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PubMed2001

Inhibition of rat PC12 cell calpain activity by glutathione, oxidized glutathione and nitric oxide.

Calpain, a calcium activated neutral protease, is involved in mediating neurotoxicity resulting from conditions of oxidative stress and free radical formation, such as hypoxia and ischemia. Nitric oxide (NO) may also be involved in modulating the cytotoxic effects of oxidative stress. We investigated the roles of reduced glutathione (GSH), oxidized glutathione (GSSG), and NO in modulating calpain activity in PC12 cells. Cell extracts were treated with GSSG, GSH, or the NO-donor S-nitroso-N-acetylpenicillamine. Calpain activity was determined by means of a fluorescent assay. Non-linear regression analysis was used to determine the type of inhibition (competitive, uncompetitive, or non-competitive). GSH displayed uncompetitive inhibition, with K(i)=7.0+/-2.0 mM (Mean+/-SEM) while GSSG exhibited competitive inhibition with K(i)=2.5+/-0.3 mM. NO was an irreversible inhibitor of calpain activity. These results suggest that both GSH and GSSG may be important physiological modulators of calpain activity.

Animals↗
PubMed1992

Glutathione homeostasis and turnover in the totally hepatectomized rat: evidence for a high glutathione export capacity of extrahepatic tissues.

Glutathione (GSH) homeostasis and turnover were investigated in totally hepatectomized (HX) rats. A technique is described to remove the liver totally, with preservation of the hepatic portal and vena caval vasculature. Euglycemia could be maintained with hourly infusions of 50 mg 100 g-1 b.m. of glucose after bolus i.v. injection of glucose at the same dose. The efficiency of the animal model was demonstrated by examination of paraclinical blood parameters: progressive increases in total plasma bilirubin and alkaline phosphatase activity were noted after HX; the other parameters tested were predominantly in the normal range during the observation period of 6 hours. Histological examination revealed an acute but reversible impairment of intestine and kidneys. These results indicate that the surgical procedure and postoperative care were able to secure sufficient physiological conditions for the experiments over a longer period. 3 to 6 hours after HX we observed a decreased but stable plasma GSH level in anhepatic rats (about 50% of the control value). The GSH levels of brain and kidney were not changed. With increasing time period after HX the heart and lung GSH levels were depressed. A small depression of muscle GSH concentration was observed 4 and 6 hours after HX. A progressive increase in the concentration of oxidized glutathione was seen in brain and kidney. Our observations could be indicative for a high GSH export capacity of extrahepatic tissues contributing about 50% of the total GSH influx into circulation. Probably, the skeletal musculature is an important GSH origin for plasma.

Animals↗
PubMed2000

The putative benzene metabolite 2,3, 5-tris(glutathion-S-yl)hydroquinone depletes glutathione, stimulates sphingomyelin turnover, and induces apoptosis in HL-60 cells.

In this study, we show that 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a putative metabolite of benzene, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Prior to the onset of apoptosis, TGHQ depletes intracellular glutathione (GSH) in a reactive oxygen species (ROS)-independent manner. Neutral, Mg(2+)-dependent sphingomyelinases, which are normally inhibited by GSH, are subsequently activated, as evidenced by increases in intracellular ceramide and depletion of sphingomyelin. As ceramide levels rise, effector caspase (DEVDase) activity steadily increases. Interestingly, while catalase has no effect on TGHQ-mediated depletion of GSH, this hydrogen peroxide (H(2)O(2)) scavenger does inhibit DEVDase activity and apoptosis, provided the enzyme is added to HL-60 cells before an increase in ceramide can be observed. Since ceramide analogues inhibit the mitochondrial respiratory chain, these data imply that ceramide-mediated generation of H(2)O(2) is necessary for the activation of effector caspases-3 and/or -7, and apoptosis. In summary, these studies indicate that TGHQ, and perhaps many quinol-based toxicants and chemotherapeutics, may induce apoptosis in hematopoietic cells by depleting GSH and inducing the proapoptotic ceramide-signaling pathway.

Apoptosis↗
PubMed2001

Characterization of nucleoside and DNA adducts formed by S-(1-acetoxymethyl)glutathione and implications for dihalomethane-glutathione conjugates.

S-(1-Acetoxymethyl)glutathione (GSCH(2)OAc) was synthesized and used as a model for the reaction of glutathione (GSH)-dihaloalkane conjugates with nucleosides and DNA. Previously, S-[1-(N(2)-deoxyguanosinyl)methyl]GSH had been identified as the major adduct formed in the reaction of GSCH(2)OAc with deoxyguanosine. GSCH(2)OAc was incubated with the three remaining deoxyribonucleosides to identify other possible adducts. Adducts to all three nucleosides were found using electrospray ionization mass spectrometry (ESI MS). The adduct of GSCH(2)OAc and deoxyadenosine was formed in yield of up to 0.05% and was identified as S-[1-(N(7)-deoxyadenosinyl)methyl]GSH. The pyrimidine deoxyribonucleoside adducts were formed more efficiently, resulting in yields of 1 and 2% for the GSCH(2)OAc adducts derived from thymidine and deoxycytidine, respectively, but their lability prevented their structural identification by (1)H NMR. On the basis of the available UV spectra, we propose the structures S-[1-(N(3)-thymidinyl)methyl]GSH and S-[1-(N(4)-deoxycytidinyl)methyl]GSH. Because adduct degradation occurred most rapidly at alkaline and neutral pH values, an enzymatic DNA digestion procedure was developed for the rapid hydrolysis of DNA to deoxyribonucleosides at acidic pH. DNA digests were completed in less than 2 h with a two-step method, which consisted of a 15 min incubation of DNA with high concentrations of deoxyribonuclease II and phosphodiesterase II at pH 4.5, followed by incubation of resulting nucleotides with acid phosphatase. Analysis of the hydrolysis products by HPLC-ESI-MS indicated the presence of the thymidine adduct.

DNA↗
PubMed2004

Modelling and bioinformatics studies of the human Kappa-class glutathione transferase predict a novel third glutathione transferase family with similarity to prokaryotic 2-hydroxychromene-2-carboxylate isomerases.

The Kappa class of GSTs (glutathione transferases) comprises soluble enzymes originally isolated from the mitochondrial matrix of rats. We have characterized a Kappa class cDNA from human breast. The cDNA is derived from a single gene comprising eight exons and seven introns located on chromosome 7q34-35. Recombinant hGSTK1-1 was expressed in Escherichia coli as a homodimer (subunit molecular mass approximately 25.5 kDa). Significant glutathione-conjugating activity was found only with the model substrate CDNB (1-chloro-2,4-ditnitrobenzene). Hyperbolic kinetics were obtained for GSH (parameters: K(m)app, 3.3+/-0.95 mM; V(max)app, 21.4+/-1.8 micromol/min per mg of enzyme), while sigmoidal kinetics were obtained for CDNB (parameters: S0.5app, 1.5+/-1.0 mM; V(max)app, 40.3+/-0.3 micromol/min per mg of enzyme; Hill coefficient, 1.3), reflecting low affinities for both substrates. Sequence analyses, homology modelling and secondary structure predictions show that hGSTK1 has (a) most similarity to bacterial HCCA (2-hydroxychromene-2-carboxylate) isomerases and (b) a predicted C-terminal domain structure that is almost identical to that of bacterial disulphide-bond-forming DsbA oxidoreductase (root mean square deviation 0.5-0.6 A). The structures of hGSTK1 and HCCA isomerase are predicted to possess a thioredoxin fold with a polyhelical domain (alpha(x)) embedded between the beta-strands (betaalphabetaalpha(x)betabetaalpha, where the underlined elements represent the N and C motifs of the thioredoxin fold), as occurs in the bacterial disulphide-bond-forming oxidoreductases. This is in contrast with the cytosolic GSTs, where the helical domain occurs exclusively at the C-terminus (betaalphabetaalphabetabetaalphaalpha(x)). Although hGSTK1-1 catalyses some typical GST reactions, we propose that it is structurally distinct from other classes of cytosolic GSTs. The present study suggests that the Kappa class may have arisen in prokaryotes well before the divergence of the cytosolic GSTs.

Amino Acid Sequence↗
PubMed1982

Purification and characterization of three forms of glutathione S-transferase A. A comparative study of the major YaYa-, YbYb- and YcYc-containing glutathione S-transferases.

Rat liver glutathione S-transferases have previously been defined by their elution behaviour from DEAE-cellulose and CM-cellulose as M, E, D, C, B, A and AA. These enzymes are dimeric proteins which comprise subunits of mol.wt. 22 000 (Ya), 23 500 (Yb) or 25 000 (Yc). Evidence is presented that YaYa protein, one of two previously described lithocholate-binding proteins which exhibit transferase activity, is an additional enzyme which is not included in the M, E, D, C, B, A and AA nomenclature. We therefore propose that this enzyme is designated transferase YaYa. Transferases YaYa, C, A and AA have molecular weights of 44 000, 47 000, 47 000 and 50 000 respectively and each comprises two subunits of identical size. These enzymes were purified to allow a study of their structural and functional relationships. In addition, transferase A was further resolved into three forms (A1, A2 and A3) which possess identical activities and structures and appear to be the product of a single gene. Transferases YaYa, C, A and AA each had distinct enzymic properties and were inhibited by cholate. The recently proposed proteolytic model, which attributes the presence of multiple forms of glutathione S-transferase activity to partial proteolysis of transferase AA, was tested and shown to be highly improbable. Peptide maps showed significant differences between transferases YaYa, C, A and AA. Immunotitration studies demonstrated that antisera raised against transferases YaYa and C did not precipitate transferase AA.

Animals↗
PubMed1987

Expression of human glutathione S-transferase 2 in Escherichia coli. Immunological comparison with the basic glutathione S-transferases isoenzymes from human liver.

A plasmid, termed pTacGST2, which contains the complete coding sequence of a GST2 (glutathione S-transferase 2) subunit and permits the expression of the protein in Escherichia coli was constructed. The expressed protein had the same subunit Mr as the enzyme from normal human liver and retained its catalytic function with both GST and glutathione peroxidase activity. Antiserum raised against the bacterially synthesized protein cross-reacted with all the basic GST isoenzymes in human liver. The electrophoretic mobility in agarose of the bacterially expressed isoenzyme suggested that its pI is identical with that of the cationic isoenzyme from human liver previously termed GST2 type 1. The available evidence suggests that the three common cationic isoenzymes found in human liver are the products of two very similar gene loci.

Antibodies, Bacterial↗
PubMed1989

Glutathione transferases in rat hepatoma cells. Effects of ascites cells on the isoenzyme pattern in liver and induction of glutathione transferases in the tumour cells.

Rat hepatoma cells grown intraperitoneally as an ascites tumour were analysed with respect to their contents of cytosolic glutathione transferases. In contrast with normal liver tissue, the hepatoma cells were dominated by the class Pi glutathione transferase 7-7. All the major hepatic enzyme forms were down-regulated to almost undetectable concentrations. Livers of rats bearing ascites-hepatoma cells expressed low, but significant, amounts of protein which, by electrophoretic and immunochemical properties, appeared identical with transferase 7-7. This enzyme is not detectable in normal hepatocytes. Treatment of rats with trans-stilbene oxide induced the expression of transferase 7-7 in the livers of normal rats as well as in hepatoma-cell-bearing animals. In addition, a 2-fold induction of transferase 7-7 was measured in the hepatoma ascites cells. No significant elevation of any other enzyme forms in the hepatoma cells was noted.

Animals↗
PubMed1996

Structure-activity relationships for chemical and glutathione S-transferase-catalysed glutathione conjugation reactions of a series of 2-substituted 1-chloro-4-nitrobenzenes.

Glutathione S-transferases (GSTs) constitute an important class of phase II (de)toxifying enzymes, catalysing the conjugation of glutathione (GSH) with electrophilic compounds. In the present study, Km, kcat and kcat/Km values for the rat GST 1-1-, 3-3-, 4-4- and 7-7-catalysed conjugation reactions between GSH and a series of 10 different 2-substituted 1-chloro-4-nitrobenzenes, and the second-order rate constants (ks) of the corresponding base-catalysed reactions, were correlated with nine classical physicochemical parameters (electronic, steric and lipophilic) of the substituents and with 16 computer-calculated molecular parameters of the substrates and of the corresponding Meisenheimer complexes with MeS- as a model nucleophile for GS- (charge distributions and several energy values), giving structure-activity relationships. On the basis of an identical dependence of the base-catalysed as well as the GST 1-1- and GST 7-7-catalysed reactions on electronic parameters (among others, Hammett substituent constant sigma p and charge on p-nitro substituents), and the finding that the corresponding reactions catalysed by GSTs 3-3 and 4-4 depend to a significantly lesser extent on these parameters, it was concluded that the Mu-class GST isoenzymes have a rate-determining transition state in the conjugation reaction between 2-substituted 1-chloro-4-nitrobenzenes and GSH which is different from that of the other two GSTs. Several alternative rate-limiting transition states for GST 3-3 and 4-4 are discussed. Furthermore, based on the obtained structure-activity relationships, it was possible to predict the kcat/Km values of the four GST isoenzymes and the ks of the base-catalysed GSH conjugation of 1-chloro-4-nitrobenzene.

Animals↗
PubMed1996

Role of the 3' untranslated region in the regulation of cytosolic glutathione peroxidase and phospholipid-hydroperoxide glutathione peroxidase gene expression by selenium supply.

Selenium is an essential nutrient and synthesis of selenoproteins is affected by limited selenium supply. During selenium deficiency there is a differential regulation of selenoprotein synthesis and gene expression; for example, there is a decrease in abundance of mRNA for cytosolic glutathione peroxidase (cGSH-Px) and a preservation of mRNA for phospholipid-hydroperoxide glutathione peroxidase (PHGSH-Px). This difference is not due to an alteration in the rate of transcription but might reflect differences in translation. The aim of the present work was to assess the role of cGSH-Px and PHGSH-Px 3' untranslated regions (UTRs) in the regulation of selenoprotein mRNA stability and translation by using H4-II-E-C3 cells transfected with different constructs containing a type I iodothyronine deiodinase-coding region linked to different selenoprotein mRNA 3' UTRs. Translational efficiency results showed that the efficiency of the 3' UTRs in permitting selenocysteine incorporation is similar in selenium-replete conditions but, when selenium is limiting, the 3' UTR of cGSH-Px is less efficient than the 3' UTR of PHGSH-Px. The results suggest that the 3' UTR of these selenoprotein mRNA species influences their extent of translation when selenium levels are low. The different sensitivity of the 3' UTRs to selenium deficiency can explain the differential effect that selenium deficiency has on cGSH-Px and PHGSH-Px activity and mRNA levels, stability and translation. This might be partly responsible for channelling selenium for synthesis of PHGSH-Px rather than cGSH-Px.

Animals↗
PubMed1991

Suppression of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine.

The effects of glutathione (GSH), glutathione ester (GSE), and N-acetyl-L-cysteine (NAC) on the induction of human immunodeficiency virus (HIV) expression were investigated in the chronically infected monocytic U1 cell line, a previously described cellular model for HIV latency. U1 cells constitutively express low levels of virus, which can be increased by phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and other inducers. GSH, GSE, and NAC suppressed in a dose-dependent fashion the induction of HIV expression mediated by PMA, TNF-alpha, and IL-6, in the absence of cytotoxic or cytostatic effects. Reverse transcriptase activity, inducible by PMA, TNF-alpha, or IL-6, was decreased by 80-90% after pretreatment with GSH, GSE, or NAC. The induction of total HIV protein synthesis was also decreased appreciably after pretreatment with GSH, GSE, or NAC. The accumulation of HIV mRNA was substantially suppressed after pretreatment with NAC but to a lesser extent after pretreatment with GSH or GSE. Although PMA induces the expression of TNF-alpha in U1 cells, the suppressive effect of GSH, GSE, and NAC on PMA-induced HIV expression in U1 cells was not associated with the inhibition of TNF-alpha expression. The present findings, which elucidate relationships between cellular GSH and HIV expression, suggest that therapy with thiols may be of value in the treatment of HIV infection.

Acetylcysteine↗
PubMed1985

Reaction of 2-acetylaminofluorene-N-sulfate with RNA and glutathione: evidence for the generation of two reactive intermediates with different reactivities towards RNA and glutathione.

The sulfate ester of N-hydroxy-2-acetylaminofluorene (AAF-N-sulfate) is one of the reactive intermediates of this carcinogen. This ester breaks down spontaneously to a very reactive nitrenium ion, which reacts with nucleophilic groups in protein, DNA, RNA and glutathione (GSH). Reactions involving the nitrenium ion with several nucleophiles under various conditions were studied. The adduct formation to RNA was much higher in Tris-HCI buffer than in phosphate buffer (at pH 7.4), while adduct formation to deoxy-guanosine monomers was the same in both buffers. The presence of 150 mM KCI had the same decreasing effect in both cases. Ionic strength effects may be involved in these phenomena. GSH decreased RNA adduct formation by 20-45%, while other thiols were much more effective. On the other hand, RNA did not decrease the formation of GSH conjugates from AAF-N-sulfate. The decrease in RNA adduct formation by thiols corresponded with an increase in the formation of 2-acetylaminofluorene (AAF) from AAF-N-sulfate, while no N-hydroxy-AAF was formed. These results suggest that two independent reactive intermediates are formed from AAF-N-sulfate, with different reactivities towards RNA and glutathione. Possibly these intermediates are the 'hard' triplet state nitrenium ion and the 'soft' singlet state nitrenium ion. Cysteine, cysteamine and penicillamine were most effective in the inhibition of RNA adduct formation; the extent of inhibition correlated with the extent of AAF formation. The mechanisms involved are discussed.

2-Acetylaminofluorene↗
PubMed1991

Partial sequence of human plasma glutathione peroxidase and immunologic identification of milk glutathione peroxidase as the plasma enzyme.

Plasma glutathione peroxidase (p-GSHPx) is a unique selenoglycoprotein. A hepatic cell line synthesizes both this extracellular form for secretion and the cellular form that remains within the cells. Because the two forms could be a result of post-translational modifications of a product of a single gene, we partially sequenced p-GSHPx. Purified p-GSHPx was trypsin digested, and three of the peptides were sequenced. Only one of the peptide sequences was partially homologous to a sequence found in human cellular glutathione peroxidase. Because p-GSHPx is a secreted enzyme, we determined whether GSHPx in milk (another extracellular fluid) is due to this form of the enzyme. Ninety percent of human milk GSHPx activity could be precipitated by anti-p-GSHPx-immunoglobulin G. Thus, most, if not all, GSHPx activity in milk is due to the plasma selenoprotein form of the enzyme. In milk of two North American women, 3.6% and 14.3% of selenium was associated with GSHPx.

Amino Acid Sequence↗
PubMed1990

Modulation of glutathione peroxidase expression by selenium: effect on human MCF-7 breast cancer cell transfectants expressing a cellular glutathione peroxidase cDNA and doxorubicin-resistant MCF-7 cells.

We have studied the effect of selenium on the expression of a cellular glutathione peroxidase, GSHPx-1, in transfected MCF-7 cells and in doxorubicin-resistant (Adrr) MCF-7 cells. A GSHPx-1 cDNA with a Rous Sarcoma virus promoter was transfected into a human mammary carcinoma cell line, MCF-7, which has very low endogenous cytosolic glutathione (GSH) peroxidase activity and no detectable message. The transfectant with the highest GSH peroxidase activity among the isolates, MCF-7H6, was characterized. Adrr MCF-7 cells, a subline of MCF-7 cells, also has elevated GSH peroxidase activity. GSH peroxidase expressed by MCF-7H6 and Adrr MCF-7 cells is similar to the endogenous GSHPx-1 based on molecular weight, immunoreactivity, and metabolic labeling with 75Se. MCF-7H6 and Adrr MCF-7 cells grown in Se-deficient media had 2.6 +/- 2.4 (mean +/- S.D.) and 4.2 +/- 3.6 units/mg protein of GSH peroxidase specific activity, respectively. Se supplementation increased GSH peroxidase activity in a concentration- and time-dependent fashion. Enzymatic activity reached a level of 164 +/- 62 in MCF-7H6 cells and 114 +/- 27 in Adrr MCF-7 cells within 5 days of growth in media supplemented with 30 nM Se. Northern analysis revealed that Se-deficient MCF-7H6 cells expressed 2.1 +/- 0.4-fold less GSHPx-1 mRNA than their Se-sufficient counterparts. Similarly, Se-deficient Adrr MCF-7 cells expressed 3.3 +/- 1.8-fold less GSHPx-1 mRNA than their Se-supplemented counterparts after the quantity of mRNA was normalized with beta-actin. These studies suggest that modulation of GSH peroxidase activity by Se in both MCF-7H6 transfectants expressing pRSV-GSHPx-1 and Adrr MCF-7 cells expressing endogenous GSHPx-1 occurs largely at the translational level, and to a lesser degree at the level of mRNA, possibly by stabilizing GSHPx-1 mRNA since the transfected cDNA in MCF-7H6 cells has only 5 nucleotides 5' to the AUG initiation codon.

Avian Sarcoma Viruses↗
PubMed1998

Glutathione is present in reproductive tract secretions and improves development of mouse embryos after chemically induced glutathione depletion.

We investigated the hypothesis that reduced glutathione (GSH) is present in secretions of the female reproductive tract and that this extracellular GSH may protect preimplantation mouse embryos after intracellular GSH depletion. The cleavage-stage mouse embryo cannot synthesize GSH de novo and is unable to recover from glutathione depletion in vitro. Analysis of GSH and total protein of oviduct flushings, quantified by HPLC and the Bradford method, respectively, revealed 51 nmol GSH per mg total protein. Embryos were treated with 60 microM diethyl maleate (DEM) to deplete cellular GSH. When cultured with 1 mM GSH, these embryos exhibited improved development compared to those cultured in control medium (96% vs. 87% morula [p < 0.05], 78% vs. 75% blastocyst, 58% vs. 54% expanded blastocyst, 21% vs. 17% initiating hatching blastocyst). However, intracellular GSH content of embryos was not significantly increased by the culture of DEM-treated embryos in medium containing GSH for 16, 40, or 64 h of incubation, suggesting that the embryo is not capable of taking up intact GSH. Furthermore, addition of buthionine sulfoximine (which inhibits synthesis of GSH) or acivicin (which inhibits breakdown of GSH at the membrane) to culture medium blocked the improvement in development. These data suggest that GSH in reproductive tract fluid may help protect preimplantation embryos from the adverse effects of toxicant-induced and endogenous depletion of embryonic GSH.

Animals↗
PubMed1997

Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes.

Acetophenones↗
PubMed1994

Effect of oral glutathione monoethyl ester and glutathione on circulating and hepatic sulfhydrils in the rat.

Glutathione (GSH) plays an important role in the detoxification of reactive metabolites of oxygen and xenobiotics and as a source of cysteine. Since several clinical situations characterized by low circulating and intracellular GSH have been identified, there is a growing interest in pharmacological interventions to correct a deranged sulfhydril status. Therefore, the systemic bioavailability of orally administered GSH and glutathione monoethyl ester (GSHE) was examined in the rat. Following the intraduodenal administration of 0.5 mmol/kg of GSH and GSHE there was no significant increase in the concentrations of cysteine and GSH in plasma, but hepatic cysteine and GSH increased significantly, albeit transiently. Five mmol/kg of GSH and GSHE significantly increased circulating and hepatic cysteine and GSH. Following the administration of 0.5 and 5 mmol/kg of GSHE low concentrations of the ester were found in plasma and the liver, indicating that GSHE is not readily absorbed from the gastrointestinal tract, although it is not a substrate for gamma-glutamyl-transferase. GSHE resulted in a delayed release of cysteine and GSH compared to GSH, such that the concentrations of GSH and cysteine in liver and plasma were significantly higher 2 h after administration of GSHE than after GSH. The data indicate that the bioavailability of GSH and GSHE is low in the rat. Orally administered GSH and GSHE do not affect the circulating concentrations of GSH and cysteine unless very high doses are administered, but increase hepatic cysteine and GSH at lower doses because of the efficient extraction by the liver of cysteine originating from the breakdown of GSH and GSHE in the gut.

Administration, Oral↗
PubMed2003

Studies on the chemical reactivity of diclofenac acyl glucuronide with glutathione: identification of diclofenac-S-acyl-glutathione in rat bile.

Diclofenac, a nonsteroidal anti-inflammatory drug, is metabolized to a reactive acyl glucuronide that has been proposed to mediate toxic adverse drug reactions associated with its use. In the present study, we examined the ability of diclofenac acyl glucuronide (D-1-O-G) to transacylate glutathione (GSH) in vitro in buffer and in vivo in rats. Thus, in vitro reactions of D-1-O-G (100 microM) with GSH (10 mM) at pH 7.4 and 37 degrees C showed a linear time-dependent formation of diclofenac-S-acyl-glutathione (D-SG, 3 microM/h) through 60 min of incubation, reaching a maximum of 3.7 microM after 2 h of incubation. The major reaction that occurred was acyl migration of D-1-O-G (t1/2, 54 min) to less reactive isomers. The D-SG thioester product was shown to be unstable by degrading primarily to 1-(2,6-dichlorophenyl)indolin-2-one and by hydrolysis to diclofenac. After administration of diclofenac to rats (200 mg/kg), bile was collected and analyzed for D-SG by liquid chromatography-tandem mass spectrometry. Results indicated the presence of D-SG, which was confirmed by coelution with synthetic standard and by its tandem mass spectrum. When the reactivity of D-SG (100 microM) was compared with D-1-O-G (100 microM) in vitro in reactions with N-acetylcysteine (NAC, 10 mM), results showed the quantitative reaction of D-SG with NAC after 30 min of incubation, whereas only approximately 1% of D-1-O-G reacted to form diclofenac-S-acyl-NAC at the same time point. Results from these studies indicate that GSH reacts with D-1-O-G in vitro, and presumably in vivo, to form D-SG, and that the product D-SG thioester is chemically more reactive in transacylation-type reactions than the D-1-O-G metabolite.

Animals↗