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PubMed1992

Brefeldin A binds to glutathione S-transferase and is secreted as glutathione and cysteine conjugates by Chinese hamster ovary cells.

Radioactively labeled brefeldin A was used to probe for proteins that interact with this metabolite. The most prominent protein labeled after in vivo incubation of Chinese hamster ovary cells with [3H]brefeldin A turned out to have an apparent molecular mass of 26 kDa. Radioactive peptides derived from the [3H]brefeldin A-labeled protein showed sequence identity with glutathione S-transferases, and immunoblotting after two-dimensional gel electrophoresis confirmed this result. In addition, Chinese hamster ovary cells convert the antibiotic to its glutathionyl and cysteinyl derivatives and secrete them rapidly into the medium. From these findings we conclude that detoxification of the antibiotic in mammalian cells occurs via the glutathion S-transferase system. This may explain the often observed reversibility of brefeldin A's action on the steady state of organelles in mammalian cells.

Amino Acid Sequence↗
PubMed1991

Glutathione peroxidase and glutathione S-transferase, class alpha, in rat intestine. Immunohistochemical and immunoblotting studies on changes in expression of these antilipoperoxidative enzymes during normal development.

Glutathione peroxidase (GSH-PO) and glutathione S-transferase (GST), class alpha, showing GSH-PO-like enzymatic activity, were localized immunohistochemically in frozen sections of rat intestine in order to elucidate changes in the expression of these antilipoperoxidative enzymes during normal development. The direct immunoperoxidase method was performed using specific rabbit antibodies (Fab fragments) against the enzymes purified from rat liver. Immunoreactive GSH-PO and GST-alpha were demonstrated in the intestinal villous epithelial cells. In the duodenum, GSH-PO was positive during the period from 19 days of gestation to 1 week after birth, while GST-alpha was negative during this period. Two weeks after birth, positivity for GST-alpha appeared, and GSH-PO became undetectable. In the ileum, both of the enzymes were observed until 2 weeks of age, but after weaning their expression disappeared. These immunohistochemical findings were confirmed by immunoblot analysis using intestinal tissue extracts. To evaluate environmental effects on the expression of these enzymes, germ-free animals, common bile duct-ligated rats, and Hank's solution-fed infant rats were prepared. No remarkable alterations in the immunohistochemical localization pattern were observed. Since the switching of enzyme expression around the time of weaning was not influenced by these experimentally induced environmental conditions, it appears that these enzymatic changes are genetically predetermined.

Aging↗
PubMed1991

Influence of glutathione S-transferases on cellular glutathione determination by flow cytometry using monochlorobimane.

Intracellular glutathione (GSH) levels for seven mammalian cell lines (four human tumors, two rodent, one monkey) were determined by flow cytometry following staining with monochlorobimane (MBCl), and the results were compared with GSH levels measured by the Tietze assay. The mean fluorescence intensity for all but the two rodent lines did not correlate with GSH levels determined biochemically. Good agreement between the two assays was observed for the rodent lines following depletion of GSH by buthionine sulfoximine, but the level of GSH depletion achieved in the human and monkey lines was always underestimated by MBCl/flow cytometry. These discrepancies were not resolved by increasing stain concentration or staining time. Total glutathione S-transferase (GST) activity and GST isozyme profiles were determined for each of the cell lines. Western analysis with antibodies raised against rat Ya, Yb1, and Yc and human pi isozymes revealed that the rodent cell lines expressed abundant alpha (Ya, Yc subunits) and mu (Yb1 subunits) class isozymes. In contrast, GST-pi was the predominant isozyme detected in the human tumor cell lines and Cos-7 monkey cells. Michaelis-Menten analysis with purified GSTs from rat liver as well as purified human placental (pi) GST revealed that the conjugation of MBCl and GSH catalyzed by the alpha (1-1 and 2-2) and mu (3-3 and 3-4) class GST isozymes was approximately 10 and 80 times more efficient than was conjugation by the GST pi form, respectively. These data indicate that the GST-catalyzed conjugation of GSH and MBCl is isozyme dependent and that MBCl is a relatively poor substrate for the pi isozyme. As a consequence of this isozyme rate differential, the MBCl/flow cytometry technique for GSH quantitation must be applied cautiously, particularly with human tumor cells, many of which have been shown to have high GST-pi activity. Application to other cell types should also be made after careful characterization of GSH levels and GST isozyme composition and only after comparison with other independent assays of GSH concentration.

Animals↗
PubMedDate not supplied

[The effect of nicotinamide on the activity of the glutathione- glutathione reductase system in subcellular fractions of regenerating rat liver].

Experiments on white rats have shown that growth rates of the glutathione reductase activity and reduced glutathione concentration in the cytoplasmic fraction of the generating liver tissue and especially in the mitochondrial one are more pronounced with an increase of the nicotinamide dose from 50 mg/kg to 150 mg/kg, than after administration of nicotinamide in a dose of 300 mg/kg. Higher doses of nicotinamide (500 mg/kg) produce less pronounced changes in these parameters.

Animals↗
PubMed1988

Regional distribution of glutathione peroxidase and Glutathione-S-transferase in adult and premature human retinas.

The activities of glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GSH-S-tase) were investigated in adult and premature human retinas. The measurements were done in the vascular and avascular regions of premature retinas at gestational age of 22-33 weeks and in the central, mid-peripheral, and far peripheral regions of mature retinas from the age of 1 month to 73 years. Among the premature infants, those who survived for greater than 24 hours were supplemented with alpha-tocopherol (vitamin E) on a periodic basis. The vascular and avascular regions of premature retinas had higher activities of GSH-Px when compared to the central and far peripheral regions of mature retinas. Infants surviving more than 24 hr had higher activities of GSH-S-tase in the avascular region than infants who survived less than 24 hr. Survival did not affect either enzyme activity in the vascular regions. Mature retinas showed a decrease in GSH-Px specific activity with age, but no age-related changes in GSH-S-tase were observed. These data demonstrate that premature infants are born with relatively high levels of GSH-Px and GSH-S-tase.

Adolescent↗
PubMed1984

Effect of steroidal sex hormones on the sex-related differences in the hepatic activities of gamma-glutamyltranspeptidase, glutathione S-transferase and glutathione peroxidase in rats.

The activities of gamma-glutamyltranspeptidase (gamma-GTP) and glutathione peroxidase (GSH-Px) were higher in female than in male rat liver. Glutathione S-transferase (GST) activities were conversely higher in males than females. Estradiol administration to castrated rats enhanced the gamma-GTP activity. Castration produced a significant increase in GSH-Px activities which was reduced to the control level by subsequent testosterone treatment. Ovariectomized rats had markedly increased the GST activity. These results suggest that GSH-related enzymes studied here may be subject to modulation by androgenic and estrogenic influences.

Animals↗
PubMed1983

Changes in activities of glutathione peroxidase and glutathione reductase during chemical hepatocarcinogenesis in the rat.

Changes in the activities of rat hepatic glutathione (GSH) peroxidase (GSH-Px) and GSH reductase (GR) were investigated during the induction of preneoplastic gamma-glutamyltransferase (gamma-GT)-positive foci and hyperplastic nodules by the administration of diethylnitrosamine followed by N-2-fluorenylacetamide. Activities of GSH-Px towards both cumene hydroperoxide (cumene-OOH) and hydrogen peroxide markedly decreased at an early stage of the above hepatocarcinogenesis, but the activity towards cumene-OOH again increased with the appearance of the foci and nodules, which were detectable as an increased gamma-GT activity. It was demonstrated by CM-Sephadex column chromatography and immunochemically that the increased activity towards cumene-OOH alone is due to the increased GSH-Px activity of GSH S-transferase B. GR activity and the total glutathione content in the liver also increased with increased preneoplastic foci and nodules. These results are discussed in connection with the protection mechanism(s) of these cell populations against damage by active oxygen and lipid peroxides produced in chemical hepatocarcinogenesis.

Animals↗
PubMed1995

Glutathione S-transferase isoenzymes and glutathione in renal cell carcinoma and kidney tissue.

Many studies have established the role of the glutathione S-transferases (GSTs) and glutathione (GSH) in the neoplastic process and the drug resistance of tumor. Using isoelectric focusing we separated different forms of GSTs in 28 renal cell carcinomas (RCCs) and in morphologically unchanged adjacent kidney. In addition we determined in RCCs and adjacent kidney the level of GSH and the activities of enzymes participating in synthesis and uptake of this thiol compound. We found higher activity of acidic GSTs and higher level of GSH in RCCs versus kidney. Therefore we suggest that both parameters may play the significant role in the well known phenomenon of intrinsic cytostatic drug resistance of RCC. We also observed the elevation of GSH synthetase activity in tumor tissues in comparison to the kidneys. It may indicate that GSH synthetase, catalysing the final step in GSH synthesis, may participate in the elevation of GSH concentration in RCCs. In this work we also compared the tested parameters in RCCs in relation to the size and local extent of primary tumor (T). We found significantly lower activity of gamma-glutamyl transpeptidase (GGT) as well as GSH synthetase in the group of T3 and T4 tumors than in T2 tumors. However, no substantial differences in GSH concentrations were observed between these distinguished groups.

Adult↗
PubMed1994

Functions of glutathione and glutathione disulfide in immunology and immunopathology.

Even a moderate increase in the cellular cysteine supply elevates the intracellular glutathione (GSH) and glutathione disulfide (GSSG) levels and potentiates immunological functions of lymphocytes in vitro. At low GSSG levels, T cells cannot optimally activate the immunologically important transcription factor NF kappa B, whereas high GSSG levels inhibit the DNA binding activity of NF kappa B. The effects of GSSG are antagonized by reduced thioredoxin (TRX). As the protein tyrosine kinase activities p56lck and p59fyn are activated in intact cells by hydrogen peroxide, they are likely targets for GSSG action. These redox-regulated enzymes trigger signal cascades for NF kappa B activation and transduce signals from the T cell antigen receptor, from CD4 and CD8 molecules, and from the IL-2 receptor beta-chain. The effector phase of cytotoxic T cell responses and IL-2-dependent functions are inhibited even by a partial depletion of the intracellular GSH pool. As signal transduction is facilitated by prooxidant conditions, we propose that the well-known immunological consequences of GSH depletion ultimately may be results of the accompanying GSSG deficiency. As HIV-infected patients and SIV-infected rhesus macaques have, on the average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we also hypothesize that AIDS may be the consequence of a GSSG deficiency as well.

Amino Acid Sequence↗
PubMed1993

Pneumocystis carinii pneumonia in HIV infected patients: effects of the diseases on glutathione and glutathione disulfide.

Human immunodeficiency virus (HIV) infection is associated with altered levels of glutathione (GSH) in cells and extracellular fluids. GSH is essential for lymphocyte proliferation and inhibits HIV replication. Therefore, determination of GSH and glutathione disulfide (GSSG) levels could be useful as indicators of the progression of the disease. Thyroid hormone levels are altered in acquired immuno-deficiency syndrome (AIDS), such that thyroid hormone might be a useful prognostic indicator of the severity of AIDS. Pneumocystis carinii pneumonia (PCP) is a debilitating disease of the lung that can accompany HIV infection. The effects of pulmonary infections were assessed in AIDS patients on thyroid hormone, GSH, GSSG levels and other parameters. Two groups of AIDS patients were selected, a group with PCP and a control group with other respiratory diseases. GSH was evaluated in plasma, pulmonary lavage fluid, pulmonary biopsy tissue and buccal cells. Levels of GSSG in pulmonary lavage fluid were higher in PCP patients than in controls, which suggests that PCP patients suffer from oxygen radical toxicity in their lungs. PCP patients may have altered plasma GSH utilization such that damaged lung tissue may become less efficient at using plasma GSH. Patients with PCP may have altered CD4 cell functions such that thyroid hormone levels do not correlate with CD4 cell counts. Patients with AIDS and secondary infections of the lung were found to have altered GSH redox states, probably indicative of physiologic adaptation to AIDS.

AIDS-Related Opportunistic Infections↗
PubMed1995

Developmental changes in the cellular distribution of glutathione and glutathione S-transferases in the murine nervous system.

The distribution of glutathione (GSH) and glutathione S-transferases (GSTs) in the adult rat brain is cell-type specific, but their cellular distribution in the developing central nervous system is unknown. In the present study, GSH distribution in the mouse nervous system was visualized by mercury orange histochemistry and class-specific GSTs were localized by immunohistochemistry at ages E13 to PN30. Both neuronal and glial progenitor cells stain uniformly positive for GSH at E13. Spinal anterior horn neurons become GSH-negative by E17, at which time neurons and glia in other CNS regions are still GSH-positive. By PN5, most neurons have lost GSH staining and are surrounded by GSH-rich neuropil, ependyma, and vasculature. Olfactory mitral and granule cells, cerebellar granule cells, and dorsal root ganglion (DRG) neurons retain consistently high levels of GSH throughout development and into adulthood. Immunoreactivity to alpha-class GST antisera is not observed in the CNS until PN10, when very weak staining becomes apparent in the pia, ependyma, choroid plexus and neurons throughout the brain and spinal cord. Immunoreactivity to mu-GST is observed in neurons and astrocytes (but not oligodendrocytes), pia, ependyma, and choroid plexus throughout the brain by PN10. pi-GST immunoreactivity is observed in all cells of the embryonic nervous system. Postnatally, it is found in neurons and oligodendrocytes (but not astrocytes) in all regions of the brain and spinal cord as well as in pia, ependyma, and choroid plexus. The neurons and satellite cells of the DRG are immunoreactive to alpha-, mu-, and pi-GST antisera at all time points examined. The developmental changes in the cellular distribution of GSH and GSTs suggest that enzymatic conjugation and antioxidant activities may also be cell specific during brain development.

Animals↗
PubMed1996

Busulfan-glutathione conjugation catalyzed by human liver cytosolic glutathione S-transferases.

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human liver cytosol, purified human liver GST, and cDNA-expressed GST-alpha 1-1. Human liver microsomes and cytosol were incubated with 40 microM busulfan and 1 mM GSH. Cytosol catalyzed the formation of the GSH-busulfan tetrahydrothiophenium ion (THT+) in a concentration-dependent manner, whereas microsomes lacked activity. The total and spontaneous rates of THT+ formation increased with pH (pH range, 6.50-7.75), with the maximum difference at pH 7.4. Due to the limited aqueous solubility of busulfan, a K(m) for busulfan was not determined. The intrinsic clearance (Vmax/K(m)) of busulfan conjugation was 0.167 microliter/min/mg with 50-1200 microM busulfan and 1 mM GSH. GSH Vmax and K(m) for busulfan conjugation were 30.6 pmol/min/mg and 312 microM, respectively. Ethacrynic acid (0.03-15 microM) inhibited cytosolic busulfan-conjugating activity with 40 microM busulfan and 1 mM GSH. Enzyme-mediated THT+ formation was decreased 97% by 15 microM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-alpha 1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 microliters/min/mg, respectively. Busulfan is a GST substrate with a high K(m) relative to concentrations achieved clinically (1-8 microM).

Antineoplastic Agents, Alkylating↗
PubMed1996

Conjugation of glutathione with a toxic metabolite of valproic acid, (E)-2-propyl-2,4-pentadienoic acid, catalyzed by rat hepatic glutathione-S-transferases.

The hepatotoxic metabolite of the anticonvulsant drug valproic acid (VPA), namely (E)-2-propyl-2,4-pentadienoic acid (E)-2,4-diene VPA), is known to react with glutathione (GSH) in vivo. Although glutathione-S-transferase (GST) was suspected of being the catalyst for this conjugation reaction, this was yet to be confirmed. In this study, GST activities were detected in the hepatic cytosolic and sonic-disrupted mitoplast fractions isolated from male Sprague-Dawley rats by using 1-chloro-2,4-dinitrobenzene as a substrate. An elevation of GST activities by 45 to 100% was observed after pretreatment of rats with phenobarbital (PB). Subsequently, these apparent GST activities were examined for their effects on the in vivo conjugation of GSH with N-acetyl-S-((E)-2-propyl-2,4-pentadienoyl)cysteamine (2,4-diene VPA-NACA), a structural mimic of (E)-2,4-diene VPA coenzyme A thioester. Reaction products were identified and quantitated by combined liquid chromatography-tandem mass spectrometry. The GST-mediated conjugation of GSH with 2,4-diene VPA-NACA produced two structural isomers via either 5,6- or 1,6-addition of GSH. Only the 1,6- addition product was found for the spontaneous conjugation reaction (control). Quantitatively, GSH conjugates formed in the cytosolic fraction were 23-fold that of control. An additional 1.5-fold enhancement was observed in the cytosolic fraction from PB-treated rats. The production of the GSH conjugates was increased by 2-fold for reactions involving the sonic-disrupted mitoplasts, either from untreated or PB-treated rats. Partially purified GST was found to catalyze the conjugation reactions in a fashion similar to that of the isolated subcellular fractions. No reaction with GSH could be detected for the free acid form of (E)-2,4-diene VPA. As was the case with the in vitro data, two structural isomers of GSH conjugates were detected in the bile of rats that received (E)-2,4-diene VPA. These results indicate that in vivo production of the GSH conjugates of (E)-2,4-diene VPA is most likely catalyzed by GST enzymes, with the esterified diene being essential for the conjugation reaction. In a separate experiment, 2,4-diene VPA-NACA was observed to alkylate reduced oxytocin through one or both cysteine residues. Thus, the toxicity of (E)-2,4-diene VPA might be produced via either GST-promoted depletion of cellular GSH, or a direct modification of key proteins, or both.

Animals↗
PubMed1997

Inhibition of glutathione conjugation by glutathione analogues in the perfused rat liver. Effect of esterification on the potency of gamma-L-glutamyl-alpha-(D-2-aminoadipyl)-N-2-heptylamine.

To assess the role of GST's (glutathione S-transferases) in the (de)toxification of their substrates, an in vivo active inhibitor based on the structure of glutathione (GSH), gamma-L-glutamyl-alpha-(D-2-aminoadipyl)-N-2-heptylamine monoethyl ester (Et-R-Hep), was developed. To increase its effectivity, analogues esterified with alkyl chains of varying lengths and one diesterified derivative (DiEt-R-Hep) were synthesized. The unesterified analogue, R-Hep, was also tested. Their isoenzyme selectivity was characterized using purified rat GST isoenzymes. Furthermore, the extent of inhibition of the GSH conjugation of (RS)-2-bromoisovalerylurea (BIU) was evaluated in rat liver cytosol, isolated hepatocytes, and in liver perfusions. All compounds inhibited Alpha- (1-1 and 2-2) more effectively than Mu (3-3 and 4-4) class GSTs; Pi-(5-5) and Theta (7-7) classes were minimally inhibited. The unesterified R-Hep was the most effective inhibitor towards purified isoenzymes; its Ki value towards GST 3-3 (S-BIU as substrate) was 27 microM. The mono ethyl ester derivative, Et-R-Hep (Ki 270 microM for 3-3), was the most potent inhibitor in hepatocytes and in the perfused liver: 50 microM inhibited the conjugation of (S)-BIU by 50%. Longer ester chains or diesterification did not increase the inhibitory potency; R-Hep had less inhibitory activity. In all systems, only the (S)-enantiomer of BIU, which is conjugated mainly by Alpha class GSTs, was inhibited, confirming Alpha isoenzyme selective inhibition.

Animals↗
PubMedDate not supplied

[Glutathione peroxidase and glutathione reductase activity in the rat liver after the administration of sodium selenite].

Sodium selenite 24h after its single administration to rats causes an increase in the activity of glutathione peroxidase and glutathione reductase in the liver tissue. 6 h after the selenium administration the enzymes activity does not differ from the control. Doses of 0.15, 0.3, 0.5 and 1 mg of selenium per 1 kg of the animal weight were investigated. 0.3 mg proved to be the least effective dose. An increase in the enzyme activity after administering 0.5 mg of selenium is retained for 6 days and 14 days after it does not differ from the control. The liver relative weight 24 h after administration of 0.5 mg of selenium per 1 kg of animal weight proved to be higher but three days later it did not differ from the control. After administering selenium in a dose of 1 mg/kg the liver relative weight was higher for 6 days. Actinomycin D administered in a dose of 0.5 mg/kg simultaneously with selenite prevents the rise in the enzyme activity and relative weight of the liver caused only by a single injection of selenium in the same dose.

Animals↗
PubMedDate not supplied

Glutathione and glutathione S-transferases in human squamous cell carcinomas of the larynx and GSTM1 dependent risk.

BACKGROUND: The aim of the present study was to establish the risk of squamous cell carcinoma (SCC) of the larynx associated with the congenital absence of glutathione S-transferase M1 (GSTM1), and to describe the expression of the isoenzymes GSTA1/2, GSTP1-1, and GSTM1 and glutathione (GSH) content in healthy and tumoral larynx tissue. MATERIAL AND METHODS: Blood samples from 160 SCC male patients and 158 controls were phenotyped for GSTM1 by ELISA. Using 37 paired samples (normal and tumour specimens) from cancer patients we carried out a descriptive study of enzyme activity by ELISA (GSTs) and Ellman's as say (GSH) RESULTS: GSTM1 null phenotype was more common in the SCC group than in controls (OR 1.9, CIs 1.18-3.05, p = 0.004). Total GST activity was higher in tumour samples than in matched healthy tissue (2.2-fold, p-0.00001), being largely determined by GSTP1-1 (1.9-fold increased in malignant tissue; p = 0.0003). The GSH content was also significantly higher in SCC than in normal mucosa (1.9-fold, p = 0.0007). CONCLUSIONS: We confirmed the GSTM1-dependent risk for larynx cancer among smokers. The overexpression of the GST/GSH system in tumours reported here indicates their possible role in chemoresistance to pharmacological therapy.

Carcinoma, Squamous Cell↗
PubMedDate not supplied

Impact of dietary supplement of Crassostrea gigas extract (JCOE) on glutathione levels and glutathione S-transferase activity in rat tissues.

Male Sprague Dawley rats received various amounts of extract of Crassostrea gigas by gavage every day for 2 weeks or one month. At these times, groups of animals were sacrificed and samples of major organs analyzed for levels of glutathione (GSH) and glutathione S-transferase (GST) activities. Following the two week protocol, GSH levels were significantly increased in the mucosa of the large intestine; at one month the small intestine and spleen were elevated. GST activity increased in liver under both schedules and at one month, activity was also elevated in kidney and small intestine. Since the Crassostrea gigas extract contains high levels of a variety of important amino acids, it is concluded that biologically available peptides are taken up in target organs and stimulate GSH metabolism. Enhanced levels of GSH and associated enzymes may contribute to a more effective detoxification phenotype, thus providing enhanced chemoprotective capacity.

Animals↗
PubMed2000

Characterization of dansylated glutathione, glutathione disulfide, cysteine and cystine by narrow bore liquid chromatography/electrospray ionization mass spectrometry.

A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the identity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and glutathione disulfide (GSSG). Cysteine, GSH, CSSC and GSSG are present at low concentrations in rainbow trout (Oncorhynchus mykiss) liver cells. Initially, hepatic cells were sampled from a suspension culture and disrupted upon addition of 10% perchloric acid. The reduced thiols present in the cell extracts were acetylated to prevent dimerization and then the C and GSH species were derivatized with dansyl chloride for fluorescence detection. An LC system using a weak anion exchange column (AE) with fluorescence detection (FLD) was used for sensitive routine analysis; however, it produced peaks of unknown origin in addition to the expected analytes. Analytes were then separated on a C18 RP-LC system using a water/acetonitrile gradient with 0.2% formic acid, and detected using LC/ESI-MS at 3.5 KV which produced an intense ion with a minimum limit of detection of less than 0.5 pmole injected (>10:1 signal-to-noise (S/N). Subsequently, fractions of effluent from the AE-LC/FLD system were analyzed by LC/ESI-MS to confirm the presence of the target analytes in routine cell extracts. Monodansylated GSSG was identified as a product that could possibly affect the quantification of GSH and GSSG.

Animals↗