Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone)

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochondrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thiol-disulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.     

Diurnal fluctuation and pharmacological alteration of mouse organ glutathione content

Mouse liver glutathione content showed a diurnal variation with a maximum GSH + 2 GSSG content at 6 to 10 a.m. of 62 +/- 8 nmole per mg protein and a minimum of 42 +/- 7 at 6 p.m. Starvation for more than 24 hr decreased the hepatic glutathione content to 22 +/- 3 nmole/mg protein and abolished the diurnal rhythm. Artificial reversal of the feeding habit of the animals reversed the diurnal rhythm. Kidney, spleen and lung glutathione contents showed no such rhythm. The organ glutathione content decreased by 50% or more upon starvation. The increase of the liver glutathione content by injection of either free or liposomally entrapped GSH to starved animals was not dependent on the time of administration. The physiological maximum level could not be exceeded by this treatment. It was not possible to influence the glutathione content of kidney, lung or intestine by glutathione injections in either form. Intravenous injections of equimolar doses of 2,3-dimercaptopropanol, 2-mercaptoethanesulfonic acid, N-2-mercaptopropionylglycine, D-penicillamine, or cysteamine did not lead to any significant change in liver, kidney, spleen or lung glutathione contents 2 hr after administration. Intravenously given N-acetylcysteine, methionine, GSH or GSSG restored liver glutathione levels of starved animals to the contents observed in the fed state. The diurnal hepatic variation of GSH caused by the food intake habit of the animals may limit the capacity of the intracellular detoxication system.     

Possible regulation mechanism of microsomal glutathione S-transferase activity in rat liver

After rats were injected with the reduced glutathione (GSH) depletor phorone (diisopropylidene acetone, 250 mg/kg, i.p.), there was a significant increase in microsomal glutathione S-transferase activity in the liver. The maximum activity was observed 24 hr after injection and was about 2-fold that of the control activity. Diethylmaleate (500 mg/kg, i.p.) had the same effect. Twenty-four hours after phorone injection (250 mg/kg, i.p.), the concentrations of GSH and oxidized glutathione (GSSG) in the liver were increased about 2-fold. Under the same conditions, the level of mixed disulfides with microsomal proteins (GSS-protein) was also increased. Further, the activity of microsomal glutathione S-transferases was increased by the in vitro addition of disulfide compounds such as GSSG, cystine and homocystine, and the activity increased by GSSG was reduced to control levels by incubating with the corresponding sulfhydryl compounds such as GSH, cysteine and homocysteine respectively. Thus, microsomal glutathione S-transferase activity appears to be regulated by the formation and/or cleavage of a mixed disulfide bond between the sulfhydryl group present in the enzyme and GSSG. Therefore, the increase of microsomal glutathione S-transferase activity after phorone injection may be due to the formation of a mixed disulfide bond between the sulfhydryl group in the enzyme and GSSG.     

Effect of selenium on glutathione metabolism. Induction of gamma-glutamylcysteine synthetase and glutathione reductase in the rat liver

The effect of sodium selenite (Na₂SeO₃, Se) on cellular glutathione metabolism was examined, particularly with respect to its ability to alter the activities of γ-glutamylcysteine synthetase and glutathione disulfide (GSSG) reductase. The treatment of rats with Se (5, 10 and 20 μmoles/kg) caused time- and dose-dependent increases in the activities of the synthetase and the reductase in the liver. The activity of γ-glutamylcysteine synthetase, the rate-limiting enzyme of the glutathione (GSH) biosynthesis, was particularly susceptible to Se treatment. The Se-mediated increases in the activities of the above enzymes were inhibited by puromycin and the increases could not be elicited in vitro. Selenium treatment caused time-dependent perturbations in the levels and ratio of GSSG and GSH in the liver. When compared to the control animals, rats treated for 3 hr with 10 and 20 μmoles Se/kg showed increased cellular levels of GSSG; in contrast, 24 hr after Se treatment the concentration of GSH was increased significantly. The activity of γ-glutamyl transpeptidase, which catalyzes the initial reaction in GSH breakdown, was unaltered by Se treatment. Repeated administration of low doses of Se (7.0 μmoles/kg, three times) also increased the activities of the reductase and the synthetase as well as the cellular levels of hepatic GSH and GSSG. It is suggested that the Se-mediated increases in the activities of γ-glutamylcysteine synthetase and GSSG-reductase represent cellular responses to Se-mediated perturbations in the levels and ratio of GSH and GSSG.     

Increase in hepatic mixed disulphide and glutathione disulphide levels elicited by paraquat

Paraquat (1 mM), when added to isolated haemoglobin-free perfused rat liver, leads to an increase of intracellular mixed disulphides from 1.3 μmole GSH equivalents per g wet weight in the controls to 2.5 μmole/g. This raises the proportion of mixed disulphides to total glutathione equivalents from about 0.2 at the beginning of the perfusion to about 0.4. The mixed disulphides are predominantly protein-bound, with low molecular weight compounds being quantitatively negligible.The content of intracellular glutathione disulphide (GSSG) is increased from 17 nmole/g in the controls to 38 nmole/g in the presence of paraquat. In addition, there is an increased rate of release of GSSG into the extracellular (biliary) space, reported previously.It is suggested that, in a reaction catalysed by thioltransferase(s), the rise in GSSG is correlated with the rise in mited disulphides (reaction 1). Occupancy of potential cellular mixed disulphide sites is about $\text{1}\text{2}$ in the controls, and rises to about $\text{2}\text{3}$ in the presence of paraquat.The ratio of cellular contents, NADPH/NADP⁺, is decreased from 5.1 in the controls to 2.3 in the presence of paraquat, while the sum of NADPH plus NADP⁺ remains unaltered.The perturbation in the glutathione status may be related to metabolic effects such as the stimulation of the pentose-phosphate pathway activity, and possibly also to the expression of toxic effects.     

A simultaneous liquid chromatography/mass spectrometric assay of glutathione,cysteine, homocysteine and their disulfides in biological samples

A liquid chromatography/mass spectrometric (LC/MS) method was developed for simultaneous detection and quantitation of glutathione (GSH), glutathione disulfide (GSSG), cysteine (CysSH), homocysteine (HCysSH) and homocystine in biological samples (rat brain, lung, liver, heart, kidneys, erythrocytes and plasma). Thiols were derivatized with a large excess of Ellman's reagent, a thiol-specific reagent, to ensure an instantaneous and complete derivatization. The derivatization blocked the oxidation of the thiols to disulfides, preventing errors caused by thiol oxidation. The samples were then analyzed by LC/MS. The method provides a highly selective and sensitive assay for these endogenous thiols and their corresponding disulfides. The detection limits for GSH, GSSG, CysSH, HCysSH and homocystine were 3.3, 3.3, 16.5, 29.6 and 14.9 pmol, respectively. An attempt for cystine analysis was unsuccessful due to earlier elution of the compound and strong interferences caused by other endogenous compounds. This method will be a useful tool in the investigation of the roles of these important thiol-containing compounds and their corresponding disulfides in physiological and pathological processes.     

Increased biliary secretion and loss of hepatic glutathione in rat liver after nifurtimox treatment

Treatment of rats with nifurtimox, a nitrofuran derivative widely used for the treatment of Chagas' disease, induced a time- and dose-dependent depletion of liver glutathione, maximal effects being obtained with 200 mg nifurtimox/kg body weight. Extra release of both oxidized (GSSG) and reduced (GSH) glutathione into bile contributed to this depletion. Glutathione excretion into bile accounted for only part of liver glutathione loss, thus indicating that, in addition to the GSH-peroxidase reaction (resulting in GSSG generation), other glutathione-related processes were involved in nifurtimox detoxification. Bile flow, bile salt excretion, liver lipid conjugated diene content, liver glutathione reductase and glutathione peroxidase activities, and serum alanine aminotransferase (ALAT) activity were not affected by the nifurtimox treatment, thus ruling out widespread damage of the liver cell by nifurtimox. Nevertheless, the extra GSH release in the nifurtimox-treated rats may indicate an alteration of the hepatocyte membrane.     

2-Amino-5-chlorophenol toxicity in renal cortical slices from Fischer 344 rats: effect of antioxidants and sulfhydryl agents

A rapid inhibition of protein synthesis is observed when isolated rat hepatocytes are incubated in the presence of 0.25-0.5 mM of tert-butyl hydroperoxide (tBOOH). Such an inhibition occurs in the absence of a cytolytic effect by tBOOH. Iron chelators (o-phenanthroline and desferrioxiamine), protected against oxidative cell death, but they did not modify the inhibition of protein synthesis caused by tBOOH (0.5 mM), suggesting that free radicals are less implicated in such an impairment. Electron micrographs of hepatocytes under oxidative stress show disaggregation of polyribosomes but not oxidative alterations, such as blebs or mitochondrial swelling. Protein synthesis inhibition is accompanied by a decrease in reduced glutathione (GSH) and an increase in glutathione disulfide (GSSG) and the level of protein S-thiolation (protein mixed disulfides formation). Such an increase of GSSG appears as a critical event since diethylmaleate (DEM) at 0.2 mM reduced GSH content by more than 50% but did not affect either GSSG content or protein synthesis. The addition of exogenous GSH and N-acetylcysteine (NAC) to tBOOH-treated hepatocytes significantly reduced the formation of protein mixed disulfides and restored the depressed protein synthesis either completely or partially. We suggest that S-thiolation of some key proteins may be involved in protein synthesis inhibition by tBOOH.     

7-Glycidoxycoumarin (GOC): a fluorophotometric epoxide substrate for the assay of glutathione S-transferase activity

7- Glycidoxycoumarin ( GOC ), a new fluorophotometric epoxide substrate for glutathione S-transferase (GSH TFase ), was conjugated regiospecifically with GSH at pH 6.5 in rat liver cytosol to yield S-(2-hydroxy-3-(7'- coumaroxy )-1-propyl)glutathione which was isolated by HPLC and identified with an authentic specimen by 13C NMR spectroscopy. The conjugation product formed in the incubation media consisting of GOC , GSH, and 9000 g supernatant fractions from various tissues of the rat, was directly determined by photometry of fluorescence emission at 388 nm at an excitation wavelength of 328 nm after removal of the unreacted substrate and its enzymic hydrolysis product, 7-(1',2'-dihydroxy-3'-propoxy)coumarin, by simple extraction with isobutyl alcohol in the presence of a saturating amount of sodium chloride. Stability of GOC at pH 6.5 markedly retarded its autoconjugation with GSH and made the fluorophotometric method sensitive enough to assay small GSH TFase activities in gel column chromatographic fractions as well as in various tissues of the animals. Apparent Km and Vmax for GOC in rat liver cytosol were 55 microM and 7.41 nmole/mg protein/min, respectively. GSH conjugation of GOC was catalyzed by at least two isozymes, E and AA, of hepatic GSH TFases .     

Mechanisms of glutathione disulfide efflux from erythrocytes

Glutathione (GSH) plays numerous critical protective roles in the erythrocyte and GSH turnover is likely an important factor in regulating susceptibility to oxidative stress and toxins. Efflux of glutathione disulfide (GSSG) from erythrocytes is an important component in the regulation of GSH levels; however, little is known of the mechanisms involved. We hypothesize that multidrug resistance associated protein 1 (MRP1) is responsible, in part, for GSSG transport from erythrocytes. To test this, we determined the levels of MRP1 protein in erythrocyte membranes from healthy adults and compared them with intracellular levels of GSH. MRP1 levels varied substantially from person to person and were inversely correlated with levels of GSH (r = -0.39, P < 0.05). In contrast, activity levels of glutamyl cysteine ligase, the rate limiting GSH biosynthetic enzyme, were unrelated to GSH levels. To directly determine the role of MRP1 in GSSG transport, in vitro studies were conducted examining the effects of MRP1 inhibitors MK571 and verapamil on GSSG efflux. Both compounds resulted in significant but not complete inhibition (20-53%) of GSSG efflux from cells. Overall, these findings support a role for MPR1 in the regulation of erythrocyte GSH levels through the transport and elimination of GSSG from cells.     

The role of glutathione and changes in thiol homeostasis in cultured lung cells exposed to ozone

Cells of an alveolar type II cell-line (A549) were exposed to ozone, using an in vitro exposure model. In this study, attention was focused on the cellular glutathione system. It was demonstrated that cellular levels of both reduced (GSH) and oxidized glutathione (GSSG) were significantly reduced after exposure of the cells to ozone. When A549 cells were incubated with methionine sulfoximine and diethylmaleate, glutathione levels were depleted, and the cells showed a marked increase in sensitivity towards ozone. Some of the possible mechanisms by which the observed effects might be explained were investigated. It was shown that glutathione lost from the cells was not incorporated into "mixed disulfides", but could be detected in the surrounding medium. Furthermore, it was shown that A549 cells do not contain any detectable glutathione peroxidase activity. Therefore it was concluded that glutathione peroxidase-catalysed reduction of lipid peroxides could not be responsible for the observed protective role of glutathione. Finally some other mechanisms by which glutathione might accomplish its antioxidant effect are discussed.     

Simultaneous spectrophotometric determination of oxidized and reduced glutathione in human and rabbit red cells

An improved spectrophotometric procedure for oxidized and reduced glutathione determination in erythrocytes is described. The method is based upon a reaction using Ellman's reagent. It gave recoveries of 99 and 90% for both GSH and GSSG. The use of oxidized glutathione as an internal standard makes it accurate for simultaneous assay. The method offers the advantage of not having to use alkylation products to prevent oxidation of GSH during protein precipitation. Data are presented to demonstrate reliability and simplicity of rapid estimation of GSH and GSSG.     

Protective effect of N-acetyl-L-cysteine against maneb induced oxidative and apoptotic injury in Chinese hamster V79 cells

The role of antioxidant N-acetyl-L-cysteine (NAC) in protection against cellular changes triggered by maneb during in vitro exposure was investigated in cultured Chinese hamster V79 cells. We observed high apoptotic activity and high oxidative stress induced by exposure to maneb evidenced by a statistically significant increase in lipid peroxidation (measured as TBARS - thiobarbituric acid reactive substances) as well as a decrease of glutathione (GSH) and glutathione disulfide (GSSG) ratio (GSH/GSSG). Maneb did not exhibit any effect on protein oxidation (measured by protein carbonyls content). NAC suppressed cellular changes induced by maneb in V79 cells. NAC pre-treatment prevented TBARS production and significantly decreased the number of apoptotic cells. However, protective effect of NAC on GSH and GSSG levels has been shown only in cells exposed to lower concentration of maneb (100 μM).     

Effects of glycerol-induced acute renal failure on tissue glutathione and glutathione-dependent enzymes in the rat

The activities of tissue glutathione (reduced and oxidized) and glutathione-dependent enzymes such as glutathione S-transferase (GSH S-transferase), glutathione reductase (GSSG reductase) and glutathione peroxidase (GSH-Px) were determined for control and uremic rats. Acute renal failure (ARF) was produced by glycerol-water injection. Cytosolic and microsomal GSH S-transferase activity in the kidney was decreased by 38% and 15%, respectively. Hepatic microsomal GSH S-transferase was also decreased by 40% in uremic rats. GSH-Px activity was decreased by 51% in the cytosolic fraction and 33% in the microsomal fraction in the kidney, but was not affected in the liver and whole blood. GSSG reductase activity was also decreased by 48% in the cytosolic fraction in the kidney of uremic rats. In whole blood, however, GSSG reductase activity was increased by 12-fold (0.66 +/- 0.12 mumol NADPH oxidized/min/ml blood in the control; 8.03 +/- 3.29 mumol NADPH oxidized/min/ml blood in uremia). Although the total glutathione concentrations were not significantly affected, the GSSG/GSH ratio, which is an indication of oxidative stress, was significantly increased in the liver and whole blood of uremic rats. In addition to the decreases in hepatic and renal GSH S-transferase activities, which is important in drug disposition, ARF caused decreases in GSSG reductase and GSH-Px activity, which are essential for the protection against lipid peroxidation.     

Analysis of solutions containing glutathione and inorganic nitrite: application to nitroglycerin metabolism studies

Nitroglycerin (GTN) is metabolized to 1,2-dinitroglycerin (1,2-GDN) and 1,3-dinitroglycerin (1,3-GDN) in vivo and in liver homogenates. 1,2-GDN and 1,3-GDN are converted to isomers of glyceryl mononitrate (GMN) in vivo. The denitration reactions yield inorganic nitrite (NO(-)(2)) which is oxidized to inorganic nitrate (NO(-)(3)). Denitration involves utilization of glutathione (GSH). In attempting to use the Bratton-Marshall assay for NO(-)(2) in studies of GTN metabolism in vitro, and in attempting to use Ellman's reagent for GSH in the same research, apparent concentrations of both NO(-)(2) and GSH were noticed lower than anticipated. Apparent mutual interference by NO(-)(2) and GSH in their respective assays was then found. Development of a specific liquid chromatographic method for measurement of NO(-)(2), NO(-)(3), GSH and oxidized glutathione (GSSG) permitted the study of the interaction of NO(-)(2) and GSH, which yielded NO(-)(3) and GSSG.     

The anticonvulsive effect of glutathione in mice

To explore the role of glutathione as a neuromodulator, we investigated effects of reduced (GSH) and oxidized glutathione (GSSG) on drug-induced convulsions in mice. Intracerebroventricular administration of GSH or GSSG (10-300 nmol) did not produce convulsions. When GSH was administered prior to subcutaneous administration of pentylenetetrazol (80 mg/kg), it significantly inhibited pentylenetetrazol-induced convulsions. The inhibitory effect of GSH was dose dependent, and mimicked by GSSG. In addition, neither GSH nor GSSG affected convulsions induced by subcutaneous administration of N-methyl-DL-aspartate (400 mg/kg). These findings suggest that glutathione has a specific anticonvulsive effect.     

Effects of diethyldithiocarbamate and nickel chloride on glutathione and trace metal concentrations in rat liver

Concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG) and 4 trace metals (Ni, Cu, Mn, Zn) were measured in livers from rats treated with sodium diethyldithiocarbamate (DDC, 0.67 or 1.33 mmol/kg, i.m.) and NiCl2 (0.25 or 0.50 mmol/kg, s.c.), singly or in combination. In rats treated with DDC or NiCl2, singly, hepatic GSH was diminished at 4 h and returned to control levels (or slightly above) at 17 h. In rats that received DDC plus NiCl2, hepatic GSH was not diminished at 4 h after increased 1.4-1.8-fold at 17 h. Hepatic GSSG was diminished at 4 h after NiCl2 treatment and returned to control values at 17 h; hepatic GSSG did not differ from control values at 4 h or 17 h after treatment with DDC, alone or combined with NiCl2. Hepatic Ni was below the detection limit (approximately 20 nmol/g) in control and DDC-treated rats; hepatic Ni was increased to 53 +/- 26 (S.D.) nmol/g at 17 h after treatment with NiCl2 alone, and was increased 6-fold (308 +/- 63 nmol/g) in rats that received Ni plus DDC. Under the same conditions, hepatic Zn was increased 33% or 41%, respectively, in rats that received NiCl2 or DDC, singly, and was not further increased by combined treatment; hepatic Cu and Mn concentrations were unaffected by NiCl2 or DDC, singly, but were diminished in rats that received NiCl2 and DDC. This study suggests: (a) that increased hepatic uptake of Ni is largely responsible for the synergistic induction of heme oxygenase activity in rats treated with NiCl2 and DDC; and (b) that increased hepatic uptake of Zn contributes to the induction of hepatic metallothionein by NiCl2 and DDC.     

Early decreases in pulmonary, hepatic and renal glutathione levels in response to cadmium instillation into rat trachea

After instillation of cadmium (Cd) into the rat trachea, reduced and oxidized glutathione (GSH and GSSG) levels in the lung, liver and kidney were studied in relation to Cd concentrations and metallothionein (Mt) contents. Rats instilled with Cd developed haemorrhagic pneumonia, which deteriorated with a marked swelling of the lung throughout the experimental period of 48 h. The total glutathione (GSH + GSSG) level in the organs decreased after 6 h to 60-70% of the control levels. The decreased glutathione level was never restored to the control level within 48 h in the lung, and was possibly due to the pneumonia. Completely recovered glutathione was seen in other organs. The GSSG level did not decrease significantly in the lung or liver but lowered significantly after 12 h and 24 h. The GSSG fraction in the amount of total glutathione was 10% or more in the lung and 5% or less in the liver or kidney. This finding indicated that the total glutathione level was mainly changed by the decrease in the GSH fraction. Cadmium in the lung increased to 7.3 ppm 3 h after Cd instillation and decreased to 2.5 ppm within 48 h. Cadmium in the liver and kidney gradually increased with time, and after 48 h reached 1.1 and 2.3 ppm, respectively. This indicated a transportation of Cd from the lung to these organs. Moreover, the early stage of Cd accumulation coincided with the total glutathione decrease in the organs. After Cd instillation, pulmonary, hepatic and renal Mt started to increase at 3 or 6 h, and markedly increased at 24 h or later.(ABSTRACT TRUNCATED AT 250 WORDS)     

Life span profiles of glutathione and acetaminophen detoxification

Our previous finding of a glutathione (GSH) deficiency in aging or senescent mice suggested that a concomitant decrease in detoxification capacity also may occur. To test this, mice at different biological stages of the life span (growth, maturity, aging or senescence) were injected with various doses of acetaminophen (APAP), and GSH depletion and recovery rates were determined. At intervals for 24 hr, samples of blood and other tissues were obtained, processed, and analyzed for reduced GSH, glutathione disulfide (GSSG), cysteine, and cystine using HPLC with dual electrochemical detection. In the uninjected controls, the GSH concentration decreased about 30% in all tissues of the aging mouse, but the GSSG cysteine, and cystine levels were unchanged during the life span. APAP administration depleted liver and lung GSH contents in a dose- and time-dependent manner. Four hr after APAP administration, hepatic GSH levels of all ages had decreased 70-80%. After 24 hr, the GSH levels of the young, growing (3-6 months), and mature (12 months) mice recovered to 94 and 66%, respectively, of the controls. In contrast, the level in aging (31 months) mice rose only 41%, a lower recovery that was correlated with their decreased GSH content. The lungs of old mice also were GSH-deficient but differed from liver, for there was less GSH depletion by APAP and no decrease in GSH recovery. Thus, these findings demonstrate clearly the occurrence of decreased detoxification capacity in the GSH-deficient, aging mouse.