GSSG Release Research Articles

P 012 - The Enzymatic Nature of Ascorbate Recycling

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Taurodeoxycholate-induced intestinal injury is modulated by oxidative stress-dependent pre-conditioning like mechanisms

Mechanisms by which hydrophobic bile salts cause tissue changes below their critical micellar concentration (CMC, 1–2 mM) and above (4–8 mM) remain poorly understood. In this study, rat colonic mucosa was exposed to different concentrations of taurodeoxycholate (TDC), t-butyl-hydroperoxide ( t-BH) or glutathione ester with or without pre-incubation with 2 mM TDC. Exposure to 2 mM TDC was associated with 10% higher tissue levels of total glutathione (GSH, basal values: 33.7 ± 3.3 nmol/mg prot). With TDC 8 mM, GSH decreased to 16.4 ± 2.3 nmol/mg prot ( P < 0.05), oxidized glutathione (GSSG) increased by 60% ( P < 0.05), glutathione peroxidase (GSH-Px) and reductase activities were threefold increased, protein carbonyls fourfold increased, protein sulfhydrils decreased by 78%, lactate dehydrogenase (LDH) and GSSG release in the incubation medium were sixfold higher. In 2 mM TDC pre-treated tissues, the subsequent incubation with 8 mM TDC induced a lower loss of tissue GSH, and a lower release of LDH and GSSG. Pre-incubation with 2 mM TDC partly protected against t-BH toxicity, while glutathione ester protected against 8 mM TDC toxicity. In conclusion, TDC exposure causes opposite effects depending on CMC: induction of antioxidant protective systems including glutathione system (pre-conditioning effect) was observed with TDC below CMC, oxidative damages pointing to decreased mucosal detoxification potential with above CMC.

Prominent cardioprotective effects of third generation beta blocker nebivolol against anthracycline-induced cardiotoxicity using the model of isolated perfused rat heart

Nebivolol is a cardioselective beta-blocker (BB) currently used for the treatment of hypertension. It has mild vasodilating properties attributed to its interaction with the L-arginine/nitric oxide pathway, a property not shared by other BBs. Carvedilol is a nonselective ß-adrenergic receptor antagonist that also blocks α1-adrenergic receptors and is a potent antioxidant. Anthracyclines (ANTs), daunorubicin and doxorubicin, are commonly used in the treatment of several tumours, but their cardiac toxicity prevents their use at maximum myelotoxic doses, representing an important problem. In this study, we have evaluated the role of these BBs administered in combination with ANTs (daunorubicin and doxorubicin) on a reduction in cardiac toxicity. The combination of BB and ANTs has reduced the release of GSSG and GSH; in particular, co-treatment with nebivolol to ANTs has shown a significant reduction. The total integrated creatine kinase and troponin T activities were improved by BB and ANTs co-treatment. A significant reduction of their release was observed when hearts were treated with nebivolol. Cardiac tissue activity of gluthatione reductase was not significant and similar among experimental groups. In contrast, gluthatione peroxidise, Mn-superoxide dismutase and nitrite/nitrate release were increased after co-treatment with nebivolol. Finally, three parameters have been used to evaluate the cardiac toxicity of ANTs: the left ventricular pressure developed under a constant perfusion pressure (LVDP), the rate of variation of this parameter during systole (contractility) (LV/dt) max and during diastole (relaxation) (LV(dP/dt) min. Combination with BB has shown a reduction in cardiac toxicity; in particular, nebivolol has exerted the most significant cardioprotective effect.

Early postoperative function of the heart after coronary artery bypass grafting is not predicted by myocardial necrosis and glutathione-associated oxidative stress

Background To investigate whether cardioplegia-related myocardial necrosis, lactate and glutathione release are predictive for early postoperative cardiac function after coronary artery bypass grafting (CABG). Methods Twelve patients with stabile angina scheduled for elective CABG were included. Myocardial release of troponin I (Tn I), creatine kinase MB isoenzyme mass (CK-MB), oxidized glutathione (GSSG) and lactate in blood were measured before cardioplegia, and up to 20 min thereafter. Cardiac function was assessed for 12 postoperative hours. Results Release of Tn I and CK-MB peaked at 20 min (− 14.5 ± 24.1 ng/ml and − 23.9 ± 30.6 ng/ml, respectively) and lactate at 1 min of reperfusion (− 1.5 ± 0.6 mmol/l). Significant GSSG release occurred at 5 min, with concomitant increase of glutathione redox ratio. The changes were not correlated to ischemic time. Cardiac index was increased after CPB and remained higher than preoperative value until the first postoperative morning. No correlations between postcardioplegic heart function and markers of tissue injury were found. Conclusions The extent of myocardial reversible and irreversible injury does not predict early postoperative contractile function of the heart.

Effects of anisotonicity on pentose-phosphate pathway, oxidized glutathione release and t-butylhydroperoxide-induced oxidative stress in the perfused liver of air-breathing catfish, Clarias batrachus.

Both hypotonic exposure (185 mOsmol/l) and infusion of glutamine plus glycine (2 mmol/l each) along with the isotonic medium caused a significant increase of 14CO2 production from [1-14C]glucose by 110 and 70%, respectively, from the basal level of 18.4 +/- 1.2 nmol/g liver/min from the perfused liver of Clarias batrachus. Conversely, hypertonic exposure (345 mOsmol/l) caused significant decrease of 14CO2 production from [1-14C]glucose by 34%. 14CO2 production from [6-14C]glucose was largely unaffected by anisotonicity. The steady-state release of oxidized glutathione (GSSG) into bile was 1.18 +/- 0.09 nmol/g liver/min, which was reduced significantly by 36% and 34%, respectively, during hypotonic exposure and amino acid-induced cell swelling, and increased by 34% during hypertonic exposure. The effects of anisotonicity on 14CO2 production from [1-14C]glucose and biliary GSSG release were also observed in the presence of t-butylhydroperoxide (50 mmol/l). The oxidative stress-induced cell injury, caused due to infusion of t-butylhydroperoxide, was measured as the amount of lactate dehydrogenase (LDH) leakage into the effluent from the perfused liver; this was found to be affected by anisotonicity. Hypotonic exposure caused significant decrease of LDH release and hypertonic exposure caused significant increase of LDH release from the perfused liver. The data suggest that hypotonically-induced as well as amino acid-induced cell swelling stimulates flux through the pentose-phosphate pathway and decreases loss of GSSG under condition of mild oxidative stress; hypotonically swollen cells are less prone to hydroperoxide-induced LDH release than hypertonically shrunken cells, thus suggesting that cell swelling may exert beneficial effects during early stages of oxidative cell injury probably due to swelling-induced alterations in hepatic metabolism.

Changes in glutathione status and the antioxidant system in blood and in cancer cells associate with tumour growth in vivo

The relationship among cancer growth, the glutathione redox cycle and the antioxidant system was studied in blood and in tumour cells. During cancer growth, the glutathione redox status (GSH/GSSG) decreases in blood of Ehrlich ascites tumour-bearing mice. This effect is mainly due to an increase in GSSG levels. Two reasons may explain the increase in blood GSSG: (a) the increase in peroxide production by the tumour that, in addition to changes affecting the glutathione-related and the antioxidant enzyme activities, can lead to GSH oxidation within the red blood cells; and (b) an increase of GSSG release from different tissues into the blood. GSH and peroxide levels are higher in the tumour cells when they proliferate actively, however GSSG levels remain constant during tumour growth in mice. These changes associate with low levels of lipid peroxidation in plasma, blood and the tumour cells. The GSH/GSSG ratio in blood also decreases in patients bearing breast or colon cancers and, as it occurs in tumour-bearing mice, this change associates with higher GSSG levels, especially in advanced stages of cancer progression. Our results indicate that determination of glutathione status and oxidative stress-related parameters in blood may help to orientate cancer therapy in humans.

A Short Burst of Oxygen Radicals at Reflow Induces Sustained Release of Oxidized Glutathione from Postischemic Hearts

Oxygen radical generation induced by postischemic reperfusion can overwhelm endogenous radical scavenging systems, resulting in “oxidative stress.” Release of oxidized glutathione (GSSG) upon reflow has been taken as evidence for the occurrence of oxidative stress in postischemic hearts. However, demonstration that GSSG release is due to oxygen radicals and not to other consequences of ischemia/reperfusion is lacking. To address this issue, isolated rabbit hearts underwent 30 min of global ischemia at 37°C. At reflow, control hearts were perfused with standard buffer for 45 min ( n = 8); treated hearts received the oxygen radical scavenger superoxide dismutase (hSOD) for 15 min, followed by 30 min of standard perfusion ( n = 8). During reperfusion control hearts showed a prominent release of GSSG, which peaked 5 min after reflow. Interestingly, GSSG release was still significantly elevated 45 min into reperfusion, at a time when oxygen radical generation has long ceased. In contrast, in hSOD-treated hearts GSSG release was negligible. Prevention of oxidative stress was also associated with significantly greater recovery of function. Thus, GSSG release occurs in postischemic hearts as a direct consequence of oxygen radical generation, and it may outlast the initial oxidant load.

Blood Glutathione as an Index of Radiation-Induced Oxidative Stress in Mice and Humans

The effect of x-rays on GSH and GSSG levels in blood was studied in mice and humans. An HPLC method that we recently developed was applied to accurately determine GSSG levels in blood. The glutathione redox status (GSH/GSSG) decreases after irradiation. This effect is mainly due to an increase in GSSG levels. Mice received single fraction radiotherapy, at total doses of 1.0 to 7.0 Gy. Changes in GSSG in mouse blood can be detected 10 min after irradiation and last for 6 h within a range of 2.0–7.0 Gy. The highest levels of GSSG (20.1 ± 2.9 μM), a 4.7-fold increase as compared with controls) in mouse blood are found 2 h after radiation exposure (5 Gy). Breast and lung cancer patients received fractionated radiotherapy at total doses of 50.0 or 60.0 Gy, respectively. GSH/GSSG also decreases in humans in a dose–response fashion. Two reasons may explain the radiation-induced increase in blood GSSG: (a) the reaction of GSH with radiation-induced free radicals resulting in the formation of thyl radicals that react to produce GSSG; and (b) an increase of GSSG release from different organs (e.g., the liver) into the blood. Our results indicate that the glutathione redox ratio in blood can be used as an index of radiation-induced oxidative stress. © 1997 Elsevier Science Inc.

Protection of myocytes against free radical-induced damage by accelerated turnover of the glutathione redox cycle.

The primary defence mechanism of myocytes against peroxides and peroxide-derived peroxyl and alkoxyl radicals is the glutathione redox cycle. The purpose of the present study was to increase the turnover rate of this cycle by stimulating the glutathione peroxidase catalysed reaction (2GSH-->GSSG), the glutathione reductase catalysed reaction (GSSG-->2GSH), or both. Neonatal rat heart cell cultures were subjected to a standardized protocol of oxidative stress using 80 mumol.l-1 cumene hydroperoxide (CHPO) for 0-90 min. The consequences of this protocol were described in terms of cellular concentrations of GSH, GSSG, NADPH and ATP, formation of malondialdehyde (MDA), release of GSSG and of ATP catabolites, depression of contraction frequency, cellular calcium overload, and enzyme release. Trolox-C, an analogue of vitamin E, accelerated the glutathione peroxidase reaction leading to lowering of GSH concentration and the GSH/GSSG ratio, less MDA formation, diminished negative chronotropy, delayed calcium overload, and less enzyme release. Glucose was used to accelerate the glutathione reductase reaction by supplying NADPH, leading to higher GSH concentration and a higher GSH/GSSG ratio, less MDA formation, diminished negative chronotropy, unchanged development of calcium overload, and less enzyme release. As a full turn of the glutathione redox cycle involves both the peroxidase and the reductase reactions, the combination of Trolox-C and glucose was superior to either of the two alone: 90 min following addition of CHPO together with Trolox-C and glucose, the GSH concentration and the GSH/GSSG ratio were almost normal, MDA formation was extremely low, calcium overload was markedly delayed, and enzyme release hardly occurred at all. Cells remained beating in the observation period of 30 min. We conclude that the capacity of the glutathione redox cycle to withstand oxidative stress can be increased by stimulation of either the peroxidase reaction or the reductase reaction, and that optimal redox cycling is achieved by stimulation of both reactions.

Oxidative stress during reperfusion of human hearts: potential sources of oxygen free radicals

The aim was to examine the role of neutrophil activation in the genesis of oxidative stress during the early phases of reperfusion after ischaemia in patients subjected to aortocoronary bypass grafting. Ten selected patients were studied. All had normal ejection fraction and normal left ventricular end diastolic pressures before operation. Each patient required at least three grafts, so that the duration of aortic crossclamping exceeded 30 min, the minimum ischaemic period required to detect oxidative stress upon reperfusion. Oxidative stress was assessed by measuring the formation and release of oxidised glutathione (GSSG) in the coronary sinus 1 min before and 3 min after the start of the cardiopulmonary bypass, and then 1, 5, 10, and 20 min after removal of the aortic clamp, and again 5 and 10 min after the end of the cardiopulmonary bypass. The arterial-coronary sinus difference for neutrophils, elastase-alpha 1 protease complex (elastase), and creatine phosphokinase was also monitored at the same intervals. Before clamping GSSG was undetectable in arterial and coronary sinus blood. There was no significant arterial-coronary sinus difference for neutrophils or elastase [53(SEM 66) cell.ml-1 and 1.10(2.49) micrograms.litre-1, respectively[. Five minutes after re-establishment of coronary blood flow, there was both a release of GSSG into the coronary sinus [arterial-coronary sinus difference: 11(2.6) nmol.dl-1] and an accumulation of neutrophils in the heart [arterial-coronary sinus difference: 262(33), P < 0.01 cell.ml-1], whereas no elastase release from the heart was measured [arterial-coronary sinus difference 7.6(4.46) microgram.litre-1, NS]. The arterial levels of elastase increased progressively during the operation from 48(5) microgram.litre-1 (preclamping) to 405(62) microgram.litre-1, P < 0.01 (end of the cardiopulmonary bypass). These data indicate that, in man, neutrophils do accumulate in the myocardium during early reperfusion. However, they are not activated when oxidative stress occurs. It is unlikely that the neutrophil localisation in the heart has pathological significance in the production of oxygen free radicals during early reperfusion. Free radical accumulation in the coronary vessels may contribute to disorders of coronary flow associated with reperfusion.

K(+)-linked release of oxidized glutathione induced by tert-butyl hydroperoxide in perfused rat liver is independent of lipid peroxidation and cell death.

The tert-butyl hydroperoxide (BHP)-induced release of oxidized glutathione (GSSG) and K+ was studied in relation to lipid peroxidation and cell death using isolated perfused rat livers. Infusion of BHP into the perfused liver resulted in an early and simultaneous release of GSSG and K+ and a sustained release of thiobarbituric-acid-reactive substances (TBARS) into the effluent perfusate, which was followed by further prenecrotic leakage of K+ followed by lactic dehydrogenase (LDH). These actions of BHP were not significantly affected by cutting or ligating the bile duct, and they were potentiated by omitting Ca2+ from the perfusion medium. Co-infusion of desferrioxamine, propyl gallate and diethyldithiocarbamate suppressed TBARS release as well as the later leakage of K+ and LDH. Desferrioxamine was also effective under Ca(2+)-free conditions. N,N'-diphenyl-p-phenylenediamine inhibited TBARS release, but it was not protective against cell death, although there was some delay. The action of dithiothreitol was only moderate. On the other hand, leakage of TBARS, K+ (prenecrotic) and LDH was enhanced by cysteamine and beta-mercaptoethanol and most markedly enhanced by ferrous iron. However, none of these agents markedly affected the early release of GSSG and K+. These observations, which support our previous findings, suggest that the early and coupled sinusoidal efflux of GSSG and K+ caused by BHP is independent of lipid peroxidation and cell death and that they represent a physiological mechanism of GSSG release. The results also suggest that lipid peroxidation is not the sole cause of BHP-induced cell death.

Endogenous hydroperoxide formation, cell volume and cellular K+ balance in perfused rat liver.

Addition of benzylamine (0.5 mM) to isolated perfused rat liver led to a net release of K+ of 10.5 +/- 0.3 mumol/g, which was accompanied by a decrease in liver mass by 9.3 +/- 0.4% and a decrease of the intracellular water space by 13.7 +/- 0.6%, suggestive of hepatocellular shrinkage. Benzylamine had no effect on the perfusion pressure, and there was a close relationship between benzylamine-induced net K+ release and the accompanying decrease in liver mass. Benzylamine-induced net K+ release was sensitive to inhibition of monoamine oxidase by pargyline and increased with benzylamine flux through monoamine oxidase, suggesting its dependence on intracellular H2O2 formation. In line with this, infusion of H2O2 (but not of benzaldehyde, the other product of benzylamine metabolism) stimulated net K+ release from the liver. However, at a given H2O2 load K+ release was about 2-3-fold higher when H2O2 was generated intracellularly during the oxidation of benzylamine, as compared with exogenously delivered H2O2. Inhibition of catalase by 3-amino-1,2,4-triazole (0.2 mM) significantly increased the benzylamine-induced net K+ release as well as the benzylamine-induced release of GSSG into bile, but had no effect on benzylamine oxidation at monoamine oxidase. In the presence of Ba2+ (1 mM) or in Ca(2+)-free perfusions, the benzylamine-induced net K+ efflux was diminished by 60-70% or about 30%, respectively. This was not explained by the 20-30% decrease in flux through monoamine oxidase observed under these conditions. The results suggest that metabolic generation of H2O2 inside the liver leads to a net K+ efflux and subsequent hepatocellular shrinkage. Net K+ efflux under these conditions is enhanced when catalase is inhibited, suggesting that the rate of both intracellular H2O2 generation and degradation can modulate cellular K+ balance and cellular volume. The data support the idea that oxidative stress may affect hepatocellular functions also by lowering the hepatocellular hydration state.

Malondialdehyde and glutathione production in isolated perfused human and rat hearts.

A number of studies show the relation between oxygen-derived free radicals and cardiac ischemia/reperfusion injury. However, little is known about oxidative stress in the human heart, which can be measured by oxidized glutathione (GSSG) and malondialdehyde (MDA) formation. Furthermore, data on MDA production by rat hearts are controversial, possibly because of the use of the aspecific thiobarbituric acid assay. Therefore, GSSG and MDA were measured, with colorimetric and high-performance liquid chromatographic assays, respectively, in buffer-perfused explanted human hearts and normal rat hearts made temporarily ischemic. Human hearts received cardioplegia; rat hearts were studied in a control and an ischemic group with or without cardioplegia. Baseline GSSG release was < 0.01 nmol.min-1.g wet wt-1 in both species. During reperfusion, GSSG release from human hearts and from ischemic and cardioplegic/ischemic rat hearts peaked at 0.24 +/- 0.12, 1.1 +/- 0.4, and 0.19 +/- 0.04 nmol.min-1.g-1, respectively. MDA was undetectable (< 0.02 nmol.min-1.g-1) in the effluent of both species and in human hearts (< 4 nmol/g protein). Rat heart reduced glutathione levels decreased 32% as a consequence of cardioplegia and ischemia. Cardioplegia induced a 41% (P = .08) decrease in rat heart MDA content, whereas cumene hydroperoxide increased it 3.6 times (P < .01). Thus, after ischemia human and rat hearts release GSSG, indicating that oxidative stress has occurred. Apparently, lipid peroxidation takes place in normal rat hearts, decreases after cardioplegia, but does not increase after ischemia/reperfusion. Human hearts lack MDA under normoxic and ischemic conditions. This novel finding seems to reflect a low MDA-forming potential in both situations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of medium pH on glutathione redox cycle in cultured human umbilical vein endothelial cells

Impairments of the glutathione redox cycle in cultured endothelial cells under acidic pH conditions were measured. Glutathione-dependent H 2O 2-degrading activities decreased by 20% ( P < .01) at pH 6 and by 51% ( P < .01) at pH 4 compared with activities at pH 7.4 1 hour after a change with fresh medium. Intracellular reduced glutathione (GSH) content increased by 85% ( P < .01) following the change with pH 7.4 medium. Such increases in GSH content were impaired after exposure to acidic medium. After exposure to 500 μmol/L H 2O 2, intracellular GSH content decreased by 61% compared with the level obtained in the absence of H 2O 2 at pH 7.4 ( P < .01). Compared with the level at pH 7.4, the H 2O 2-induced decrease in intracellular GSH content was 32% lower ( P < .01) at pH 6 and did not change at all at pH 4. After exposure to 500 μmol/L H 2O 2, the intracellular oxidized glutathione (GSSG) content increased by 160% at pH 7.4 ( P < .01), 370% at pH 6 ( P < .01), and 90% at pH 4 compared with treatment without H 2O 2, respectively. After exposure to 500 μmol/L H 2O 2, the release of GSSG from cells at pH 6 decreased by 38% compared with the value found at pH 7.4 ( P < .05), and the release at pH 4 completely disappeared. Both glutathione peroxidase (GPO) and glutathione reductase activities decreased as a function of a decrease in pH from 7.4 to 4. The results (1) indicate that the glutathione redox cycle in cultured endothelial cells exposed to acidic conditions is profoundly impaired because of a decrease in GSH content, GPO and glutathione reductase activities, and GSSG transport out of the cells; and (2) suggest that impairment of active oxygen degradation under acidic conditions may induce endothelial cell dysfunction.

Decreased Oxidized Glutathione with Aerosolized Cyclosporine Delivery

Cyclosporine immunosuppression remains vital for successful lung transplantation. Cyclosporine also functions as a membrane active biological response modifier and has been noted to have a variable effect on ischemia-reperfusion (I/R) injury in various tissues. Glutathione plays an important role in the endogenous antioxidant defense system; plasma oxidized glutathione (GSSG) levels are useful as a sensitive indicator of in vivo oxidant stress and I/R injury. Lung transplantation results in ischemia, followed by a period of reperfusion, potentially producing functional injury. This study was designed to evaluate the effect of cyclosporine on oxygen radical generation in a model of single-lung transplantation. Single-lung transplantation was performed in 12 mongrel puppies, with animals assigned to receive either intravenous or aerosolized cyclosporine. Arterial blood and bronchoalveolar lavage fluid (BALF) samples were obtained to determine GSSG levels via a spectrophotometric technique. Samples were obtained both prior to and following the revascularization of the transplanted lung. Whole blood and tissue cyclosporine levels were determined via an high-performance liquid chromatography technique 3 hr following the completion of the transplant. Aerosolized cyclosporine administration resulted in greatly decreased arterial plasma and BALF GSSG levels, whole blood cyclosporine levels, and equivalent tissue cyclosporine levels when compared to intravenous cyclosporine delivery. These findings support the hypothesis that the transplanted lung is a source of GSSG production and release into plasma. Additionally, these findings suggest that cyclosporine may have a direct antioxidant effect on pulmonary tissue, with this activity occuring at the epithelial surface, an area susceptible to oxidant injury.

Effect of anisotonic cell-volume modulation on glutathione-S-conjugate release, t-butylhydroperoxide metabolism and the pentose-phosphate shunt in perfused rat liver.

1. Addition of 1-chloro-2,4-dinitrobenzene to isolated perfused rat liver results in the rapid formation of its glutathione-S-conjugate [S-(2,4-dinitrophenyl)glutathione], which is released into both, bile and effluent perfusate. Anisotonic perfusion did not affect total S-conjugate formation, but release of the S-conjugate into the perfusate was increased (decreased) following hypertonic (hypotonic) exposure at the expense of excretion into bile. Stimulation of S-conjugate release into the perfusate following hypertonic exposure paralleled the time course of volume-regulatory net K+ uptake. 2. Basal steady-state release of oxidized glutathione (GSSG) into bile was 1.30 +/- 0.12 nmol.g-1.min-1 (n = 18) during normotonic (305 mOsmol/l) perfusion and was 3.8 +/- 0.3 nmol.g-1.min-1 in the presence of t-butylhydroperoxide (50 mumol/l). Hypotonic exposure (225 mOsmol/1) lowered both, basal and t-butylhydroperoxide (50 mumol/l)-stimulated GSSG release into bile by 35% and 20%, respectively, whereas hypertonic exposure (385 mOsmol/l) increased. Anisotonic exposure was without effect on t-butylhydroperoxide removal by the liver. GSSG release into bile also decreased by 33% upon liver-cell swelling due to addition of glutamine plus glycine (2 mmol/l, each). 3. Hypotonic exposure led to a persistent stimulation 14CO2 production from [1-14C]glucose by about 80%, whereas 14CO2 production from [6-14C]glucose increased by only 10%. Conversely, hypertonic exposure inhibited 14CO2 production from [1-14C]glucose by about 40%, whereas 14CO2 production from [6-14C]glucose was unaffected. The effect of anisotonicity on 14CO2 production from [1-14C]glucose was also observed in presence of t-butylhydroperoxide (50 mumol/l), which increased 14CO2 production from [1-14C]glucose by about 40%. 4. t-Butylhydroperoxide (50 mumol/l) was without significant effect on volume-regulatory K+ fluxes following exposure to hypotonic (225 mOsmol/l) or hypertonic (385 mOsmol/l) perfusate. Lactate dehydrogenase release from perfused rat liver under the influence of t-butylhydroperoxide was increased by hypertonic exposure compared to hypotonic perfusions. 5. The data suggest that hypotonic cell swelling stimulates flux through the pentose-phosphate pathway and diminishes loss of GSSG under conditions of mild oxidative stress. Hypotonically swollen cells are less prone to hydroperoxide-induced lactate dehydrogenase release than hypertonically shrunken cells. Hypertonic cell shrinkage stimulates the excretion of glutathione-S-conjugates into the sinusoidal circulation at the expense of biliary secretion.

Effects of ischemia and reperfusion on cardiac tolerance to oxidative stress

Oxidative stress may affect cardiac function and metabolism. Oxidants are normally inactivated by reacting with reduced glutathione (GSH), with resulting formation and release of oxidized glutathione (GSSG). However, ischemia might affect glutathione metabolism. This might render ischemic hearts less resistant against subsequent oxidant injury during reperfusion, and it might also affect the reliability of GSSG measurements as a means to investigate oxidative stress in reperfused hearts. We compared the metabolic and functional consequences of an oxidant load in control rabbit hearts and in hearts reperfused after 30 min of normothermic total ischemia. In controls, H2O2 infusion (H2O2; 5-30 microM) induced a dose-dependent stimulation of GSSG release and a progressive impairment of cardiac function. At these doses, H2O2 challenge of postischemic hearts resulted in biochemical and functional changes identical to those observed in controls. Release of lactate dehydrogenase (LDH) and of GSH was negligible, similar in both groups. In additional experiments, infusion of H2O2 at a much higher dose (200 microM) elicited a further increase in GSSG release from both groups, although GSSG concentrations were lower in postischemic hearts. The functional effects of the 200 microM H2O2 infusion were similar in both groups, all hearts showing rapid and irreversible deterioration of function. Occurrence of irreversible cell injury was also manifested by a large release of LDH and GSH to a similar extent in both groups. These data demonstrate that cardiac tolerance toward oxidants is largely unaffected by a relatively brief episode of severe ischemia and indicate that GSSG release can be reliably used to investigate oxidative stress in reperfused hearts.

Myocardial sulfhydryl pool alterations occur during reperfusion after brief and prolonged myocardial ischemia in vivo.

Myocardial sulfhydryl (SH)-containing compounds, including reduced glutathione (GSH), are both defenses against and potential markers of reactive oxygen metabolite injury during ischemia and reperfusion. We examined the alterations in GSH and other myocardial SH pools during reperfusion in anesthetized dogs exposed to brief (15 minutes, n = 7) or prolonged (90 minutes, n = 6) regional ischemia caused by occlusion of the left anterior descending artery. Ninety minutes of ischemia followed by 5 hours of reperfusion, which resulted in myocardial necrosis of 43.9 +/- 4.0% of the area at risk, caused a 22% reduction in total myocardial SH groups (p less than 0.01), a 57% decrease in nonprotein myocardial SH groups (p less than 0.01), a 56% decrease in GSH (p less than 0.01), and a 62% decrease in non-GSH, nonprotein SH groups (p less than 0.02). However, protein SH groups were not significantly reduced (12% decrease, p = NS). Also, myocardial release of GSH and oxidized glutathione (GSSG) into the coronary venous effluent occurred during early reperfusion. In contrast, 15 minutes of ischemia, followed by 30 minutes of reperfusion, did not alter myocardial total SH groups, protein SH groups, or GSH (9% decrease, p = NS); nor was there reperfusion release of GSH or GSSG. However, even with brief ischemia, nonprotein SH groups decreased 23% (p less than 0.05), due mainly to a 59% decrease in the non-GSH, nonprotein SH pool (p less than 0.05). These changes after brief ischemia occurred without alterations in myocardial GSSG or the GSH/GSSG ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione disulfide as index of oxidant stress in rat liver during hypoxia

Formation of glutathione disulfide (GSSG) was used as an index of reactive oxygen generation in the isolated perfused liver of male Fischer rats during normoxia and hypoxia. Low oxygen tension may affect GSSG formation, rereduction, and transport mechanisms. The effect of short-term hypoxia (15 min) on the biliary and sinusoidal transport of GSSG was tested with the glutathione S-conjugates of sulfobromophthalein and 1-chloro-2,4-dinitrobenzene. Hypoxia inhibited S-conjugate excretion through both pathways by 15-20%. tert-Butyl hydroperoxide (75 microM tBHP) or diquat (200 microM) in the perfusate increased hepatic GSSG release by 430 and 1,550%, respectively, and increased the tissue GSSG content by 47 and 124%, respectively, under normoxia. Hypoxia reduced the stimulated GSSG export by 38 (tBHP) and 83% (diquat) and also caused an additional increase of the tissue GSSG content by 112% during tBHP infusion but caused a reduction by 32% during diquat infusion. Inhibition of the biliary export of GSSG and S-conjugates is mainly compensated by the sinusoidal efflux. Therefore, it is concluded that hypoxia reduces GSSG formation predominantly through suppression of reactive oxygen formation with only marginal effects on the biliary and sinusoidal excretion mechanism. Thus hepatic GSSG formation is a sensitive indicator of oxidant stress during normoxia and hypoxia. Because single parameters may vary considerably, simultaneous monitoring of GSSG in bile, perfusate, and tissue is essential for qualitative and quantitative estimation of reactive oxygen formation.