Regulation of the synthesis of superoxide dismutases in rat lungs during oxidant and hyperthermic stresses

Heat shock proteins are induced at normal temperatures by oxidants and during reoxygenation following hypoxia. We now report cyanide-resistant O2 consumption increased 30-50% in rat lungs exposed to heat shock or reoxygenation following hypoxia. The synthesis of Cu,Zn superoxide dismutase, but not Mn superoxide dismutase, was increased in rat lung slices by in vivo hyperthermia (39 degrees C), by in vitro heat shock (41 degrees C), and during incubation of lung slices with the Cu chelator diethyldithiocarbamate, which decreased the activity of Cu,Zn superoxide dismutase. The heat shock-induced increase in Cu,Zn superoxide dismutase developed 2 h later than the induction of heat shock proteins and was not blocked by actinomycin D. The rates of synthesis of both superoxide dismutases were decreased 50% by hypoxia and failed to increase during reoxygenation. During hypoxia the activity of Cu,Zn superoxide dismutase decreased about 50%, but the activity of Mn superoxide dismutase remained unchanged. We conclude that hyperthermia increases the synthesis of Cu,Zn superoxide dismutase, the synthesis of Cu,Zn superoxide dismutase and Mn superoxide dismutase are not coordinately regulated by hyperthermia or by the oxidant stress produced by lowering the activity of Cu,Zn superoxide dismutase, and the synthesis of heat shock proteins and Cu,Zn superoxide dismutase are regulated at different levels of gene expression.     

Oxygen detoxification in the strict anaerobic archaeon Archaeoglobus fulgidus: superoxide scavenging by neelaredoxin

Archaeoglobus fulgidus is a hyperthermophilic sulphate-reducing archaeon. It has an optimum growth temperature of 83 degrees C and is described as a strict anaerobe. Its genome lacks any homologue of canonical superoxide (O2.-) dismutases. In this work, we show that neelaredoxin (Nlr) is the main O2.- scavenger in A. fulgidus, by studying both the wild-type and recombinant proteins. Nlr is a 125-amino-acid blue-coloured protein containing a single iron atom/molecule, which in the oxidized state is high spin ferric. This iron centre has a reduction potential of +230 mV at pH 7.0. Nitroblue tetrazolium-stained gel assays of cell-soluble extracts show that Nlr is the main protein from A. fulgidus which is reactive towards O2.-. Furthermore, it is shown that Nlr is able to both reduce and dismutate O2.-, thus having a bifunctional reactivity towards O2.-. Kinetic and spectroscopic studies indicate that Nlr's superoxide reductase activity may allow the cell to eliminate O2.- quickly in a NAD(P)H-dependent pathway. On the other hand, Nlr's superoxide dismutation activity will allow the cell to detoxify O2.- independently of the cell redox status. Its superoxide dismutase activity was estimated to be 59 U mg-1 by the xanthine/xanthine oxidase assay at 25 degrees C. Pulse radiolysis studies with the isolated and reduced Nlr proved unambiguously that it has superoxide dismutase activity; at pH 7.1 and 83 degrees C, the rate constant is 5 x 106 M-1 s-1. Besides the superoxide dismutase activity, soluble cell extracts of A. fulgidus also exhibit catalase and NAD(P)H/oxygen oxidoreductase activities. By putting these findings together with the entire genomic data available, a possible oxygen detoxification mechanism in A. fulgidus is discussed.     

Generation of hydrogen peroxide during the oxidation of L-phenylalanine by Proteus mirabilis isolated membranes

In the process of L-phenylalanine oxidation by Proteus mirabilis cytoplasmic membrane, hydrogen peroxide was produced at a rate corresponding to 1-3 per cent of the total electron flow (30-110 nmoles min-1mg-1). Peroxide was estimated using a fluorimetric assay with horseradish peroxidase, or by anodic oxidation on a platinum electrode. When using the former method, superoxide dismutase decreased the apparent yield of peroxide, a fact suggesting that H2O2 was in part the dismutation product of superoxide radicals. However the superoxide dismutase effect could be an artefact due to the generation of some superoxide during the peroxidatic reaction in the assay. Adrenaline was the reagent used for the detection of superoxide. There was no significant emergence of superoxide as the result of phenylalanine oxidation by the membrane (specific activity lower than 1-2 nmoles min-1mg-1). Thus it seemed that superoxide was not an intermediate for the bulk of H2O2 formed in this system. According to the results, peroxide was probably formed at a stage of electron transport earlier than the cytochrome level. The membrane phenylalanine dehydrogenase could be a site where peroxide was evolved in these experiments.     

The rate of reaction of superoxide radical ion with oxyhaemoglobin and methaemoglobin.

Superoxide radical ions (O2-) produced by the radiolytic reduction of oxygenated formate solutions and by the xanthine oxidase-catalysed oxidation of xanthine were shown to oxidize the haem groups in oxyhaemoglobin and reduce those in methaemoglobin as in reactions (1) and (2): (see articles) Reaction (1) is suppressed by reaction (8) when [O2-]exceeds 10 muM, but consumes all the O2- generated in oxyhaemoglobin solutions when [oxyhaemoglobin] greater than 160 muM and [O2-]less than 1 nM at pH 7. The yield of reaction (2) is also maximal in methaemoglobin solutions under similar conditions, but less than one haem group is reduced per O2- radical. From studies of (a) the yield of reactions (1) and (2) at variable [haemoglobin] and rates of production of O2-, (b) their suppression by superoxide dismutase, and (c) equilibria observed with mixtures of oxyhaemoglobin and methaemoglobin, it is shown that k1/k2=0.7 +/- 0.2 and k1 = (4 +/- 1) X 10(3) M-1-S-1 At pH7, and k1 and k2 decrease with increasing pH. Concentrations and rate constants are expressed in terms of haem-group concentrations. Concentrations of superoxide dismutase observed in normal erythrocytes are sufficient to suppress reactions (1) and (2), and hence prevent the formation of excessive methaemoglobin.     

Superoxide dismutase and Fenton chemistry. Reaction of ferric-EDTA complex and ferric-bipyridyl complex with hydrogen peroxide without the apparent formation of iron(II).

A ferric-EDTA complex, prepared directly from FeCl3 or from an oxidized ferrous salt, reacts with H2O2 to form hydroxyl radicals (.OH), which degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, hydroxylate benzoate to form fluorescent dihydroxy products and react with 5,5-dimethylpyrrolidine N-oxide (DMPO) to form a DMPO-OH adduct. Degradation of deoxyribose and benzoate and the hydroxylation of benzoate are substantially inhibited by superoxide dismutase and .OH-radical scavengers such as formate, thiourea and mannitol. Inhibition by the enzyme superoxide dismutase implies that the reduction of the ferric-EDTA complex for participation in the Fenton reaction is superoxide-(O2.-)-dependent, and not H2O2-dependent as frequently implied. When ferric-bipyridyl complex at a molar ratio of 1:4 is substituted for ferric-EDTA complex (molar ratio 1:1) and the same experiments are conducted, oxidant damage is low and deoxyribose and benzoate degradation were poorly if at all inhibited by superoxide dismutase and .OH-radical scavengers. Benzoate hydroxylation, although weak, was, however, more effectively inhibited by superoxide dismutase and .OH-radical scavengers, implicating some role for .OH. The iron-bipyridyl complex had available iron-binding capacity and therefore would not allow iron to remain bound to buffer or detector molecules. Most .OH radicals produced by the iron-bipyridyl complex and H2O2 are likely to damage the bipyridyl molecules first, with few reacting in free solution with the detector molecules. Deoxyribose and benzoate degradation appeared to be mediated by an oxidant species not typical of .OH, and species such as the ferryl ion-bipyridyl complex may have contributed to the damage observed.     

Interactions between metals, ligands, and oxygen in the autoxidation of 6-hydroxydopamine: mechanisms by which metal chelation enhances inhibition by superoxide dismutase

Transition metal ions and superoxide participate in different autoxidations to a variable extent. In the reaction of 6-hydroxydopamine (6-OHDA) with oxygen at pH 7.0 or 8.0, addition of 5 to 300 U/ml superoxide dismutase inhibited autoxidation by up to 96% at the highest concentrations. Superoxide dismutase at concentrations of 5-20 U/ml inhibited by less than 40% when present alone, but inhibited by over 99% in the presence of desferrioxamine or histidine. EDTA also enhanced the inhibition by 20 U/ml superoxide dismutase to 86%, even though EDTA accelerated the autoxidation of 6-OHDA when present alone or with desferrioxamine. In contrast, other ligands, such as ADP or phytic acid, had little or no effect on inhibition by superoxide dismutase. Proteins such as albumin, cytochrome oxidase, or denatured superoxide dismutase also enhanced inhibition by active superoxide dismutase from less than 40% to over 90%. Evidently, in the presence of redox active metals, autoxidation occurs by inner sphere electron transfer, presumably within a ternary 6-OHDA.metal.oxygen complex. This mechanism does not involve free O2-. and is not inhibited by superoxide dismutase. On the other hand, the presence of certain ligands (including proteins) diminishes the ability of trace metals to exchange electrons with 6-OHDA or oxygen by an inner sphere mechanism. These ligands render autoxidation dependent on propagation by O2-. and therefore inhibitable by superoxide dismutase. Previously conflicting reports that superoxide dismutase alone inhibits 6-OHDA autoxidation are thus explicable on the basis that at sufficient concentration the apoprotein coordinates trace metals in such a way to preclude inner sphere metal catalysis.     

Effects of superoxide dismutase, dithiothreitol and formate ion on the inactivation of papain by hydroxyl and superoxide radicals in aerated solutions.

Losses in enzyme activity and sulphydryl content have been studied in aerated papain solutions containing formate, superoxide dismutase and dithiothreitol. Both formate and dithiothreitol converted .OH to .O2-, whereas superoxide dismutase completely suppressed the inactivation by .O2-. Using results from all three systems, the fraction of .O2- reactions with papain that caused inactivation of the enzyme was 0.33 +/- 0.07. The results also showed that the fraction of .OH reactions, which cause inactivation of papain, is significantly higher in aerated than in oxygen-free solutions.     

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.     

Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.

Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.     

Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.     

Characteristics of mutagenesis by glyoxal in Salmonella typhimurium: contribution of singlet oxygen

The characteristics of mutagenesis by glyoxal in Salmonella tester strains TA100 and TA104, and particularly a possible role of active oxygen species, were investigated. Glyoxal was converted into a non-mutagenic chemical with glutathione (GSH) by glyoxalase I, and the mutagenic activity was enhanced by the depletion of intracellular GSH. Glyoxal caused the reduction of nitro blue tetrazolium, which was suppressed by the addition of 2,5-diphenylfuran, superoxide dismutase (SOD) and catalase (CAT), scavengers of singlet oxygen (1O2), superoxide radical (O2-) and hydrogen peroxide (H2O2), respectively. However, only the 1O2 scavenger almost completely suppressed the mutagenic activity of glyoxal. Mutagenicity assays using strains pretreated with N,N-diethyldithiocarbamate of a SOD inhibitor and strains with low levels of SOD and CAT indicated that the mutagenesis by glyoxal was independent of intracellular levels of SOD and CAT, though glyoxal itself repressed them. Therefore, all the results suggest that 1O2 formed from glyoxal is related to its mutagenesis, but that neither O2- nor H2O2 is intracellularly predominantly related to it. The action of glyoxal against SOD and CAT, and the formation of glyoxal adducts with amino acids as their components are also discussed.     

Superoxide release from interleukin-1B-stimulated human vascular cells: in situ electrochemical measurement.

Release of superoxide anion by cultured vascular cells was investigated with the use of selective microelectrodes. Local concentration of superoxide anion (O2*-) was followed by differential pulse amperometry on a carbon microfiber at 0.1 V/SCE. The oxidation current allows O2*- detection in the 10(-8) M concentration range without interference of the other major oxygen species. Interleukin-1beta-stimulated O2*- release that progressively increased to reach local concentrations at the cell membrane level of 76 +/- 11 nm 40-60 min after stimulation in human cord vein endothelial cells, and 131 +/- 18 nm 1-2 h after stimulation in internal mammary artery smooth muscle cells. In the two types of cells, the O2*- oxidation signal was suppressed in the presence of superoxide dismutase. Spontaneous O2*-release from unstimulated cells was undetectable. These results demonstrate that selective microelectrodes allow direct and real-time monitoring of local O2*- released from vascular endothelial as well as from smooth muscle cells submitted to an inflammatory stimulus.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes: evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 X g particulate fraction of the postnuclear supernatant obtained frm guinea-pig phagocytosing poymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O2- generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH. 2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1:1. 3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O2- generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation. 4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O2- formed during the oxidation of NADPH. 5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reactin at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH. 6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH. 7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible. 8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase. 9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O2; an enzymatic, apparently non-univalent, O2 reduction and a non-enzymatic chain reaction. 10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O2 is optimal at pH 7.0 with 1 mM NADPH.     

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-.     

Effect of superoxide dismutase on the autoxidation of various hydroquinones--a possible role of superoxide dismutase as a superoxide:semiquinone oxidoreductase

The autoxidation of DT-diaphorase-reduced 1,4-naphthoquinone, 2-OH-1,4-naphthoquinone, and 2-OH-p-benzoquinone is efficiently prevented by superoxide dismutase. This effect was assessed in terms of an inhibition of NADPH oxidation (over the amount required to reduce the available quinone), O2 consumption, and H2O2 formation. Superoxide dismutase also affects the distribution of molecular products -hydroquinone/quinone-involved in autoxidation, by favoring the accumulation of the reduced form of the above quinones. In contrast, the rate of autoxidation of DT-diaphorase-reduced 1,2-naphthoquinone is enhanced by superoxide dismutase, as shown by increased rates of NADPH oxidation, O2 consumption, and H2O2 formation and by an enhanced accumulation of the oxidized product, 1,2-naphthoquinone. These findings suggest that superoxide dismutase can either prevent or enhance hydroquinone autoxidation. The former process would imply a possible new activity displayed by superoxide dismutase involving the reduction of a semiquinone by O2-.. This activity is probably restricted to the redox properties of the semiquinones under study, as indicated by the failure of superoxide dismutase to prevent autoxidation of 1,2-naphthohydroquinone.     

Multiple actions of superoxide dismutase: why can it both inhibit and stimulate reduction of oxygen by hydroquinones?

Superoxide dismutase can either inhibit or stimulate autoxidation of different hydroquinones, suggesting multiple roles for O2.-. Inhibitory actions of superoxide dismutase include termination of O2.(-)-propagated reaction chains and metal chelation by the apoprotein. Together, chelation of metals and termination of O2.(-)-propagated chains can effectively prevent reduction of oxygen. Chain termination by superoxide dismutase can thus account for negligible accumulation of H2O2 without invoking a superoxide:semiquinone oxidoreductase activity for this enzyme. One stimulatory action of superoxide dismutase is to decrease thermodynamic limitations to reduction of oxygen. Whether superoxide dismutase inhibits or accelerates an autoxidation depends on the reduction potentials of the quinone and the availability of metal coordination for inner sphere electron transfers.     

The roles of superoxide anion and methylene blue in the reductive activation of indoleamine 2,3-dioxygenase by ascorbic acid or by xanthine oxidase-hypoxanthine

To clarify the roles of superoxide anion (O2.-) and methylene blue in the reductive activation of the heme protein indoleamine 2,3-dioxygenase, effects of xanthine oxidase-hypoxanthine used at various oxidase concentration levels as an O2.- source and an electron donor on the catalytic activity of the dioxygenase have been examined in the presence and absence of either methylene blue or superoxide dismutase using L- and D-tryptophan as substrates. In the absence of methylene blue, initial rates of the product N-formylkynurenine formation are enhanced in parallel with the xanthine oxidase level up to approximately 100 and approximately 50% of the apparent maximal activity (approximately 2 s-1) for L- and D-Trp, respectively. Superoxide dismutase effectively inhibits the reactions by 80-98% for both isomers. Additions of methylene blue (25 microM) help to maintain the linearity of the product formation that would be rapidly lost a few minutes after the start of the reaction without the dye, especially for L-Trp. Additions of methylene blue also enhance the activity to the maximal level for D-Trp. In the presence of methylene blue, the inhibitory effects of superoxide dismutase are considerably decreased with the increase in xanthine oxidase concentration, and at near maximal dioxygenase activity levels superoxide dismutase is totally without effect. In separate anaerobic experiments leuco-methylene blue, generated either by photoreduction or by ascorbate reduction, is shown to be able to reduce the ferric dioxygenase up to 25-40%. Substrate Trp and heme ligands (CO, n-butyl isocyanide) help to shift a ferric form----ferrous form equilibrium to the right. Thus, under aerobic conditions leuco-methylene blue might similarly be able to reduce the dioxygenase in the presence of an electron donor with the aid of substrate and O2. These results strongly suggest that indoleamine 2,3-dioxygenase can be activated through different pathways either by O2.- or by an electron donor-methylene blue system. For the latter case, the dye is acting as an electron mediator from the donor to the ferric dioxygenase.