Oxidized glutathione stimulated the amyloid formation of alpha-synuclein

alpha-Synuclein is the major filamentous constituent of Lewy bodies found in Parkinson's disease (PD). The amyloid formation of alpha-synuclein was significantly facilitated by oxidized glutathione (GSSG) as the lag period of the aggregation kinetics was shortened by 2.5-fold from its absence. Reduced glutathione (GSH), on the other hand, did not influence the lag phase although it increased the final amyloid formation. The GSSG stimulation was specific for not only alpha-synuclein but also its intactness. The preferred GSSG interaction of alpha-synuclein to GSH was also demonstrated with dissociation constants of 0.53 and 43.5 mM, respectively. It is suggested that the oxidative stress favoring the GSSG generation from GSH could result in the augmented amyloid formation of alpha-synuclein, which ought to be related to the pathogenesis of PD.     

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.     

Induction of PR-1 accumulation accompanied by runaway cell death in the lsd1 mutant of Arabidopsis is dependent on glutathione levels but independent of the redox state of glutathione

The lesions simulating disease (lsd) mutants of Arabidopsis spontaneously develop hypersensitive-response-like lesions in the absence of pathogens. To address the function of the redox regulator glutathione in disease resistance, we examined the relationship between endogenous glutathione and PR-1 accumulation using one of these mutants, lsd1, as a disease resistance model. Lesion formation on lsd1 was suppressed by weak light and initiated by the subsequent transition to normal light. The application of buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis, suppressed conditionally induced runaway cell death and expression of the PR-1 gene, suggesting that glutathione regulates the conditional cell death and PR-1 gene expression. The application of reduced (GSH) or oxidized (GSSG) glutathione to lsd1 upregulated the level of total glutathione ([GSH]+[GSSG]) accompanied by hastened accumulation of PR-1, and the basal level of total glutathione in lsd1 was higher than that in wild-type plants. The glutathione redox state defined as [GSH]/([GSH]+[GSSG]) decreased following the conditional transition, but the suppression of this decrease by the application of GSH did not inhibit the accumulation of PR-1. Taken together, conditional PR-1 accumulation in lsd1 is regulated not by the redox state but by the endogenous level of glutathione.     

Microtubule disassembly and morphologic alterations induced by 1-chloro-2,4-dinitrobenzene, a substrate for glutathione S-transferase

1-chloro-2,4-dinitrobenzene (CDNB), a potent substrate for glutathione S-transferase, is known to rapidly deplete cellular glutathione (GSH) via conjugate formation. Treatment of quiescent 3T3 cells with 5 uM CDNB results in disassembly of microtubules (MT) within 1 hr as revealed by indirect immunofluorescence microscopy. In addition, CDNB treatment also induces dramatic morphologic alterations similar to those mediated by colchicine. Furthermore, taxol prevents both MT disassembly and morphologic changes normally occurring in CDNB as well as colchicine-treated cells. The mechanism of CDNB-mediated MT disassembly in vivo and its possible relationship to cellular GSH metabolism are under current studies.     

Redox regulation of neutral sphingomyelinase-1 activity in HEK293 cells through a GSH-dependent mechanism

Phospholipases are essential enzymes in cellular signalling processes such as cellular differentiation, proliferation and apoptosis. Based on its high degree of homology with sequences of prokaryote SMases, a type of Mg(2+)-dependent PLC (nSMase-1) was recently discovered which displayed strong redox dependence for activity in vitro [F. Rodrigues-Lima, A.C. Fensome, M. Josephs, J. Evans, R.J. Veldman, M. Katan (2000), J. Biol. Chem. 275 (36) 28316-28325]. The aim of this work was to test the hypothesis that glutathione could be a natural regulator of nSMase-1 activity ex vivo. We studied how altering glutathione levels and redox ratio modulate nSMase-1 activity in a HEK293 cell line that ectopically overexpressed the nSMase-1 gene. Diminishing total glutathione with BSO without altering significantly the GSH/GSSG ratio did not affect nSMase-1 activity. Treatment of cells with diamide produced a transient decrease of total glutathione and a sharp, but also transient, decrease of the GSH/GSSG ratio. Under these conditions, nSMase-1 activity was temporarily activated and then returned to normal levels. Simultaneous treatment with BSO and diamide that resulted in permanent decreases of total glutathione and GSH/GSSG redox ratio produced a sustained activation of nSMase-1 activity. Taken together, these data indicate that altering the GSH/GSSG ratio by increasing GSSG or decreasing GSH levels, but not the total concentration of glutathione, modulates nSMase-1 activity. Our findings are the first evidence supporting the ex vivo regulation of nSMase-1 through a redox glutathione-dependent mechanism.     

Glutathione metabolism under the influence of hydroperoxides in the lactating mammary gland of the rat. Effect of glucose and extracellular ATP

Tert-butyl hydroperoxide decreases GSH and total free glutathione (GSH+2GSSG) contents of acini from lactating mammary glands. The decrease in total free glutathione can be explained by an increase in mixed disulfide formation and by excretion of GSS G to the extracellular medium, and subsequent degradation catalyzed by gamma-glutamyl transpeptidase. Low concentrations of glucose prevented the changes in glutathione levels induced by the peroxide. In the presence of extracellular ATP, glucose did not prevent these changes. However, incubations with the peroxide, did not alter the rate of other metabolic pathways by acini.Abbreviations used GSH Reduced glutathione - GSSG Glutathione disulfide - GSSR Glutathione mixed disulfide - GGT Gamma-glutamyl transpaptidase - tbOOH Tert-butyl hydroperoxide     

Interaction of menadione (2-methyl-1,4-naphthoquinone) with glutathione

The interaction of menadione with reduced glutathione (GSH) led to a removal of menadione and formation of menadione-GSH conjugate and glutathione disulfide (GSSG). The changes in thiol level were essentially biphasic with an initial rapid decrease in GSH and appearance of GSSG (less than 1 min) followed by secondary less pronounced changes. The interaction of menadione and GSH caused an oxygen uptake and both superoxide anion radical and hydrogen peroxide were produced during the reaction, the amount dependent on the GSH/menadione ratio. Catalase did not protect against the initial decrease in GSH level but markedly inhibited the secondary changes while superoxide dismutase had little effect. These results suggest that the initial changes in thiol level are the result in part of a redox reaction between menadione and GSH as well as conjugate formation, whilst the secondary changes reflect conjugate formation and the activity of other oxidants such as hydrogen peroxide. The potential biological significance of this reaction was investigated using hepatocytes depleted of reduced pyridine nucleotides and thus not able to perform enzyme-catalyzed reduction of menadione. In these cells menadione induced GSSG formation at a rate similar to that observed in control cells. This suggests that quinone-induced oxidative challenge caused by the chemical interactions of a quinone and glutathione may have biological relevance.     

Increased efflux rather than oxidation is the mechanism of glutathione depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Incubation of isolated hepatocytes in the presence of either the parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its putative toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) led to a depletion of intracellular reduced glutathione (GSH), which was mostly recovered as glutathione disulfide (GSSG). However, both MPTP- and MPP+-induced glutathione perturbances were relatively unaffected by the prior inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), suggesting that intracellular oxidation was not the major mechanism involved in the GSH loss. Inclusion of cystine in the incubation mixtures revealed a time-dependent formation of cysteinyl glutathione (CySSG), indicating that an increased efflux was mostly responsible for the MPTP- and MPP+-induced GSH depletion. Therefore, the measurement of GSSG, which is apparently formed extracellularly, was not associated with oxidative stress.     

Ageing of tomato seeds involves glutathione oxidation

The effect of seed ageing on the oxidation of reduced glutathione (GSH) and the role of GSH oxidation in ageing-induced deterioration were studied in seeds of tomato ( Lycopersicon esculentum Mill. cv. Lerica, Moneymaker and Cromco). Both long-term storage at 15°C/30% relative humidity (RH) and artificial ageing at 20°C/75% RH, 30°C/45% RH and 60°C/45% RH resulted in a marked loss of GSH and a simultaneous, though not proportional, increase in its oxidized form GSSG. The glutathione thiol-disulfide status shifted towards a highly oxidized form, while the total glutathione pool decreased. The extent of GSH oxidation differed between ageing conditions and was not directly related to the extent of seed deterioration. Thiobarbituric acid-reactive substances did not increase in ageing tomato seeds, suggesting that lipid peroxidation did not take place. Hydration of seeds, either upon imbibition in water or by priming in an osmotic solution, resulted in a rapid decrease in GSSG, a shift of the glutathione redox couple to a mainly reduced status and an increase in the glutathione pool, in both control and aged seeds. The results indicate that, in tomato seeds, (1) seed ageing involves GSH oxidation into GSSG, which is indicative of oxidative stress, (2) ageing does not affect the GSSG reduction capacity upon subsequent imbibition, and (3) the lowered viability of aged seeds cannot directly be ascribed to the decreased GSH pool or To the highly oxidized glutathione redox status.     

Blood glutathione oxidation during human exercise

To examine the effects of increased O2 utilization on the glutathione antioxidant system in blood, eight moderately trained male volunteers were exercised to peak O2 consumption (VO2peak) and for 90 min at 65% of VO2peak on a cycle ergometer. Blood samples were taken during exercise, and for up to 4 days of recovery from submaximal exercise. During exercise to VO2peak, blood reduced glutathione (GSH) and total glutathione [GSH + oxidized glutathione (GSSG)] did not change significantly. Lactate (L), pyruvate (P), and L/P increased significantly from rest values (P less than 0.01). During prolonged submaximal exercise, GSH decreased 60% from control, and GSSG increased 100%. Total glutathione, glucose, pyruvate, and lactate concentrations and L/P did not change significantly during sustained exercise. During recovery, GSH and GSH/GSSG increased from exercise levels and significantly overshot preexercise levels, reaching maximum values after 3 days. Oxidation of GSH during submaximal exercise and its reduction in recovery suggest increased formation of active O2-. species in blood during physical exercise in moderately trained males.     

Quantitation of glutathione and its oxidation products in erythrocytes by multiple-label stable-isotope dilution

A multiple-label stable isotope dilution assay for quantifying glutathione (GSH), glutathione disulfide (GSSG), and glutathione sulfonic acid in erythrocytes was developed. As the internal standards, [¹³C₃,¹⁵N]glutathione, [¹³C₄,¹⁵N₂]glutathione disulfide, and [¹³C₃,¹⁵N]glutathione sulfonic acid were used. Analytes and internal standards were detected by LC–MS/MS after derivatization of GSH with iodoacetic acid and dansylation of all compounds under study. The calibration functions for all analytes relative to their respective isotopologic standards revealed slopes close to 1.0 and negligible intercepts. As various labelings of the standards for GSH and GSSG were used, their simultaneous quantitation was possible, although GSH was partly oxidized to its disulfide during analysis. The degree of this artifact formation of GSSG was calculated from the abundance of the mixed disulfide formed from unlabeled GSH and its respective standard. Thus, the detected GSSG amount could be corrected for the artifact amount. In this way, the amount of GSSG in erythrocytes was found to be less than 0.5% of the GSH concentration. Similar to GSSG, the detected amount of glutathione sulfonic acid was found to be formed at least in part during the analytical process, but the degree could not be quantified.     

Effects of sulfite on glutathione S-sulfonate and the glutathione status of lung cells

A mechanistic study was performed to elucidate the biochemical events connected with the cocarcinogenic effect of sulfur dioxide (SO2). Glutathione S-sulfonate (GSSO3H), a competitive inhibitor of the glutathione S-transferases, forms in lung cells exposed in culture to sulfite, the hydrated form of SO2. Changes in glutathione status (total GSH) were also observed during a 1-h exposure. Some cells were pretreated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione reductase. In human lung cells GSSO3H formed in a concentration-dependent manner, while glutathione (GSH) increased and glutathione disulfide (GSSG) decreased as the extracellular sulfite concentration was increased from 0 to 20 mM. The ratio of GSH/GSSG increased greater than 5-fold and the GSH/GSSO3H ratio decreased to 10 with increasing sulfite concentration. GSSO3H formed in rat lung cells exposed to sulfite, with no detectable effect on GSH and GSSG. GSSO3H also formed from cellular GSH mixed disulfides. GSSO3H formed rapidly, reaching its maximum value in 15 min. The viability of both cell types was unaffected except at 20 mM sulfite. GSSO3H incubated with human lung cells did not affect cellular viability. BCNU inhibited cellular GSSO3H reductase to the same extent as GSSG reductase. These results indicate that GSSO3H is formed in cells exposed to sulfite, and could be the active metabolite of sulfite responsible for the cocarcinogenic effect of SO2 by inhibiting conjugation of electrophiles by GSH.     

Effect of 1-methyl-4-phenylpyridinium on glutathione in rat pheochromocytoma PC 12 cells

We investigated the effect of the selective dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) on glutathione redox status and the generation of reactive oxygen intermediates (ROI) in rat pheochromocytoma PC 12 cells in vitro. Treatment with MPP+ (250 microM) led to a 63% increase of reduced glutathione (GSH) after 24 h, while a 10-fold higher concentration of MPP+ (2.5 mM) depleted cellular GSH to 12.5% of control levels within that time. Similarly, the complex I-inhibitor rotenone induced a time-dependent loss of GSH at 1 and 10 microM, whereas treatment with lower concentrations of rotenone (0.1, 0.01 microM) increased cellular GSH. Both MPP+ and rotenone increased cellular levels of oxidised glutathione (GSSG) and the higher concentrations of both compounds led to an elevated ratio of oxidised glutathione (GSSG) vs total glutathione (GSH + GSSG) indicating a shift in cellular redox balance. MPP+ or rotenone did not induce the generation of ROI or significant elevation of intracellular levels of thiobabituric acid reactive substances (TBARS) for up to 48 h. Our data suggest that MPP+ has differential effects on glutathione homeostasis depending on the degree of complex I-inhibition and that inhibition of complex I is not sufficient to generate ROI in this paradigm.     

External GSSG enhances intracellular glutathione level in isolated cardiac myocytes

The addition of external GSSG at concentrations in the range 50-500 microM produces in isolated adult rat heart myocytes an increase of GSH level and only a slight increase of GSSG level. On the contrary, external GSH at the above same indicated concentrations did not change the cell glutathione pool. The pretreatment of the cells with diethylamaleate depleted the myocytes of glutathione and enhanced the GSSG-induced replenishment effect on GSH level. On the contrary, the addition of GSH did not increase the concentration of cell glutathione. The level of cell GSH in diethylmaleate-treated myocytes was not increased after 30 min of incubation with cysteine, or acetylcysteine. The GSSG induced-stimulation on GSH level was not inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. On the contrary, this stimulatory effect was inhibited by N, N-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of glutathione reductase, or partially, by the remotion of glucose from the incubation medium. These results support the idea that the isolated adult rat heart myocytes are able to utilize external GSSG in order to increase the intracellular glutathione pool, probably through the reduction of the imported GSSG to GSH.     

Mechanism of an insect glutathione S-transferase: kinetic analysis supporting a rapid equilibrium random sequential mechanism with housefly I1 isoform

The steady-state kinetics of glutathione S-transferase I1 (GST I1) from housefly Musca domestica expressed in Escherichia coli were investigated with glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Concentrations of the varied substrates were from 0.03 to 1 mM for GSH and 0.05 to 1 mM for CDNB. Within this range, Michaelis-Menten behaviour was observed and convergent straight lines in double reciprocal plots excluded a ping-pong kinetic mechanism. Instead, data were consistent either with rapid-equilibrium random or with steady-state ordered sequential mechanisms because of abscissa convergence. Discrimination was achieved by studying the reaction with another electrophilic partner, p-nitrophenyl-acetate (PNPA). Concentrations of PNPA and GSH varied within the ranges 0.5 to 10 mM and 0.03 to 0.6 mM, respectively. The complete set of data supports the proposal of a rapid-equilibrium random-sequential model with strictly independent sites for GSH and CDNB or PNPA. Kinetic parameters are thus true dissociation equilibrium constants with values of 0.15 mM for GSH, 0.15 mM for CDNB, and 7 mM for PNPA. Analysis of the inhibition by the product (S-(2,4-dinitrophenyl)-glutathione, 10 to 100 microM), on the coupling reaction between GSH and CDNB with either GSH (0.05 to 0.5 mM, CDNB 0.2 mM) or CDNB (0.05 to 0.5 mM, GSH 0.2 mM) varied, consistent with the proposed mechanism. Binding of product to the free enzyme excludes GSH (competitive inhibition pattern with Kp = 12 microM) but only slightly hinders binding of CDNB. Binding free energies, together with the inhibition pattern, suggest that the non-peptidic moiety of product interacts with an alternative sub-site within the large open pocket accommodating the various electrophilic substrates. These results lead us to propose a model for intra-pocket shifting of the non-peptidic moiety upon product formation which contributes to the product release.     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E(GSSG/2 GSH)) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E(CySS/2 Cys)). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a 'thiol-disulphide redox environment' (E(thiol-disulphide)), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E(CySS/2 Cys) to E(thiol-disulphide) in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Glutathione and glutathione disulfide affect adventitious root formation and growth in tomato seedling cuttings

To study the relationship between glutathione and rooting, tomato seedling cuttings, grown on basal- or on auxin-supplemented media, were treated with the reduced (GSH) or oxidized (GSSG) form of this antioxidant. In turn, the consequences of the depletion of GSH pool on rooting were tested using l-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis. Effects of the aforementioned treatments on rooting response were assessed. GSH treatment promoted root formation on cuttings grown on both basal- and auxin-supplemented media. Whereas GSSG did not affect the number of roots formed by cuttings grown on basal medium, it strongly enhanced the rooting stimulatory effect of auxin treatment. GSH depletion resulting from BSO application did not change the number of roots formed. All the tested compounds, namely GSH, GSSG, BSO and auxin, had a strong inhibitory effect on the elongation of regenerated roots. Supplementing the rooting medium with glutathione efficiently increased the GSH level in the rooting zones, while addition of BSO led to a strong decrease in endogenous GSH level. Neither of the treatments affected the level of GSSG. Exogenous auxin affect neither GSH nor GSSG levels in rooting zones; however, in the regenerated roots, GSH level was significantly higher when the organs were formed on auxin-supplemented medium. Patterns of GSH distribution in the roots regenerated on basal- and auxin-enriched media were studied using the GSH-specific dye monochlorobimane and confocal laser scanning microscopy. GSH was found in the root apical meristem and in the elongation zone. Auxin did not change the GSH distribution; however, the number of fluorescent cells was higher when roots were regenerated on auxin-supplemented medium.     

Unequivocal evidence in support of the nonenzymatic redox coupling between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid.

Experiments were performed to evaluate the nonenzymatic reaction between glutathione (GSH) and dehydroascorbic acid (DHA). Though both ascorbic acid and glutathione disulfide (GSSG) are formed from this reaction, previous work has focused almost exclusively on measurements of ascorbic acid. In contrast, there is very little information about the formation of GSSG under the same conditions as those used to produce ascorbic acid. The emphasis on ascorbic acid stems from the fact that a spectrophotometric technique is available for its measurement, whereas 1H-NMR or an amino acid analyzer has been used to measure GSSG. The present experiments use a simple, rapid method for accurately and precisely measuring the concentrations of GSSG in a solution. The spectrophotometric (340 nm) procedure uses NADPH and glutathione reductase; analysis time is very short, many replicate samples can be tested and as little as 0.05-0.1 mM GSSG can be detected. Using this method, it is shown that there is an equimolar production of GSSG and ascorbic acid from GSH and DHA and that the decrease in GSH is stoichiometrically related to the increase in the concentration of GSSG. The present findings provide additional insight into the interaction between the GSH/GSSG redox couple and the ascorbic acid/DHA redox couple.     

A study of the intracellular effects of glutathione by 1H-spin echo NMR of intact human erythrocytes

The effect of adding either reduced (GSH) or oxidized (GSSG) glutathione to intact human erythrocytes was investigated by 1H-spin echo NMR, which allows direct observation of relatively concentrated low molecular weight compounds within intact cells. A specific region of the spectrum was affected by addition of GSH, with the appearance of new peaks that were diagnostic of an increase of intracellular GSH. These changes did not occur in hemolysates, and did not involve extra-cytosol GSH either free or membrane-bound. These results indicate that the intracellular redox balance of glutathione is shifted toward the reduced state by exogenous glutathione, possibly via a signal transferring system of the cell membrane.     

The interaction of reduced glutathione with active oxygen species generated by xanthine-oxidase-catalyzed metabolism of xanthine

The interaction of reduced glutathione (GSH) with active oxygen species generated during xanthine-oxidase-catalyzed metabolism of xanthine was investigated. The only GSH-derived product detected in this system was oxidized glutathione (GSSG). Catalase inhibited the oxidation of GSH to GSSG by more than 80%, whereas superoxide dismutase exerted a smaller but significant inhibition of GSSG formation. Hydroxyl radical (OH) scavengers or desferrioxamine (1 mM) had no effect on GSSG formation. Using EPR spectroscopy and the spin trap 5,5-dimethylpyrroline-N-oxide (DMPO), the production of superoxide was observed by the detection of a DMPO-OOH radical adduct. This spectrum was altered by the inclusion of GSH (5 - 20 mM) in the reaction mixture, indicating the generation of a different radical species consistent with DMPO-glutathionyl radical adduct generation.