Effects of 4-hydroxynonenal on isolated hepatocytes. Studies on chemiluminescence response, alkane production and glutathione status.

The effect of 4-hydroxy-2,3-trans-nonenal, a diffusible product of lipid peroxidation, on isolated hepatocytes was evaluated with two non-invasive techniques measuring low-level chemiluminescence and alkane evolution. Oxygen-induced low-level chemiluminescence and ethane and n-pentane formation by hepatocytes is enhanced over 7-fold in the presence of 4-hydroxynonenal (2 mM). Glutathione-depleted hepatocytes show a higher increase than controls in both low-level chemiluminescence and alkane formation upon supplementation with 4-hydroxynonenal. The effects on both parameters are diminished by vitamin E pretreatment of rats and are absent under anaerobiosis. At variance with chemiluminescence and alkane formation, 4-hydroxynonenal does not elicit a concomitant increase in malonaldehyde or diene-conjugate formation. Addition of 4-hydroxynonenal to a suspension of hepatocytes causes a rapid loss of cellular glutathione in the form of a glutathione conjugate with the alkenal as observed with high-pressure liquid-chromatographic analysis. The reaction between glutathione and 4-hydroxynonenal proceeds also spontaneously in vitro at 1:1 stoichiometry. The cellular effects of 4-hydroxynonenal evaluated by low-level chemiluminescence and alkane formation are independent of the formation of a glutathione conjugate and seem to rely on the remaining not-bound 4-hydroxynonenal. The sensitivity of 4-hydroxynonenal-enhanced chemiluminescence and alkane formation to free-radical quenchers suggests the participation of a free-radical propagation process.     

Effect of L-2-oxothiazolidine-4-carboxylate administration on glutathione and cysteine concentrations in guinea pig liver and kidney

Effect of L-2-oxothiazolidine-4-carboxylate administration on the glutathione and cysteine concentrations in liver and kidney was studied in the guinea pig. Liver glutathione concentration increased significantly by 21 to 29% at one to three hours after the intraperitoneal injection of 5 mmol of sodium L-2-oxothiazolidine-4-carboxylate per kg of body weight. Cysteine concentration did not change significantly. In contrast to the liver, a significant increase in cysteine and also a significant decrease in glutathione concentrations were observed in the kidney. Incubation of L-2-oxothiazolidine-4-carboxylate with liver and kidney homogenates resulted in cysteine formation of 1.21 and 0.56 mumol per g of fresh tissue per 30 min, respectively. These results seem to indicate that, in the liver, L-2-oxothiazolidine-4-carboxylate administration resulted in the formation of cysteine, which was utilized for glutathione synthesis. In the kidney, it seems to be suggested that the administration of this compound accelerated glutathione turnover.     

Role of glutathione in the formation of the active form of the oxygen sensor FNR ([4Fe-4S].FNR) and in the control of FNR function.

The oxygen sensor regulator FNR (fumarate nitrate reductase regulator) of Escherichia coli is known to be inactivated by O2 as the result of conversion of a [4Fe-4S] cluster of the protein into a [2Fe-2S] cluster. Further incubation with O2 causes loss of the [2Fe-2S] cluster and production of apoFNR. The reactions involved in cluster assembly and reductive activation of apoFNR isolated under anaerobic or aerobic conditions were studied in vivo and in vitro. In a gshA mutant of E. coli that was completely devoid of glutathione, the O2 tension for the regulatory switch for FNR-dependent gene regulation was decreased by a factor of 4-5 compared with the wild-type, suggesting a role for glutathione in FNR function. In isolated apoFNR, glutathione could be used as the reducing agent for HS- formation required for [4Fe-4S] assembly by cysteine desulfurase (NifS), and for the reduction of cysteine ligands of the FeS cluster in FNR. Air-inactivated FNR (apoFNR without FeS) could be reconstituted to [4Fe-4S].FNR by the same reaction as used for apoFNR isolated under anaerobic conditions. The in vivo effects of glutathione on FNR function and the role of glutathione in the formation of active [4Fe-4S].FNR in vitro suggest an important role for glutathione in the de novo assembly of FNR and in the reductive activation of air-oxidized FNR under anaerobic conditions.     

Oxygen toxicity in the perfused rat liver and lung under hyperbaric conditions.

1. In the lung and liver of tocopherol-deficient rats, the activities of glutathione peroxidase and glucose 6-phosphate dehydrogenase were increased substantially, suggesting an important role for both enzymes in protecting the organ against the deleterious effects of lipid peroxides. 2. Facilitation of the glutathione peroxidase reaction by infusing t-butyl hydroperoxide caused the oxidation of nicotinamide nucleotides and glutathione, resulting in a concomitant increase in the rate of release of oxidized glutathione into the perfusate. Thus the rate of production of lipid peroxide and H2O2 in the perfused organ could be compared by simultaneous measurement of the rate of glutathione release and the turnover number of the catalase reaction. 3. On hyperbaric oxygenation at 4 X 10(5)Pa, H2O2 production, estimated from the turnover of the catalase reaction, was increased slightly in the liver, and glutathione release was increased slightly, in both lung and liver. 4. Tocopherol deficiency caused a marked increase in lipid-peroxide formation as indicated by a corresponding increase in glutathione release under hyperbaric oxygenation, with a further enhancement when the tocopherol-deficient rats were also starved. 5. The study demonstrates that the primary response to hyperbaric oxygenation is an elevation of the rate of lipid peroxidation rather than of the rate of formation of H2O2 or superoxide.     

The generation of oxygen radicals: a positive signal for lymphocyte activation

The induction of ornithine decarboxylase (ODC) activity in lymphocytes is associated with activation and the initiation of cellular proliferation. ODC is also an essential component in tumor promotion. Phorbol myristic acetate (PMA) is a mitogen for lymphocytes, but can also promote tumor formation. Tumor promotion is linked to the generation of free radicals induced by PMA. Modulation of intracellular glutathione is associated lymphocyte activation and in protection of cells from damage due to oxygen radicals. We examined the interaction between ODC activity and intracellular glutathione concentrations in EL4 murine lymphoblastoid cells. The intracellular glutathione concentration could be augmented in EL4 cells when cultured with the cysteine delivery agents 2-oxothiazolidine 4-carboxylate (OTC) and 2-mercaptoethanol (2-ME) and suppressed with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO). OTC and 2-ME suppressed ODC activity in fresh serum and PMA-activated EL4 cells. BSO had no effect on ODC activity of EL4 cells cultured in the presence of PMA. While both OTC and 2-ME augmented the total intracellular glutathione concentration, PMA enhanced only the level of oxidized glutathione. To determine if the mechanism by which PMA or fresh serum altered intracellular glutathione and ODC activity was through the generation of oxygen radicals, EL4 cells were cultured with free radical scavengers. The nonpermeant electron acceptor potassium ferricyanide, and the H2O2 scavenger catalase, lowered ODC activity in both serum-stimulated and PMA-activated EL4 cells. Similarly, incubation of EL4 cells with either potassium ferricyanide or catalase elevated intracellular glutathione concentrations. These data suggest that (a) modulation of intracellular glutathione in the EL4 lymphoblastoid cell line alters ODC activity induced by fresh serum and by the mitogen PMA; (b) activation of EL4 cells by PMA alone alters intracellular glutathione metabolism, which may be associated with its role as a mitogen in lymphocyte activation; and (c) the generation of free radicals in EL4 cells may play a positive role in cellular activation.     

Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase

Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.     

Fast product formation and slow product release are important features in a hysteretic reaction mechanism of glutathione transferase T2-2.

The reaction mechanism of rat glutathione transferase T2-2 has been studied using pre-steady-state and steady-state kinetics. Several parts of the catalytic cycle including binding of substrates, product formation, and product release were investigated. Under saturating conditions, a two-step product release was found to be rate limiting in the enzyme-catalyzed reactions between the nucleophilic substrate glutathione and either of the two electrophilic substrates 1-menaphthyl sulfate and 4-nitrobenzyl chloride. The rate constant for pre-steady-state product formation on rat glutathione transferase T2-2 has an observed pK(a) value of 5.7 apparently due to ionization of the sulfhydryl group of glutathione. This rate constant is approximately 2 orders of magnitude higher than k(cat) at pH values of >6. It can be predicted from the pH dependence that product formation would be the sole rate-limiting step at pH values of <3. A hysteretic mechanism of rGST T2-2 is proposed based on a slow conformational transition detected in pre-steady-state displacement experiments.     

Metabolism and cytotoxicity of eugenol in isolated rat hepatocytes

The metabolism and toxic effects of eugenol (4-allyl-2-methoxyphenol) were studies in isolated rat hepatocytes. Incubation of hepatocytes with eugenol resulted in the formation of conjugates with sulfate, glucuronic acid and glutathione. The major metabolite formed was the glucuronic acid conjugate. Covalent binding to cellular protein was observed using [3H]eugenol. Loss of intracellular glutathione and cell death were also observed in these incubations. Concentrations of 1 mM eugenol caused a loss of over 90% of intracellular glutathione and resulted in approximately 85% cell death over a 5-h incubation period. The loss of the majority of glutathione occurred prior to the onset of cell death (2 h). The effects of eugenol were concentration dependent. The addition of 1 mM N-acetylcysteine to incubations containing 1 mM eugenol was able to completely prevent glutathione loss and cell death as well as inhibit the covalent binding of eugenol metabolites to protein. Conversely, pretreatment of hepatocytes with diethylmaleate to deplete intracellular glutathione increased the cytotoxic effects of eugenol. These results demonstrate that eugenol is actively metabolized in hepatocytes and suggest that the cytotoxic effects of eugenol are due to the formation of a reactive intermediate, possibly a quinone methide.     

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.     

Methyl linoleate ozonide as a substrate for rat glutathione S-transferases: reaction pathway and isoenzyme selectivity

The 9,10-mono-ozonide of methyl linoleate was shown to be a substrate for rat hepatic cytosolic, rat lung cytosolic and rat hepatic microsomal glutathione S-transferases (GST). The activities of lung cytosol and liver microsomes with methyl linoleate ozonide (MLO) were found to be high relative to the activity demonstrated by liver cytosol, as compared with their respective activities towards 1-chloro-2,4-dinitrobenzene (CDNB). Only a slight catalytic activity towards the ozonide was noticed for rat lung microsomes. Isoenzyme 2-2 exhibited the highest specific activity (208 nmol/min/mg) when isoenzymes 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 7-7 were compared. This isoenzyme accounts for approx. 25% of cytosolic GST protein in rat lung, while in rat liver it represents approx. 9%. This may partly explain the high activity towards the ozonide noticed for rat lung cytosol. No stable conjugates were formed as products of the reaction of MLO with glutathione; although two glutathione-conjugates were noticed on TLC, they were only formed as intermediate compounds. Coupling of an aldehyde dehydrogenase assay or a glutathione reductase assay to the GST-catalyzed conjugation, demonstrated that oxidized glutathione and aldehydes are formed as the major products in the reaction. To further confirm the formation of aldehydes, the products of the GST-catalyzed reaction were incubated with 2,4-dinitrophenylhydrazine, which resulted in hydrazone formation. In conclusion, the activity of the GST towards the ozonide of methyl linoleate is similar to their peroxidase activity with lipid hydroperoxides as substrates.     

Purification and properties of gamma-glutamylcyclotransferase from human erythrocytes.

1. GAMMA-Glutamylcyclotransferase was purified 10000-fold from human erythrocytes. 2. The purification steps involved fractionation with (NH4)(2)SO(4) and chromatography on Sephadex G-75, DEAE-cellulose and hydroxyapatite. The purified enzyme was found to be homogeneous on density-gradient polyacrylamide-gel electrophoresis. 3. The maximum reaction rate was observed at pH9.0 and the apparent Km value for gamma-glutamyl-L-alanine was 2.2mM. 4. The molecular weight (25250) of the purified enzyme agreed well with the value (25500) in fresh haemolysates, indicating no apparent structural modification of the enzyme during purification. However, rapid processing of the blood through the initial (NH4)(2)SO(4) and Sephadex-chromatography steps was required to prevent formation of a high-molecular-weight aggregate with substantially lower specific activity. 5. gamma-Glutamylcyclotransferase catalyses the formation of 5-oxoproline from gamma-glutamyl dipeptides. The role of this enzyme in erythrocytes is of particular interest, because gamma-glutamyl-L-cysteine serves as a substrate for both gamma-glutamylcyclotransferase and glutathione synthetase. Thus the cyclotransferase could modulate glutathione synthesis.     

The formation of styrene glutathione adducts catalyzed by prostaglandin H synthase. A possible new mechanism for the formation of glutathione conjugates

The metabolism of styrene by prostaglandin hydroperoxidase and horseradish peroxidase was examined. Ram seminal vesicle microsomes in the presence of arachidonic acid or hydrogen peroxide and glutathione converted styrene to glutathione adducts. Neither styrene 7,8-oxide nor styrene glycol was detected as a product in the incubation. Also, the addition of styrene 7,8-oxide and glutathione to ram seminal vesicle microsomes did not yield styrene glutathione adducts. The peroxidase-generated styrene glutathione adducts were isolated by high pressure liquid chromatography and characterized by NMR and tandem mass spectrometry as a mixture of (2R)- and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione. (1R)- and (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione were not formed by the peroxidase system. The addition of phenol or aminopyrine to incubations, which greatly enhances the oxidation of glutathione to a thiyl radical by peroxidases, increased the formation of styrene glutathione adducts. We propose a new mechanism for the formation of glutathione adducts that is independent of epoxide formation but dependent on the initial oxidation of glutathione to a thiyl radical by the peroxidase, and the subsequent reaction of the thiyl radical with a suitable substrate, such as styrene.     

Stereochemistry of the microsomal glutathione S-transferase catalyzed addition of glutathione to chlorotrifluoroethene

The stereochemistry of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation was studied in rat liver cytosol, microsomes, N-ethylmaleimide-treated microsomes, 9000g supernatant fractions, purified rat liver microsomal glutathione S-transferase, and isolated rat hepatocytes. The absolute configuration of the chiral center generated by the addition of glutathione to chlorotrifluoroethene was determined by degradation of S-(2-chloro-1,1,2-trifluoroethyl)glutathione to chlorofluoroacetic acid, followed by derivatization to form the diastereomeric amides N-(S)-alpha-methylbenzyl-(S)-chlorofluoacetamide and N-(S)-alpha-methylbenzyl-(R)-chlorofluoroacetamide, which were separated by gas chromatography. Native and N-ethylmaleimide-treated rat liver microsomes, purified rat liver microsomal glutathione S-transferase, rat liver 9000g supernatant, and isolated rat hepatocytes catalyzed the formation of 75-81% (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione; rat liver cytosol catalyzed the formation of equal amounts of (2R)- and (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione. In rat hepatocytes, microsomal glutathione S-transferase catalyzed the formation of 83% of the total S-(2-chloro-1,1,2-trifluoroethyl)glutathione formed. These observations show that the microsomal glutathione S-transferase catalyzes the first step in the intracellular, glutathione-dependent bioactivation of the nephrotoxin chlorotrifluoroethene.     

Sequential enzyme-catalyzed metabolism of 4-nitrotoluene to S-(4-nitrobenzyl)glutathione

[14C]-4-Nitrotoluene was metabolized by rat liver postmitochondrial supernatant containing NADPH, reduced glutathione and a sulfate activating system to 4-nitrobenzyl alcohol, 4-nitrobenzyl sulfate, and S-(4-nitrobenzyl) glutathione. Formation of both sulfur-containing metabolites was dependent on the presence of a sulfate activating system. These results suggest that the glutathione conjugate was derived from 4-nitrobenzyl sulfate. Reaction of 4-nitrobenzyl sulfate with glutathione was not detected in pH 7.4 buffer, but rat liver cytosol catalyzed the formation of the glutathione conjugate from 4-nitrobenzyl sulfate. These results show that 4-nitrotoluene is metabolized in rat liver by sequential side chain oxidation, sulfation, and glutathione conjugation. Furthermore, they indicate that, unlike certain other arylmethyl sulfates, 4-nitrobenzyl sulfate is not highly reactive.     

In vivo reversal of glutathione deficiency and susceptibility to in vivo dexamethasone-induced apoptosis by N-acetylcysteine and L-2-oxothiazolidine-4-carboxylic acid, but not ascorbic acid, in thymocytes from gamma-glutamyltranspeptidase-deficient knockout mice.

Cellular glutathione is released during apoptosis and may play a role in the regulation of the mitochondrial permeability transition pore. The question of whether only cytosolic glutathione is important in apoptosis, or whether mitochondrial glutathione also plays a role, was investigated using gamma-glutamyltranspeptidase-deficient knockout mice. Thymocytes from these mice were found to have both glutathione pools diminished and they were more susceptible to dexamethasone (DEX)-induced apoptosis. Supplementation with N-acetylcysteine (NAC) and L-2-oxothiazolidine-4-carboxylic acid replenished both glutathione pools and provided protection from apoptosis. Ascorbate supplementation was beneficial to the mitochondrial glutathione pool, but apoptosis was not prevented. NAC supplementation caused an increase in reactive oxygen species formation and cardiolipin oxidation, but had no adverse affect on the amount of apoptotic cells. Our results suggest that the glutathione status is an important factor in apoptosis and indirect evidence indicates that the cytosolic pool of glutathione may be important in DEX-induced apoptosis, with mitochondrial events being secondary, and may reflect the execution phase.     

In vitro-refolding of a single-chain Fv fragment in the presence of heteroaromatic thiols

The aim of the present work was to explore the use of heteroaromatic thiol compounds, namely derivatives of pyridine and pyrimidine, as redox reagents for the in vitro-refolding of a recombinantly expressed single-chain Fv fragment (scFvOx). The mixed disulfide of scFvOx with glutathione was used as a starting material, while reduced glutathione, 4-mercaptopyridine, 2-mercaptopyrimidine, 2-mercaptopyridine N-oxide, and the mercaptobenzene derivative thiosalicylic acid, respectively, served as catalysts for the formation of native disulfide bonds during renaturation. In contrast to thiosalicylic acid, and despite their significantly lower thiol pKa values, none of the heteroaromatic thiol compounds accelerated the apparent kinetics of in vitro-refolding compared to the naturally occurring peptide glutathione. However, significantly improved renaturation yields were observed in the presence of 4-mercaptopyridine and 2-mercaptopyrimidine, demonstrating the usefulness of aromatic thiol compounds as reagents for the in vitro-refolding of antibody fragments.     

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.     

Lap4, a vacuolar aminopeptidase I, is involved in cadmium-glutathione metabolism

In Saccharomyces cerevisiae, accumulation of cadmium-glutathione complex in cytoplasm inhibits cadmium absorption, glutathione transferase 2 is required for the formation of the complex and the vacuolar gamma-glutamyl transferase participates of the first step of glutathione degradation. Here, we proposed that Lap4, a vacuolar amino peptidase, is involved in glutathione catabolism under cadmium stress. Saccharomyces cerevisiae cells deficient in Lap4 absorbed almost 3-fold as much cadmium as the wild-type strain (wt), probably due to the lower rate of cadmium-glutathione complex synthesis in the cytoplasm. In wt, but not in lap4 strain, the oxidized/reduced GSH ratio and the Gtt activity increased in response to cadmium, confirming that the mutant is deficient in the synthesis of the complex probably because the degradation of vacuolar glutathione is impaired. Thus, under cadmium stress, Lap4 and gamma-glutamyl transferase seem to work together to assure an efficient glutathione turnover stored in the vacuole.     

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants

The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme.     

Evidence for the generation of hydroxyl radicals from a chromium(V) intermediate isolated from the reaction of chromate with glutathione.

The formation of hydroxyl radicals from a chromium(V) complex isolated from the reaction of glutathione with chromate has been demonstrated in spin trapping experiments using dimethylsulfoxide and 3,5-dibromo-4-nitrosobenzene sulfonate. Mechanisms for the formation of radicals in such systems are discussed. These results help to explain the ability of solutions containing chromate and glutathione to cause strand breaks in DNA.