Absence Of Superoxide Dismutase Research Articles

Superoxide and hydrogen peroxide induce CD18-mediated adhesion in the postischemic heart

A burst of endothelial derived oxidants including hydrogen peroxide (H 2O 2) and superoxide (·O 2 −) occurs on reperfusion of ischemic tissues that directly causes injury; however, it is not known if this also triggers further injury due to subsequent leukocyte adhesion and adhesion molecule expression. Therefore, studies were performed in an isolated heart model developed to enable study of the role of isolated cellular and humoral factors in the mechanism of postischemic injury. Isolated rat hearts were subjected to 20 min of 37°C-global ischemia followed by reperfusion with polymorphonuclear leukocytes (PMNs) and plasma in the presence or absence of superoxide dismutase (SOD), 200 U/ml, or catalase, 500 U/ml. Measurements of contractile function, coronary flow, high-energy phosphates, free radical generation, and PMN accumulation were performed. Adhesion molecule expression was measured on the surface of effluent PMNs by fluorescence flow cytometry and within the tissue using immunohistochemistry. SOD or catalase treatment resulted in 2- to 3-fold higher recoveries of contractile function, coronary flow, and high energy phosphates. EPR spin trapping measurements demonstrated that SOD totally quenched the free radical generation observed upon reperfusion while catalase prevented the formation of hydroxyl and alkyl radicals derived from superoxide. SOD or catalase treatment decreased PMN accumulation in the reperfused heart and prevented the marked upregulation of CD18 expression seen after reperfusion. These experiments demonstrate that in addition to their direct antioxidative actions, SOD and catalase each decrease PMN adhesion and CD18 expression resulting in marked suppression of PMN-mediated injury in the postischemic heart. Thus, endothelial derived H 2O 2 and ·O 2 − further amplify postischemic injury by triggering CD18 expression on the surface of PMNs leading to increased PMN adhesion within the heart.

Superoxide anion radical generation during the oxidation of various amines by diamine oxidase.

Diamine oxidase (DAO) or histaminase is an enzyme which deaminates histamine and several aliphatic amines to their corresponding aldehydes. Hydrogen peroxide and ammonia are side products of this reaction. The purpose of the present work was to evaluate if determination of produced hydrogen peroxide reflects DAO activity or if intermediate formation of the superoxide radical could be a reason for lack of correspondence between oxygen uptake and hydrogen peroxide production at different pH. Superoxide radical formation was determined by cytochrome c reduction in the presence and absence of superoxide dismutase (SOD). Oxygen uptake was measured with an oxygen electrode and hydrogen peroxide production by a spectrophotometric method. At pH 6.6 there was no superoxide production, but at pH 7.4 there was some, and it increased markedly at pH 9.5. Oxygen uptake also increased with increasing pH, especially with histamine as substrate. These results lead us to suggest that the mechanism of action of DAO involves the intermediate generation of superoxide radicals.

Impairment of endothelium-dependent dilation in rabbit renal arteries by oxidized lipoprotein(a). Role of oxygen-derived radicals.

Hyperlipoproteinemia is associated with impairment of nitric oxide (NO)-mediated, endothelium-dependent dilation in renal arteries. In the present study, we assessed and compared the effects of human lipoprotein(a) and LDL on endothelium-dependent and -independent dilation in vitro. Dilator responses were detected in isolated, saline-perfused, preconstricted arterial segments by a photoelectric device. Acetylcholine-induced, endothelium-dependent dilator responses of rabbit renal arteries were not significantly attenuated after 150 minutes of incubation with native lipoprotein(a) (30 and 100 micrograms/mL). However, exposure to in vitro oxidized lipoprotein(a) (150 minutes, 30 and 100 micrograms/mL) suppressed acetylcholine-induced dilator responses in a dose-dependent manner. At similar concentrations, native and oxidized LDL had no effect. Endothelium-independent dilations induced by the NO-donor sodium nitroprusside were also impaired by oxidized lipoprotein(a), whereas forskolin-induced dilator responses were unaffected, indicating that smooth muscle dilator capacity was not impaired. Attenuation of dilator responses by oxidized lipoprotein(a) was potentiated in the presence of superoxide dismutase (SOD). The SOD effect was completely blunted by coincubation with catalase (100 U/mL) or deferoxamine. In the absence of SOD, catalase or deferoxamine had no effect on dilator responses. Using a chemiluminescence assay, we could detect increased O2- production by arteries pretreated with oxidized lipoprotein(a), which suggested that enhanced NO inactivation by O2- could be the underlying mechanism for impairment of endothelium-dependent dilations. These data indicate that oxidized lipoprotein(a) impairs endothelium-dependent dilation and is more potent than oxidized LDL in this effect. The mechanism of the impairment may involve formation of O2- and inactivation of NO.

Superoxide contributes to the rapid inactivation of specific secondary donors of the photosystem II reaction center during photodamage of manganese-depleted photosystem II membranes.

The role of superoxide in the mechanism of photoinactivation of the secondary donors of the reaction center of photosystem II membranes depleted of Mn by extraction with NH2OH plus EDTA (NH2OH/EDTA-PSII) was assessed. EPR analyses (g = 2 region) in continuous light, optical kinetic spectrophotometric analyses of P680+ and Car+, and AT-band emission measurements were made after various durations of weak and strong light treatment of NH2OH/EDTA-PSII in the presence and absence of superoxide dismutase, or of PSII electron acceptors to suppress superoxide formation. Additionally, flash-induced variable fluorescence of chlorophyll a and the capabilities of the membranes of photooxidize Mn2+ (in the presence of H2O2) via a high-affinity site (Km approximately 180 nM) and to carry out the photoactivation of the Mn-cluster were determined. In the absence of any additions to the NH2OH/EDTA-PSII membranes which were highly depleted of Mn, weak light treatment caused rapid (t1/2 approximately 20 s) and parallel losses of (a) the approximately 10 microseconds phase of P680+ reduction, which reflects the TyrZ-->P680+ reaction, (b) the amplitude of chlorophyll a variable fluorescence, (c) the capability to accumulate the TyrZ(+)-radical in continuous light, and (d) the capability to photooxidize Mn2+/H2O2 in continuous light. As reported previously [Blubaugh et al. (1991) Biochemistry 30, 7586-7597], a dark-stable 12-G-wide featureless EPR signal centered at g = 2.004 was formed rapidly during illumination. This signal previously was tentatively identified as a Car+ radical and was suggested to contribute to the quenching of chlorophyll a variable fluorescence and to the slowing of the TyrZ-->P680+ reaction. However, we failed to detect Car+ formation by sensitive optical spectrophotometry and obtained no definable evidence for either a quencher of fluorescence other than P680+ itself or a slowing of the TyrZ-->P680+ reaction. Addition of a saturating concentration (96 units/mL) of superoxide dismutase diminished the rate of photodamage(s) by approximately 30-fold, but did not abolish it completely. Superoxide dismutase similarly suppressed strong light-induced photodamages, causing the loss of capability to photooxidize Mn2+/H2O2, to carry out photoactivation, and to generate the AT-band emission as well as TyrZ+ EPR signal. In contrast to others, we found no evidence that the initial target(s) of photodamage is (are) different in weak versus strong light treatment. The totality of the results suggests that the initial event in either weak light or strong light photodamage of NH2OH/EDTA-PSII is a decoupling of the redox activity of TyrZ from P680.(ABSTRACT TRUNCATED AT 400 WORDS)

Stabilization of Purified Human 5-Lipoxygenase with Glutathione Peroxidase and Superoxide Dismutase

Human 5-lipoxygenase (5LO) becomes very unstable after purification. Commonly used methods for protein stabilization could not prevent this inactivation. However, addition of small amounts of glutathione peroxidase (0.15 μg/ml) and superoxide dismutase (1 μg/ml) to the solution of purified 5LO (300-500 μg/ml) stabilized the enzyme during storage. The protected 5LO maintained full activity for at least 12 days at 25°C, while 50% of the activity was lost within 10 h without protection. Glutathione peroxidase alone also preserved the activity of 5-lipoxygenase; however, the effect declined rapidly in the absence of superoxide dismutase. 2-Mercaptoethanol was the most efficient hydrogen donor substrate for glutathione peroxidase in the protection of 5LO. Catalase was less effective as a stabilizing agent, and ebselen, a synthetic glutathione peroxidase-mimicking compound, did not protect 5LO. Since many metal ion binding proteins are susceptible to H 2O 2 inactivation, this method could be useful also for the stabilization of other proteins.

Characterization of the electron acceptors of old yellow enzyme: mechanistic approach to the mode of one electron transfer from the enzyme to menadione or dyestuffs.

Molecular oxygen or cytochrome c has been described as the electron acceptor of the reaction of old yellow enzyme with NADPH. In this study, menadione was found to be a sensitive electron acceptor of the reaction under aerobic as well as anaerobic conditions. The Km value of menadione for old yellow enzyme is as low as 2-3 x 10(-7) M in the presence or absence of superoxide dismutase. The rate enhancement of the cytochrome c reduction of old yellow enzyme with NADPH was about eight times in the presence of menadione. The rate increment was slightly higher under aerobic than anaerobic conditions. The rate enhancement by menadione enabled sensitive determination of the enzyme activity in the assay system, which contained NADPH, cytochrome c, menadione, and old yellow enzyme. In the reaction course, the semiquinone species of menadione was trapped by the reaction with t-butyl-alpha-phenylnitrone. The radical adduct was detected on EPR. The dyestuff, 2,6-dichlorophenolindophenol, was found to be reduced ineffectively even in the presence of menadione; moreover, it was inhibitory in the NADPH consumption reaction. Methylene blue or Lauth's violet, known to be capable of semiquinone formation, also behaved, like menadione, as a mediator of electron transport to cytochrome c. On the basis of the experimental results, the occurrence of the one electron transfer of the old yellow enzyme reaction was emphasized.

Caco-2 cell metabolism of oxygen-derived radicals.

Reactive oxygen metabolites have been associated with gastrointestinal injury and may play a role as mediators of inflammation. The effect of oxygen metabolites on Caco-2 cell viability (trypan blue exclusion and 51Cr release), hexose monophosphate shunt activity, and glutathione was assessed. Caco-2 cells were incubated with amino acid oxidase, xanthine oxidase, menadione, and t-butylhydroperoxide in the presence and absence of superoxide dismutase, catalase, mannitol, and butylated hydroxytoluene. With amino acid oxidase, trypan blue exclusion decreased (P < 0.01) and 51Cr release, oxidized glutathione, and shunt activity increased (P < 0.05). The addition of catalase attenuated these changes. Trypan blue exclusion decreased (P < 0.005) and 51Cr release, oxidized glutathione, and shunt activity increased (P < 0.01) with xanthine oxidase. The addition of superoxide dismutase caused a further increase in 51Cr release, oxidized glutathione, and shunt activity (P < 0.01), which was prevented by the addition of catalase or mannitol. t-Butylhydroperoxide did not effect 51Cr release or trypan blue exclusion, but oxidized glutathione and shunt activity increased (P < 0.01). The increase in shunt activity was prevented by preincubation with butylated hydroxytoluene (P < 0.01). Menadione did not alter trypan blue exclusion or 51Cr release, but caused an increase in oxidized glutathione and shunt activity (P < 0.001). The increase in shunt activity was attenuated by preincubation with butylated hydroxytoluene (P < 0.001). Menadione also caused a depletion of total glutathione.(ABSTRACT TRUNCATED AT 250 WORDS)

Inverse relationship between the induction of human sperm capacitation and spontaneous acrosome reaction by various biological fluids and the superoxide scavenging capacity of these fluids.

Capacitation of spermatozoa is essential for fertilization, and can be induced by various agents or biological fluids. Previous reports have shown that foetal cord serum (FCS) and the superoxide anion trigger human sperm hyperactivation and capacitation, and that superoxide dismutase (SOD) prevents these processes. We investigated: (1) the capacity of seminal plasma (SP) and follicular fluid (FF) (whole, or fractionated into high and low molecular weight components), in the presence or absence of SOD, to induce the spontaneous acrosome reaction (no stimulant needed, AR) and capacitation (as measured by the lysophosphatidyl-choline-induced AR, LPC-AR); (2) a possible relationship between the levels of AR and capacitation obtained with these biological fluids and the superoxide scavenging capacity of the same fluids. The highest levels of LPC-AR were obtained with FF ultrafiltrate (48 +/- 6%), followed by SP ultrafiltrate (31.9 +/- 0.8%), FF (30 +/- 5%), dialysed FF (27 +/- 4%), and finally, by FCS ultrafiltrate (23 +/- 1%), SP (21 +/- 1%) and dialysed SP (18.9 +/- 0.8%). A similar order of potency for the fluids existed when sperm AR was studied, the levels of AR observed ranging from 16 +/- 2% to 5.3 +/- 0.8% after incubation with FF ultrafiltrate and SP respectively. None of these treatments had detrimental effects on sperm motility. In the presence of SOD, there was always an important reduction (52-86%) of the AR and LPC-AR observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat enterocyte injury by oxygen-dependent processes

Neutrophils migrate to areas of inflammation and, when stimulated, produce O2−, H2O2, and other reactive O2 metabolites. To assess the effects of stimulated neutrophils on enterocytes, rat enterocytes were incubated with peripheral neutrophils. To assess cell viability, trypan blue exclusion and lactate dehydrogenase and protein release were measured. When 106 enterocytes/mL were incubated with 2.5 × 105 neutrophils/mL stimulated with phorbol myristate acetate, trypan blue exclusion decreased and lactate dehydrogenase and protein release increased. With the addition of 0.10 mg/mL of Superoxide dismutase, trypan blue exclusion further decreased and lactate dehydrogenase and protein release increased. This suggests that H2O2- or H2O2/ O2 -derived metabolites are more damaging to isolated enterocytes than O2−1. To test this hypothesis, enterocytes were incubated with xanthine and increasing concentrations of xanthine oxidase in the presence and absence of Superoxide dismutase. With increasing concentrations of xanthine oxidase, the cell number decreased and protein release increased. With the addition of Superoxide dismutase, fewer cells were present, suggesting that cell lysis occurred. Protein release was further increased by the addition of Superoxide dismutase. Enterocytes were then incubated with leucine and increasing concentrations of amino acid oxidase. With increasing concentrations of amino acid oxidase, trypan blue exclusion decreased and protein and lactate dehydrogenase release increased. These effects were ameliorated by the addition of 500 IU catalase/mL. These data suggest that O2− and H2O2, whether created by stimulated neutrophils or an enzymegenerating system, are damaging to isolated enterocytes. Superoxide dismutase did not offer enterocytes protection.

QUINONE SENSITIZED ELECTRON TRANSFER PHOTOOXIDATION OF NUCLEIC ACIDS: CHEMISTRY OF THYMINE AND THYMIDINE RADICAL CATIONS IN AQUEOUS SOLUTION*

The 2-methyl-1,4-naphthoquinone (MQ) sensitized photooxidation of nucleic acid derivatives has been studied by laser flash photolysis and steady state methods. Thymine and thymidine, as well as other DNA model compounds, quench triplet MQ by electron transfer to give MQ radical anions and pyrimidine or purine radical cations. Although the pyrimidine radical cations cannot be directly observed by flash photolysis, the addition of N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD) results in the formation of the TMPD radical cation via scavenging of the pyrimidine radical cation. The photooxidation products for thymine and thymidine are shown to result from subsequent chemical reactions of the radical cations in oxygenated aqueous solution. The quantum yield for substrate loss at limiting substrate concentrations is 0.38 for thymine and 0.66 for thymidine. The chemistry of the radical cations involves hydration by water leading to C(6)-OH adduct radicals of the pyrimidine and deprotonation from the N(1) position in thymine and the C(5) methyl group for thymidine. Superoxide ions produced via quenching of the quinone radical anion with oxygen appear to be involved in the formation of thymine and thymidine hydroperoxides and in the reaction with N(1)-thyminyl radicals to regenerate thymine. The effects of pH were examined in the range pH 5-8 in both the presence and absence of superoxide dismutase. Initial C(6)-OH thymine adducts are suggested to dehydrate to give N(1)-thyminyl radicals.

Role of oxygen radicals in phenazine mediated ATP synthesis obtained with spinach chloroplasts: Toxicity of OH • radicals

Superoxide anions and hydroxyl radicals are produced during the reduction of oxygen by reduced phenazine methosulphate or pyocyanine. Chloroplasts synthesize ATP when this reaction occurs. It is shown that orthophosphate, in the presence or absence of superoxide dismutase, increases the dismuta- tion of superoxide anions with a concomitant production of hydroxyl radicals. Superoxide dismutase was found to inhibit the ATP synthesis obtained with chloroplasts in darkness. In contrast, it is shown that superoxide dismutase with catalase, which inhibit the production of hydroxyl radicals, enhance ATP synthesis. Similarly the presence of methional, a scavenger of hydroxyl radicals, increases ATP synthesis. The possible participation of the superoxide anion in orthophosphate dehydroxylation, and the inhibitory action of the hydroxyl radical on phosphorylation, are discussed.

Role of oxygen radicals in phenazine mediated ATP synthesis obtained with spinach chloroplasts: Toxicity of OH radicals

Superoxide anions and hydroxyl radicals are produced during the reduction of oxygen by reduced phenazine methosulphate or pyocyanine. Chloroplasts synthesize ATP when this reaction occurs. It is shown that orthophosphate, in the presence or absence of superoxide dismutase, increases the dismutation of superoxide anions with a concomitant production of hydroxyl radicals. Superoxide dismutase was found to inhibit the ATP synthesis obtained with chloroplasts in darkness. In contrast, it is shown that superoxide dismutase with catalase, which inhibit the production of hydroxyl radicals, enhance ATP synthesis. Similarly the presence of methional, a scavenger of hydroxyl radicals, increases ATP synthesis. The possible participation of the superoxide anion in orthophosphate dehydroxylation, and the inhibitory action of the hydroxyl radical on phosphorylation, are discussed.

The properties of reducing agents released by treatment of Solanum tuberosum with elicitors from Phytophthora infestans

Tuber and leaf discs of Solanum tuberosum were treated with germination fluid from Phytophthora infestans cystospores and with several biotic and abiotic elicitors. The subsequent production of reducing agents was determined by the ability of the various treated tuber and leaf discs to reduce cytochrome c. The occurrence of superoxide (O 2 −) among the reducing agents was determined by measuring the reduction of cytochrome c in the presence and absence of superoxide dismutase (SOD). Although several of the treatments induced the production of reducing agents, very little superoxide was detected in any of the treatments. Reducing agents were present when the discs were treated with germination fluid. Control experiments revealed that these reducing agents were derived solely from the germination fluid in levels comparable to those found in the absence of tuber or leaf discs. The reducing agents in the germination fluid were found to be large molecules (> 10000 mol. wt units), and their ability to reduce cytochrome c was enhanced by boiling or by treatment with NADH. The treatment of tuber discs with arachidonic acid, digitonin, or AgNO 3 was shown to induce the production of reducing agents. Nearly identical levels of reducing agents were induced by treatment of tuber discs with fatty acids other than arachidonic acid (oleic, linolenic, and eicosapentaenoic acids). The presence of calcium had very little effect on the induction of reducing agents by tuber discs. When leaf discs were also treated with various elicitors, only digitonin and AgNO 3 induced the production of reducing agents. These results do not support the hypothesis that superoxide is involved in the resistance interaction between S. tuberosum and P. infestans.

Photosensitization with α-terthienyl: The formation of superoxide ion in aqueous media

It is well known that α-terthienyl generates singlet oxygen in organic solvents with high quantum yields. In an aqueous medium, the production of superoxide radical-anion is readily detected by comparing the reduction of ferricytochrome c or nitro blue tetrazolium in the presence and in the absence of superoxide dismutase. Electron transfer reactions from the electronically excited sensitizer are also detected in an argon atmosphere.

Auto-oxidation of dialuric acid, divicine and isouramil: Superoxide dependent and independent mechanisms

The toxicity of dialuric acid to pancreatic β cells, and the haemolytic action of divicine and isouramil involve auto-oxidation and redox cycling reactions. Divicine and isouramil are produced on hydrolysis of the fava bean glycosides, vicine and convicine. The mechanism of auto-oxidation of the three compounds as well as the acid hydrolysis product of vicine (provisionally assigned the structure 2-amino-4,5,6-trihydroxypyrimidine) has been studied. All four pyrimidines auto-oxidized rapidly at neutral pH, generating H 2O 2 by an O 2 −-dependent chain mechanism. Superoxide dismutase inhibited the initial oxidation, but inhibition was transitory, and after a lag period rapid oxidation occurred. The lag period varied with pH, temperature and pyrimidine concentration, and was much shorter for isouramil and divicine than for dialuric acid and acid-hydrolysed vicine. The initial rate of dialuric acid oxidation was greater and the acceleration less pronounced than with the other pyrimidines. A mechanism common to all four pyrimidines has been shown by kinetic analysis to account for nearly all the observations in the presence and absence of superoxide dismutase. Autocatalysis in the latter case is attributed mainly to the reactions reduced pyrimidine + oxidized pyrimidine ai 2 pyrimidine radical pyrimidine radical + O 2 → oxidized pyrimidine + O 2 − Rate constants for these and other reactions are reported. At pH7.4 and 37° the lag period before 100 μM acid-hydrolysed vicine underwent rapid oxidation was approx. 15 min. Isouramil and divicine at an equivalent concentration gave lags of < 1 min, which became less at higher concentrations. Thus intracellular superoxide dismutase should provide only transitory protection against the oxidation products of dialuric acid, divicine or isouramil. Prolonged protection should only be achieved if accumulation of oxidized pyrimidine is also prevented.

A kinetic model of superoxide production from single pulmonary alveolar macrophages

A kinetic model was developed to describe the production of superoxide (O2-) by single pulmonary alveolar macrophages (PAM). Model predictions were compared with experimental results obtained from single rat PAM. The O2- was quantified by measuring the reduction of nitro blue tetrazolium (NBT) to a diformazan precipitate (NBTH2) from video-recorded images of individual cells. The kinetic model considered three reactions: 1) the production of extracellular O2- from the reduction of oxygen by NADPH oxidase using intracellular NADPH as the substrate, 2) the subsequent dismutation of O2- to form H2O2, and 3) the reaction of O2- and NBT to form diformazan. NBT specificity for O2- was analyzed by comparing results in the presence and absence of superoxide dismutase (SOD) that catalyzes the dismutation of O2- to H2O2. Measured PAM heterogeneity was accounted for by varying the concentration of intracellular NADPH, its rate of depletion, and the concentration of intracellular NADPH oxidase in the kinetic model. Model predictions compared favorably with experimental results except when SOD was present. This discrepancy may be due to diffusional limitations because NBT is a relatively small molecule (818 mol wt) compared with SOD (34,000 mol wt). In addition, the cell surface is both ruffled and negatively charged, which may introduce steric hindrances and/or electrostatic effects, since SOD is also negatively charged, whereas NBT is positively charged.

Detection of oxygen radicals during reperfursion of intestinal cell in vitro

The nitroxide OXANO· (2-Ethyl-2,5,5-trimethyl-3-oxazolidinoxyl) which in its reduced form, OXANOH (2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine), is capable of reacting with short-lived radicals, forming a secondary stable radical, was used for ESR-detection of radical production in isolated cells. The properties of OXANO· and OXANOH in terms of stability in cellular and subcellular systems, membrane permeability and effects on cellular viability were evaluated. Ischemia and reperfusion was simulated in vitro in a preparation of cells from rat intestinal mucosa by incubation at high density (4 × 10 8 cells/ml) under an atmosphere of nitrogen for 25 min and resuspended with fresh oxygenated buffer containing 5 mM Oxanoh. A significant increase in radical formation during the 15 min reperfusion period studied was obtained in cells exposed to ischemia compared to control cells incubated at normal density under an atmosphere of oxygen . The addition of 5 μM of the scavenging enzyme superoxide dismutase reduced the radical formation by 50%. The sequence of the superoxide formation was calculated as the difference in radical production in the presence and absence of superoxide dismutase.

Glutathione-mediated redox cycling of alloxan: Mechanisms of superoxide dismutase inhibition and of metal-catalyzed OH formation

The mechanism of the reaction between alloxan and GSH has been studied in the presence and absence of Superoxide dismutase. Excess GSH reduced alloxan to dialuric acid, which underwent subsequent autoxidation, thus establishing a redox cycle in which O 2 and GSH in excess of the alloxan concentration were consumed. The major reaction products were H 2O 2 and GSSG. At each cycle, a small fraction of the alloxan reacted with GSH to form a 305 nm-absorbing adduct that gradually accumulated. In the presence of SOD, alloxan was reduced by GSH, but increasing concentrations of GSH progressively inhibited redox cycling as shown by decreased rates of O 2 uptake and GSH oxidation. With GSH: alloxan or dialuric acid molar ratios of >8–10:1, redox cycling was almost completely suppressed. A mechanism based on known reactions of GSH and dialuric acid is proposed. Alloxan and GSH, with an iron chelate present as catalyst, caused the hydroxylation of salicylate, an indicator of hydroxyl radical production. Hydroxylation was inhibited by catalase but not by Superoxide dismutase, and it is attributed to the Fenton reaction in which the ferric catalyst is reduced by dialuric acid.

The effects of dihydroxyfumarate on isolated rabbit papillary muscle function: evidence for an iron dependent non-hydroxyl radical mechanism.

To delineate the active free radical species mediating the toxic effects of autoxidizing dihydroxyfumarate (DHF), isolated rabbit right ventricular papillary muscles were exposed to 4.5 mM DHF in the presence of FeCl3, ADP and bovine albumin. In the absence of free radical scavengers a 47.3 +/- 11.5% (mean +/- standard deviation) depression in contractile force was noted over 60 minutes. Neither the combination of superoxide dismutase (SOD) 3,200 u/cc and catalase (CAT) 2,950 u/cc nor mannitol 0.1 M provided statistically significant protection. Deferoxamine mesylate (DFX) 10 mg/cc (15 mM) did provide significant protection of muscle function both in the presence and absence of SOD and CAT (p less than 0.01). The degree of protection conferred by DFX alone was statistically similar to that of DFX with SOD and CAT. This data suggests the involvement of an iron-oxygen complex not dependent on superoxide or hydrogen peroxide for its formation and not readily scavenged by mannitol. The perferryl ion may be representative of such a species. Alternatively, a reactive complex similar to the 'Crypto-OH' radical proposed by Youngman may be formed by the reaction of DHF with iron and oxygen.

Superoxide dismutase and oxygen toxicity defenses in the genus Neisseria.

Among aerotolerant cells, Neisseria gonorrhoeae is very unusual because despite its obligately aerobic lifestyle and frequent isolation from purulent exudates containing polymorphonuclear leukocytes vigorously evolving O2- and H2O2, it contains no superoxide dismutase (SOD). Strains (14) of N. gonorrhoeae were compared with each other and with strains of Neisseria meningitidis, Neisseria mucosa, and Neisseria subflava under identical growth conditions for their contents of the oxy-protective enzymes catalase, peroxidase, and SOD, as well as respiratory chain proteins and activity. The absence of SOD from N. gonorrhoeae strains was demonstrated under a variety of oxygen-stress conditions. The neisserial species showed very different SOD, catalase, and peroxidase profiles. These profiles correlated well with the tolerance of the species to various intra- and extracellular oxygen insults. The high tolerance of N. gonorrhoeae for extracellular O2- and H2O2 appeared to be due to very high constitutive levels of peroxidase and catalase activity combined with a cell envelope impervious to O2-. Nevertheless, N. gonorrhoeae 19424 was much more sensitive to an intracellular flux of O2- than were the other (SOD-containing) neisserial species. The responses of N. gonorrhoeae and N. meningitidis respiratory and oxy-protective enzymes to growth under high and low oxygen tensions were followed, and a novel response, the apparent repression of the respiratory chain intermediates, respiration, and SOD, peroxidase, and catalase activity, was observed. The gonococcal catalase was partially purified and characterized. The results suggest that the very active terminal oxidase, low pO2 natural habitat, O2-stable catalase, and possibly the high glutathione content of the organism explain its aerobic survival in the absence of SOD.