Characterization of sulfur-centered radical intermediates formed during the oxidation of thiols and sulfite by peroxynitrite. ESR-spin trapping and oxygen uptake studies

Using a novel phosphorylated spin trap, 5-diethoxy-phosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), an analog of the commonly used trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO), we have investigated the reactions of sulfur-centered radicals produced from the oxidation of thiols and sulfite by peroxynitrite. The predominant species trapped in all cases are the corresponding sulfur-centered radicals, i.e. glutathionyl radical (GS) from glutathione (GSH), N-acetyl-DL-penicillamine thiyl radical (S-NAP) from N-acetyl-DL-penicillamine (NAP) and sulfate anion radical (SO3-) from sulfite. These radicals consume molecular oxygen forming either peroxyl or superoxide anion radicals. GS, S-NAP, and (SO3-)-derived radicals react with ammonium formate to form the carbon dioxide anion radical (CO2-). Further support of spin adduct assignments and radical reactions are obtained from photolysis of S-nitrosoglutathione and S-nitroso-N-acetyl-DL-penicillamine. We conclude that the direct reaction of peroxynitrite with thiols and sulfate forms thiyl and sulfate anion radicals, respectively, by a hydroxyl radical-independent mechanism. Pathological implications of thiyl radical formation and subsequent oxyradical-mediated chain reactions are discussed. Oxygen activation by thiyl radicals formed during peroxynitrite-mediated oxidation of glutathione may limit the effectiveness of GSH against peroxynitrite-mediated toxicity in cellular systems.     

Lipoxygenase-mediated glutathione oxidation and superoxide generation

Soybean lipoxygenase-mediated cooxidation of reduced glutathione (GSH) and concomitant superoxide generation was examined. The oxidation of GSH was dependent on the concentration of linoleic acid (LA), GSH, and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 0.42 mM LA, 2 mM GSH, and 50 pmole of enzyme/mL. The GSH oxidation was linear up to 10 minutes and exhibited a pH optimum of 9.0. The reaction displayed a Km of 1.49 mM for GSH and Vmax of 1.35 +/- 0.02 mumoles/min/nmole of enzyme. Besides LA, arachidonic and gamma-linolenic acids also supported the lipoxygenase-mediated GSH oxidation. Hydrogen peroxide and 13-hydroperoxylinoleic acid supported GSH cooxidation, but to a very limited extent. Oxidized glutathione (GSSG) was identified as the major product of the reaction based on the depletion of nicotinamide-adenine dinucleotide 3'-phosphate (NADPH) in the presence of glutathione reductase. The GSH oxidation was accompanied by the reduction of ferricytochrome c, which can be completely abolished by superoxide dismutase (SOD), suggesting the generation of superoxide anion radicals. Under optimal conditions, the rate of superoxide generation (measured as the SOD-inhibitable reduction of ferricytochrome c) was 10 +/- 1.0 nmole/min/nmole of enzyme. These results clearly suggest that lipoxygenase is capable of oxidizing GSH to GSSG and simultaneously generating superoxide anion radicals, which may contribute to oxidative stress in cells under certain conditions.     

Clarification of the relationship between free radical spin trapping and carbon tetrachloride metabolism in microsomal systems

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/.CCl3) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/.CO2-) or the glutathione (GSH) and CCl4-dependent PBN radical adduct (PBN/[GSH-.CCl3]). Inclusion of PBN/.CCl3 in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-.CCl3] or PBN/.CO2- radical adducts. Microsomes alone or with GSH had no effect on the PBN/.CCl3 radical adduct. Addition of NADPH to a microsomal system containing PBN/.CCl3 presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/.CCl3. We conclude that PBN/.CCl3 is not metabolized into either PBN/[GSH-.CCl3] or PBN/.CO2- in microsomal systems.     

Biotransformation of 4-dimethylaminophenol: reaction with glutathione, and some properties of the reaction products

4-Dimethylaminophenol (DMAP) forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. In solutions of purified human hemoglobin, quick binding of oxidized DMAP to the globin moiety of hemoglobin terminates this reaction. Reduced glutathione in high concentrations, as in the red cell, substantially diminished binding of oxidized DMAP to hemoglobin by formation of S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)-tris-glutathione (tris-(GS)-DMAP), which does not form ferrihemoglobin. In the presence of reduced glutathione, DMAP disappeared more rapidly from hemoglobin solutions than in its absence. The formation of tris(GS)-DMAP in red cells was found to be of importance for the termination of catalytic ferrihemoglobin formation by DMAP in vivo. With low concentrations of GSH, DMAP in hemoglobin solutions formed another conjugate, (GS)-DMAP, S,S(2-dimethylamino-5-hydroxy-1,3-phenylene)-bis-glutathione. Similar to DMAP, bis(GS)-DMAP catalyzed the formation of ferrihemoglobin. As the oxidized bis(GS)-DMAP was bound to hemoglobin more slowly and to a lesser extent, it produced more ferrihemoglobin than DMAP. In contrast to the reactions of DMAP with hemoglobin, hydrogen peroxide and superoxide radicals are involved in the ferrihemoglobin formation by bis(GS)-DMAP. The radicals accelerate the oxidation of bis(GS)-DMAP and thereby the ferrihemoglobin formation.     

Nitric oxide trapping of tyrosyl radicals generated during prostaglandin endoperoxide synthase turnover. Detection of the radical derivative of tyrosine 385

Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alia nitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.     

The protective effect of vitamin E, idebenone and reduced glutathione on free radical mediated injury in rat brain synaptosomes

In the present study the effect of ascorbate (0.8 mM)/iron (2.5 microM) on lipid and protein oxidation, in Synaptosomes isolated from rat brain cortex, was evaluated. Vitamin E, idebenone and reduced glutathione were used as free radicals scavengers, in order to analyze the mechanism involved in ascorbate/iron-induced oxidative stress. An increased formation of reactive oxygen species (ROS) in the cytosol and in the mitochondria was observed, in ascorbate/iron treated synaptosomes. Idebenone (50 microM) prevented the increased formation of ROS in both synaptosomal compartments, vitamin E (150 microM) protected partially this formation in mitochondria, whereas reduced glutathione (250 microM) (GSH) was ineffective. After ascorbate/iron treatment an increase in lipid peroxidation occurred as compared to control, which was completely inhibited by idebenone. A decrease in protein-SH content was also observed, and it was prevented by Vitamin E, idebenone and GSH. When synaptosomes were treated with ascorbate/iron the levels of GSH decreased, and the levels of oxidized glutathione (GSSG) increased as compared to controls under these conditions. Glutathione peroxidase activity was unchanged, whereas an inhibition of glutathione reductase activity was observed. These data suggest that the increased formation of free radicals in synaptosomes leads to lipid and protein oxidation, the role of the endogenous GSH being essential to protect protein thiol-groups against oxidative damage in order to maintain enzyme activity.     

Reactive oxygen species produced in metal-catalyzed oxidation of bis(trifluoromethyl)disulfide and protection by ZE

Bis(trifluoromethyl)disulfide (TFD), used as an industrial fumigant, was found to generate a thiyl free radical as seen by EPR/spin trapping. Oxygen appears to be an absolute requirement for radical production. The results obtained in this investigation implicate the production of thiyl and reactive oxygen species (ROS), superoxide radical anion and hydroxyl radicals, during TFD autoxidation. The rate of production of these free radical intermediates was found to increase in the presence of iron(III) and copper(II). In addition, the metal ion chelator DETAPAC and ROS scavengers ethanol, mannitol, and PEG-SOD/catalase were found to inhibit free radical production. Reactive oxygen species were not formed when a high-potency zinc plus antioxidant, ZE caps, was present. These results provide support for the pro-oxidation of TFD and a protective role for zinc.     

Studies on the stability of oxygen radical spin adducts of a new spin trap: 5-methyl-5-phenylpyrroline-1-oxide (MPPO)

A new spin trap, 5-methyl-5-phenylpyrrolin-1-oxide (MPPO), has been evaluated with respect to the intrinsic stabilities of the hydroxyl and superoxide (or hydroperoxyl) radical spin adducts. Hydroxyl or superoxide radicals were generated using various sources in the presence of MPPO, and the hydroxyl or superoxide radical spin adduct of MPPO was detected by EPR spectroscopy. The time course of spontaneous decay of the EPR signal from hydroxyl or superoxide spin adducts followed first-order kinetics and the half-life was dependent on the pH of the medium. At pH 7.4 the half-life times are 76.4 and 5.7 min for the hydroxyl and hydroperoxyl/superoxide spin adducts, respectively. Structural factors which could influence the decay rates are also discussed.     

Electronic spin resonance detection of superoxide and hydroxyl radicals during the reductive metabolism of drugs by rat brain preparations and isolated cerebral microvessels

A spin trapping technique was used to analyze by electron spin resonance (ESR) the formation of oxygen-derived free radicals during the cerebral reductive metabolism of xenobiotics able to undergo a single electron reduction, i.e. quinones, pyridinium compounds and nitroheterocyclics. Paraquat, menadione and nitrofurazone were used as model compounds of these three classes of molecules. ESR spectra indicative of superoxide and hydroxyl radical formation were obtained by incubation of brain homogenates directly within the ESR cavity at 37 degrees C for each of the three molecules tested. These signals were dependent on nucleotide cofactors, and increased in a time-dependent manner. The NADPH and NADH dependent free radical production was further characterized in brain microsomal and mitochondrial fractions, respectively. By using various combinations of reactive species inactivating enzymes (superoxide dismutase, catalase), a metal chelator (deferoxamine), and an hydroxyl trapping agent (dimethylsulfoxide), it was shown that (1) the primary radical generated was the superoxide anion; and (2) a significant production of the hydroxyl radical also occurred, that was secondary to the superoxide anion production. Consistent signals indicative of the production of both oxygen-derived free radicals were obtained when isolated cerebral microvessels which constitute the blood-brain barrier were incubated with the model molecules. This is of particular toxicological relevance, because this barrier represents a key element in the protection of the brain, and is in close contact with blood-born exogenous molecules.     

Photoreduction of the fluorescent dye 2'-7'-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements

The photoreduction of 2'-7'-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (lambda > 300 nm) 2'-7'-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/*OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.     

MtDNA mutations associated with sideroblastic anaemia cause a defect of mitochondrial cytochrome c oxidase

Ethanol has been shown to be oxidized to a free radical metabolite, the 1-hydroxyethyl radical (HER). Interaction of HER with cellular antioxidants may contribute to the known ability of ethanol administration to lower levels of GSH and alpha-tocopherol. Experiments were carried out to establish a model system for the generation of HER and to study its interaction with GSH, ascorbic acid and alpha-tocopherol. A standard reaction for formation of azo-compounds using acetaldehyde and hydroxylamine-O-sulfonic acid was applied for the synthesis of 1,1'-dihydroxyazoethane (CH3CH(OH)-N=N-CH(OH)CH3). Although stable at -70 degrees C, thermal decomposition of this compound at room temperature was shown to produce HER, detected by EPR spectrometry as the PBN/HER or DMPO/HER spin adducts, and validated by computer simulation. GSH, present at the beginning of the experiment, inhibited formation of the PBN/HER signal. However, GSH did not cause any decay of pre-formed PBN/HER spin adduct. GSH was consumed in the presence of the HER-generating system in a reaction largely reversed by addition of NADPH plus glutathione reductase. Ascorbate also inhibited formation of the PBN/HER spin adduct and rapidly reduced the pre-formed adduct. HER amplified the oxidation of ascorbate, which was associated with the formation of the semidehydroascorbyl radical. Alpha-tocopherol was also consumed in the presence of HER. Production of HER in intact HepG2 cells by the redox cycling of 2,3-dimethoxy-1,4-naphthoquinone was associated with consumption of GSH. These data demonstrate the use of a simple chemical system for the controlled, continuous formation of HER and indicate that cellular antioxidants such as GSH, ascorbate, and alpha-tocopherol, interact with HER. The ability of agents such as ascorbate to reduce the PBN/HER spin adduct to EPR-silent product(s) may mask the quantitative detection of HER in biological systems.     

Free radical enhancement promotes leucocyte recruitment through a PAF and LTB4 dependent mechanism

In the present investigation we studied the concerted role of superoxide anion, platelet activating factor (PAF) and leukotriene B4 (LTB4) in the mechanism that results in polymorphonuclear leucocyte accumulation induced by oxygen free radicals in rat pancreas. This was done by comparing the effects of a PAF antagonist (BN-52021), a LTB4 inhibitor (MK-886) and superoxide dismutase (SOD) in a experimental rat model of inflammation elicited by the oxygen free radicals induced via infusion of xanthine/xanthine oxidase. Also, the effect of independent LTB4 infusion has been studied. The results show that increases in polymorphonuclear cell infiltration (evaluated by tissue histology), myeloperoxidase and LTB4 levels induced in pancreas by infusion of xanthine/xanthine oxidase were abolished by the administration of either the PAF antagonist, the LTB4 inhibitor, or SOD. The fact that BN-52021 could prevent neutrophil recruitment and LTB4 synthesis suggests that PAF is a necessary step for subsequent LTB4 synthesis and polymorphonuclear leucocyte accumulation.     

Myoglobin-induced oxidative damage: evidence for radical transfer from oxidized myoglobin to other proteins and antioxidants

Reaction of equine Fe(III) myoglobin with H2O2 gives rise to an Fe(IV)-oxo species at the heme center and protein (globin)-derived radicals. Studies have shown that there are two (or more) sites for the protein-derived radical: at tyrosine (Tyr-103) or tryptophan (Trp-14). The latter radical reacts rapidly with oxygen to give a Trp-derived peroxyl radical. The formation of both the tyrosine phenoxyl radical and the tryptophan-derived peroxyl species have been confirmed in the present study; the latter appears to be the major initial radical, with the phenoxyl radical appearing at longer reaction times, possibly via secondary reactions. We have investigated, by EPR spectroscopy, the reactivity of the Trp-14 peroxyl radical with amino acids, peptides, proteins, and antioxidants, with the aim of determining whether this species can damage other targets, i.e., whether intermolecular protein-to-protein radical transfer and hence chain-oxidation occurs, and the factors that control these reactions. Three amino acids show significant reactivity: Tyr, Trp, and Cys, with Cys the least efficient. Evidence has also been obtained for (inefficient) hydrogen abstraction at peptide alpha-carbon sites; this may result in backbone cleavage in the presence of oxygen. The myoglobin Trp-14 peroxyl radical has been shown to react rapidly with a wide range of proteins to give long-lived secondary radicals on the target protein. These reactions appear to mainly involve Tyr residues on the target protein, although evidence for reaction at Trp has also been obtained. Antioxidants (GSH, ascorbate, Trolox C, vitamin E, and urate) react with the myoglobin-derived peroxyl radical; in some cases antioxidant-derived radicals are detected. These reactions are only efficient at high antioxidant concentrations, suggesting that protein-to-protein damage transfer and protein chain-oxidation may occur readily in biological systems.     

Ribonucleotide reductases and radical reactions

Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to deoxyribonucleotides. They play a pivotal role in the regulation of DNA synthesis and are targets for antiproliferative drugs. Ribonucleotide reductases are unique enzymes in that they all require a protein radical for activity. Class I nonheme iron RNRs (mammals, plants, Escherichia coli) use a tyrosyl/cysteinyl radical pair, class II adenosylcobalamin RNRs (prokaryotes, archaea) a cysteinyl radical, class III iron-sulphur RNRs (facultative anaerobes) a glycyl radical. Here we describe the reactivity of these radicals with respect to the natural ribonucleotide substrates as well as to a variety of enzyme inhibitors, radical scavengers, nitric oxide, superoxide radicals and substrate analogues.     

Spin trapping of azidyl and hydroxyl radicals in azide-inhibited rat brain submitochondrial particles

Succinate-driven respiration in azide-inhibited rat brain submitochondrial particles (smps) produces azidyl and hydroxyl radicals that were detected by spin trapping with 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Production of radicals required succinate and oxygen and was eliminated by heat denaturation, which indicates that radical production is a result of respiration. The concentrations of both DMPO/.OH and DMPO/.N3 were decreased by addition of catalase to the smps, which indicates that H2O2 is involved in radical production. In the absence of azide anion, DMPO/.OH was not detected in the same system, even after five additions of succinate over a period of 24 h. It is proposed that azide inhibition of cytochrome c oxidase results in increased production of superoxide, which is efficiently converted to hydrogen peroxide by membrane-bound superoxide dismutase. Hydrogen peroxide activates endogenous peroxidase to react with azide anion forming azidyl radical, which damages the peroxidase, resulting in decreased production of azidyl radical with successive additions of succinate. Hydroxyl radical is produced from the hydrogen peroxide that is not removed by peroxidase. The increased production of superoxide in the azide-inhibited system suggests that loss of cytochrome c oxidase activity can lead to increased radical production if other proteins in the respiratory chain remain active. In the azide-inhibited system, reaction of azide anion with H2O2-activated endogenous peroxidase and spin-trapping of the resulting azidyl radical is a convenient monitor of H2O2 production.     

Active oxygen generation as a possible mechanism of selenium toxicity

Selenium plays an important role in scavenging active oxygen (AO) species as an essential constituent of glutathione peroxidase. On the other hand, several reports proposed a possible induction of toxic AO by selenium compounds in vitro. However, some of these experiments including ours, were revealed to conclude on the basis of experimental artifacts, and to have problems in the interpretation of data. Methods or principles so far used for the detection of AO species generated by selenium compound were measurement of chemiluminescence from lucigenin or luminol by AO species, the spectrophotometric analysis of reduction of ferricytochrome c or nitroblue tetrazolium (NBT) by superoxide anion (O2-), electron spin resonance (ESR) spectra using dimethylpyrroline oxide (DMPO) as a spin trapping agent, the deoxyribose decomposition by hydroxyl radical (HO.), the salicylate hydroxylation by HO., and the strand breakage of DNA by AO. Many of these methods together with their principles seem to have some defects which prevent clear conclusion as stated below. (i) Lucigenin was found to mediate the formation of O2- in the presence of selenite and reduced glutathione (GSH). Therefore, lucigenin is not a suitable reagent. (ii) Luminol may also mediate O2- generation in the presence of HO.. (iii) ferricytochrome c can be reduced to ferrocytochrome c in the mixture of selenite and GSH in the absence of oxygen. Moreover, the spectrophotometric method is interfered by turbidity of elemental selenium formed under some conditions in the reaction mixture containing selenite and GSH. (iv) NBT is also reduced by selenium compounds in the absence of O2. (v) ESR signals of AO species were obtained in the reaction mixture containing selenite and GSH, or in the solution of hydrogen selenide in the presence of O2. However, selenide decomposed spin adduct of DMPO with HO. (DMPO-OH). Therefore, the intensity of the signals is not quantitative. (vi) CuZn-SOD is not necessarily a good tool to prove the involvement of O2- because it enhanced HO. generation in the reaction mixture containing selenite and GSH. Thus, we would like to emphasize that carefully designed experiments are required to further identify the molecular species of active oxygen induced by selenium compounds.     

Glandular odontogenic cyst: report of a case and review of the literature

The reactions of hydroxylamine 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine hydrochloride (TEMPONE-H) with peroxynitrite, superoxide and peroxyl radicals were studied. It was shown that under these reactions TEMPONE-H is oxidized into a stable nitroxide 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidi-noxyl (TEMPONE). The reactivity of TEMPONE-H towards reactive oxygen species was compared with the spin traps DMPO and TMIO as well as with DMSO and SOD. The rate constants of reactions of TEMPONE-H with peroxynitrite and superoxide radicals were 6 x 10(9) M(-1)s(-1) and 1.2x10(4) M(-1)s(-1), respectively. Using TEMPONE-H the sensitivity in the detection of peroxynitrite or superoxide radical was about 10-fold higher than using the spin traps DMPO or TMIO. Thus, TEMPONE-H may be used as a spin trap in chemical and biological systems to quantify peroxynitrite and superoxide radical formation.     

Lipid peroxidation and antioxidant status in the liver, erythrocytes, and serum of rats after methanol intoxication

Lipid peroxidation products measured as a malondialdehyde and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), and concentrations of ascorbic acid, alpha-tocopherol, and glutathione (GSH) were measured in the liver, erythrocytes, and serum of rats 6, 14, and 24 h and 2, 5, and 7 d after treatment with 3 g methanol/kg. GSH-Px and GSSG-R activities, GSH level, and ascorbate concentration in the liver, erythrocytes, and blood serum were significantly decreased. In addition, SOD and alpha-tocopherol in erythrocytes were diminished, while malondialdehyde (MDA) in liver, erythrocytes, and serum were elevated. Further, erythrocyte counts, hemoglobin levels, hematocrit, and mean corpuscular volume (MCV) were reduced. These results indicate that methanol intoxication in rats leads to an increase in the lipid peroxidation and impairment in the antioxidant mechanisms in liver, erythrocytes, and blood serum.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-1-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (.OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the .OH generation. UV and O2 consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H2O2 did not generate any significant amount of .OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H2O2-->Co(II) + .OH + OH-] seems responsible for .OH generation. H2O2 is produced from O2.- via dismutation, O2.- is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or beta-ananyl-3-methyl-L-histidine alters, its oxidation-reduction potential and makes Co(II) capable of generating .OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H2O2-->Co(III) + .OH + OH-]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and beta-ananyl-3-methyl-L-histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.