UMLS:C1260386 - FACTA Search

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Query: UMLS:C1260386 (GSH)
38,102 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exposure of yeast glutathione reductase (GR) in vitro to S-(N-methylcarbamoyl)glutathione (SMG) and S-(N-methylcarbamoyl)cysteine (SMC), two carbamoylating metabolites of methylisocyanate (MIC), led to a time-dependent, irreversible loss of enzyme activity (50-90%) over a period of 3 hr. The extent of inhibition was dependent upon the concentration of these carbamate thioester conjugates (0.1 to 1.0 mM) and on the presence of NADPH (100 microM). Omission of NADPH markedly attenuated the inhibitory effects of both SMG and SMC, while oxidized glutathione (GSSG), the natural substrate of the enzyme, protected against the inhibition. Parallel experiments with the antineoplastic drug N,N'-bis-(2-chloroethyl)-N-nitrosourea (BCNU), a carbamoylating agent which is known to inhibit GR selectively, gave results that were similar to those obtained with the above conjugates. When analogs of SMG and SMC labeled with 14C in the carbamoyl group were incubated with GR, radioactivity became bound covalently to the enzyme. These findings, together with the results of kinetic experiments on the release of GSH from SMG and cysteine from SMC, suggested that while both conjugates inhibit GR by carbamoylation of an active-site thiol(s), SMG exhibits a greater affinity for the active site than SMC. In contrast to the studies with GR, SMG and SMC failed to inhibit either glutathione-S-transferase (GST) or glutathione peroxidase (GPO) enzymes in vitro. It is concluded, therefore, that these conjugates most likely inhibit GR by carbamoylating free thiol groups in the active site of this enzyme, which are absent (or inaccessible) at the active-site of GST and GPO.
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PMID:Selective and irreversible inhibition of glutathione reductase in vitro by carbamate thioester conjugates of methyl isocyanate. 816 49

The alkylating agent BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] can be inactivated through denitrosation reactions catalyzed by both cytosolic and microsomal enzymes. While previous studies have identified a class mu glutathione S-transferase [rat transferase 4-4 (Yb2)] as a major catalyst of the cytosolic denitrosation reaction, the enzymatic catalysts of BCNU denitrosation in microsomal membranes have not been identified. In the present study, both NADPH and glutathione (GSH) were found to support BCNU denitrosation catalyzed by isolated rat liver microsomes. Treatment of rats with the microsomal enzyme inducers phenobarbital and dexamethasone increased NADPH-dependent liver microsomal BCNU denitrosation up to fivefold without major effect on the GSH-dependent denitrosation activity. Although the NADPH-dependent activity was fully inhibited by antibody to NADPH-P450 reductase, purified NADPH-P450 reductase catalyzed BCNU denitrosation at rates that could only account for approximately 2-3% of the microsomal activity. Other experiments, including selective inhibition of NADPH-dependent microsomal BCNU denitrosation by chemical and antibody inhibitors of cytochrome P450, competitive inhibition of P450-catalyzed cyclophosphamide and ifosfamide activation by BCNU, and reconstitution of the denitrosation reaction by purified P450 enzyme 2B1 (major phenobarbital-inducible P450 form), established an important role for cytochrome P450 in BCNU denitrosation. By contrast, GSH-dependent microsomal BCNU denitrosation was unaffected by cytochrome P450 inhibitors, but was inhibited, with varying degrees of selectivity, by the microsomal glutathione S-transferase inhibitors ethacrynic acid, bromosulfophthalein, and indomethacin. These studies establish that BCNU inactivation can be catalyzed by two independent microsomal enzyme systems and suggest that therapeutically useful improvements in BCNU antitumor activity might be achieved through differential inhibition of these enzyme systems in tumor as compared to extratumoral sites.
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PMID:Denitrosation of the anti-cancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea catalyzed by microsomal glutathione S-transferase and cytochrome P450 monooxygenases. 827 24

The antitumor agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) is known to be unstable in aqueous solution, and to degrade spontaneously to reactive alkylating and carbamoylating intermediates. Whereas the alkylating component is believed to be responsible for the antitumor effects of this drug, it has been speculated that the carbamoylating species 2-chloroethyl isocyanate (CEIC) may mediate some of the serious adverse effects of BCNU therapy. In order to determine whether CEIC is released from BCNU in vivo, rats were administered an ip injection of the drug and a targeted search was made by ionspray LC-MS/MS techniques for the glutathione (GSH) conjugate of CEIC in bile and for the corresponding N-acetylcysteine (NAC) adduct in urine. Both of these S-linked conjugates were identified on the basis of their HPLC and MS/MS characteristics, which were identical to those of the respective reference compounds prepared by synthesis. Quantitative studies indicated that, following an ip dose of BCNU (24 mg kg-1), excretion of the GSH conjugate in bile over 4 h accounted for 3.90 +/- 0.64% of the administered dose, while excretion of the mercapturic acid derivative in urine over 24 h accounted for a further 18.1 +/- 3.3% (n = 4). Experiments conducted in vitro demonstrated that the S-linked conjugates of CEIC were of limited stability under simulated physiological conditions, decomposing to generate free GSH and NAC. In addition, both adducts inhibited rat liver glutathione reductase in vitro, when they were essentially equipotent to BCNU.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glutathione and N-acetylcysteine conjugates of 2-chloroethyl isocyanate. Identification as metabolites of N,N'-bis(2-chloroethyl)-N-nitrosourea in the rat and inhibitory properties toward glutathione reductase in vitro. 831 60

The effects of postmaturational aging on the toxicity of diquat, a redox cycling compound, were investigated in hepatocytes that were isolated from mature (6 months) and old (27 months) male Fischer 344 rats and pretreated with 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU), an inhibitor of glutathione reductase. The hepatocytes were incubated for 2 hr with 0, 0.5, or 2.0 mM diquat dibromide, and samples were taken at various time points for measurements of glutathione, glutathione disulfide, thiobarbituric acid reactive substances, lactate dehydrogenase leakage, protein sulfhydryl groups, and protein carbonyl groups. Diquat cytotoxicity was intensified in hepatocytes of old rats compared with those of mature rats, and the enhanced toxicity was associated with increased lipid peroxidation and protein carbonyl formation. However, the enhanced toxicity in old rat hepatocytes was also accompanied by a decrease in diquat-induced GSH oxidation and there was no difference in protein sulfhydryl loss. Concentrations of total nonheme iron and low-molecular-weight chelatable Fe2+, measured with ferene as the chromogen, were several times higher in freshly isolated hepatocytes of old rats than in those of mature rats. We hypothesize that the age-associated enhancement of diquat toxicity could be due to an increased availability of iron for reaction with diquat-generated hydrogen peroxide and for stimulation of lipid and protein oxidation.
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PMID:Diquat-induced oxidative damage in BCNU-pretreated hepatocytes of mature and old rats. 838 45

Murine leukemia L1210 cells grown for 5-7 d in the presence of 1% serum without added selenium [Se(-) cells] expressed < 5% of the glutathione peroxidase (GPX) activity of selenium-supplemented controls [Se(+) cells]. Clonogenic survival assays indicated that t-butyl hydroperoxide (t-BuOOH) is much more toxic to Se(-) cells (LC50 approximately 10 microM) than to Se(+) or selenium-repleted [Se(-/+)] cells (LC50 approximately 250 microM). Hypersensitivity of Se(-) cells to t-BuOOH was partially reversed by treating them with Ebselen, a selenoperoxidase mimetic; thus, selenoperoxidase insufficiency was probably the most serious defect of Se deprivation. Cytotoxicity of t-BuOOH was inhibited by desferrioxamine and by alpha-tocopherol, indicating that redox iron and free radical intermediates are involved. Elevated sensitivity of Se(-) cells to t-BuOOH was accompanied by an increased susceptibility to free radical lipid peroxidation, which became even more pronounced in cells that had been grown in arachidonate (20:4, n-6) supplemented media. That glutathione (GSH) is required for cytoprotection was established by showing that Se(+) cells are less resistant to t-BuOOH after exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. Coupled enzymatic assays indicated that Se(+) or Se(-/+) cells metabolize t-BuOOH 20-25 times more rapidly than Se(-), consistent with the measured difference in GPX activities of these cells. Correspondingly, when challenged with t-BuOOH, Se(+) cells showed an initial loss of GSH and elevation of GSSG that exceeded that of Se(-) cells. It was further shown that like Se(-) cells, BSO- or BCNU-treated Se(+) cells metabolize t-BuOOH more slowly than nontreated controls. These results clearly indicate that selenoperoxidase action in the glutathione cycle is a vital element in cellular defense against toxic hydroperoxides.
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PMID:Selenoperoxidase-mediated cytoprotection against the damaging effects of tert-butyl hydroperoxide on leukemia cells. 845 83

Intracellular glutathione (GSH) content was measured by flow cytometry using monochlorobimane (mBCl) and by the enzymatic assay in a set of 6 sublines of murine L1210 leukemia cells made resistant to DNA-interacting agents having distinct mechanisms of action: L-phenylalanine mustard (L-PAM), 1,3-bis(2-chloroethyl)-I-nitrosourea (BCNU), cisplatin (DDP), N-deformyl-N-(4-N,N-bis(2-chloroethylamino) benzoyl) distamycin A (FCE 24517), doxorubicin (DX) and 3'-deamino-3' (2-methoxy-4-morpholinyl)-doxorubicin (FCE 23762). A significant correlation was demonstrated between the mean intracellular mBCl fluorescence values measured by flow cytometry and levels of GSH measured by the classical enzymatic assay, despite the possible influence of glutathione-S-transferases and of other thiols on the mBCl fluorescence. Although less specific, the flow cytometric method is more informative than the enzymatic assay, allowing detection of fluorescence distributions, which we proved to be characteristic of each subline. In order to assess a procedure enabling a quantitative analysis to be made of intercellular GSH heterogeneity, we propose the use of appropriate thresholds and parameters of the mBCl flow cytometric distribution. By use of this analysis procedure, distinct types of alterations, with respect to the heterogeneity distribution of the parental L1210 cell line, have been evidenced in resistant cells. A uniform increase in mBCl fluorescence was observed among cells of the sublines resistant to L-PAM and FCE-24517. The mean mBCl fluorescence increase in sublines resistant to DX and DDP was due to a higher number of cells with fairly high mBCl fluorescence, but still within the range spanned by the parental cell line. A less heterogeneous mBCl fluorescence distribution was found in the L1210 subline resistant to FCE 23762, which was, however, similar to a cloned sensitive line. Though GSH was linked to the principal cause of drug resistance only in the L-PAM-resistant cell line, alterations in heterogeneity, as detected by mBCl fluorescence distributions, were found in 5 out of 6 resistant lines.
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PMID:Intracellular glutathione heterogeneity in L1210 murine leukemia sublines made resistant to DNA-interacting anti-neoplastic agents. 850 18

The effect of acetaminophen (APAP) exposure on the formation of oxidized glutathione (GSSG) was investigated in cultured mouse hepatocytes to determine if oxidative damage is involved in the toxicity of this drug. Incubations of hepatocytes for 24 hr with 1 mM APAP produced a time-dependent loss of cell viability which was preceded by depletion of reduced glutathione (GSH) and an increase in GSSG formation. Pretreatment with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) (0.1 mM) for 30 min, which irreversibly inhibited glutathione reductase (GSSG-Rd) activity, increased the extent of GSSG formation produced by APAP exposure and potentiated its cell killing. Pretreatment of hepatocytes with 20 mM deferoxamine (DFO) for 1 hr to chelate ferric iron decreased GSSG formation and cell killing produced by APAP. Pretreatment with BCNU or DFO did not affect APAP oxidation as determined by the formation of the APAP-GSH conjugate or the covalent binding of APAP metabolites to cellular protein. Hence, increasing the susceptibility of hepatocytes to an oxidative stress with BCNU increased both the formation of GSSG and cell killing produced by APAP. Conversely, decreasing their susceptibility to an oxidative stress by chelating iron with DFO decreased GSSG formation and cell injury. It follows that APAP toxicity involves oxidative processes that occur early in the poisoning process and are a major factor contributing to injury in these cells.
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PMID:Oxidative stress in cultured hepatocytes exposed to acetaminophen. 851 69

O6-Methyl-2'-deoxyguanosine (O6-MedG), a novel inhibitor of O6-alkylguanine-DNA alkyltransferase (O6-AGT), has been synthesized. The ability of O6-MedG to deplete the O6-AGT activity in leukemia L1210 and melanoma B16 cells in vivo has been studied. After intraperitoneal administration of O6-MedG to mice bearing leukemia L1210 or melanoma B16, the activity of O6-AGT in tumour cells decreased by 50%. Pretreatment of leukemia L1210 bearing mice with O6-MedG (200 mg/kg) 24 hours prior to ACNU (15 mg/kg) administration resulted in six out of seven 60-day survivors. Treatment of mice with ACNU (15 mg/kg) alone increased the life span by 200%. Treatment of melanoma B16 bearing mice with O6-MedG and 3 hours thereafter with ACNU resulted in a 50% inhibition of tumour growth, whereas the inhibiting effect of ACNU alone was 16%. There was no difference in leukemia growth when L1210/BCNU bearing mice were treated with O6-MedG followed by ACNU treatment. In vivo ACNU (15 mg/kg) produced a deep and prolonged inhibition of DNA, RNA and protein synthesis in leukemia L1210 cells. The DNA synthesis in leukemia L1210/BCNU cells was shown to recover more rapidly than in L1210 cells. The activities of DNA-polymerases alpha and beta and, especially, of O6-AGT were elevated in ACNU-resistant leukemia cells as compared with ACNU-sensitive cells. The activation of some repairing enzymes, such as O6-AGT, DNA-polymerases alpha and beta as well as increased levels of GSH may play a role in the development of drug resistance to ACNU.
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PMID:[Modulation of the antitumor activity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoure a by O(6)-methyl-2'-deoxyguanosine--a new inhibitor of O(6)-alkylguanine-DNA-alkyltransferase]. 856 57

The nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is an alkylating agent that causes oxidative stress and subsequently death of renal proximal tubular cells (PTC). In this paper the role of inhibition of the glutathione redox cycle (GSH-reductase (GRd) and -peroxidase (GPx) in the development of DCVC-induced oxidative cell injury is described. DCVC inhibited both GRd and GPx activity in PTC. Inhibition occurred already after 10 min incubation while at that time point lipid peroxidation and cell death had not started yet; the antioxidant N,N-diphenyl-p-phenylenediamine did not prevent inhibition of GRd and Gpx- inhibition of L-cysteine S-conjugate beta-lyase-mediated formation of reactive metabolites using aminooxyacetic acid, which prevented covalent binding to cellular macromolecules, was associated with prevention of the DCVC-induced inhibition of both enzymes. Covalent binding of reactive metabolites of [35S]DCVC to several cellular proteins was found, including proteins which had molecular weights identical to subunits of GPx and GRd. An inhibitor of GRd, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), potentiated the oxidative cell injury caused by DCVC, whereas BCNU itself did not use oxidative stress and cell death. The thiol-reducing compound dithiothreitol prevented the oxidative cell injury whereas oxidation of cellular thiols with diamide potentiated the DCVC-induced oxidative stress and cell death. Moreover, incubation with (R,S)-3-hydroxy-4-pentenoic acid (HPA), which depletes mitochondrial GSH, potentiated the DCVC-induced oxidative cell injury. Neither diamide nor HPA affected the covalent binding and inhibition of the GSH-redox cycle. Together, the data suggest that the inhibition of GRd and GPx, presumably caused by binding of reactive metabolites of DCVC, impairs the cellular antioxidant system, which seems causally related to DCVC-induced oxidative cell injury.
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PMID:Alkylation-induced oxidative cell injury of renal proximal tubular cells: involvement of glutathione redox-cycle inhibition. 861 98

Glutathione (GSH) depletion by buthioninine sulfoximine (BSO) is being explored clinically as a means of enhancing the efficacy of cancer chemotherapy. We investigated the kinetics of GSH depletion and altered gamma-L-glutamyl-L-cysteine synthetase (gamma-GC-S) gene expression in two human malignant glioma cell lines, HBT5 and HBT28, and examined how these relate to GSH resynthesis and changes in DNA interstrand cross-link induction and cytotoxicity of 1,3-bis(2-chloroethyl)-nitrosourea (BCNU). GSH content was 54 and 126 nmol/mg/protein in HBT 5 and HBT 28, respectively, and after a 24-hr exposure to 100 microM BSO was decreased by 95% in HBT 5 and 91% in HBT 28. Basal gamma-GC-S enzyme activity in HBT 28 was twice that in HBT 5, and steady state gamma-GC-S gene transcripts were 2.6-fold higher in HBT 28 than in HBT 5, with no apparent amplification or rearrangement of the gene in either cell line. BSO exposure (100 microM) for 24 hr increased gamma-GC-S gene transcripts by 1.7-fold in HBT 5 and 2.8-fold in HBT 28. After BSO removal, the rate of GSH resynthesis in HBT 28 was twice that in HBT 5. Continuous BSO exposure increased the level of BCNU-induced DNA interstrand cross-links, and cytotoxicity was significantly higher in cells exposed continuously to BSO than in cells with only a 24-hr BSO preexposure. This increase was, however, greater in HBT 28 than in HBT 5. These findings indicate significant heterogeneity in the effects of BSO on gamma-GC-S gene expression and in the ability of BSO to sensitize tumors and cell lines to BCNU. The data also suggest that by preventing GSH resynthesis, a greater level of cytotoxicity is achieved with continuous BSO exposure than with BSO preexposure alone.
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PMID:Buthionine sulfoximine induction of gamma-L-glutamyl-L-cysteine synthetase gene expression, kinetics of glutathione depletion and resynthesis, and modulation of carmustine-induced DNA-DNA cross-linking and cytotoxicity in human glioma cells. 864 39

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