Superoxide dismutase enhances the formation of hydroxyl radicals in the reaction of 3-hydroxyanthranilic acid with molecular oxygen.

Superoxide dismutase (SOD) enhanced the formation of hydroxyl radicals, which were detected by using the e.s.r. spin-trapping technique, in a reaction mixture containing 3-hydroxyanthranilic acid (or p-aminophenol), Fe3+ ions, EDTA and potassium phosphate buffer, pH 7.4. The hydroxyl-radical formation enhanced by SOD was inhibited by catalase and desferrioxamine, and stimulated by EDTA and diethylenetriaminepenta-acetic acid, suggesting that both hydrogen peroxide and iron ions participate in the reaction. The hydroxyl-radical formation enhanced by SOD may be considered to proceed via the following steps. First, 3-hydroxyanthranilic acid is spontaneously auto-oxidized in a process that requires molecular oxygen and yields superoxide anions and anthranilyl radicals. This reaction seems to be reversible. Secondly, the superoxide anions formed in the first step are dismuted by SOD to generate hydrogen peroxide and molecular oxygen, and hence the equilibrium in the first step is displaced in favour of the formation of superoxide anions. Thirdly, hydroxyl radicals are generated from hydrogen peroxide through the Fenton reaction. In this Fenton reaction Fe2+ ions are available since Fe3+ ions are readily reduced by 3-hydroxyanthranilic acid. The superoxide anions do not seem to participate in the reduction of Fe3+ ions, since superoxide anions are rapidly dismuted by SOD present in the reaction mixture.     

The involvement of biological quinones in the formation of hydroxyl radicals via the Haber-Weiss reaction

The present investigation was made to evaluate biologically relevant quinones as possible catalysts in the generation of hydroxyl radicals from hydrogen peroxide and superoxide radicals. ESR spectra demonstrated that menadione, plastoquinone, and ubiquinone derivatives could all be reduced to their semiquinone forms by electron transfer from superoxide radicals. Reductive homolytic cleavage of H₂O₂ was observed to be dependent upon the presence of semiquinones in the reaction medium. Our data strongly support the concept that quinones normally involved in physiological processes may also play a role as catalysts of the Haber-Weiss reaction in the biological generation of hydroxyl radicals.     

Superoxide-dependent formation of hydroxyl radicals: Detection of hydroxyl radicals by the hydroxylation of aromatic compounds

A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O₂^$\text{?}$, and H₂O₂. In the presence of iron salts, O₂^$\text{?}$ and H₂O₂ can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described.     

Possible role of hydroxyl radicals in the oxidation of dichloroacetonitrile by Fenton-like reaction

Dichloroacetonitrile (DCAN), is a member of haloacetonitrile group and detected in drinking water supplies as a by-product of chlorination process. The mechanism of DCAN-induced carcinogenesis is believed to be mediated by oxidative bioactivation of DCAN molecules. The present study was designed to investigate if reactive oxygen species (ROS), similar to that generated in biological systems, are capable of oxidative activation of DCAN. A model ROS generation system (Fenton-like reaction; Fe2+ and H2O2) that predominantly produces hydroxyl radical (OH*) was used. DCAN oxidation was monitored by the extent of cyanide (CN-) release. The results indicate that DCAN was markedly oxidized by this system, and the rate of oxidation was dependent on DCAN concentration. Four-fold increase in H2O2 concentration (50-200 mM) resulted in a 35-fold increase in CN- release. The rates of DACN oxidation in presence of various transition metals were in the following order; iron>copper>titanium. DCAN oxidation was enhanced significantly by the addition of vitamin C and sulfhydryl compounds such as glutathione, N-acetyl-L- cysteine, and dithiothreitol (10 mM) to 140, 130, 145 and 136% of control, respectively. Addition of H2O2 scavenger; catalase or iron chelator; desferrioxamine (DFO) resulted in a significant decrease in CN- release 47 and 41% of control, respectively. Addition of various concentrations of the free radical scavengers, DMSO, or mannitol, to the incubation mixtures caused a significant decrease in DCAN oxidation, 32 and 50% of control, respectively. Michaelis-Menten kinetic analysis of the rates of this reaction, with or without inhibitors, indicated that ROS mediated oxidation of DCAN was inhibited by catalase (Ki = 0.01 mM)>DFO (0.02 mM) > mannitol (0.09 mM) > DMSO (0.12 mM). In conclusion, our results indicate that DCAN is oxidized by a ROS-mediated mechanism. This mechanism may have an important role in DCAN bioactivation and DCAN-induced genotoxicity at target organs where multiple forms of ROS generating systems are abundant.     

Superoxide Dismutase Enhanced the Formation of Hydroxyl Radicals in a Reaction Mixture Containing Xanthone under UVA Irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 μM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O₂, H₂O₂, and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O₂ ^?·.     

Enhanced production of hydroxyl radicals by the xanthine-xanthine oxidase reaction in the presence of lactoferrin

The generation of hydroxyl radicals by the xanthine-xanthine oxidase reaction (C. Beauchamp and I. Fridovich (1970) J. Biol. Chem. 245, 4641-1616) has been shown to be increased by iron-saturated lactoferrin isolated from pig neutrophils. Hydroxyl radical production, measured by EPR spin trapping and by ethylene production from alpha-keto-gamma-methiol butyric acid, has been demonstrated to be produced by a Fenton-type Haber-Weiss reaction catalysed by lactoferrin. The possibility that lactoferrin catalyses such a reaction in vivo is considered.     

Detection of 2-deoxy-D-ribose radicals generated by the reaction with the hydroxyl radical using a rapid flow-ESR method

ESR spectrum of the short-lived radicals derived from 2-deoxy-D-ribose by the reaction with the hydroxyl radical (HO*) was measured using a rapid flow method. A dielectric mixing resonator was used for the measurement, which made it possible to measure the highly sensitive ESR spectra of the radicals with a lifetime of the order of milliseconds. A complex spectrum was obtained and the spectral simulation was done to show that it was the superposition of the signals due to five radicals (I-V). Three of them were those formed by the dehydrogenation with the HO* at C-1 (I), C-3 (II), and C-4 (III) positions of the 2-deoxy-D-ribose molecule. The other two (IV and V) were carbonyl-conjugated radicals formed by the elimination of a water molecule from III and II. The results showed that dehydrogenation occurred randomly at the positions where hydroxyl groups are attached, but the most preferred position was C-3 and the radical position moved from C-3 to C-4 by the elimination of water molecule.     

In vivo formation of hydroxyl radicals following intragastric administration of ferrous salt in rats

Accidental poisoning by oral iron preparations is a serious problem in young children. We investigated the formation of hydroxyl radicals (.OH) in rats after intragastric instillation of ferrous sulfate. .OH was detected via its reaction with intragastrically administered 2-keto-4-methylthiobutyrate to generate ethylene gas. Ascorbic acid is typically present in oral iron preparations in order to facilitate absorption by maintaining iron in the reduced state. However, ascorbate possesses two properties that can affect .OH, recycling of oxidized iron to the ferrous state augments .OH production, while ascorbate in high concentration scavenges .OH. In experiments conducted in vitro, both actions were evident, depending upon the concentration of ascorbate. In parallel experiments conducted in vivo, the scavenging action of ascorbate was more prominent. Experiments in vitro with .OH-scavengers (dimethylsulfoxide, ethanol) and with the enzyme, catalase, confirmed both the presence of .OH and its dependence upon generated hydrogen peroxide during the oxidation of ferrous salt by molecular oxygen. Hydroxyl radicals (and/or reactive higher oxidation states of iron) may play a role in tissue damage after accidental overdose of oral iron.     

Evidence against the generation of free hydroxyl radicals from the interaction of copper,zinc-superoxide dismutase and hydrogen peroxide

Prior spin trapping studies reported that H(2)O(2) is metabolized by copper,zinc-superoxide dismutase (SOD) to form (.)OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free (.)OH or (.)OH scavenged off the Cu(2+) catalytic site. To distinguish whether (.)OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different (.)OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging (.)OH as the spin trap 5, 5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging (.)OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free (.)OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free (.)OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free (.)OH. EPR studies of the copper center demonstrated that while high H(2)O(2) concentrations induce release of Cu(2+), the magnitude of spin adducts produced by free Cu(2+) was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H(2)O(2) and SOD. Thus, SOD and H(2)O(2) oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound (.)OH not release of (.)OH from the enzyme.     

Superoxide dismutase inhibits the superoxide-driven Fenton reaction at two different levels. Implications for a wider protective role

Superoxide dismutase (SOD) completely inhibits the damage caused by a ferric-EDTA chelate in the presence of a superoxide-generating system. In this reaction superoxide is enzymically dismuted to hydrogen peroxide. Since hydrogen peroxide and a ferric-EDTA chelate are themselves a hydroxyl radical-generating system, it follows that SOD must also protect against damage done by this reaction. The ability of SOD to inhibit damage to deoxyribose caused by hydrogen peroxide and a ferric-EDTA chelate is experimentally demonstrated in this paper.     

A mechanistic study of the formation of hydroxyl radicals induced by horseradish peroxidase with NADH

During the oxidation of NADH by horseradish peroxidase (HRP-Fe(3+)), superoxide (O(-)(2)) is produced, and HRP-Fe(3+) is converted to compound III. Superoxide dismutase inhibited both the generation of O(-)(2) and the formation of compound III. In contrast, catalase inhibited only the generation of O(-)(2). Under anaerobic conditions, the formation of compound III did not occur in the presence of NADH, thus indicating that compound III is produced via formation of a ternary complex consisting of HRP-Fe(3+), NADH and oxygen. The generation of hydroxyl radicals was dependent upon O(-)(2) and H(2)O(2) produced by HRP-Fe(3+)-NADH. The reaction of compound III with H(2)O(2) caused the formation of compound II without generation of hydroxyl radicals. Only HRP-Fe(3+)-NADH (but not K(+)O(-)(2) and xanthine oxidase-hypoxanthine) was able to induce the conversion of metmyoglobin to oxymyoglobin, thus suggesting the participation of a ternary complex made up of HRP-Fe(2+…)O(2)(…)NAD(.) (but not free O(-)(2) or H(2)O(2)) in the conversion of metmyoglobin to oxymyoglobin. It appears that a cyclic pathway is formed between HRP-Fe(3+), compound III and compound II in the presence of NADH under aerobic conditions, and a ternary complex plays the central roles in the generation of O(-)(2) and hydroxyl radicals.     

Superoxide dismutase activity in the BB rat: a dynamic time-course study

Diabetes produced spontaneously in the BB rat is similar to that observed in multiple low dose streptozocin-induced diabetes, both being characterized histologically by a lympho-monocytic infiltrate in the pancreatic islets (insulitis). Recent studies indicated that streptozocin acts through peroxidative patterns sensitive to superoxide dismutase (SOD) activity. We therefore conducted a time-course study to evaluate if SOD activity in the islets of Langerhans is related to the onset of diabetes in BB rats with varying degree of diabetes. It was found that SOD activity does not change with age nor with the onset of diabetes. However SOD activity in the islets of BB rats was significantly lower than in the control Wistars. This lower SOD activity may be a proneness factor that favors the development of the diabetic syndrome.     

Identification of iron Superoxide dismutase and a copper/zinc Superoxide dismutase enzyme activity within the marine cyanobacterium Synechococcus sp. WH 7803

Abstract Three constitutive forms of Superoxide dismutase activity have been demonstrated in the cyanobacterial marine picoplankter Synechococcus sp. WH 7803 using polyacrylamide gel activity staining techniques. A protein which gave a positive non-haem iron stain on native polyacrylamide gels exhibited N-terminal similarity to both the iron Superoxide dismutase and the manganese Superoxide dismutase of Escherichia coli . The metal prosthetic group of each of the three activity bands was characterised by analysing their differential sensitivities to 5 mM H₂O₂, 2 mM cyanide and 2 mM of the copper chelator diethyldithiocarbamate. Three distinct Superoxide dismutase activities were observed, an iron Superoxide dismutase, a copper/zinc Superoxide dismutase and a third form which has not been identified. Growth of Synechococcus cells in ASW medium containing no added iron resulted in no alteration in the activity of the iron Superoxide dismutase. Growth of cultures in the absence of copper or zinc resulted in differential changes in the activities of the copper/zinc Superoxide dismutase and the unidentified Superoxide dismutase.     

Histidinyl radical formation in the self-peroxidation reaction of bovine copper-zinc superoxide dismutase.

In the absence of suitable oxidizable substrates, the peroxidase reaction of copper-zinc superoxide dismutase (SOD) oxidizes SOD itself, ultimately resulting in its inactivation. A SOD-centered free radical adduct of 2-methyl-2-nitrosopropane (MNP) was detected upon incubation of SOD with the spin trap and a hydroperoxide (either H(2)O(2) or peracetic acid). Proteolysis by Pronase converted the anisotropic electron paramagnetic resonance (EPR) spectrum of MNP/(center dot)SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine + HO(center dot)) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/(center dot)SOD, suggesting that the latter was centered on a histidine residue. An additional hyperfine coupling was detected when histidine specifically (13)C-labeled at C-2 of the imidazole ring was used, providing evidence for trapping at that atom. All of the experimental spectra were convincingly simulated assuming hyperfine couplings to 2 nearly equivalent nitrogen atoms and 2 different protons, also consistent with trapping at C-2 of the imidazole ring. Free histidinyl radical consumed oxygen, implying peroxyl radical formation. MNP-inhibitable oxygen consumption was also observed when cuprous SOD but not cupric SOD was added to a H(2)O(2) solution. Formation of 2-oxohistidine, the stable product of the SOD-hydroperoxide reaction, required oxygen and was inhibited by MNP. These results support formation of a transient SOD-peroxyl radical.