Role Of Superoxide Research Articles

Role of superoxide in radiation-killing of [formula omitted] and in thymine release from thymidine

The role of superoxide and hydroxyl radicals in gamma-radiation-killing of Escherichia coli K12 was studied in aerated suspensions supplemented with formate, phosphate, superoxide dismutase, catalase and saturated with nitrous oxide. Nitrous oxide, which converts e − aq to •OH, caused decreased radiosensitivity. On the other hand, formate, which results in conversion of •OH to •O 2 − , resulted in an increased radiosensitivity. The results implicated •O 2 − as a major cause of radiation-mediated cell-killing. The addition of the enzymes, superoxide dismutase or catalase to the E. coli suspensions prior to and during irradiation had no effect on cell survival, indicating that the biologically significant site of generation and action of •O 2 − is an intracellular one. Further studies were undertaken to examine the role of superoxide in DNA damage. The release of thymine from the DNA base, thymidine was studied as a result of gamma-irradiation and of chemically generated superoxide (using KO 2 in dimethyl sulfoxide). Thymine was identified by HPLC and mass spectrometry. C-13 NMR analysis of the reaction mixture of thymidine with KO 2 in dimethyl sulfoxide provided evidence for attack of •O 2 − at the ribosyl Cl′ atom.

The role of superoxide anion and hydrogen peroxide in glomerular injury induced by puromycin aminonucleoside in rats.

1. The nephrotic syndrome was induced in inbred female Wistar rats by the intravenous injection of puromycin aminonucleoside (PA) (5 mg/100 g body weight). 2. One group (n = 12) received superoxide dismutase (SOD) (15 mg/kg body weight), a second group (n = 12) received polyethylene glycol coupled catalase (PEG-catalase) (5000 i.u./kg body weight) and the third (n = 9) saline (150 mmol/l NaCl) via the intraperitoneal route, in addition to the PA. 3. SOD and PEG-catalase reduced the 24 h urine protein on days 8 and 15 compared with unmodified puromycin treated animals and this difference was significant on day 15 for SOD (P less than 0.05) and for PEG-catalase (P less than 0.01). Glomerular filtration rate, as measured by the creatinine clearance, was lower in the PEG-catalase group but did not differ significantly from the saline treated group. 4. These data suggest that superoxide anion and hydrogen peroxide, or their reaction products, are involved in the glomerular injury of puromycin nephropathy.

Implication of active oxygen species in the direct-acting mutagenicity of tea

The present study shows that the l-arabinose resistance test with Salmonella typhimurium detects that freshly infused tea is highly mutagenic in the absence of mammalian microsomal activation. Both the mutagenesis protocol (preincubation test) and the additional genetic characteristics of the bacterial tester strain (excision repair deficiency, normal lipopolysaccharide barrier and the presence of plasmid pKM101) were critical factors in the optimal induction by tea of forward mutations to l-arabinose resistance. The TA104 strain — a histidine auxotroph specific to oxidative mutagens — was the most sensitive tester strain of the Ames test to the direct-acting mutagenicity of tea. In comparison with strain TA104, the sensitivity of the Ara forward mutation test was 18 times higher, one cup of tea (200 ml) inducing 3 × 10 6 Ara R mutants. More than 90% of the mutagenicity of 150μl of a fresh tea infusion, or that of the equivalent amount (1.32 mg) of the corresponding lyophilized residue, was suppressed by 10 units of catalase. In contrast to catalase, superoxide dismutase was rather ineffective. These results indicate that hydrogen peroxide is produced in tea solutions, playing an essential role in its mutagenicity. In comparison, the role of superoxide anion seems negligible. Like catalase, the chelating agent DETAPAC showed a protective effect with respect to the mutagenicity of tea, suggesting the additional implication of hydroxyl radicals.

Oxygen radicals are generated by dye-mediated intracellular photooxidations: A role for superoxide in photodynamic effects

Representative thiazines, xanthenes, acridines, and phenazines photosensitized the oxidation of reduced pyridine nucleotides and reduced glutathione when illuminated with low intensity visible light. Photooxidation resulted in oxygen consumption and in superoxide generation, assayed as the Superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. The major pathway of electron transfer involved dye reduction rather than singlet oxygen-mediated oxidation of the substrate, as demonstrated by the relative insensitivity of the oxidation to inhibition by sodium azide and by the observable bleaching of the dye. Hydrogen peroxide was a stable end product of photooxidation. Photosensitive dyes were photoreduced intracellularly. These dyes were transported across the membranes of Escherichia coli B and stimulated a light- and concentration-dependent increase in the cyanide-insensitive respiration. Dyes reduced intracellularly subsequently diffused out of the cell where they reduced extracellular cytochrome c. The photosensitive dyes examined in this study exhibited a light-dependent bacteriostatic effect on E. coli B grown in nutrient broth, manifested as an increased lag prior to growth. Restoration of growth coincided with increased levels of SOD, and the intracellular level of SOD correlated with the level of illumination, the dye concentration, and the reactivity of the dye to NADH in vitro. The thiazine dye, toluidine blue o, imposed a light- and oxygen-dependent lethality on E. coli B grown in glucose minimal medium. Toxicity was relieved by hydroxyl radical scavengers, and their ability to protect the cells was proportional to their reactivity with the hydroxyl radical. The results indicate that oxygen radicals and related species mediate photodynamic effects in E. coli B.

Acetaldehyde-mediated hepatic lipid peroxidation: Role of superoxide and ferritin

Evidence in alcoholics as well as in experimental models support the role of hepatic lipid peroxidation in the pathogenesis of alcohol-induced liver injury, but the mechanism of this injury is not fully delineated. Previous studies of the metabolism of ethanol by alcohol dehydrogenase revealed iron mobilization from ferritin that was markedly stimulated by superoxide radical generation by xanthine oxidase. Peroxidation of hepatic lipid membranes (assessed as malondialdehyde production) was studied during in vitro alcohol metabolism by alcohol dehydrogenase. Peroxidation was initiated by acetaldehyde-xanthine oxidase, stimulated by ferritin, and inhibited by superoxide dismutase or chelation or iron with desferrioxamine. In conclusion, lipid peroxidation may be initiated during the metabolism of ethanol by alcohol dehydrogenase by an iron-dependent acetaldehyde-xanthine oxidase mechanism.

Lucigenin-dependent chemiluminescence in mononuclear phagocytes. Role of superoxide anion

Lucigenin-dependent chemiluminescence in mononuclear phagocytes. Role of superoxide anion

Lucigenin-dependent chemiluminescence in mononuclear phagocytes. Role of superoxide anion

A method for the measurement of reactive oxygen species generated by activated mononuclear phagocytes by use of lucigenin-dependent chemiluminescence was developed. Opsonized zymosan was used as a stimulant to evaluate the chemiluminescence response of monocytes. A cell-free system in which superoxide anion and hydrogen peroxide were produced by the xanthine-xanthine oxidase reaction was used to examine the role of these radical species in the excitation of lucigenin, and to standardize the chemiluminescence method. It was found that the light emission in lucigenin-dependent chemiluminescence was evoked by the superoxide anion, but other radicals may also be active.

The Role of Superoxide Anion and Lysosomal Enzymes in Anti‐Listerial Activity of Elicited Peritoneal Macrophages

The in vitro effect of superoxide anion and lysosomal enzyme activity on the killing of Listeria monocytogenes EGD (listeria) by peritoneal macrophages (PM) was investigated. Generation of superoxide anion by PM stimulated with phorbol myristate acetate (PMA) was significantly increased by intraperitoneal injection of Lactobacillus casei YIT 9018 (LC9018) or Corynebacterium parvum (CP), but not by injection of peptone. However, superoxide anion generation by LC9018-elicited PM stimulated with listeria was not increased any more than that by peptone-elicited PM, and generation of superoxide anion by the PM was affected by the difference in stimuli. The killing of listeria by LC9018- or CP-elicited PM in vitro was significantly less than that by peptone-elicited PM or resident PM. Significant correlation was observed between the anti-listerial activity of PM and the intracellular killing of listeria by PM. On the other hand, beta-glucuronidase and beta-N-acetyl-D-glucosaminidase activities of LC9018-elicited, CP-elicited, or resident PM were significantly weaker than those of peptone-elicited PM, and no significant correlation was observed between the increase in beta-glucuronidase and beta-N-acetyl-D-glucosaminidase activities and the increase in anti-listerial activity. These results suggest that increase in the activity of lysosomal enzymes such as beta-glucuronidase and beta-N-acetyl-D-glucosaminidase is not correlated with the anti-listerial activity of PM, and that superoxide anion has very little effect on the anti-listerial activity of PM in vitro.

Superoxide mediates the toxicity of paraquat for Chinese hamster ovary cells.

The roles of superoxide and H2O2 in the cytotoxicity of paraquat were assessed in Chinese hamster ovary cells. Neither catalase nor superoxide dismutase inhibited the loss of ability to form colonies when added to the medium. When introduced into the cells, superoxide dismutase but not catalase inhibited the toxicity of paraquat. That superoxide dismutase acted by its known catalytic action is shown by the loss of inhibition when the enzyme was inactivated by H2O2 before being introduced into the cells. The lack of inhibition by catalase, by dimethyl sulfoxide, and by desferoxamine suggests that the toxicity is not mediated by a reaction between H2O2 and superoxide to engender the hydroxyl radical. Exposure of Chinese hamster ovary cells to paraquat may be a suitable means to determine the effects of superoxide anion in cultured cells and the ways in which cells can resist this toxic action.

Treatment of radiofibrosis with liposomal superoxide dismutase. Preliminary results of 50 cases.

Well and long established radio-fibroses have been treated successfully with a liposomal encapsulated bovine copper superoxide dismutase. After a short treatment (three weeks intramuscular injection of 5 mg twice a week) regression of the fibrosis is stable. The average size is reduced by one third and significant softening occurs in 82% of the cases. Efficiency is independent of the time between radiotherapy (origin of the fibrosis) and treatment with liposomal SOD. Complete regression even after this limited treatment is seen in cases of chronic prefibrotic inflammatory syndromes and prophylactic action in cases where the probability of fibrosis formation is certain appears to be successful. The roles of superoxide and superoxide dismutase are discussed.

Modulation of Lens-Induced Uveitis by Superoxide Dismutase

Recent studies on reactive oxygen metabolites have suggested that these products may be important mediators in the early tissue damage that develops from immunopathologic inflammations. The superoxide anion appears to be the principal product of the respiratory burst. In order to determine the effects of removing superoxide with superoxide dismutase on modulating the inflammation in experimental phacoanaphylactic endophthalmitis, the animals with this experimental disorder were treated with superoxide dismutase. The treatment resulted in significant reduction of choroidal inflammation, retinal edema and vasculitis, suggesting phlogogenic role of superoxide in the ocular immune-complex disease.

Role of superoxide in the N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine by the rat liver cytochrome P-450 system.

The N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine (N-hydroxyphentermine, MPPNHOH) and the N-hydroxylation of 2-methyl-1-phenyl-2-propylamine (phentermine) by reconstituted systems that contained cytochromes P-450 purified from rat liver microsomes were demonstrated. The oxidation of MPPNHOH, but not of phentermine, could also be mediated by a superoxide and hydrogen peroxide generating system that contained xanthine and xanthine oxidase. Superoxide dismutase completely inhibited the oxidation of MPPNHOH by the xanthine/xanthine oxidase system and inhibited by 70% the oxidation mediated by a reconstituted cytochrome P-450 oxidase system. The majority of the microsomal oxidation was inhibited by an antibody raised against the major isozyme of cytochrome P-450 purified from livers of phenobarbital-pretreated rats. 2-Methyl-2-nitroso-1-phenylpropane (MPPNO) was found to be an intermediate in the overall oxidation of MPPNHOH to 2-methyl-2-nitro-1-phenylpropane (MPPNO2). Superoxide dismutase appeared to inhibit the first step, the conversion of MPPNHOH to MPPNO. These observations are accounted for by a sequence of two mechanistically distinct P-450-mediated oxidations. In the first reaction, N-hydroxylation of phentermine occurs by a normal cytochrome P-450 pathway. The formed hydroxylamine then uncouples the cytochrome P-450 system to generate superoxide and hydrogen peroxide. The superoxide oxidizes MPPNHOH to MPPNO which is then oxidized to MPPNO2, the ultimate product. This superoxide-mediated oxidation represents another pathway for N-oxidation by cytochrome P-450.

Mast cell and neutrophil interactions: A role for superoxide anion and histamine

Histamine inhibits superoxide anion (O-2) production from human neutrophils stimulated by N-formylmethionyl-leucyl-phenylalanine (FMLP). The effects of histamine are dose-dependent and competitively antagonized by cimetidine. When passively sensitized rat serosal mast cells and human neutrophils are mixed together, O-2 production from FMLP-activated granulocytes is significantly reduced, following mast cell degranulation by acetylcholine. These inhibitory effects can be counteracted by cimetidine. Exposure of non-sensitized rat mast cells to FMLP-stimulated human neutrophils causes histamine release. These results suggest bidirectional control mechanisms between mast cells and neutrophils, that further stress the role of histamine in regulating inflammatory processes.

Role of superoxide and hydrogen peroxide in cell lysis during irradiation in vitro of Ehrlich ascitic carcinoma cells in the presence of melanin.

The reactive species involved in the cell lysis during ultraviolet irradiation of Ehrlich ascitic carcinoma cells in the presence of red hair melanin (RHM) were investigated by determining 51Cr release from labeled cells. Cysteine at 1 mM in the presence of RHM increased the cell lysis during the incubation in the dark as well as during irradiation; this lysis was enhanced by superoxide dismutase (SOD). Catalase abolished the dark reaction and inhibited the cysteine-induced increase of cell lysis during irradiation. The cell lysis by the superoxide-generating xanthine oxidase system was not significantly increased by SOD, but was significantly decreased by nitroblue tetrazolium and completely abolished by catalase. The cell lysis induced by the supernatants obtained from the suspensions of RHM either irradiated alone or with cysteine was abolished by catalase. Sediments of irradiated RHM when incubated in the dark with the cells did not release 51Cr. Irradiation of the cells in the presence of the same sediments produced lysis which was not inhibited by catalase. These studies suggest that superoxide per se is not toxic to the cells, but the H2O2 formed by dismutation of superoxide produces cell lysis either directly or by generating OH through Fenton-type reactions. A large part of the cell lysis seen during irradiation of cells in the presence of RHM is not due to H2O2, but may possibly be due to the melanin free radicals formed during irradiation.

Mediators and mechanisms II: Modulation of phagocytosis-associated chemiluminescence (CL) response of PMN and macrophages during acute inflammation. Role of superoxide anion (O 2−) and prostanoids

Mediators and mechanisms II: Modulation of phagocytosis-associated chemiluminescence (CL) response of PMN and macrophages during acute inflammation. Role of superoxide anion (O 2−) and prostanoids

Absence of a Role for Superoxide Anion, Hydrogen Peroxide and Hydroxyl Radical in Endothelium-Mediated Relaxation of Rabbit Aorta

We tested the hypothesis that the endothelium-dependent relaxation of rabbit thoracic aorta in vitro is mediated by reduced metabolites of oxygen. Helical vascular strips were contracted with either norepinephrine or phenylephrine. Oxygen metabolites, generated by the xanthine oxidase reaction, completely relaxed norepinephrine-induced contractile tone but not tone induced by phenylephrine. A mixture of oxygen metabolite scavengers (superoxide dismutase, catalase and mannitol) eliminated the relaxation induced by the xanthine oxidase products. Acetylcholine caused a dose-dependent and endothelium-dependent relaxation of the strips; this was not inhibited by the presence of the scavengers. We conclude that reduced oxygen metabolites have little direct effect on rabbit aortic smooth muscle in vitro, although they indirectly but specifically relax norepinephrine-induced tone, presumably by oxidation of norepinephrine. Oxygen metabolites do not appear to mediate the endothelium-dependent relaxation response of this tissue to acetylcholine.

Superoxide generation by NADPH-cytochrome P-450 reductase: The effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by Superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe 3+ and EDTA-Fe 3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O 2 ⨪ production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O 2 ⨪ generation by xanthine oxidase. Although both EDTA-Pe 3+ and DTPA-Fe 3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe 3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe 3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O 2 ⨪ generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe 3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O 2 ⨪ generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe 3+ was demonstrated to be independent of O 2 ⨪ generation by the reductase.

Role of superoxide anion in host cell injury induced by mycoplasma pneumoniae infection. A study in normal and trisomy 21 cells.

The role of Mycoplasma pneumoniae-generated superoxide and hydrogen peroxide in inducing host cell injury was studied in normal and trisomy 21 human cells. As a result of M. pneumoniae infection, catalase activity in infected normal skin fibroblasts and ciliated epithelial cells decreased by 74-77% as compared with uninfected controls. Addition of superoxide dismutase to the infected cultured cells totally prevented the inhibition whereas addition of catalase or catalytically inactivated superoxide dismutase had no protective effect. Trisomy 21 erythrocytes and cultured skin fibroblasts in which CuZn-superoxide dismutase content is 50% greater than in normal cells were infected by M. pneumoniae. The inhibition of catalase activity in these cells was 7-33% and 0-20.5%, respectively, as compared with 65-72% and 48-68% inhibition in normal infected controls. Following M. pneumoniae infection, the levels of malonyldialdehyde, an indicator for membrane lipid peroxidation were raised in trisomy 21 cultured fibroblasts by 10-32% while in normal cells malonyldialdehyde increased by 140-870%. Externally added superoxide dismutase, but not catalase, reduced the extent of lipid peroxidation in normal infected cells. Lactate dehydrogenase release from normal infected cells was time correlated with the increase in their malonyldialdehyde formation. It is suggested that superoxide generated during M. pneumoniae infection is involved in the inhibition of host cell catalase activity. The inactivation of this cellular antioxidative defense mechanism results in progressive oxidative damage to the M. pneumoniae-infected cells.

Studies on the phenazine methosulphate--tetrazolium salt capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. III. The role of superoxide in tetrazolium reduction.

A study was made of the involvement of superoxide anions in the aerobic reduction of tetrazolium salts by NAD(P)H and phenazine methosulphate (PMS). On the basis of experiments with superoxide dismutase two mechanisms of tetrazolium reduction could be distinguished--one in which fully reduced PMS (PMSH) is the reducer and one in which superoxide anion is the reducer of tetrazolium salts. It is proposed that superoxide anions is formed after a PMSH-PMS+ dismutation reaction. The relative contributions of the two distinct pathways to tetrazolium salt reduction are controlled by the PMS redox state and the oxygen tension. The consequences of the presence of superoxide anions and scavengers of superoxide anions for quantitative dehydrogenase cytochemistry are discussed.

Studies on the Role of Reactive Oxygen Species in Mediating Lipid Peroxide Formation in Epidermal Microsomes of Rat Skin

The role of superoxide, hydrogen peroxide, and singlet oxygen in mediating nonenzymic and NADPH-supported enzymic lipid peroxidation in skin microsomes was investigated. Incubation of skin microsomes with NADPH and/or Fe+3-ADP or ascorbate resulted in the formation of lipid peroxides. The epidermis was the major target site for microsomal lipid peroxide formation in skin. Enzymic peroxidation of epidermal microsomes required NADPH and was oxygen-dependent. Addition of the nonenzymic catalysts, Fe+3 and ADP, to the enzymic peroxidation system had an additive effect on the generation of lipid peroxide in epidermal microsomes. Epidermal microsomal lipid peroxidation was inhibited by singlet oxygen quenchers such as dimethylfuran, histidine, and beta-carotene. Hydroxyl ion scavengers such as mannitol, benzoate, or ethyl alcohol and the enzymic scavenger of superoxide, superoxide dismutase, were all ineffective in this respect. Addition of EDTA, Mn+2, cytochrome c+3, and catalase to the NADPH-supported enzymic peroxidation system resulted in strong inhibition of lipid peroxide formation in skin. Glutathione or epidermal cytosol added alone or in combination to the NADPH-supported incubation system enhanced peroxidation of microsomal lipids. Vitamin E (alpha-tocopherol) inhibited lipid peroxidation. These results indicate that singlet oxygen may mediate lipid peroxide formation in epidermal microsomes.