Increased survival of skin flaps by scavengers of superoxide radical

Elevation of rat abdominal skin flaps, followed by ligation and division of the left inferior neurovascular pedicle, resulted in only a 40% survival of the area normally perfused by the ligated artery and vein. Superoxide dismutase (SOD) (EC 1.15.1.1) administered i.v. (20,000 U/kg) 30 min before flap elevation increased survival to 52%. SOD derivatized with polyethylene glycol, which increases circulating half-life, was more effective, increasing survival to 80%. This protective effect resulted from the catalytic activity of the derivatized enzyme because inactivation by treatment with H2O2 eliminated its effect on skin flap survival. An equimolar mixture of Desferal and MnCl2, which catalyzes the dismutation of O2- in vitro, improved survival to 72%. Desferal-Fe3+, which lacks in vitro SOD activity, or Mn2+ alone did not affect the survival of skin flaps, but Desferal alone was nearly as effective as the Desferal-Mn2+ mixture. This effect of Desferal may result from acquisition of and subsequent removal of iron in vivo. These results support the view that the superoxide radical or a product derived from it plays a role in limiting the survival of island skin flaps.     

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

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

Protection of Chinese hamster ovary cells from paraquat-mediated cytotoxicity by a low molecular weight mimic of superoxide dismutase (DF-Mn)

Paraquat exerts a cytotoxic effect of Chinese hamster ovary cells in culture via the superoxide radical (O₂⁻. We have described a superoxide dismutase (SOD) mimic based on manganese (DF-Mn) which consists of a one-to-one complex between desferrioxamine B (Desferal) and MnO₂. It is a small molecular weight molecule, easy to prepare and possesses considerable stability. It is now shown to protect mammalian cells from paraquat toxicity. Thus, 20 μM DF-Mn affords up to complete protection against the cytotoxicity of 200 μM paraquat in Chinese hamster ovary cells. Desferrioxamine B or MnO₂ alone gave no protection. MnCl₂ or catalase provided little or no protection against the paraquat, respectively. Equivalent amounts of human Cu-Zn SOD in terms of activity, also provided no protection. Copper diisopropylsalicylate (CuDIPS) provided limited, yet significant, protection, but this is explained in terms other than SOD activity. Finally, at higher concentrations, purified human SOD, exerts a limited toxicity as well as a protective ability against paraquat (similar to DF-Mn) both of which are eliminated upon heat denaturation of the enzyme. It appears that the SOD mimic, DF-Mn, can enter mammalian cells and can protect against the cytotoxic effects of O₂⁻.     

Adhering lung macrophages produce superoxide demonstrated with desferal-Mn(IV)

It has been shown that H2O2, the dismutation product of O2., is produced at cell-surface interfaces. Nevertheless, the relationships between the degree of attachment itself, type of surface, and O2. production are not clear. Superoxide production can be measured by the O2.-dependent reduction of nitroblue tetrazolium to an insoluble formazan. Superoxide dismutase (SOD) may be unable to scavenge O2. produced between alveolar macrophages (AM) and a surface. Desferal-Mn(IV) (Des-Mn), a low molecular weight mimic of SOD, is protective against paraquat toxicity in vivo, presumably because of specificity for O2-. Using that assumption, Des-Mn was used to measure O2. production that occurred during adherence of AM. AM suspensions were placed on fibronectin-coated glass coverslips or uncoated glass coverslips or non-stick tissue culture plates. Adherence to the surfaces varied with fibronectin greater than glass greater than non-stick and the percent formazan positive cells was 60, 24, and 4, respectively. With SOD present, the percentage of formazan positive cells were 40, 17, and 2; however, in the presence of Des-Mn the percent stained cells was 4, 4, and 0. When phorbol myristate acetate (PMA) was added during adherence, the percent of formazan positive cells was 82, 57, and 44, respectively. With PMA, Des-Mn was able to inhibit 88-100% of formazan staining whereas SOD inhibition decreased more markedly with increasing adherence. These results indicated that the degree of attachment correlated with both the degree of NBT reduction and the relative effectiveness of Des-Mn versus SOD to scavenge O2..     

Protective effect of Dunaliella salina (Volvocales, Chlorophyta) against experimentally induced fibrosarcoma on wistar rats

The beta-carotene-yielding microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne's medium was harvested and lyophilized. Fibrosarcoma was induced in rats by 20-methylcholanthrene. 0.5 g and 1.0 g of lyophilized D. salina powder was administered to the rats orally through carboxy methyl cellulose. Cisplatin was administered along with vitamin E to compare the protective effect of D. salina against fibrosarcoma. Administration of D. salina decreased the levels of cholesterol and lactate dehydrogenase as well as the activities of catalase, superoxide dismutase, serum aspartate aminotransaminase, serum alanine aminotransferase, when compared to control. A significant reduction in the levels of hepatic and renal RNA and DNA was observed in the sarcoma rats when treated with D. salina powder. Histopathological studies of tumor tissues showed regenerative and regressive changes. beta-carotene globules isolated from the powder of Dunaliella salina confirmed the presence of 9-cis-beta-carotene and all-trans-beta-carotene.     

Combined action of paraquat and superoxide on the peroxidation of detergent-dispersed linolenic acid.

We investigated the peroxidative effect of paraquat and active oxygens on detergent-dispersed linolenic acid in phosphate buffer (pH 7.5) from the malondialdehyde (MDA) level. Our complete system and further inclusion of catalase were effective in stimulating MDA formation. On the other hand, xanthine oxidase (XOD) or paraquat omission, superoxide dismutase (SOD) inclusion or anaerobic incubation inhibited the formation of MDA. Ferrous ion was weakly associated with phosphate of the buffer, forming a complex, and the release of ferrous ion from the complex intensified the MDA levels with the complete and catalase inclusion systems. The electron paramagnetic resonance (EPR) spectra using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that superoxide, produced immediately after the addition of XOD, played a crucial role. We could obtain a DMPO-OOH signal at the starting stage whenever MDA stimulation was observed. The omission of paraquat, however, produced no increase in MDA level in spite of an appearance of DMPO-OOH signal, indicating that paraquat also plays an important role. On the other hand, Desferal, a ferric chelator, showed a concentration-dependent inhibition effect. There was an immediate strong intensity of DMPO-OOH and paraquat signals. We did not, however, observe MDA stimulation at 250 microM Desferal, which confirms that ferrous ion plays an essential role in the lipid peroxidation. These results indicate a combined action of paraquat (or its radical) and superoxide on the accessibility of ferrous ion, including its release from the complex with phosphate, which may be an endogenous chelator. The possibility of ternary complex participation is also discussed.     

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.     

Comparative study of the enzymatic defense systems against oxygen-derived free radicals: the key role of glutathione peroxidase

Human WI-38 diploid fibroblasts have been cultivated under high toxic O2 pressure, and their survival curves are reported. Superoxide dismutase, catalase, or glutathione peroxidase provided some protection when injected in the cells exposed to O2. This protective effect, recorded after 3 or 4 days of incubation, was the most pronounced when cells were injected just before oxygen exposure. Quantitative injection assays have been performed for the three enzymes. Surprisingly, glutathione peroxidase was found to be much more effective than both catalase and superoxide dismutase, the latter being particularly inefficient.     

Comparison of the effects of the superoxide dismutase mimetics EUK-134 and tempol on paraquat-induced nephrotoxicity

Paraquat-induced nephrotoxicity involves severe renal cell damage caused by reactive oxygen species (ROS), specifically via increasing concentrations of superoxide anions in the kidney. Recently, superoxide dismutase (SOD) mimetics (SODm) have been developed that display safe SOD activities but which also possess additional antioxidant enzyme (e.g., catalase) or ROS-scavenging activities. The aim of this study was to compare the effects of two such SODm, specifically, EUK-134, a SODm with catalase activity, and tempol, a SODm with ROS-scavenging properties, on paraquat-induced nephrotoxicity of renal NRK-52E cells. Incubation with paraquat (1 mM) for 24 h reduced cell viability and increased necrosis significantly. Paraquat also generated significant quantities of superoxide anions and hydroxyl radicals. Both EUK-134 (10-300 microM) and tempol (0.3-1.0 mM) were able to improve cell viability and reduced paraquat-induced cell death significantly via dismutation or scavenging of superoxide anions and reduced hydroxyl radical generation. The data presented here suggest that SODm such as EUK-134 and tempol, which possess additional catalase and/or ROS-scavenging activities, can significantly reduce renal cell damage caused by paraquat. These effects were evident at concentrations which avoid the pro-oxidant activities associated with higher concentrations of SOD. Such SODm could therefore prove to be beneficial as therapies for paraquat nephrotoxicity.     

The role of oxygen radicals in dye-mediated photodynamic effects in Escherichia coli B

Photosensitive dyes representative of the thiazines, xanthenes, acridines, and phenazines mediated phototoxicity in Escherichia coli B. The observed phototoxicity was sensitizer-, light-, and oxygen-dependent and is therefore a photodynamic effect. Hydroxyl radical scavengers conferred protection against the photodynamic action of all of the representative dyes. The extent of protection was dependent on the concentration of scavenger and on the in vitro reactivity of the scavenger with the hydroxyl radical. Exogenous superoxide dismutase and catalase partially protected the cells against the dye-mediated phototoxicity, and prior induction of intracellular superoxide dismutase and catalase by growth in glucose minimal medium containing manganese and paraquat substantially protected E. coli B against the photodynamic action of all of the dyes examined. Combinations of protective treatments against the phototoxicity of all four classes of dyes, including superoxide dismutase and catalase preinduction and addition of extracellular superoxide dismutase and catalase or the addition of hydroxyl radical scavengers, provided nearly complete protection against the oxygen-dependent component of dye-mediated lethality. E. coli B grown in glucose minimal medium containing manganese and photosensitive dyes induced manganese superoxide dismutase. The extent of induction was correlated with the dyes' ability to photooxidize NADH in vitro. Thus, oxygen radicals are primarily responsible for the oxygen-dependent toxicity of the photosensitive dyes examined, and one adaptive response of E. coli B to a dye-mediated oxidative threat is to induce superoxide dismutase.     

Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique. the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase

Yeast glutathione reductase catalyzes an NADPH-dependent reduction of the herbicide paraquat in vitro. The single-electron reduced paraquat radical reacts with O2 to generate the superoxide radical, O2.-. Hydroxyl radicals (OH.) can also be detected in this assay system by their reaction with phenol to form diphenols, as assayed quantitatively by a highly specific and sensitive method employing gas-liquid chromatography. Formation of hydroxyl radicals can be virtually completely suppressed by catalase and partially suppressed by superoxide dismutase. The role of hydroxyl radicals and superoxide in paraquat toxicity in vivo is discussed.     

Dismutation of superoxide radicals by ceruloplasmin--details of the mechanism

Like superoxide dismutase (SOD), human ceruloplasmin (Cp) scavenges superoxide anion radicals injected into the solution with the aid a high-voltage generator, hydrogen peroxide being the product of reaction. The O2-/H2O2 ratio is close to 2:1. The dismutase activity of Cp is about 1500 times lower than that of Cu, Zn-SOD isolated from human erythrocytes. The dismutation of O2- accomplished by SOD, "free" copper ions, native Cp or partly copper-depleted Cp, is inhibited with equal efficiency by cyanide. All the copper ions of the multicopper catalytic center of Cp are not essentially required for the dismutation of O2-, since the enzyme depleted of all type 2 Cu2+ and partly of type 1 Cu2+ lost none of its dismutase activity. Type 1 copper ions of Cp seem to play the leading role in the one-electron transfer occurring upon dismutation of O2-.     

Photoinhibition of manganese enzymes: insights into the mechanism of photosystem II photoinhibition

Evidence has recently been presented that photoinhibition of photosystem II (PSII) is triggered by absorption of light by the oxygen-evolving manganese cluster. To get insight into the effects of light on enzymes containing manganese or other transition metal cofactors, the photosensitivities of Mn catalase, Mn superoxide dismutase, the haem (Fe)-containing bovine liver catalase, and CuZn superoxide dismutase were investigated. Glucose oxidase was studied as an example of an enzyme that does not have a metal cofactor. Sensitivities of these five enzymes to UVC, UVA, and visible light were compared in anaerobic conditions. The Mn(III)-oxo-Mn(III)-containing Mn catalase was found to be more sensitive to both visible and UV light than bovine liver catalase. Furthermore, the action spectrum of photoinhibition of Mn catalase was found to be fairly similar to that of photoinhibition of PSII. The Mn(II)-containing Mn superoxide dismutase was sensitive to UVC light and somewhat sensitive to UVA light, while only UVC light caused some inhibition of CuZn superoxide dismutase. Glucose oxidase was the least photosensitive of the enzymes studied. The photosensitivity of Mn enzymes supports the hypothesis that the oxygen-evolving manganese complex of PSII can be damaged by UV and visible light absorbed by its Mn(III) or Mn(IV) ions.     

Carbon tetrachloride toxicity on Escherichia coli exacerbated by superoxide

The effects of carbon tetrachloride (CCl4) and paraquat on the growth of Escherichia coli were investigated. Paraquat at 10 mM caused some inhibition of growth of E. coli in trypticase-soy-yeast extract medium. CCl4 enhanced growth inhibition by paraquat in a concentration-dependent manner. In the absence of paraquat, CCl4 had no effect on growth rate or on surviving cell numbers at stationary phase. CCl4 did not prevent the induction of manganese-superoxide dismutase by paraquat. Under anaerobic conditions, CCl4 and paraquat exhibited no effect on E. coli. In the presence of Mn(II) and paraquat, intracellular superoxide dismutase was markedly induced and protected E. coli against the toxicity of CCl4 and paraquat. The reactive free radical CCl3OO-, which can be formed from the reaction of O2- with CCl4, may cause cell damage. The growth-inhibiting effects of polyhalides in the presence of paraquat followed the order CBrCl3 greater than CCl4 greater than CHCl3 greater than CH2Cl2, which is in accord with that of the reaction rates of these compounds with O2- and with their hepatotoxicities. These results suggest that O2- plays a role in the hepatotoxicity of polyhalides.     

Induction of superoxide dismutases in Escherichia coli by manganese and iron.

Growth of Escherichia coli B in simple media enriched with Mn(II) resulted in the elevation of the manganese-containing superoxide dismutase, whereas growth in such medium enriched with iron caused increased content of the iron-containing superoxide dismutase. Enrichment of the medium with Co(II), Cu(II), Mo(VI), Zn(II), or Ni(II) had no effect. The inductions of superoxide dismutase by Mn(II) or by Fe(II) were dioxygen dependent, but these metals did not affect the CN- -resistant respiration of E. coli B and did not influence the increase in the CN- -resistant respiration caused by paraquat. Mn(II) and paraquat acted synergistically in elevating the superoxide dismutase content, and Mn(II) reduced the growth inhibition imposed by paraquat, E. coli grown in the complex 3% Trypticase soy broth (BBL Microbiology Systems)-0.5% yeast extract-0.2% glucose medium contained more superoxide dismutase than did cells grown in the simple media and were less responsive to enrichment of the medium with Mn(II) or Fe(II). Nevertheless, in the presence of paraquat, inductions of superoxide dismutase by these metals could be seen even in the Trypticase-yeast extract-glucose medium. On the basis of these observations we propose that the apo-superoxide dismutases may act as autogenous repressors and that Mn(II) and Fe(II) increase the cell content of the corresponding enzymes by speeding the conversion of the apo- to the holoenzymes.     

Catalase and superoxide dismutase in Escherichia coli

We assessed the roles of intrabacterial catalase and superoxide dismutase in the resistance of Escherichia coli to killing by neutrophils. E. coli in which the synthesis of superoxide dismutase and catalase were induced by paraquat 10-fold and 5-fold, respectively, did not resist killing by neutrophils. When bacteria were allowed to recover from the toxicity of paraquat for 1 h on ice and for 30 min at 37 degrees C, they still failed to resist killing by neutrophils. Induction of the synthesis of catalase 9-fold by growth in the presence of phenazine methosulfate did not render E. coli resistant to killing by either neutrophils or by H2O2 itself. The lack of protection by intrabacterial catalase from killing by neutrophils could not be attributed to an impermeable bacterial membrane; the evolution of O2 from H2O2 was no less rapid in suspensions of E. coli than in lysates. The failure of intrabacterial catalase or superoxide dismutase to protect bacteria from killing by neutrophils might indicate either that the flux of O-2 and H2O2 in the phagosome is too great for the intrabacterial enzymes to alter or that the site of injury is at the bacterial surface.     

Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants

An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide. Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E. coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide. This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins. Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity. Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.     

The effect of paraquat on the peroxide metabolism enzymes in erythrocytes of freshwater fish species

The toxic effects of 10 ppm paraquat in vivo on the enzymes superoxide dismutase (SOD), catalase (C), peroxidase (P), glutathione peroxidase (GSH-Px) and on lipid peroxidation (LP) were estimated in erythrocytes of the carp, the tench and the crucian carp. Paraquat caused activity enhancement of the peroxide metabolism enzymes and increase of the lipid peroxidation in the carp and the crucian carp. The enzyme activities and lipid peroxidation were dependent on the species and on the length of the exposure to paraquat.     

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.