Glutathione Isopropyl Ester Research Articles

Performance management, appraisals, and revalidation: quantity analysis and quality control for UK GPs

In this study the effects of S-nitrosothiols, in particular S-nitrosoglutathione (GSNO), were evaluated with regard to their bronchodilating properties, both after infusion via the pulmonary circulation and after inhalation, in the isolated perfused and ventilated guinea pig lung. Infused GSNO induced bronchorelaxation of lungs that were precontracted with methacholine. During a 15-min period of single-passage perfusion with GSNO (10 microM), maximally 10% was taken up and/or degraded by the lung. A spontaneous breakdown of GSNO in the perfusion buffer was also observed, which was partially accompanied by the formation of nitrite. Low levels of nitric oxide (NO) were detected in the perfusion buffer when GSNO was present. This was due to the presence of contaminating transition metals, because EDTA and 2,29-dipyridyl largely reduced the formation of NO. The NO-scavenging agents oxyhemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide abolished levels of NO in the buffer but did not abolish GSNO-induced bronchodilation. The effects of infused GSNO are therefore attributed to an action of the intact S-nitrosothiol and not to NO released from GSNO in the perfusion buffer. Similarly, perfusion with S-nitrosated glutathione isopropyl ester, cysteinyl glycine, N-acetyl-L-cysteine or N-acetyl-D,L-penicillamine, but not with nitrosated bovine serum albumin or sodium nitrite, was found to induce bronchodilation. Inhalation of nebulized GSNO induced bronchodilation of methacholine-precontracted lungs with a rapid onset of action, although it was a less potent bronchodilator than salbutamol. The results show that infused or inhaled S-nitrosothiols have bronchodilating properties in the isolated perfused and ventilated guinea pig lung.

Inhibition of Protein Phosphorylation in Macrophages by Antioxidants.

It has been shown that a wide variety of antioxidants inhibits macrophage scavenger and polylactosamine receptor activities. The activity of macrophage scavenger receptors to take up oxidized low density liporprotein is also inhibited by protein kinase inhibitors but enhanced by protein phosphatase inhibitors. We studied whether the antioxidants including lipid-soluble phenolic, water-soluble phenolic, glutathione-related and ascorbic acid-related antioxidants were effective to inhibit protein phosphorylation of macrophages. Lower than 100 μM of α-tocopherol, probucol, propyl gallate, nordihydroguaiaretic acid, curcumin, quercetin and Trolox inhibited total protein phosphorylation of thioglycollate-induced mouse macrophages as assessed by H332PO4 incorporation. Lower than 1 mM of α-tocopherol, probucol, butylated hydroxytoluene, curcumin, catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, glutathione, glutathione isopropyl ester, N-acetylcysteine, ascorbic acid, erythorbic acid and dehydroascorbic acid inhibited protein tyrosine phosphorylation of macrophages in the presence of vanadate, a protein tyrosine phosphatase inhibitor, as assessed by Western-blot analysis using anti-phosphotyrosine IgG monoclonal antibody. Most of the antioxidants inhibited protein tyrosine phosphorylation of epidermal growth factor (EGF) receptor of A431 cells in a cell-free system. Oxidative satus of macrophages examined by using dihydrorhodamine 123, and all the catechins reduced their oxidative status. Above results suggest that oxidative stress in macrophages maintain or enhance protein phosphorylation, and that antioxidants reduce protein phosphorylation and the receptor activities by reducing oxidative status.

Protein thiol oxidation by haloperidol results in inhibition of mitochondrial complex I in brain regions: comparison with atypical antipsychotics

Usage of ‘typical’ but not ‘atypical’ antipsychotic drugs is associated with severe side effects involving extrapyramidal tract (EPT). Single dose of haloperidol caused selective inhibition of complex I in frontal cortex, striatum and midbrain (41 and 26%, respectively) which was abolished by pretreatment of mice with thiol antioxidants, α-lipoic acid and glutathione isopropyl ester, and reversed, in vitro, by disulfide reductant, dithiothreitol. Prolonged administration of haloperidol to mice resulted in complex I loss in frontal cortex, hippocampus, striatum and midbrain, while chronic dosing with clozapine affected only hippocampus and frontal cortex. Risperidone caused complex I loss in frontal cortex, hippocampus and striatum but not in midbrain from which extrapyramidal tract emanates. Inhibition of the electron transport chain component, complex I by haloperidol is mediated through oxidation of essential thiol groups to disulfides, in vivo. Further, loss of complex I in extrapyramidal brain regions by anti-psychotics correlated with their known propensity to generate side-effects involving extra-pyramidal tract.

Inhibition of the Activity of Mouse Macrophage Scavenger Receptors by Antioxidants.

Effect of exogenously added antioxidants on the activity of the mouse macrophage scavenger receptors for oxidized low density lipoprotein (oxLDL) was investigated. Binding of oxLDL-coated erythrocytes and/or 125I-oxLDL to a monolayer of thioglycollate-induced peritoneal macrophages preincubated with antioxidants at 37°C for 2 h was examined. The binding was effectively or slightly decreased by preincubation of macrophages with probucol, butylated hydroxytoluene, α-tocopherol, sesamol, propyl gallate, quercetin, nordihydroguaiaretic acid, epicatechin gallate, epigallocatechin gallate (EGCg), glutathione isopropyl ester (GSH-Pr), N-acetylcysteine, ascorbic acid, erythorbic acid, and Desferal at concentrations lower than 100 μM. The binding was slightly enhanced by preincubation of macrophages with buthionine sulfoximine. The antioxidants and related compounds inhibited the formation of foam cells induced by oxLDL in the prolonged incubation for 24 h. Glutathione, oxidized glutathione and dehydroascorbic acid inhibited the foam cell formation, Total cholesterol accumulation induced by oxLDL in the prolonged incubation was reduced by the phenolic antioxidants. The number of scavenger receptors of macrophages was unchanged upon treatment with EGCg and GSH-Pr. The results indicate that the antioxidants at relatively high concentrations inhibited macrophage functions, binding of oxLDL, foam cell formation, and accumulation of cholesterol, and suggest that the scavenger receptor activity of macrophages was exerted by certain oxidative mechanisms.

Water-soluble antioxidants inhibit macrophage recognition of oxidized erythrocytes.

Effect of exogenously added water-soluble antioxidants on the mouse macrophage lectin-like receptor activity for oxidized erythrocytes was investigated. A monolayer of thioglycollate-induced mouse peritoneal macrophages was preincubated with each of the antioxidants at 37 degrees C for 1 h, and the binding for mouse erythrocytes oxidized with ADP-chelated Fe(III) was examined. The binding was decreased by preincubation of macrophages with ascorbic acid-related compounds including ascorbic acid, erythorbic acid and dehydroascorbic acid in a dose-dependent fashion at relatively high concentrations above 10 microM. The binding was similarly decreased by preincubation of macrophages with catechin compounds including epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate in a dose-dependent fashion at 0.01-100 microM. The binding was more effectively decreased by preincubation of macrophages with thiol-related compounds including glutathione, oxidized glutathione, glutathione isopropyl ester and N-acetylcysteine in a dose dependent fashion at relatively low doses below 1 microM. These results showed that water-soluble antioxidants especially glutathione and its derivatives reduced the ability of macrophages to bind oxidized erythrocytes, suggesting that the activity of lectin-like receptors of macrophages for oxidized erythrocytes was regulated by oxidative mechanisms.

Inhibition of selenite cataract by S-diethylsuccinyl glutathione isopropyl ester

Previous studies have demonstrated that glutathione derivatives can partially prevent loss of hepatic glutathione levels, in vivo, during periods of oxidative stress. Since cataracts in animal model systems and in humans are thought to be the direct result of oxidative insult, the following study tested the possibility that treatment with these glutathione analogues may affect the progression of lens opacification. Glutathione esters were tested for their ability to inhibit the selenite-induced cataract in rats. The S-alkyl glutathione ester Et(2)Sc-GS-iPr, but not a similar glutathione derivative S-succinyl glutathione (Sc-GS), at 0.5 mmoles/kg body weight, had anti-cataract activity in the selenite-induced cataract of rats. Analysis of lenses from treated and untreated animals demonstrated that Et(2)Sc-GS-iPr partially prevented the loss of reduced glutathione levels. The results demonstrate for the first time that an S-alkylated glutathione ester has anti-cataractogenic potential in the selenite-treated rat, and suggest possible mechanisms involving glutathione in the prevention of this lens opacification.

Inhibition of selenite cataract by S-diethylsuccinyl glutathione isopropyl ester

Purpose. Previous studies have demonstrated that glutathione derivatives can partially prevent loss of hepatic glutathione levels, in vivo, during periods of oxidative stress. Since cataracts in animal model systems and in humans are thought to be the direct result of oxidative insult, the following study tested the possibility that treatment with these glutathione analogues may affect the progression of lens opacification. Methods. Glutathione esters were tested for their ability to inhibit the selenite-induced cataract in rats. Results. The S-alkyl glutathione ester Et 2 Sc-GS-iPr, but not a similar glutathione derivative S-succinyl glutathione (Sc-GS), at 0.5 mmoles/kg body weight, had anti-cataract activity in the selenite-induced cataract of rats. Analysis of lenses from treated and untreated animals demonstrated that Et 2 Sc-GS-iPr partially prevented the loss of reduced glutathione levels. Conclusions. The results demonstrate for the first time that an S-alkylated glutathione ester has anti-cataractogenic potential in the selenite-treated rat, and suggest possible mechanisms involving glutathione in the prevention of this lens opacification.

Protection of cultured gastric cells against tert-butyl hydroperoxide by glutathione isopropyl ester

Oxidants are involved in the pathogenesis of a variety of gastrointestinal disorders. Intracellular GSH protects rat gastric cells against oxidants. We examined whether GSH isopropyl ester (GSH ester) can protect against oxidants and whether a system of GSH ester uptake is present in these cells; we also investigated a possible role of GSH in inhibiting lipid peroxidation. Cytotoxicity was quantified by 51 Cr release. Lipid peroxidation was assessed by measuring malondialdehyde production. tert-Butyl hydroperoxide caused a dose-dependent increase in 51 Cr release. Treatment with GSH ester attenuated the toxicity of tert-butyl hydroperoxide. Incubation with GSH ester enhanced the cellular GSH content, which was prevented by dl-buthionine-[ S, R]-sulfoximine, an inhibitor of γ-glutamylcysteine synthetase. GSH ester prevented tert-butyl hydroperoxide-induced lipid peroxidation, corresponding with the degree of protection. Therefore, we concluded that GSH isopropyl ester protects gastric cells against oxidants through the accumulation of intracellular GSH. This protection is mediated in part by the prevention of hydroperoxide-induced lipid peroxidation. However, the gastric epithelial system of GSH ester uptake appears distinctly different from those observed in hepatocytes, lymphoid cells and fibroblasts in terms of mediation of γ-glutamylcysteine synthetase activity.

Toxicity of fotemustine in rat hepatocytes and mechanism-based protection against it

Fotemustine is a relatively novel DNA-alkylating 2-chloroethyl-substituted N-nitrosourea (CENU) drug, clinically used for the treatment of disseminated malignant melanoma in different visceral and non-visceral tissues. Thrombocytopenia has been observed in patients treated with fotemustine and liver and renal toxicities as well. In this study, firstly the metabolism of fotemustine was investigated in vitro and secondly the undesired cytotoxicity of fotemustine as well as different ways of protection against it. In rat hepatocytes, chosen as a model system, fotemustine was shown to cause lactate dehydrogenase (LDH) leakage, glutathione (GSH) depletion, GSSG-formation and lipid peroxidation (LPO). A reactive metabolite, DEP-isocyanate, is most likely responsible for these undesired cytotoxic effects. Based on the observed cytotoxicity mechanisms, chemoprotection with several sulfhydryl-containing nucleophiles and antioxidants was investigated. The sulfhydryl nucleophiles, GSH, N-acetyl- l-cysteine (NAC) and glutathione isopropylester (GSH-IP) protected almost completely against fotemustine-induced LDH-leakage and LPO. NAC and GSH protected partly against fotemustine-induced GSH-depletion. The antioxidant, vitamin E protected completely against fotemustine-induced LPO, but only partly against fotemustine-induced LDH-leakage and not against GSH-depletion. Ebselen, a peroxidase-mimetic organoselenium compound, did not show protective effects against the cytotoxicity of fotemustine, possibly because GSH is required for the bioactivation of ebselen. It is concluded that co-administration of sulfhydryl nucleophiles, in particular NAC and GSH-IP, possibly in combination with antioxidants, such as vitamin E, are effective against the toxicity of fotemustine in vitro. It might, therefore, be worthwhile to investigate the cytoprotective potency of these agents against undesired toxicities of fotemustine in vivo as well.

Glutathione isopropyl ester reduces UVB-induced skin damage in hairless mice.

The protective effect of administration of glutathione (GSH) isopropyl ester on photodamage, such as lipid peroxidation, inflammation and tumorigenesis induced by UV exposure (290-400 nm, max. 312 nm), was investigated using hairless mice. Pretreatment with 20 mg/kg GSH isopropyl ester prevented the increases of thiobarbituric acid-reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, which were caused by a single dose (15 kJ/m2) of UV irradiation. The level of epidermal GSH in skins of the GSH ester-treated mice was maintained within normal limits. When mice were exposed to UV at a dose of 2 kJ/m2, three times weekly, skin tumors developed in all of them after 25 weeks. The formation of skin tumors was significantly inhibited by administration of 10 mg/kg GSH ester prior to each UV irradiation for 25 weeks. Moreover, the increases of cutaneous TBARS and serum sialic acid in the tumor-bearing mice were also prevented by continuous pretreatment with GSH ester. Even after 24 weeks, the epidermal GSH content of the pretreated mice was mostly retained compared to nonirradiated mice. However, administration of GSH prior to acute or chronic UV irradiation had no effect on the UV-induced damage. The present results suggest that the protection from photodamage afforded by pretreatment with GSH ester is due to maintenance of a normal GSH level.

Protective effect of S-(1,2-dicarboxyethyl)glutathione, an intrinsic tripeptide in liver, heart and lens, and its esters on acetaminophen-induced hepatotoxicity in rats.

The administration of acetaminophen (APAP, 500 mg/kg, i.p.) produced liver necrosis and increased aspartate aminotransaminase (AST) activity in serum. The pretreatment of S-(1,2-diethoxycarbonyl)glutathione isopropyl ester (DCE-Et-GS iPr, 0.5 mmol/kg, p.o.) prevented hepatic necrosis and the elevation of serum AST activity by 99.9%. DCE-Et-GS iPr inhibited APAP-induced hepatotoxicity much more strongly than reduced glutathione (GSH), DCE-GS and other esters of DCE-GS. To clarify this protective effect, the hepatic GSH concentration was determined 2h after APAP administration. It was found that the DCE-Et-GS iPr administration significantly inhibited the GSH depletion caused by APAP, suggesting that the protective effect of DCE-Et-GS iPr on APAP-induced hepatotoxicity was due, at least in part, to the retention of hepatic GSH level.

Cardiac [formula omitted] ATPase activity and its relation to myocardial glutathione status: Studies in the rat

Cardiac Na + K + ATPase (which has previously been suggested to be regulated by a thiol-dependent process) is inactivated during ischemia. Moreover, myocardial glutathione (which controls the redox state of protein-thiol groups) is depleted during ischemia and thus may contribute to the changes in Na + K + ATPase activity. The objectives of the present study were to use the rat to: (1) pharmacologically manipulate myocardial glutathione to mimic ischemia-reperfusion-induced glutathione depletion and (2) determine (in the absence of ischemia) the relationship between myocardial glutathione content and Na + K + ATPase activity. Tissue glutathione was depleted by injecting rats with diethylmaleate (0, 55, 110, 215, 430 or 860 mg/kg; i.p.) 30 min before study. Total glutathione content fell from 1.72±0.03 to 1.66±0.04, 1.50±0.05, 0.93±0.03, 0.23±0.04 and 0.21±0.02 μmol/g wet weight, respectively. There was a linear correlation ( r=0.96) between Na + K + ATPase activity and glutathione content in diethylmaleate-treated animals. A separate group of animals were treated with phorone (0, 25, 50, 100, 150, 200 or 250 mg/kg; i.p. 120 min before study) which also depletes glutathione. Myocardial glutathione fell from its control value of 1.74±0.03 to 1.52±0.04, 1.35±0.06, 1.14±0.05, 0.84±0.04, 0.64±0.04, 0.54±0.03 μmoles/g wet weight, respectively. In these animals Na + K + ATPase activity was also linearly-related ( r=0.98) to glutathione content. We also characterized the temporal relationship between diethylmaleate-induced glutathione depletion and Na + K + ATPase activity. Diethylmaleate (216 mg/kg; i.p.) was given to rats and at various times (0–200 min), after administration the hearts were removed and assayed for glutathione. During the first 30 min after administration glutathione fell from 1.74±0.03 to its lowest value of 0.90±0.04 μmol/g wet weight. It then progressively recovered to within control levels by 150 min after administration. Na + K + ATPase activity paralleled the recovery of glutathione status. In additional studies, the cell-permeant glutathione analogue YM 737 (glutathione isopropyl ester: 1 mmol/kg, i.p.) accelerated the recovery of tissue glutathione and evoked a commensurate and parallel increase in the rate of recovery of Na + K + ATPase activity. Our studies suggest there is a close coupling between tissue glutathione content and Na + K + ATPase activity and that this may be an important factor in ischemia and reperfusion-induced changes in Na + K + ATPase activity.

Protective effects of glutathione isopropyl ester on the sensitivity of cultured cells to UVB irradiation.

The effect of glutathione (GSH) isopropyl ester on cellular sensitivity to UVB irradiation was investigated in HeLaS3 cells. Pretreatment with 0.1-0.5 mM GSH isopropyl ester for 4 h significantly inhibited the decrease of thymidine (TdR) incorporation caused by UVB irradiation at a dose of 500 J/m2, whereas pretreatment with a high dose (1 mM) had no effect. The colony formation ability of the pretreated cells (0.3 mM) was significantly better than that of cells that received irradiation only. When the cells were treated with GSH isopropyl ester, their intracellular GSH level increased dose-dependently over a 4 h period, suggesting that GSH isopropyl ester was transported into the cells and there converted to GSH. Within 2 min of exposure, the intracellular GSH level depleted rapidly to about 75% of that in non-irradiated normal cells. In contrast, the GSH level in cells pretreated with 0.3 mM GSH isopropyl ester was maintained at the same level as that in normal cells, indicating that the maintenance of intracellular GSH level is due to converted GSH from GSH isopropyl ester. These results clearly show that intracellular GSH is involved in cell protection against photodamage, and that GSH isopropyl ester is a useful antioxidant for protection against photooxidative injury.

Induction of heat shock 70 mRNA by cadmium is mediated by glutathione suppressive and non-suppressive triggers

The induction mechanism of heat shock 70 (Hsp 70) gene by cadmium was investigated. In human amniotic WISH cells, Hsp 70 was induced by cadmium in a dose- and time-dependent manner. Cadmium-induced Hsp70 mRNA levels were enhanced 3- to 4-fold after depletion of intracellular glutathione (GSH) by either diethylmeleate or buthionine sulfoximine. Under the conditions, hydrogen peroxide might increase in the absence of substrate for glutathione peroxidase. We found that exogenous hydrogen peroxide alone induced Hsp70 which was further enhanced significantly after GSH-depletion by diethylmaleate. On the other hand, treatment of cells by diethyldithiocarbamate, and inhibitor of superoxide dismutase, induced Hsp70 2-fold over the level of control. This induction was further stimulated by cadmium even in the presence of GSH. Furthermore, a 4-fold increase of intracellular GSH by the treatment of cells with glutathione isopropyl ester did not diminish the cadmium-induced Hsp70. Gel mobility shift assays of nuclear extracts, from these differently treated cells, with oligonucleotide containing a promoter region of Hsp70 gene revealed that the levels of Hsp70 mRNA observed in the present study corresponded to the changes of transcription. These results imply that the induction of Hsp70 mRNA by cadmium is mediated at least partly via reactive oxygen species and attenuated by cellular GSH and that some part of cadmium-induced Hsp70 can not be eliminated by GSH, suggesting that multiple signals are functioning for this induction.

Glutathione isopropyl ester (YM737) inhibits the progression of X-ray-induced cataract in rats.

The effect of glutathione (GSH) isopropyl ester on the progression of X-ray-induced cataract was investigated in rats. Intraperitoneal administration of 20 mg/kg GSH isopropyl ester, three times weekly, 1 day after a single irradiation dose delayed the progression of X-ray-induced cataracts significantly. The amount of non-protein SH groups and the Na+/K+ ratio in the lenses of drug-treated rats were maintained at the normal levels even 27 weeks after irradiation. Posttreatment with the drug resulted in a significantly lower level of malondialdehyde in the irradiated lenses than in the nontreated lenses. When 500 mg/kg GSH-isopropyl ester was administered by i.p. injection to normal rats, the GSH-ester was detected in plasma and aqueous humor after 15 min. In the lenses of the GSH-isopropyl ester-injected rats, the GSH level was 120% of that in the non-treated rats after 4 h, suggesting that GSH-isopropyl ester is transported from the aqueous humor to the lens and there converted to GSH after about 4 h. Our observations lead us to conclude that the delay of X-ray-induced lens opacity progression is due to maintenance of normal lenticular GSH levels achieved by post-irradiation administration of GSH-isopropyl ester. However, continuous administration of 100 mg/kg after irradiation had no effect on the progression of cataracts induced by X-rays.

Determination of glutathione isopropyl ester in rat, dog and human blood by high-performance liquid chromatography with fluorescence detection

A HPLC method is developed for the determination of glutathione isopropyl ester, a drug for the treatment of cerebral vascular disease, in rat, dog and human blood. The blood is deproteinized with sulphosalicylic acid and the clear supernatant treated with a thiol-specific fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F) in borate buffer pH 7.5 at 30°C. The derivatization of glutathione isopropyl ester with SBD-F is markedly enhanced by the addition of dimethyl sulphoxide, and is complete in 30 min. The fluorescent derivatives of glutathione isopropyl ester and the internal standard, glutathione ethyl ester are separated from those of endogenous thiols such as cysteine and glutathione on a reversed-phase column. The method is simple and selective with a detection limit of 0.05 μg ml −1. Blood concentrations of glutathione isopropyl ester in rats, dogs and humans after intravenous administration are determined using the method.

Glutathione enhancement in various mouse organs and protection by glutathione isopropyl ester against liver injury

Intraperitoneal administration of glutathione isopropyl ester to fasted, male NMRI mice dose dependently increased the glutathione concentration in various organs. Administration of 1 g kg glutathione isopropyl ester led to the following increases: liver 166%; lung 164%; heart 121% after 4 hr; and brain 133% after 6 hr. Spleen, kidney, muscle, serum and blood cell glutathione were not affected by the treatment. Pretreatment with glutathione isopropyl ester was found to protect against paracetamolor allyl alcohol-induced liver damage. Following treatment with the ester a significant correlation between protection against liver damage and enhancement of liver glutathione content was obtained. The dose dependence of this protection was studied.

Glutathione in human melanoma cells: Effects of cysteine, cysteine esters and glutathione isopropyl ester

Thiols are of great importance for the regulation of many cellular functions including metabolism, transport and cell protection. In this study the usefulness of l-cysteine methyl and octyl esters, of N,S-diacetyl- l-cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems was investigated in the human IGR 1 melanoma cell line. The l-cysteine methyl and octyl esters proved to be highly toxic to the cells. Treatment of the cultures with 1 mM N,S-diacetyl- l-cysteine methyl ester or 3 mM glutathione isopropyl ester for 24 h resulted in marked elevation of the cellular glutathione level without apparent or with slight cell loss, respectively. Thus the administration of the latter two compounds seems to be suitable for inducing GSH elevation in the cultured melanoma cells.