Submolecular adventures of brain tyrosine: what are we searching for now?

This overview summarizes recent findings on the role of tyrosyl radical (TyrO(*)) in the multitudinous neurochemical systems of brain, and theorizes on the putative role of TyrO(*) in neurological disorders [Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS)]. TyrO(*) and tyrosine per se can interact with reactive oxygen species (ROS) and reactive nitrogen species (RNS) via radical mechanisms and chain propagating reactions. The concentration of TyrO(*), ROS and RNS can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive as well, and this can induce damage directly (by lipid peroxidation) or indirectly (by proteins oxidation and/or nitration), potentially causing apoptotic neuronal cell death or autoschizis. Evidence of lesion-induced neuronal oxidative stress includes the presence of protein peroxides (TyrOOH), DT (o,o'-dityrosine) and 3-NT (3-nitrotyrosine). Mechanistic details of protein- and enzymatic oxidation/nitration in vivo remain unresolved, although recent in vitro data strongly implicate free radical pathways via TyrO(*). Nitration/denitration processes can be pathological, but they also may play: 1). a signal transduction role, because nitration of tyrosine residues through TyrO(*) formation can modulate, as well the phosphorylation (tyrosine kinases activity) and/or tyrosine hydroxylation (tyrosine hydroxylase inactivation), leading to consequent dopamine synthesis failure and increased degradation of target proteins, respectively; 2). a role of "blocker" for radical-radical reactions (scavenging of NO(*), NO(*)(2) and CO(3)(*-) by TyrO(*)); 3). a role of limiting factors for peroxynitrite formation, by lowering O(2)(*-) formation, which is strongly linked to the pathogenesis of neural diseases. It is still not known if tyrosine oxidation/nitration via TyrO(*) formation is 1). a footprint of generalized stress and neuronal disorders, or 2). an important part of O(2)(*-) and NO(*) metabolism, or 3). merely a part of integral processes for maintaining of neuronal homeostasis. The full answer to these questions should be of top research priority, as the problem of increased free radical formation in brain and/or imbalance of the ratios ROS/RNS/TyrO(*) may be all important in defining whether oxidative stress is the critical determinant of tissue and neural cell injury that leads to pathological end-points.

Possible role of salsolinol quinone methide in the decrease of RCSN-3 cell survival.

The endogenous dopamine-derived neurotoxin salsolinol was found to decrease survival in the dopaminergic neuronal cell line RCSN-3, derived from adult rat substantia nigra in a concentration-dependent manner (208 microM salsolinol induced a 50% survival decrease). Incubation of RCSN-3 cells with 100 micro;M dicoumarol and salsolinol significantly decreased cell survival by 2.5-fold (P < 0.001), contrasting with a negligible effect on RCHT cells, which exhibited nearly a 5-fold lower nomifensine-insensitive dopamine uptake. The levels of catalase and glutathione peroxidase mRNA were decreased when RCSN-3 cells were treated with 100 microM salsolinol alone or in the presence of 100 microM dicoumarol. In vitro oxidation of salsolinol to o-quinone catalyzed by lactoperoxidase gave the quinone methide and 1,2-dihydro-1-methyl-6,7-isoquinoline diol as final products of salsolinol oxidation as determined by NMR analysis. Evidence of the formation of salsolinol o-semiquinone radical has been provided by ESR studies during one-electron oxidation of salsolinol catalyzed by lactoperoxidase.

The possible role of one-electron reduction of aminochrome in the neurodegenerative process of the dopaminergic system.

We present for discussion a possible molecular mechanism explaining the formation of reactive oxygen species involved in the neurodegenerative process of dopaminergic system in Parkinson's disease. This new hypothesis involves one-electron reduction of aminochrome to o-semiquinone radical, which seems to be the reaction responsible for neurodegenerative process of dopaminergic system. Leukoaminochrome o-semiquinone is extremely reactive with oxygen, which reoxidizes by reducing oxygen to superoxide radicals. Superoxide radicals enzymatically or spontaneously dismutate to dioxygen and hydrogen peroxide which is a precursor of hydroxyl radicals. ESR-experiments have showed that aminochrome o-semiquinone is extremely reactive in the presence of oxygen compared to dopamine o-semiquinone. In addition, the antioxidant enzymes superoxide dismutase and catalase play a prooxidant role by increasing the autoxidation rate and formation of superoxide radicals. One electron reduction of aminochrome to o-semiquinone can be performed by flavoenzymes which use NADPH and NADH as electron donator. The ability of aminochrome o-semiquinone to autoxidize in the presence of oxygen gives rise to a redox cycling process which will continue until oxygen, NADH and/or NADPH are depleted. Depletion of NADPH will prevent glutathione reductase from reducing glutathione, which is one of the main antioxidants in the cell. In addition depletion of NADH will prevent the formation of ATP in the electron transport chain in the mitochondria. Two antioxidants, probably, neuroprotective reactions are also discussed.

Evidence for peroxidative oxidation of substituted piperidine nitroxides, acting as apoptosis inducers in Yoshida Sarcoma cells in vivo.

The results presented herein clearly indicate that nitroxide derivatives--free radicals are effective as substrates for one-electron oxidation in the peroxidase cycle involving hydrogen peroxide, which have been the subject of considerable controversy. This oxidation is catalyzed enzymatically and it might occur in tumor cells (in vivo) where the level of ROS (H2O2 and O2.-) is increased. The result of this reaction involving hydrogen peroxide is the obligative formation of the oxo-ammonium cation involved in the superoxide dismutase-mimic reaction of nitroxides with superoxide and/or in reaction with H2O2 leading to superoxide formation and regeneration of the parent nitroxide molecule. The efficiency of this enzymatically catalyzed oxidation of nitroxide(s) depends on the structure of the substituent in position 4 of nitroxide ring as follows: -OCH3 > -NHCOCH3 > -NHCOCH2CH3. Notably, the reduced nitroxide salt was not substrate for peroxidatic oxidation clearly indicating the importance of the free radical moiety of the nitroxide molecule. These findings may have some relevance in the recent investigations of antioxidant properties/mechanisms of nitroxides. Based on these considerations we hypothesize that the administration of oxidizable free radical nitroxide compounds--antioxidants may be a useful strategy in the treatment and investigations of cancer diseases. An in vivo study ("Screening test of chemicals employing Yoshida Sarcoma animals") was carried out to verify whether the structure and/or the chain length of substituent of oxidizable nitroxide derivatives--antioxidants could influence their apoptotic activity. The results reported in this study are encouraging as we found a limited correlation between the molecular oxidative properties of nitroxides under study, their structure and antitumor (apoptotic) action. In conclusion, this work demonstrates that investigation of the structure-dependent oxidation of antioxidatively acting nitroxides can become a very important step in their future screening and selection for applications in vivo and in vitro.

The paradoxical apoptotic effects of novel nitroxide antioxidants on Yoshida sarcoma cells in vivo: a commentary.

Here we show for the first time that the model nitroxide derivatives, free radical or its reduced piperidinium salt, suppressed cytotoxicity of ROS (O2 and H2O2) generated outside the cells (B14 line, model for neoplastic phenotype) in ***. The nitroxides prevented the decrease in the number of *** caused by exogenous O2- and H2O2 at concentrations which were not themselves cytotoxic. In the present study, we have also shown that a very substantial difference in the cell response occurred when the model rat tumor cells (Yoshida Sarcoma ascites) were treated in vivo with six novel synthesized nitroxide antioxidants. A number of tumor cells displayed morphological characteristics of apoptosis. This effect was comparable to those observed for other nitroxyls under similar experimental conditions. Since the increase in the ROS generation followed by apoptotic changes of nuclei is the consistent recent finding in various experimental models of apoptosis, one fundamental question was raised: why nitroxide antioxidants paradoxically act as apoptosis inducers in vivo? Taking together the results presented here and in our previous works, it seems reasonable to suggest that nitroxide-antioxidants improve the endogenous "antioxidants reserve" and action can induce a reductive stress as opposed to an oxidative stress, triggering a cascade of dose-dependent processes involving indirectly an antioxidant mechanism(s) and resulting in the apoptotic death of cancer cells in vivo. The SAR (structure activity relationship) revealed that either the substituent structure at 4-position of the nitroxide ring or its oxidation state are determinant for the degree of the observed differences in the apoptotic potency of nitroxide derivates in vivo.

Tempicol-3, a novel piperidine-N-oxide stable radical and antioxidant, with low toxicity acts as apoptosis inducer and cell proliferation modifier of Yoshida Sarcoma cells in vivo.

The novel nitroxyl, Tempicol-3 (nitroxide-N-oxide) was synthesized and its capacity to act as a scavenger of hydroxyl radicals was tested. The concentration-dependent reducibility of this novel compound was also examined and compared with those of previously characterized nitroxides, Tempo and Tempace. The cytotoxicity of Tempicol-3 in vitro was measured by the modified tetrazolium assay (MTT), using, model cells for neoplastic phenotype (mouse NIH 3T3 fibroblast line). The ability of Tempicol-3 to act as an antitumor agent in vivo was also investigated in a pharmacological test, using rats bearing 3-day old Yoshida Sarcoma (promotion phase of the disease). Our results clearly indicated that Tempicol-3 acts as an effective and promising hydroxyl radical scavenger-antioxidant. Structure- and concentration-dependent bioreduction of Tempicol-3 by ascorbic acid may account for some of its biological effects, causing modulation of the antioxidant status of cells. The presence of one nitrone moiety per molecule of Tempicol-3 caused a significant decrease in nitroxide cytotoxicity as compared with Tempo, in vitro. The results clearly confirmed that the toxic effect could result either from the presence or structure of substituent(s) at position 4 of the free radical piperidine moiety. It can be stated that Tempicol-3 is a lowtoxicity nitroxide, which could be effective in providing antioxidative activity. We have also observed that lowtoxic Tempicol-3, at m.e.d. (minimal effective dose) suppressed tumorigenesis, acting as a cell proliferation modifier and apoptosis inducer in vivo. This work provides the base for further investigations on nitroxide-N-oxide derivatives since the serious question remains to be solved as to what is the molecular mechanism of action of the nitroxide-N-oxides.

Reactive oxygen species and reactive nitrogen species: relevance to cyto(neuro)toxic events and neurologic disorders. An overview.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are formed under physiological conditions in the human body and are removed by cellular antioxidant defense system. During oxidative stress their increased formation leads to tissue damage and cell death. This process may be especially important in the central nervous system (CNS) which is vulnerable to ROS and RNS damage as the result of the brain high O(2) consumption, high lipid content and the relatively low antioxidant defenses in brain, compared with other tissues. Recently there has been an increased number of reports suggesting the involvement of free radicals and their non-radical derivatives in a variety of pathological events and multistage disorders including neurotoxicity, apoptotic death of neurons and neural disorders: Alzheimer's (AD), Parkinson's disease (PD) and schizophrenia. Taking into consideration the basic molecular chemistry of ROS and RNS, their overall generation and location, in order to control or suppress their action it is essential to understand the fundamental aspects of this problem. In this presentation we review and summarize the basics of all the recently known and important properties, mechanisms, molecular targets, possible involvement in cellular (neural) degeneration and apoptotic death and in pathogenesis of AD, PD and schizophrenia. The aim of this article is to provide an overview of our current knowledge of this problem and to inspire experimental strategies for the evaluation of optimum innovative therapeutic trials. Another purpose of this work is to shed some light on one of the most exciting recent advances in our understanding of the CNS: the realisation that RNS pathway is highly relevant to normal brain metabolism and to neurologic disorders as well. The interactions of RNS and ROS, their interconversions and the ratio of RNS/ROS could be an important neural tissue injury mechanism(s) involved into etiology and pathogenesis of AD, PD and schizophrenia. It might be possible to direct therapeutic efforts at oxidative events in the pathway of neuron degeneration and apoptotic death. From reviewed data, no single substance can be recommended for use in human studies. Some of the recent therapeutic strategies and neuroprotective trials need further development particularly those of antioxidants enhancement. Such an approach should also consider using combinations of radical(s) scavengers rather than a single substance.

Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide.

The reactivities of glutathione, cysteine, cysteamine, penicillamine, N-acetylcysteine, dithiothreitol and captopril with superoxide generated from xanthine oxidase and hypoxanthine, and with reagent hydrogen peroxide, have been investigated. Rates of thiol loss on adding hydrogen peroxide, and superoxide-dependent thiol loss and oxygen uptake were measured. The relative reactivities of the different thiols with both oxidants were inversely related to the pK of the thiol group, such that at pH 7.4, penicillamine was the most reactive. N-acetylcysteine weakly reactive and no reaction was seen with captopril. For hydrogen peroxide, the calculated rate constants for the reaction with the thiolate anion all fell within the range 18-26 M(-1) s(-1). With superoxide, our results are consistent with each thiol reacting via a short chain that consumes oxygen and regenerates superoxide. Only with some of the thiols, was the consumed oxygen recovered as hydrogen peroxide. Reported values for the rate constant for the reaction of thiols with superoxide vary over four orders of magnitude, with the highest being > 10(5) M(-1) s(-1). Due to the complexity of the chain reaction, no study so far has been able to obtain accurate values and we consider the best estimates to be in the 30 to 1000 M(-1) s(-1) range.

Antiproliferative and cytotoxic effects of N,N-diethylaminoethyl ethers of flavanone oximes: a comparison with anthracycline(s), anthraquinone and nitroxides action.

Since flavanone oximes derivatives (ethers) have been shown to modulate the growth of Yoshida Sarcoma cells in vivo and to induce apoptosis, the effects of these substances on immortalized cell lines growth were examined. Cell viability and sensitivity to investigated substances was measured by the modified tetrazolium salt (MTT) assay. The antiproliferative effects were expressed as IC50 and IC90, respectively. There were very substantial differences in the dose-dependency of the observed antiproliferative and cytotoxic effects. The structure-activity relationship was evident and revealed that the substitution at B-ring of molecule seems to be an important factor in flavanone oxime (ether) potency. Compared to anticancer drugs (doxorubicin, aclarubicin and mitoxantrone) flavanone oximes displayed cytotoxicity at considerably higher concentrations. The antiproliferative action of the investigated model nitroxides depended on the free radical part of the molecule. N-hydroxy derivative (reduced cation form) did not influence cells proliferation and nor display any cytotoxicity at the applied range about 60 times higher than those of flavanone derivatives. Taken together it seems reasonable to suggest that flavanone oxime(s) (ethers) as compared with antracycline(s), anthraquinone and nitroxides might be especially good candidates for in the future development of new therapeutic techniques.

Anticancer potential of N,N-diethylaminoethyl ethers of flavanone oximes: a comparison with mitoxantrone action on rat Yoshida sarcoma cells in vivo.

This study was performed to evaluate the anticancer abilities of four biologically active N,N-diethylaminoethyl ethers of flavanone oximes against rat Yoshida Sarcoma cells in vivo, and to investigate the mechanism(s) involved. The effects were compared with those of anthraquinone drug (mitoxantrone) action. The presented results provide the first evidence that all the investigated substances induce programmed cell death (apoptosis) of Yoshida Sarcoma cells in vivo. On interpretative grounds, the administration of investigated flavanone derivatives in the promotion phase of the disease led to both growth inhibition (cell cycle perturbation) and apoptosis. A correlation was found between structure of the substituent(s) at B-ring of substances and the revealed anticancer activity. The data suggest that flavanone derivatives (oxime ethers) besides their antiradical, antioxidant and radioprotector properties observed before, may act as promising anticancer agents acting in the promotion phase of disease. This finding prompted us to consider the development of a new strategy: modulation of effects using combination therapy involving mitoxantrone and flavanone oximes.

Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product.

In the last ten years, there has been an important increase in interest in quercetin action as a unique antioxidant, but its putative role in numerous prooxidant effects is also being continually updated. The mechanism underlying this undesirable ability seems to involve its metabolic oxidoreductive activation. Based on the structural properties of quercetin, we have investigated whether its catechol moiety may be the potential tool for revealed toxicity. We demonstrated, with an ESR spin-stabilization technique coupled to conventional spectrophotometry, that o-semiquinone and o-quinone are indeed the products of enzymatically catalyzed oxidative degradation of quercetin. The former radical might serve to facilitate the formation of superoxide and depletion of GSH, which could confer a specificity of its prooxidative action in situ. The observed one-electron reduction of o-quinone may enrich the semiquinone pool, thereby magnifying its effect. The two-electron reduction of quinone can result in constant resupply of quercetin in situ, thereby also modulating another pathway of its known biological activities. We have also tried to see whether the intracellular oxidative degradation of quercetin can be confirmed under the controlled conditions of model monolayer cell cultures. The results are indicative of the intracellular metabolic activation of quercetin to o-quinone, the process which can be partially associated with the observed concentration-dependent cytotoxic effect of quercetin.

TTT, a novel free radical spin trap, induces apoptosis in sarcoma Yoshida cells in vivo: comparative investigations of its cytotoxicity in vitro.

In light of our previous SAR studies on nitroxides acting as less toxic anticancer agents, antioxidants and radioprotectors, we designed and tested, in vivo an in vitro, a new triradical spin trap -N,N',N"-tris-(l-oxyl-2,2,6,6-tetramethylpiperidine-4-yl)-1,3, 5-tnazine-2,4,6-triamine (TTT). The ability of TTT to act as an antitumor agent in vivo was investigated in pharmacological tests. The administration of TTT to rats bearing 3 day-old Sarcoma Yoshida (promotion phase of the disease) led to induction of apoptotic cell(s) death. Our results clearly indicated the suggested involvement of the free radical moiety of piperidine ring(s), thus indicating that the anticancer activity of nitroxide(s) may involve its intracellular redox reactions. To assess the relationship between the apoptotic effects of TTT in vivo and its possible cytotoxicity, we determined the relative antiproliferative and cytostatic potential of TTT in vitro as compared with this of the anticancer drugs: doxorubicin, aclarubicin and mitoxantrone, currently used in clinical practice in Poland. We found that TTT inhibits the growth and proliferation of two immortalized cell types-hamster B14 cell line and mouse NIH 3T3 fibroblasts which we used as a model for neoplastic phenotype, in a dose-dependent manner.

Metexyl (4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl) as an oxygen radicals scavenger and apoptosis inducer in vivo.

A stable nitroxide radical named Metexyl (4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl) was synthesized and its antioxidant and antitumor properties were investigated and compared with these of another nitroxide derivatives previously designed in our laboratories. Three experimental models were used: xanthine/xanthine oxidase system, pulse radiolysis and experimental rat cancer (Yoshida Sarcoma) in vivo. In this work we measured the rate constant of the reactions of Metexyl with enzymatically generated O2.- or radiolytically produced .OH. For comparison, the reactions of non radical derivative (4-acetamide-2,2,6,6-tetramethylpiperidinium acetate) or nitroxide Tempace (4-acetamide-2,2,6,6-tetramethylpiperidine-1-oxyl) with the above mentioned reactive oxygen radicals were also studied. The comparative ability of Metexyl to act as an inducer of apoptosis in vivo was also investigated in pharmacological test. The ring substituent (-OCH3) at position 4 of the Metexyl molecule had significant influence on its properties as antioxidant and apoptosis inducer. The results in this study suggest that Metexyl is a promising nitroxide antioxidant, which can induce apoptosis of tumor cells in vivo, thus providing a base for its further investigations in vitro and pharmacological applications.

Metabolic activation of dopamine o-quinones to o-semiquinones by NADPH cytochrome P450 reductase may play an important role in oxidative stress and apoptotic effects.

In this study, it is shown that considerable evidence for the possible pathway by which dopamine o-quinone, o-quinone and aminochrome can be activated metabolically by NADPH cytochrome P450 reductase to high reactive semiquinones. These findings were discussed from a mechanistic standpoint as well as in terms of potential physiological implications of dopamine o-quinones and o-semiquinones' concerted action in oxidative stress and apoptotic events.

Tempicol-2 (4-hydroxy-4-(2-picolyl)-2,2,6,6-tetramethylpiperidine-1-oxyl), a stable free radical, is a novel member of nitroxide class of antioxidants and anticancer agents.

As a part of our studies on the chemical, biochemical and pharmacological characteristics of the newly synthesized antioxidants, nitroxide derivatives, we designed a novel nitroxide, named Tempicol-2. Its capacity to act as antioxidant of potential pharmacological application was tested in three model systems: xanthine/xanthine oxidase, iron- and ascorbate Fenton reaction(s) and gamma-radiolysis. The antioxidant properties of Tempicol-2 as a function of concentration were compared with those previously characterized nitroxide derivatives Tempace and Rutoxyl which we had synthesized. The possibility of one-electron reduction of the novel substance by ascorbic acid was also examined and compared. The ability of Tempicol-2 to act as anticancer agent in vivo was also investigated in pharmacologic tests. The administration of Tempicol-2 to rats bearing 3 day-old Yoshida Sarcoma (promotion phase) led to both growth inhibition and the induction of apoptotic cells(s) death, comparable to the effects of Tempace and Rutoxyl under the same experimental conditions. Our results confirmed the suggested involvement of free radicals in the pathogenesis of model. Yoshida Sarcoma, thus indicating that anticancer activity of the investigated nitroxides may indirectly involve an antioxidant mechanism. The results reported here are encouraging as we find a limited correlation between the molecular redox properties, structure of nitroxides and their antitumor action. Tempicol-2, similarly to Tempace and Rutoxyl, is a promising antioxidant which can induce apoptosis, thus providing the basis for further investigations of the concentration and phase-dependent effects and the exact mechanisms of nitroxide(s) apoptotic action using cell line(s) model.

Molecular mechanisms of cellular injury produced by neurotoxic amino acids that generate reactive oxygen species.

There is now strong experimental evidence that the basic precursors for the synthesis of catechol(amine) and indolamine neurotransmitters, tyrosine and tryptophan can act as generators of ROS (reactive oxygen species): peroxides, superoxide and peroxyradicals. The consequences of free radicals formation from precursors during oxidative degradation process, their possible participation in electron transfer/addition reactions and chain processes involving cell antioxidant defense system were presented and discussed. Although the generation of neurotoxic ROS by tyrosine and tryptophan is accepted to occur in the presented model systems, doubts can exist as to the situation in vivo, which may be completely different and remain to be explored. The relevance of the present findings with regard to a variety of neurological diseases cannot be ignored.

Rutoxyl [rutin/4-acetamide-1-hydroxy-2,2,6,6-tetramethylpiperidinium] is a new member of the class of semi-natural products of high pharmacological potency.

A novel complex, Rutoxyl [rutin/4-acetamide-1-hydroxy-2,2,6,6-tetramethylpiperidinium] was synthesized and its structure and anticancer activity were investigated. The results reported here are consistent with our idea, that the formation of such a complex of two biologically active molecules: polyphenolic flavonoid antioxidant (Rutin) and nitroxylamine of nitroxide antioxidant (Tempace), stabilized by hydrogen bond(s) can result in a supra-additive properties.

Evidence for antiradical and antioxidant properties of four biologically active N,N-diethylaminoethyl ethers of flavanone oximes: a comparison with natural polyphenolic flavonoid (rutin) action.

The antiradical and antioxidant activities of four biologically active N,N-diethyloaminoethyl ethers of flavanone oximes (N,N-DEAEFo) were investigated in vitro and compared with these of polyphenolic flavonoid (rutin). Four experimental models were used: iron- and ascorbate driven Fenton systems, gamma-radiolysis, xanthine/xanthine oxidase system and diphenylpicrylhydrazyl (DPPH) radical scavenging. The results clearly indicate that N,N-DEAEFo are acting as promising antioxidants and radioprotectors comparable to rutin activities: they should prove to be useful under acute oxidative stress conditions for which both properties were required.

Tempace and troxyl-novel synthesized 2,2,6,6-tetramethylpiperidine derivatives as antioxidants and radioprotectors.

Two novel 2,2,6,6-tetramethylpiperidine derivatives (Tempace and Troxyl) were synthesized and their capacity to act as scavengers of superoxide, inhibitors of iron and ascorbate-driven Fenton reaction and radioprotectors was tested. The possibility for one-electron oxidation of novel compounds by heme-ferryl species was also examined. The results clearly indicate that Tempace and Troxyl are acting as promising antioxidants and radioprotectors thus providing a base for further investigations and pharmacological applications.

Generation of superoxide and tyrosine peroxide as a result of tyrosyl radical scavenging by glutathione.

Enzymatically generated tyrosyl radicals are effectively scavenged by reduced glutathione (GSH), thereby generating glutathione thiyl radicals and superoxide radicals, subsequently. Here, we have used horseradish peroxidase to generate tyrosyl radicals and investigated the fate of the superoxide radicals. At low GSH concentrations (with a maximum effect at 250 microM) a major reaction was between superoxide and the phenoxyl radical leading to a tyrosine peroxide. Formation of the peroxide was confirmed using a peroxide-specific colorimetric assay and detection of a new HPLC-peak. Its formation was inhibited by superoxide dismutase (SOD). The peroxide decomposed slowly in a reaction that was accelerated by GSH to give a new chromatographic peak. Increasing the GSH concentration decreased the amount of tyrosine peroxide formed and caused increases in rates of oxygen uptake and GSH oxidation. These increases were not seen in the presence of SOD and are consistent with GSH scavenging superoxide and leading to oxygen-dependent chain oxidation of GSH. Both pathways are undesirable for the cell and are effectively suppressed only if GSH as a radical scavenger acts in concert with SOD.