. Inhibition of superoxide dismutase by ethanol may increase toxic oxygen radicals in nervous tissue.     

Electron spin resonance detection of oxygen-centred radicals in murine macrophages stimulated with bacterial endotoxin

The production of oxygen radicals by Bacille-Calmette-Guerin primed mouse macrophages stimulated with bacterial endotoxin has been investigated. Superoxide radicals were spin-trapped in this system with dimethylpyrroline-N-oxide after a lag period of 20-40 minutes. The electron spin resonance signals due to the superoxide radical adduct could be inhibited by superoxide dismutase but not by catalase.     

Superoxide-dependent oxidation of melatonin by myeloperoxidase

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.     

Analysis of extracellular superoxide dismutase in fibroblasts from patients with systemic sclerosis

Systemic sclerosis (SSc) is a chronic disease of connective tissue characterized by vascular damage, autoantibody production and extensive fibrosis of skin, skeletal muscles, vessels and visceral organs. Fibrosis is a biological process involving inflammatory response and reactive oxygen species (ROS) accumulation leading to fibroblast activation. Extracellular superoxide dismutase (SOD3), a copper and zinc superoxide dismutase, which is expressed in selected tissues, is secreted into the extracellular space and catalyzes the dismutation of superoxide radical to hydrogen peroxide and molecular oxygen. Moreover, SOD3 is associated to inflammatory responses in some experimental models. In this paper we analysed, by RT-PCR and immunofluorescence, SOD3 expression and intracellular localization in dermal fibroblasts from both healthy donors and patients affected by diffuse form of SSc. Moreover, we determined SOD3 enzymatic activity in fibroblast culture medium with the xanthine/xanthine oxidase method. Increased expression of SOD3 mRNA was detected in systemic sclerosis fibroblasts (SScF), as compared to control healthy fibroblasts (HF), and SOD3 immunofluorescence staining displayed a characteristic pattern of secretory proteins in both HF and SScF. Superoxide dismutase assay demonstrated that SOD3 enzymatic activity in SScF culture medium is four times more than in HF culture medium. These data suggest that an alteration in SOD3 expression and activity could be associated to SSc fibrosis.     

Generation of superoxide anion radicals and hydrogen peroxide in the auto-oxidation of caffeic acid

Caffeic acid (5-200 mkM) reduces cytochrome c during autoxidation in potassium phosphate buffer, pH 7-8. The reduction is inhibited by superoxide dismutase, which suggests generation of superoxide anion radicals. The generation rate is 0.028-0.115 mkmoles O2- per min. Superoxide appears to be a side product of the reaction, since the autoxidation of caffeic acid itself (followed by A420) is not inhibited by superoxide dismutase. The autoxidation is accompanied by oxygen of consumption. An addition of catalase results in liberation of some part of consumed oxygen, this being indicative of accumulation of hydrogen peroxide. Caffeic acid is known to be responsible for the resistance of plants to parasites because of its toxicity. This function presumably depends on superoxide or other reactive oxygen species.     

Overexpression of extracellular-SOD in islets of nonobese diabetic mice and development of diabetes

Mice of the nonobese diabetic strain develop a progressive insulitis resulting in beta-cell destruction and diabetes. Superoxide radicals are abundantly formed by leukocytes and other mechanisms in inflammatory reactions. We here aimed to determine whether superoxide radicals contribute to the beta cell destruction in the mouse model. Transgenic nonobese diabetic mice secreting extracellular-superoxide dismutase under control of the insulin promoter were generated and the development of glucosuria monitored. The overexpression of extracellular-superoxide dismutase resulted in a 6-fold increase in the total superoxide dismutase activity of the islets. The incidence of diabetes of the transgenic mice was, however, not modified. The results suggest that superoxide radicals secreted to the extracellular space do not contribute to the beta cell destruction in the nonobese diabetic mouse model.     

Superoxide radical production during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium perchlorate.

1-Methyl-4-phenyl-2,3-dihydropyridinium perchlorate (MPDP+), an intermediate in the metabolism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, was found to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected by their ability to reduce ferricytochrome c. Superoxide dismutase inhibited this reduction in a dose-dependent manner. The rate of reduction of ferricytochrome c was dependent not only on the concentration of MPDP+ but also on the pH of the system. Thus, the rate of autoxidation of MPDP+ and the sensitivity of this autoxidation to superoxide dismutase-inhibitable ferricytochrome c reduction were both augmented, as the pH was raised from 7.0 to 10.5. The rate constant (Kc) for the reaction of superoxide radical with ferricytochrome c to form ferricytochrome c was found to be 3.48 x 10(5) M-1 s-1. The rate constant (KMPDP+) for the reaction of MPDP+ with ferricytochrome3+ c was found to be only 4.86 M-1 s-1. These results, in conjunction with complexities in the kinetics, lead to the proposal that autoxidation of MPDP+ proceeds by at least two distinct pathways, one of which involves the production of superoxide radicals and hence is inhibitable by superoxide dismutase. It is possible that the free radicals so generated could induce oxidative injury which may be central to the MPTP/MPDP(+)-induced neuropathy.     

Intermediates in the aerobic autoxidation of 6-hydroxydopamine: relative importance under different reaction conditions

Autoxidation of 6-hydroxydopamine (6-OHDA) proceeds through a balanced network of: transition metal ions, superoxide, hydrogen peroxide, hydroxyl radicals, and other species. The contribution of each to the reaction mechanism varies dramatically depending upon which scavengers are present. The contribution of each propagating intermediate increases when the involvement of others is diminished. Thus, superoxide (which is relatively unimportant when metal ions can participate) dominates the reaction when transition metal ions are bound (especially at higher pH), and it becomes essential in the simultaneous presence of catalase plus chelators. Transition metal ions participate more if superoxide is excluded; hydrogen peroxide becomes more important if both .O2- and metal ions are excluded; and hydroxyl radicals contribute more to the reaction mechanism if both H2O2 and .O2- are excluded. Superoxide dismutase inhibited strongly, by two distinct mechanisms: a high affinity mechanism (less than 13% inhibition) at catalytically effective concentrations, and a low affinity mechanism (almost complete inhibition at the highest concentrations) which depends upon both metal binding and catalytic actions. In the presence of DETAPAC catalytic concentrations of superoxide dismutase inhibited by over 98%. Conversely, metal chelating agents inhibited strongly in the presence of superoxide dismutase. When present alone they stimulated (like EDTA), inhibited (like desferrioxamine), or had little effect (like DETAPAC). Catalase which stimulated slightly but consistently (less than 5%) when added alone, inhibited 100% in the presence of superoxide dismutase + DETAPAC. However, in the absence of DETAPAC, catalase decreased inhibition by superoxide dismutase, yielding a 100% increase in reaction rate. Hydroxyl scavengers (formate, mannitol or glucose) alone produced little or no (less than 10%) inhibition, but inhibited by 30% in the presence of catalase + superoxide dismutase. Paradoxically, they stimulated the reaction in the presence of catalase + superoxide dismutase + DETAPAC.     

Superoxide free radicals are produced in glyoxysomes

The production of superoxide free radicals in pellet and supernatant fractions of glyoxysomes, specialized plant peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons, was investigated. Upon inhibition of the endogenous superoxide dismutase, xanthine, and hypoxanthine induced in glyoxysomal supernatants the generation of O₂⁻ radicals and this was inhibited by allopurinol. In glyoxysomal pellets, NADH stimulated the generation of superoxide radicals. Superoxide production by purines was due to xanthine oxidase, which was found predominantly in the matrix of glyoxysomes. The generation of O₂⁻ radicals in glyoxysomes by endogenous metabolites suggests new active oxygen-related roles for glyoxysomes, and for peroxisomes in general, in cellular metabolism.     

Changes in the levels of superoxide anion radical and superoxide dismutase during the estrous cycle of Rattus norvegicus and induction of superoxide dismutase in rat ovary by lutropin

Superoxide dismutase, which has been shown to be present in a number of tissues, exhibits cyclic changes during the reproductive cycle of rats. An inverse correlation is seen between the levels of superoxide dismutase and superoxide radical. In immature, pseudopregnant rats, primed with human Chorionic Gonadotropin, lutropin seemed to induce ovarian superoxide dismutase, which could be blocked significantly by the introduction of anti-LH serum. These results point out the specific induction of superoxide dismutase by lutropin. It is reasonable to postulate that during luteal functioning, luteinizing hormone induces superoxide dismutase which in turn seems to play a central role generating hydrogen peroxide from superoxide anion radicals. Hydrogen peroxide, thus formed, drives the peroxidase-ascorbate system, responsible for production of progesterone.     

Ethanol-induced iron mobilization: role of acetaldehyde-aldehyde oxidase generated superoxide

Superoxide radicals, a species known to mobilize ferritin iron, and their interaction with catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury. The mechanism(s) by which ethanol metabolism generates free radicals and mobilizes catalytic iron, however, is not fully defined. In this investigation the role of hepatic aldehyde oxidase in the mobilization of catalytic iron from ferritin was studied in vitro. Iron mobilization due to the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was increased 100% by the addition of aldehyde oxidase. Iron release was favored by low pH and low oxygen concentration. Mobilization of iron due to acetaldehyde metabolism by aldehyde oxidase was completely inhibited by superoxide dismutase but not by catalase suggesting that superoxide radicals mediate mobilization. Acetaldehyde-aldehyde oxidase mediated reduction of ferritin iron was facilitated by incubation with menadione, an electron acceptor for aldehyde oxidase. Mobilization of ferritin iron due to the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism of alcohol-induced liver injury.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

SUPEROXIDE DISMUTASE OF MAMMALIAN NERVOUS SYSTEM

Superoxide dismutase was assayed in portions of the nervous system (a) by inhibition of tetrazolium reduction by oxygen radicals, generated enzymically, and (b) by inhibition of tetrazolium reduction by oxygen radicals generated by oxidation of NADH in presence of phenazine methosulphate. Superoxide dismutase activity was found in beef brain, retina and adrenal medulla, as well as in brain, retina and lungs of adult and of newborn rats. Preliminary experiments with rats exposed to hyperbaric oxygen showed no alteration of enzyme activities in tissues of newborn and adult animals. The possible role of superoxide dismutase in the nervous system is discussed.     

Reactions of Adriamycin with haemoglobin. Superoxide dismutase indirectly inhibits reactions of the Adriamycin semiquinone.

The Adriamycin semiquinone produced by the reaction of xanthine oxidase and xanthine with Adriamycin has been shown to reduce both methaemoglobin and cytochrome c. In air, but not N2, both reactions were inhibited by superoxide dismutase. With cytochrome c, superoxide formed by the rapid reaction of the semiquinone with O2, was responsible for the reduction. However, even in air, methaemoglobin was reduced directly by the Adriamycin semiquinone. Superoxide dismutase inhibited this reaction by removing superoxide and hence the semiquinone by displacing the equilibrium: Semiquinone + O2 in equilibrium or formed from quinone + O2-. to the right. This ability to inhibit indirectly reactions of the semiquinone could have wider implications for the protection given by superoxide dismutase against the cytotoxicity of Adriamycin. Oxidation of haemoglobin by Adriamycin has been shown to be initiated by a reversible reaction between the drug and oxyhaemoglobin, producing methaemoglobin and the Adriamycin semiquinone. Reaction of the semiquinone with O2 gives superoxide and H2O2, which can also react with haemoglobin. Catalase, by preventing this reaction of H2O2, inhibits oxidation of oxyhaemoglobin. Superoxide dismutase, however, accelerates oxidation, by inhibiting the reaction of the semiquinone with methaemoglobin by the mechanism described above. Although superoxide dismutase has a detrimental effect on haemoglobin oxidation, it may protect the red cell against more damaging reactions of the Adriamycin semiquinone.     

Ascorbate reduces superoxide production and improves mitochondrial respiratory chain function in human fibroblasts with electron transport chain deficiencies

The present paper attempts to ascertain the role of ascorbate on the generation of superoxide radicals in skin fibroblasts of patients with deficiency of mitochondrial respiratory chain enzymes. Fibroblast cell lines were grown with or without ascorbate for the last 48 h of their growth period. The amount of superoxide radical production in cells was measured by the reduction of nitroblue tetrazolium and the activities of respiratory chain enzymes were examined in isolated fibroblast mitochondria. The results indicated a significant inverse correlation between the amount of superoxide radicals and the specific activities of complexes I-III and II-III of the respiratory chain. The ascorbate treatment of fibroblasts from control subjects did not show any effect on either superoxide radical production or respiratory chain enzymes' activities. While in patient's fibroblasts, this vitamin significantly decreased the superoxide radicals and increased the specific activities of I-III and II-III complexes but not complex IV. These observations indicate that superoxide radicals are increased in patients with deficient respiratory chain enzymes in their fibroblasts and ascorbate can prevent the loss of these enzymes by acting on the selected sites in the respiratory chain, which are related to the production of free radicals.     

Superoxide radicals and antiradical defence of propionic acid bacteria

Superoxide dismutase activity was demonstrated for 6 strains of 3 propionibacteria species. Rather high level of superoxide dismutase activity found in propionibacteria was in accordance with high level of catalase activity reported for propionibacteria previously. Both these activities were shown to have cytozolic localization. For the first time peroxidase activity was detected in gel-fractionated crude cell extracts of propionibacteria. The ability to produce superoxide radicals in NADH-dependent oxidation system was revealed for three strains of the bacteria. The level of superoxide production by the membrane particles of the propionic acid bacteria correlated with the levels of superoxide dismutase and catalase activities and was the lowest for Propionibacterium shermanii. The ability to perform monovalent oxygen reduction during succinate oxidation was not revealed. The intact cells of P. globosum, P. vannielii, P. shermanii apparently did not excrete superoxide radicals into culture fluid during respiration.     

Coenzyme Q10 depletion is comparatively less detrimental to human cultured skin fibroblasts than respiratory chain complex deficiencies

The oxidative stress possibly resulting from an inherited respiratory chain (RC) deficiency was investigated in a series of human cultured skin fibroblasts presenting either ubiquinone depletion or isolated defect of the various RC complexes. Taken as an index for superoxide overproduction, a significant induction of superoxide dismutase activity was observed in complex V-deficient fibroblasts harboring the NARP-mutation in the ATPase 6 gene. Superoxide dismutase induction was also noticed, albeit to a lesser extent, in complex II-deficient fibroblasts with a mutation in the nuclear gene encoding the flavoprotein subunit of the succinate dehydrogenase. No sign of oxidative stress could be found in ubiquinone-depleted fibroblasts. In all cases but complex IV-defect, increased oxidative stress was associated with increased cell death. In glucose-rich medium, apoptosis appeared as the main cell death process associated with all types of RC defect. However, similar to the great variations in oxidative stress associated with the various types of RC defect, we found that apoptotic features differed noticeably between defects. No indication of increased cell death was found in ubiquinone-depleted fibroblasts.     

Coenzyme Q 10 Depletion is Comparatively Less Detrimental to Human Cultured Skin Fibroblasts than Respiratory Chain Complex Deficiencies

The oxidative stress possibly resulting from an inherited respiratory chain (RC) deficiency was investigated in a series of human cultured skin fibroblasts presenting either ubiquinone depletion or isolated defect of the various RC complexes. Taken as an index for superoxide overproduction, a significant induction of superoxide dismutase activity was observed in complex V-deficient fibroblasts harboring the NARP-mutation in the ATPase 6 gene. Superoxide dismutase induction was also noticed, albeit to a lesser extent, in complex II-deficient fibroblasts with a mutation in the nuclear gene encoding the flavoprotein subunit of the succinate dehydrogenase. No sign of oxidative stress could be found in ubiquinone-depleted fibroblasts. In all cases but complex IV-defect, increased oxidative stress was associated with increased cell death. In glucose-rich medium, apoptosis appeared as the main cell death process associated with all types of RC defect. However, similar to the great variations in oxidative stress associated with the various types of RC defect, we found that apoptotic features differed noticeably between defects. No indication of increased cell death was found in ubiquinone-depleted fibroblasts.     

Response of hydroperoxidase and superoxide dismutase deficient mutants of Escherichia coli K-12 to oxidative stress

In Escherichia coli, the coordinate action of two antioxidant enzymes, superoxide dismutase and hydroperoxidase (catalase), protect the cell from the deleterious effects of oxyradicals generated during normal aerobic respiration. To evaluate the relative importance of these two classes of enzymes, strains of E. coli deficient in superoxide dismutase and (or) hydroperoxidase were constructed by generalized transduction and their physiological responses to oxygen and oxidant stress examined. Superoxide dismutase was found to be more important than hydroperoxidase in preventing oxygen-dependent growth inhibition and mutagenesis, and in reducing sensitivity to redox-active compounds known to generate the superoxide anion. However, both types of enzymes were required for an effective defense against chemical oxidants that generate superoxide radicals and hydrogen peroxide.     

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. I. Evidence for superoxide anion involvement in the oxidation of NADPH2.

1. The oxidation of NADPH2 by leucocyte granules, as measured at acid pH in the presence of Mn-2+, was found to be inhibited by superoxide dismutase. 2. Omission of Mn-2+ markedly lowered the oxidase activity at acid pH, which was still inhibited by superoxide dismutase. 3. At alkaline pH the oxidase activity was lower than at acid pH. 4. During oxidation of NADPH2 by leucocyte granules, reduction of cytochrome c occurred which was partially inhibited by superoxide dismutase. 5. It was concluded that NADPH2 oxidation occurs through an enzymatic reaction and a nonenzymatic chain reaction. Superoxide anion (O-minus-2 and NADPH- free radical would be involved in the chain reaction. The differential sensitivity of NADPH2 oxidation to superoxide dismutase in different experimental conditions (see above 1, 2 and 3) was explained on the basis of changes in the properties of the chain reaction.