Pyridine nucleotide flux and glutathione oxidation in the cultured rat conceptus.

It is proposed that protection of the developing embryo from chemical and environmental insults that produces oxidative stress requires a proper glutathione (GSH) and pyridine nucleotide status in both the embryo and extra-embryonic membranes. Modulation of pyridine nucleotide flux [NAD(H) and NAD(P)H] in the visceral yolk sac (VYS) by the thiol oxidants diamide and tert-butyl hydroperoxide (tBH) was studied in real time using microfiberoptic sensors in GD 10 rat conceptuses. Consecutive 5-min exposures to 125- and 250-microM diamide resulted in a fluorescence decrease of 14 and 32 Arbitrary Fluorescence Units (AFU). An additional consecutive exposure to 500-microM diamide caused an attenuated decrease followed by a rebound increase of 22 AFU. Consecutive 5-min exposures to tBH at 250 and 500 microM produced fluorescence decreases similar to that of 500 microM diamide, but the decreases were attenuated at 1000 microM. However, there was variability in the rebound increase. A 5-min exposure to tBH (500 microM) alone caused a fluorescence decrease of 14 AFU followed by a rebound increase of 8 AFU. The rate of fluorescence decrease was attenuated by 50% with pretreatment with the glutathione reductase (GSSG-Rd) inhibitor, BCNU (1,3, bis(2 chloroethyl)-1-nitrosourea), indicating that the decrease in surface fluorescence was probably attributable to a decrease in NADPH. Decreases in fluorescence, observed from the surface of the VYS, correlated with decreases in GSH/GSSG ratios in the embryos and the VYS. After exposure to tBH, GSH levels in conceptuses decreased at the end of 5 and 15 min, with a corresponding increase in oxidized glutathione (GSSG) at the end of 3, 5, and 15 min. Our results demonstrate that the increased production of GSSG on exposure to thiol oxidants correlates with a decrease in the reduced pyridine nucleotide, implying the presence of an active GSSG-Rd pathway in the conceptus during organogenesis, and implicating an important role of the pyridine nucleotides in the restoration of GSH homeostasis in the developing rat conceptus during organogenesis.     

Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity

N-Acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, rapidly reacts at physiological pH with glutathione (GSH) forming an acetaminophen-glutathione conjugate and stoichiometric amounts of acetaminophen and glutathione disulfide (GSSG). The same reaction products are formed in isolated hepatocytes incubated with NAPQI. In hepatocytes which have been treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) in order to inhibit glutathione reductase, the initial rise in GSSG concentration in the presence of NAPQI is maintained, whereas GSSG is rapidly reduced back to GSH in untreated hepatocytes. Oxidation by NAPQI of GSH to GSSG and the reduction of GSSG back to GSH by the NADPH-dependent glutathione reductase appear to be responsible for the rapid oxidation of NADPH that occurs in hepatocytes incubated with NAPQI in that the effect is blocked by pretreatment of cells with BCNU. When added to hepatocytes, NAPQI not only reacts with GSH but also causes a loss in protein thiol groups. The loss in protein thiols occurs more rapidly in cells pretreated with BCNU or diethylmaleate. Whereas both of these treatments enhance cytotoxicity caused by NAPQI, BCNU pretreatment has no effect on the covalent binding of [14C-ring]NAPQI to cellular proteins. Furthermore, dithiothreitol added to isolated hepatocytes after maximal covalent binding of [14C-ring]NAPQI but preceding cell death protects cells from cytotoxicity and regenerates protein thiols. Thus, the toxicity of NAPQI to isolated hepatocytes may result primarily from its oxidative effects on cellular proteins.     

Biphasic lindane-induced oxidation of glutathione and inhibition of gap junctions in myometrial cells.

The insecticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells by a mechanism involving oxidative stress. We hypothesized that oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG) and subsequent S-glutathionylation provide a mechanistic link between lindane-induced oxidative stress and lindane's inhibition of myometrial gap junction communication. Gap junction communication between cultured rat myometrial myocytes was assessed by Lucifer yellow dye transfer after microinjection. A biphasic pattern was confirmed, with dye transfer nearly abolished after 1 h of exposure to 100 microM lindane followed initially by recovery after lindane removal, and then the development 4 h after termination of lindane exposure of a delayed-onset, sustained inhibition that continued for 96 h. As measured by HPLC, cellular GSH varied over a 24-h period in a biphasic fashion that paralleled lindane-induced inhibition of dye transfer, whereas GSSG levels increased in a manner inversely related to GSH. In accordance, GSH/GSSG ratios were depressed at times when GSH and dye transfer were low. Lindane substantially increased S-glutathionylation in a concentration-dependent manner, measured biochemically by GSSG reductase-stimulated release of GSH from precipitated proteins. Furthermore, treatments that promoted accumulation of GSSG (50 microM diamide and 25 microM 1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU]) inhibited Lucifer yellow dye transfer between myometrial cells. Findings that lindane induced GSH oxidation to GSSG with increased S-glutathionylation, together with the diamide and BCNU results, suggest that oxidation of GSH to GSSG is a component of the mechanism by which lindane inhibits myometrial gap junctions.     

Role of glutathione reductase during menadione-induced NADPH oxidation in isolated rat hepatocytes

Metabolism of menadione (2-methyl-1,4-naphthoquinone) results in the rapid oxidation of NADPH within isolated rat hepatocytes. The glutathione redox cycle is thought to play a major role in the consumption of NADPH during menadione metabolism, chiefly through glutathione reductase (GSSG-reductase). This enzyme reduces oxidized glutathione (GSSG), formed via the glutathione-peroxidase reaction, with the concomitant oxidation of NADPH. To explore the relationship between GSSG-reductase and the consumption of NADPH during menadione metabolism, isolated rat hepatocyte suspensions were exposed to non-lethal and lethal menadione concentrations (100 and 300 microM respectively) following the inhibition of GSSG-reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Menadione produced a concentration-related depletion of GSH (measured as non-protein sulfhydryl content) which was potentiated markedly by BCNU. Menadione toxicity was potentiated at either concentration by BCNU based on lactate dehydrogenase leakage at 2 hr. In addition, the NADPH content of isolated hepatocytes rapidly declined following exposure to either concentration of menadione. However, at the lower menadione concentration (100 microM), the NADPH content returned to control values or above by 60 min, whereas the NADPH content of cells exposed to 300 microM menadione with or without BCNU remained depressed for the duration of the incubation. These data suggest that, although NADPH is required by GSSG-reductase for the reduction of GSSG to GSH during quinone-induced oxidative stress, this pathway does not appear to be the major route by which NADPH is consumed during the metabolism of menadione in isolated hepatocytes.     

Effect of hypoxia on tert-butylhydroperoxide-induced oxidative injury in hepatocytes

Toxicity of t-butylhydroperoxide (t-BuOOH) was studied at different steady state O2 concentrations under conditions at which O2 deficiency alone did not cause cell death. t-BuOOH-induced cell death was more rapid in hypoxic than normoxic cells; the maximal rate of cell death occurred in anoxic cells. t-BuOOH elimination was independent of O2 concentration and was complete within 15 min; t-Butanol was produced at the same rate and was the only product detected by gas chromatography. Measurement of radical production by formation of adducts of the spin-trapping agent N-tert-butylphenylnitrone showed that the amount of radicals trapped was 0.02% of the amount of peroxide added and was the same under anoxic and oxygenated (214 microM O2) conditions. These results show that the O2 dependence of t-BuOOH-induced toxicity is not related to quantitative alterations in its metabolism. Lipid peroxidation was lowest in anoxic cells and increased as the O2 concentration was increased to 1.07 mM O2, showing that enhanced toxicity during hypoxia and anoxia was not due to enhanced lipid peroxidation. In contrast, O2 deficiency impaired the ability of cells to maintain and recovery GSH and NADPH pools after addition of t-BuOOH. GSH was decreased to a greater extent in anoxic cells than in normoxic cells, and the GSH content remained lower in these cells for up to 30 min. This decrease was due both to a decrease in the rate of synthesis and to decreased supply of the NADPH needed for the reduction of GSSG. Taken together, these results show that O2 deficiency has little effect on metabolism of t-BuOOH but impairs the ability of cells to maintain cellular GSH and renders them more susceptible to injury from oxidizing agents. This suggests that oxidative injury under hypoxia or following ischemia may not require a marked stimulation in generation of oxidative species but may occur as a consequence of the impaired ability to tolerate or repair oxidative injury.     

Mechanism for the changes in levels of glutathione upon exposure of cultured mammalian cells to tertiary-butylhydroperoxide and diamide

Qualitative and quantitative changes associated with cellular glutatione (GSH) in response to oxidants were investigated in cultured Chinese hamster V79 cells. Incubation of cells with benzoylperoxide (BZP), tert-butylhydroperoxide (t-BuOOH), hydrogen peroxide or diamide for 1 h reduced the level of total GSH (GSH + GSSG). Among the oxidants, t-BuOOH and diamide caused an increase in levels of glutathione disulfide (GSSG) and a resultant increase in the ratio of the level of GSSG to the level of total GSH, suggestive of the induction within the cells of a pro-oxidant state by the oxidants. o-Phenanthroline, a chelator of divalent ion, almost completely suppressed the decrease in levels of total GSH caused by t-BuOOH while it did not suppressed either increases in levels of GSSG or increases in the ratio of the levels of GSSG to that of total GSH caused by the hydroperoxide. These results suggest that reactive oxygen radicals are involved in the decrease in levels of GSH by treatment with t-BuOOH but not in the increase in the level of GSSG. After treatment with either t-BuOOH or diamide for 1 h, the level of GSH rapidly increased to more than twice the control level during 15–45 min of post-treatment incubation. o-Phenanthroline almost completely suppressed the increase in levels of GSH caused by t-BuOOH, while it did not affect the changes caused by diamide, suggesting a difference between the mechanisms by which t-BuOOH and diamide cause increases in levels of GSH. It seems likely that reactive oxygen radicals participate not only in the decrease in levels of GSH caused by t-BuOOH but also in the rapid increase that occurs after such treatment. Hence, the first decrease in levels of GSH by the hydroperoxide may be causally related to the latter increase. The amount of [³⁵S]-cysteine taken up by cells after treatment with t-BuOOH was about one half of that taken up by control cells. By contrast, the rate of incorporation of radioactive cysteine into acid-soluble material increased to more than twice that of the controls after treatment with t-BuOOH. The increase in the rate of incorporation of [³⁵S]cysteine into acid-soluble material caused by t-BuOOH was not a consequence of inhibition by the hydroperoxide of utilization of cysteine for protein synthesis. Inhibition of protein synthesis by cycloheximide caused neither an increase in the incorporation of cysteine into acid-soluble material nor an increase in rate of biosynthesis of GSH. Incorporation of radioactive cysteine into the cysteine moiety of GSH and the disappearance of the radioactivity from the cysteine fraction were enhanced after treatment with t-BuOOH. These data indicate that biosynthesis of GSH de novo is enhanced by t-BuOOH.     

Decreased aortic glutathione levels may contribute to impaired nitric oxide-induced relaxation in hypercholesterolaemia

The aim of this study was to determine if the decrease in aortic total glutathione (GSH) levels in hypercholesterolaemia is related to the impairment of relaxation to acetylcholine (ACh) and exogenous nitric oxide (NO). Isometric tension and vascular GSH levels were measured in thoracic aortic rings from rabbits fed for 12 weeks with 0.5% cholesterol diet. Hypercholesterolaemia decreased aortic GSH levels and impaired relaxation to ACh and NO. To determine if GSH depletion impaired the response to NO, normal rabbit thoracic aorta was incubated with 1,3-bis [2-chloroethyl]-1-nitrosourea (BCNU; 0.2 mmol L(-1)), a GSH reductase inhibitor, or diazine-dicarboxylic acid bis [N, N dimethylamide] (diamide; 1 mmol L(-1)), a thiol oxidizing agent. BCNU or diamide decreased aortic GSH levels and impaired ACh and NO-induced relaxation. The effects of diamide on GSH levels and relaxation were partially prevented by co-incubation with GSH ester (GSE; 2 mmol L(-1)). Increasing GSH with GSE significantly enhanced NO-induced relaxation in aorta from both hypercholesterolaemic and normal rabbits, however relaxation of hypercholesterolaemic rabbit aorta was not restored to normal. These data suggest that other factors, perhaps related to the long-term decrease in GSH levels, are responsible for reduced NO bioactivity in hypercholesterolaemia.     

A novel biologically active seleno-organic compound--II. Activity of PZ 51 in relation to glutathione peroxidase

The anti-inflammatory compound 2-phenyl-1,2-benzoisoselenazol-3(2H)-on (PZ 51) catalysed GSSG formation from GSH in the presence of hydroperoxides in an NADPH/GSSG reductase system with the following rates (delta log GSH/min per molar selenium): 1.1 X 10(6) with H2O2, 1.2 X 10(6) with butylhydroperoxide, 1.7 X 10(6) with cumenehydroperoxide. The reaction catalysed by the sulphur analogue of PZ 51 was negligible. Similar results were obtained in a direct assay of GSH-Px activity based on GSH estimation by dithionitrobenzoate. The activation energy of the reaction was determined as 55 kJ/mol . deg in the presence of 30 mumol/1 PZ 51 compared to 36.5 kJ/mol . deg obtained in the presence of 1 nmol/1 pure GSH-Px isolated from bovine red blood cells. In mouse liver microsomes, NADPH-dependent aminopyrine dealkylation was totally inhibited in the presence of 50 mumol/1 PZ 51. In vivo experiments with Se-deficient mice showed that the Se-moiety of PZ 51 is not available for the synthesis of the selenoenzyme GSH-Px after dietary treatment or i.p. doses up to 25 mg Se as PZ 51 per kg body wt. After oral administration of labelled PZ 51, unlike with selenite, no radioactivity was incorporated into GSH-Px within 48 hr. The data suggest that several similarities between PZ 51 and the active site of GSH-Px exist, resulting in the capability of the compound to catalyse the GSH-Px reaction. An extracellular pharmacodynamic action of the drug seems likely.     

Effect of methylene blue and thiol oxidants on pancreatic islet GSH/GSSG ratios and tolbutamide mediated insulin release in vitro

Summary Methylene blue (an oxidant of NADPH), diamide (an oxidant of glutathion-SH [GSH]) and tertbutyl hydroperoxide (a substrate of glutathione peroxidase) significantly decreased the GSH content of pancreatic rat islets and decreased their GSH/GSSG ratio. They also significantly depressed the single peak insulin response to tolbutamide by the isolated perfused pancreas as well as its synergistic action with glucose in isolated pancreatic islets.These results suggest that the effect of tolbutamide alone and its synergistic action with glucose could depend on the islet NADPH and GSH. In addition it appears that augmentation of tolbutamide action by glucose in insulin release is mediated by the provision of additional NADPH and GSH through glucose metabolism.     

Inhibition of glutathione-related enzymes augments LPS-mediated cytokine biosynthesis: involvement of an IkappaB/NF-kappaB-sensitive pathway in the alveolar epithelium

The regulation of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokine biosynthesis by reduction-oxidation (redox)-sensitive enzymes involved in maintaining intracellular glutathione homeostasis was investigated in fetal alveolar type II epithelial cells (fATII). Inhibition of glutathione-oxidized disulfide reductase, which recycles GSSG --> 2GSH, by the action of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) augmented LPS-dependent secretion of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. BCNU increased [GSSG] concentration at the expense of [GSH], thereby favoring oxidation equilibrium. Inhibition of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of GSH, by the action of L-buthionine-(S,R)-sulfoximine (BSO), potentiated LPS-induced IL-1beta, IL-6 and TNF-alpha production. Similar to BCNU, BSO depleted [GSH] and induced the accumulation of [GSSG]. BCNU and BSO reduced LPS-mediated phosphorylation of inhibitory-kappaB (IkappaB-alpha), allowing its cytosolic accumulation. This effect was associated with the inhibition of the nuclear translocation of selective nuclear factor (NF)-kappaB subunits: NF-kappaB1 (p50), RelA (p65), RelB (p68) and c-Rel (p75), but not NF-kappaB2 (p52). BCNU and BSO reduced LPS-induced NF-kappaB activation as determined by the electrophoretic mobility shift DNA-binding assay. Analytical analysis of the effect of modulating the dynamic redox ratio ([GSH]+[GSSG])/[GSSG] revealed a novel role for GSSG as a disulfhydryl compound which mediates an inhibitory effect on NF-kappaB activation. It is concluded that selective modulation of redox-sensitive enzymes has an immunopharmacological potential in regulating pro-inflammatory cytokines and that the TkappaB-alpha/NF-kappaB pathway is redox-sensitive and differentially involved in mediating redox-dependent regulation of cytokine signaling.     

On the mechanisms of glutathione depletion in hepatocytes exposed to morphine and ethylmorphine

Incubation of isolated rat hepatocytes with either morphine or ethylmorphine resulted in glutathione (GSH) depletion followed by loss of cell viability. Pretreatment of cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inactivate glutathione reductase did not markedly affect the rates of GSH depletion seen in untreated cells. In contrast, hexobarbital stimulated H2O2 production in isolated liver microsomes, incubated aerobically with NADPH, whereas the effects of morphine and ethylmorphine on microsomal H2O2 production were minimal. Finally, incubation of hepatocytes with radioactively labeled morphine resulted in formation of 2 glutathione conjugates, one of which was tentatively identified as formyl glutathione. We conclude that GSH consumption during the metabolism of morphine or ethylmorphine by hepatocytes is due mainly to formation of glutathione conjugates.     

Kinetics and mechanism of the oxidation of the glutathione dimer by hypochlorous Acid and catalytic reduction of the chloroamine product by glutathione reductase

Oxidized glutathione (GSSG) reacts with two molar equivalents of HOCl/OCl- (a neutrophil-derived oxidant and a common biocide) to form the dichloro (bis-N-chloro-gamma-l-glutamyl) derivative (NDG). The reaction of less than two molar equivalents of HOCl with GSSG does not yield the unsymmetrical monochloro derivative (NCG) but rather a stoichiometric amount of NDG and GSSG. This result is explained by a faster reaction of the second equivalent of HOCl with NCG than that of the first equivalent of HOCl with GSSG. The rates of reaction of GSSG2-, GSSG3-, and GSSG4- (successive deprotonation of the ammonium groups) have been investigated, and it is clear that GSSG2- is unreactive, whereas GSSG4- is about twice as reactive as GSSG3-. Accordingly, the following mechanism is proposed (constants for 5 degrees C): H+ + OCl- = HOCl, pK1 = -7.47; GSSG2- = GSSG3- + H+, pK2 = 8.5; GSSG3- = GSSG4- + H+, pK3 = 9.5; GSSG3- + HOCl --> NCG3- + H2O, k4 = 2.7(2) x 106 M-1 s-1; GSSG4- + HOCl --> NCG4- + H2O, k5 = 3.5(3) x 107 M-1 s-1; NCG3- --> NDG4- + H+, k6 = fast; and NCG4- + HOCl --> NDG4- + H2O, k7 = fast. At physiologic pH, the k4 pathway dominates. NDG decomposes at pH 7.4 in a first-order process with kdec = 4.22(1) x 10-4 s-1 (t1/2 = 27 min). Glutathione reductase (EC 1.6.4.2) is capable of catalyzing the reduction of NDG by NADPH. The only NDG-derived product that is observed (by NMR) after the reduction by NADPH is GSH. Thus, in the presence of the GOR/NADPH system, GSH is capable of redox buffering a 3/2 mol equiv of HOCl rather than a 1/2 mol equiv as previously assumed.     

Effects of cysteine and acetaminophen on the syntheses of glutathione and adenosine 3'-phosphate 5'-phosphosulfate in isolated rat hepatocytes.

The aim of the present study was to introduce and validate a radioactive tracer method in which adenosine 3'-phosphate 5'-phosphosulfate (PAPS) and glutathione (GSH) are measured simultaneously in isolated hepatocytes. PAPS and GSH are co-substrates in sulphation and GSH conjugation, and both are dependent on sulphur deriving from cysteine. The effect of cysteine on the syntheses was investigated at non-toxic and toxic concentrations of the hepatotoxic drug acetaminophen (AA). Administration of AA trapped radioactivity (35S) in the pre-labelled PAPS and GSH pools by formation of the metabolites, AA-sulphate and AA-GSH. Turnover rates were determined from the decline of AA-sulphate and AA-GSH specific activity. Syntheses of PAPS and GSH were calculated by multiplying the rates with the concentrations of the respective co-substrates. Increasing AA concentration from non-toxic to toxic levels resulted in increased median PAPS and GSH syntheses (8 to 11 and 311 to 2218 nmol/10(6) cells/min, respectively) (P less than 0.05). Addition of cysteine did not alter median PAPS synthesis (5 to 3 nmol/10(6) cells/min) but decreased median GSH synthesis (666 to 261 nmol/10(6) cells/min) (P less than 0.05) in experiments with non-toxic AA concentrations. In experiments with toxic AA concentrations opposite effects of cysteine were seen, i.e. median PAPS synthesis was reduced (3 to 2 nmol/10(6) cells/min) (P less than 0.05) while median GSH synthesis was unchanged (23 to 16 nmol/10(6) cells/min). The present method provides a tool in which two important detoxification pathways can be measured simultaneously and the data suggest that the two pathways are regulated by substrate availability.     

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.     

The control of S-thiolation by cysteine via gamma-glutamyltranspeptidase and thiol exchanges in erythrocytes and plasma of diamide-treated rats

Protein thiol modifications including cysteinylation (CSSP) and glutathionylation (GSSP) in erythrocytes of rat treated with diamide have been reported, but mechanism and origin of CSSP formation are unknown. Experiments were performed to relate CSSP formation to GSH hydrolysis via gamma-glutamyltranspeptidase (gamma-GT) and know whether cysteine may act as deglutathionylation factor. Time-dependent variations of redox forms of glutathione and cysteine were investigated in erythrocytes, plasma, liver and kidney of diamide-treated rats (0.4 mmol/kg by infusion for 45 min followed by 135 min of washout) in the presence and absence of acivicin (10 mg/kg administered twice 1 h before diamide) a known gamma-GT inhibitor. Diamide-treated rats showed decreased concentrations of erythrocyte GSH and increased levels of GSSP and CSSP. The rate of CSSP formation was slower than that of GSSP. Besides the entity of CSSP accumulation of erythrocytes was high and equivalent to ∼ 3-fold of the normal plasma content of total cysteine. The result was paradoxically poorly related to gamma-GT activity because the gamma-GT inhibition only partially reduced erythrocyte CSSP. After gamma-GT inhibition, a large concentration fluctuation of glutathione (increased) and cysteine (decreased) was observed in plasma of diamide-treated rats, while little changes were seen in liver and kidney. There were indications from in vitro experiments that the CSSP accumulation in erythrocytes of diamide-treated rats derives from the coexistence of GSH hydrolysis via gamma-GT and production of reduced cysteine via plasma thiol exchanges. Moreover, reduced cysteine was found to be involved in deglutathionylation processes. Mechanisms of protein glutathionylation by diamide and deglutathionylation by cysteine were proposed.     

Glutathione peroxidase and glutathione reductase activities are partially responsible for determining the susceptibility of cells to oxidative stress

Different cell types response differently to toxic insult. In a previous study, it was demonstrated that the C6 glioma cell is more sensitive to Cd induced oxidative stress than the HepG2 cells. To explain the difference between the two cell lines in their response to oxidative stress, it was hypothesized that the activity of glutathione metabolizing enzymes may be different. The objective of this study is to determine the activities of glutathione peroxidase (GPx) and glutathione reductase (GR) in the two cell lines and to explain how these differences may affect the susceptibility of the two cells to oxidative stress. In the HepG2 cells, the activity of GPx was 2.24+/-0.18 micromol/mg protein/min and that for GR was 5.63+/-0.58 micromol/mg protein/min. For the C6 glioma cells, GPx and GR activities were 1.29+/-0.14 and 1.07+/-0.11 micromol/mg protein/min, respectively. Using the kinetic equilibrium: K(eq)=([GSSG]x[NADPH]x[H(+)])/([GSH](2)x[NADP(+)]), and the GSH/GSSG previously published (HepG2: 2.6 and C6 glioma: 3.6), resting NADPH/NADP(+) for the cell lines were calculated. The results showed that NADPH/NADP(+) for HepG2 cells (17.8) is higher than that in the C6 glioma cells (10.8). These data supported the notion that the reducing power (NADPH/NADP(+)) in the HepG2 cells is higher than that in the C6 glioma cell and thus, the later would be more susceptible to oxidative stress. The results also suggested that besides GSH/GSSG, the activities of GPx and GR are important in predicting tissue redox state. Applying this hypothesis to animal tissues, the ratio of the activities of the two enzymes in mouse liver, cerebral cortex, hippocampus and cerebellum were measured. It was demonstrated that the activities of GPx and GR were different in the different tissues studied. The possible correlation between enzymatic activities and the redox state in the different tissues were discussed.     

Loss of viability after disulfiram treatment without preceding depletion of intracellular GSH

Effects of disulfiram (DSF) on freshly isolated hepatocytes were examined. Its effects on the cellular reduced form of glutathione (GSH) were triphasic; GSH decreased instantly after the addition of DSF, returned to subnormal levels within 30 min, and then declined gradually. The initial decrease in GSH after DSF treatment and the subsequent recovery of GSH were accompanied by an increase and decrease in the oxidized form of glutathione (GSSG), respectively. Decreases in cell viability brought about by 0.4 mM of DSF were correlated with the later gradual decrease in GSH. The loss of viability by DSF treatment seemed to appear when the initial GSH levels became lower than approximately 5 nmole/10(6) cells. Hepatocyte toxicity of DSF was potentiated by diethylmaleate (GSH depletor) and inhibited by N-acetylcysteine (GSH biosynthesis precursor). 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSH reductase, inhibited the GSH recovery and potentiated the toxicity. Respiration of hepatocytes was also inhibited by DSF. Free sulfhydryl groups other than GSH showed similar changes to those of GSH. From these results, it seemed that DSF reacted with cellular GSH and other free sulfhydryl groups to form diethyldithiocarbamate and GSSG, GSSG was reduced back to GSH by glutathione reductase, and the decrease in the viability was dependent on the initial loss of GSH.     

Unique role of oxygen in regulation of hepatic monooxygenation and glucuronidation

The purpose of this study was to evaluate the hypothesis that NADPH supply in intact cells is regulated by oxygen tension. This was accomplished by studying monooxygenation in perfused livers from Ah locus-responsive C57BL/6J mice, where rates of monooxygenation are high. Elevation of flow rate decreases the hepatic O2 gradient and increases O2 delivery to the organ. Under these conditions, rates of p-nitroanisole O-demethylation were 2-3 times higher in perfused livers from fed or fasted mice at high (10 ml/min) compared with normal (5 ml/min) flow rates. Rates of monooxygenation were directly proportional to oxygen tension (half-maximal rates occurred with approximately 400 microM O2). On the other hand, rates were independent of oxygen concentration in isolated microsomes where NADPH was supplied in excess. The decrease in rate due to diminished O2 concentration in the intact organ could not be attributed to hypoxia, because O2 tension in the effluent perfusate exceeded 50 microM even when influent perfusate was saturated with 25% O2 and ATP/ADP ratios were in the normal range. Thus, monooxygenation of p-nitroanisole in perfused mouse liver is dependent on oxygen tension. Similarly, glucuronidation of p-nitrophenol was oxygen dependent in the intact organ but not in isolated microsomes supplemented with UDP-glucuronic acid. Taken together, these data support the hypothesis that, at high oxygen tensions (e.g., in periportal regions of the liver lobule), mitochondrial activity is increased, which in turn enhances NADPH and UDP-glucuronic acid turnover, leading to accelerated rates of monooxygenation and glucuronidation in intact cells. In support of this idea, NH4Cl, which utilizes NADPH for urea synthesis, inhibited monooxygenation in the perfused mouse liver at high but not low flow rates. Thus, important phase I and II detoxification reactions are regulated indirectly by the hepatic oxygen gradient, via mechanisms involving cofactor supply, when cytochrome P-450 is not limiting.     

Studies on the biliary efflux of GSH from rat liver due to the metabolism of aminopyrine

The biliary efflux of GSH and GSSG due to aminopyrine was studied using perfused rat livers. The infusion of 0.8 mM aminopyrine led to a rapid rise in the amount of GSH released into the bile with only a small increase in the amount of GSSG released; caval GSH + GSSG efflux was unaffected. N-Benzylimidazole, an inhibitor of cytochrome P-450, completely blocked the response while phenobarbital pretreatment of the rats doubled the rate of GSH efflux. H2O2 and selenium-containing glutathione peroxidase were not involved since livers from selenium-deficient rats perfused with aminopyrine released GSH at the same rate as control livers. Aminopyrine injected i.p. into conscious rats also stimulated biliary GSH efflux to the same extent as with perfused livers. Biliary release of GSH in the perfused livers could be duplicated by infusing formaldehyde. It is proposed that formaldehyde produced during the N-demethylation of aminopyrine by cytochrome P-450 combines reversibly with GSH to form S-hydroxymethylglutathione which is oxidized by formaldehyde dehydrogenase to S-formylglutathione. Formaldehyde formed in excess of its capacity to be metabolized enzymatically is released into the bile as S-hydroxymethylglutathione which then dissociates to its initial reactants.     

Diamide exposure, thermal resistance, and synthesis of stress (heat shock) proteins

Chinese hamster ovary (CHO) cells were treated with the thiol oxidant diamide for 1 hr at 37 degrees, incubated in diamide-free medium for 4 hr at 37 degrees, and then exposed to hyperthermic treatment (43 degrees) or assayed for the presence of 110, 90 and 66 kilodalton (kD) stress (heat shock) proteins. Cellular inactivation produced by the hyperthermic treatment was measured using colony formation as the end point. Low concentrations of diamide, which did not result in depletion of intracellular GSH, induced a moderate degree of protection against thermal toxicity but did not affect the pattern of protein synthesis. Exposure to 0.4 mM diamide, which reduced intracellular GSH concentrations by 50-60%, significantly reduced the rate of hyperthermic cellular inactivation. This occurred coincidentally with the synthesis of stress proteins of approximate molecular weights of 110, 90 and 66 kD. Furthermore, this concentration of diamide protected cells from thermal inhibition of protein synthesis. These results indicate that thiol oxidation by diamide can induce both the development of thermal resistance to cellular inactivation and the synthesis of stress proteins.