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Sample records for glutathione s-conjugate hydrolysis1woa

  1. Uncouplers of mitochondrial oxidative phosphorylation are not substrates of the erythrocyte glutathione-S-conjugate pump.

    PubMed

    Sokal, A; Bartosz, G

    1998-01-01

    Uncouplers of mitochondrial oxidative phosphorylation, dinitrophenol (DNP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), were found to stimulate Mg(2+)-ATPase activity of human erythrocyte membranes in a manner competitive with respect to 2,4-dinitrophenyl-S-glutathione (DNP-SG) which suggested that these compounds may also be substrates of the glutathione-S-conjugate pump. We confirm that the stimulation of erythrocyte membrane ATPase activity by DNP and by another uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), is competitive with respect to DNP-SG. However, we found no evidence for active transport of DNP and CCCP out of erythrocytes and demonstrate that they inhibit the low-affinity component of DNP-SG transport noncompetitively while stimulating the high-affinity DNP-SG transport (mediated by multidrug resistance-associated protein, MRP1). Implications of these findings may indicate the electrogenic nature of MRP1-mediated transport of glutathione-S conjugates and stimulation of aminophospholipid translocase (flippase) rather than the glutathione-S-conjugate pump by the uncouplers. PMID:9439589

  2. Active transport of glutathione S-conjugate in human colon adenocarcinoma cells.

    PubMed

    Zhang, K; Wong, K P

    1996-11-12

    The formation of the glutathione S-conjugate of monochlorobimane (GSH-bimane) in human colon adenocarcinoma cells was identified by HPLC-fluorimetry and its transport from the cells was found to be temperature-sensitive, saturable and ATP-dependent. The apparent K(m) and Vmax values were 2.4 +/- 0.5 nmol GSH-bimane/10(6) cells and 0.5 +/- 0.1 nmol GSH-bimane/min per 10(6) cells, respectively. This active transport of GSH-bimane was inhibited by low micromolar concentrations of classical uncouplers of oxidative phosphorylation, namely carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), carbonylcyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP). The efflux of GSH-bimane was competitively inhibited by chlorambucil (CMB) and 1-chloro-2,4-dinitrobenzene (CDNB), two other substrates of GST. This study demonstrates the presence and kinetic measurements of the glutathione S-conjugate export (GS-X) pump in human colon cancer cells, an export pump whose function has been implicated in the phenomenon of multidrug resistance. PMID:8950221

  3. Purified gamma-glutamyl transpeptidases from tomato exhibit high affinity for glutathione and glutathione S-conjugates.

    PubMed

    Martin, M N; Slovin, J P

    2000-04-01

    gamma-Glutamyl transpeptidases (gammaGTases) are the only enzymes known to hydrolyze the unique N-terminal amide bonds of reduced glutathione (gamma-L-glutamyl-cysteinyl-glycine), oxidized glutathione, and glutathione S-conjugates. Two gammaGTases (I and II) with K(m) values for glutathione of 110 and 90 microM were purified 2,977-fold and 2,152-fold, respectively, from ripe tomato (Lycopersicon esculentum) pericarp. Both enzymes also hydrolyze dipeptides and other tripeptides with N-terminal, gamma-linked Glu and the artificial substrates gamma-L-glutamyl-p-nitroanilide and gamma-L-glutamyl(7-amido-4-methylcoumarin). They transfer the glutamyl moiety to water or acceptor amino acids, including L-Met, L-Phe, L-Trp, L-Ala, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. gammaGTase I and II were released from a wall and membrane fraction of a tomato fruit extract with 1.0 M NaCl, suggesting that they are peripheral membrane proteins. They were further purified by acetone precipitation, Dye Matrex Green A affinity chromatography, and hydrophobic interaction chromatography. The two gammaGTases were resolved by concanavalin A (Con A) affinity chromatography, indicating that they are differentially glycosylated. The native and SDS-denatured forms of both enzymes showed molecular masses of 43 kD. PMID:10759537

  4. 1-Chloro-2,4-Dinitrobenzene-Elicited Increase in Vacuolar Glutathione-S-Conjugate Transport Activity.

    PubMed Central

    Li, Z. S.; Zhen, R. G.; Rea, P. A.

    1995-01-01

    Unlike most other characterized organic solute transport in plants, uptake of the model compound S-(2,4-dinitrophenyl)glutathione (DNP-GS) and related glutathione-S-conjugated by vacuolar membranes is directly energized by MgATP. Here we show that exogenous application of the DNP-GS precursor 1-chloro-2,4-dinitrobenzene (CDNB) to seedlings of Vigna radiata (mung bean) increases the capacity of vacuolar membrane vesicles isolated from hypocotyls for MgATP-dependent DNP-GS transport in vitro. Our findings are 4-fold: (a) Pretreatment of seedlings with CDNB causes a progressive increase in MgATP-dependent DNP-GS uptake by vacuolar membrane vesicles, whereas the same range of CDNB concentrations causes only marginal stimulation when the compound benoxacor [4-(dichloroacetyl)-3,4-dihydro-3-methyl-2H-1,4-benzoxazine] is included in the pretreatment solution. (b) Increased DNP-GS uptake is accompanied by a proportionate and selective increase in Vmax(DNP-GS) but not in Km(DNP-GS) or Km(MgATP). (c) CDNB-enhanced DNP-GS uptake is not accompanied by a change in the density profile or sidedness of the vacuolar membrane fraction. (d) Basal and CDNB-enhanced DNP-GS uptake are indistinguishable in terms of their inhibitor profiles. On the basis of these findings, it is inferred that pretreatment with CDNB increases the amount or recruitment of functional transporter into the vacuolar membrane and that agents such as benoxacor antagonize the effects otherwise seen with CDNB in this sytem. PMID:12228588

  5. AtMRP2, an Arabidopsis ATP binding cassette transporter able to transport glutathione S-conjugates and chlorophyll catabolites: functional comparisons with Atmrp1.

    PubMed Central

    Lu, Y P; Li, Z S; Drozdowicz, Y M; Hortensteiner, S; Martinoia, E; Rea, P A

    1998-01-01

    Three ATP binding cassette (ABC) transporter-like activities directed toward large amphipathic organic anions have recently been identified on the vacuolar membrane of plant cells. These are the Mg-ATP-energized, vanadate-inhibitable vacuolar accumulation of glutathione S-conjugates (GS conjugates), chlorophyll catabolites, and bile acids, respectively. Although each of these activities previously had been assigned to distinct pumps in native plant membranes, we describe here the molecular cloning, physical mapping, and heterologous expression of a gene, AtMRP2, from Arabidopsis thaliana that encodes a multispecific ABC transporter competent in the transport of both GS conjugates and chlorophyll catabolites. Unlike its isoform, AtMRP1, which transports the model Brassica napus chlorophyll catabolite transporter substrate Bn-NCC-1 at low efficiency, heterologously expressed AtMRP2 has the facility for simultaneous high-efficiency parallel transport of GS conjugates and Bn-NCC-1. The properties of AtMRP2 therefore establish a basis for the manipulation of two previously identified plant ABC transporter activities and provide an explanation for how the comparable transporter in native plant membranes would be systematically mistaken for two distinct transporters. These findings are discussed with respect to the functional organization of AtMRP2, the inability of AtMRP2 and AtMRP1 to transport the model bile acid transporter substrate taurocholate (despite the pronounced sensitivity of both to direct inhibition by this agent), the differential patterns of expression of their genes in the intact plant, and the high capacity of AtMRP2 for the transport of glutathionated herbicides and anthocyanins. PMID:9490749

  6. Anti-cancer-prostaglandin-induced cell-cycle arrest and its modulation by an inhibitor of the ATP-dependent glutathione S-conjugate export pump (GS-X pump).

    PubMed Central

    Ishikawa, T; Akimaru, K; Nakanishi, M; Tomokiyo, K; Furuta, K; Suzuki, M; Noyori, R

    1998-01-01

    The A and J series of prostaglandins (PGs) accumulate in the nuclei to suppress the proliferation of cancer cells. Here we report that Delta7-PGA1 methyl ester, a synthetic anti-cancer PG, increased the level of mRNA for the cyclin-dependent kinase inhibitor p21 in human leukaemia HL-60 cells. The induction of p21 was associated with the accumulation of hypophosphorylated retinoblastoma protein (pRB) and the suppression of c-myc gene expression. Since the p53 gene is deleted in HL-60 cells, the anti-cancer PG is suggested to inhibit cancer cell growth by inducing p21 via a p53-independent pathway. Unlike HL-60 cells, cisplatin-resistant HL-60/R-CP cells were insensitive to Delta7-PGA1 methyl ester. While c-myc expression was transiently suppressed, neither G1 arrest nor hypophosphorylation of pRB was observed with the anti-cancer PG. Plasma membrane vesicles from HL-60/R-CP cells showed an enhanced level of GS-X pump (ATP-dependent glutathione S-conjugate export pump) activity towards the glutathione S-conjugate of Delta7-PGA1 methyl ester (Km 110 nM). GIF-0019 ¿N-carbomethoxy-S-[5-(4-benzoylphenyl)pentyl]glutathione dimethyl ester¿, a specific inhibitor of the GS-X pump, dose-dependently enhanced the cellular sensitivity of HL-60/R-CP cells to Delta7-PGA1 methyl ester and induced G1 arrest. The GS-X pump is suggested to play a pivotal role in modulating the biological action of the anti-cancer PG. PMID:9841867

  7. Glutathione-S-conjugate transport in plants

    DOEpatents

    Rea, Philip A.; Lu, Yu-Ping; Li, Ze-Sheng

    2000-01-01

    The invention includes an isolated DNA encoding a plant GS-X pump polypeptide and an isolated preparation of a plant GS-X pump polypeptide. Also included is an isolated preparation of a nucleic acid which is antisense in orientation to a portion or all of a plant GS-X pump gene. The invention also includes a cells, vectors and transgenic plants having an isolated DNA encoding a plant GS-X pump and methods of use thereof. In addition, the invention relates to plant GS-X pump promoter sequences and the uses thereof.

  8. Glutathione

    PubMed Central

    Noctor, Graham; Queval, Guillaume; Mhamdi, Amna; Chaouch, Sejir; Foyer, Christine H.

    2011-01-01

    Glutathione is a simple sulfur compound composed of three amino acids and the major non-protein thiol in many organisms, including plants. The functions of glutathione are manifold but notably include redox-homeostatic buffering. Glutathione status is modulated by oxidants as well as by nutritional and other factors, and can influence protein structure and activity through changes in thiol-disulfide balance. For these reasons, glutathione is a transducer that integrates environmental information into the cellular network. While the mechanistic details of this function remain to be fully elucidated, accumulating evidence points to important roles for glutathione and glutathione-dependent proteins in phytohormone signaling and in defense against biotic stress. Work in Arabidopsis is beginning to identify the processes that govern glutathione status and that link it to signaling pathways. As well as providing an overview of the components that regulate glutathione homeostasis (synthesis, degradation, transport, and redox turnover), the present discussion considers the roles of this metabolite in physiological processes such as light signaling, cell death, and defense against microbial pathogen and herbivores. PMID:22303267

  9. The photochemical thiol–ene reaction as a versatile method for the synthesis of glutathione S-conjugates targeting the bacterial potassium efflux system Kef† †Electronic supplementary information (ESI) available: Further experimental details and NMR spectra. See DOI: 10.1039/c5qo00436e Click here for additional data file.

    PubMed Central

    Rasmussen, Tim; Miller, Samantha; Booth, Ian R.

    2016-01-01

    The thiol–ene coupling reaction is emerging as an important conjugation reaction that is suitable for use in a biological setting. Here, we explore the utility of this reaction for the synthesis of glutathione-S-conjugates (GSX) and present a general, operationally simple, protocol with a wide substrate scope. The GSX afforded are an important class of compounds and provide invaluable molecular tools to study glutathione-binding proteins. In this study we apply the diverse library of GSX synthesised to further our understanding of the structural requirements for binding to the glutathione-binding protein, Kef, a bacterial K+ efflux system, found in many bacterial pathogens. This system is vital to the survival of bacteria upon exposure to electrophiles, and plays an essential role in the maintenance of intracellular pH and K+ homeostasis. Consequently, Kef is an appealing target for the development of novel antibacterial drugs. PMID:27110363

  10. IN VITRO INHIBITION OF GLUTATHIONE REDUCTASE BY ARSENOTRI-GLUTATHIONE

    EPA Science Inventory

    Arsenotriglutathione, a product of the reduction of arsenate and the complexation of arsenite by glutathione, is a mixed type inhibitor of the reduction of glutathione disulfide by purified yeast glutathione reductase or the glutathione reductase activity in rabbit erythrocyte ly...

  11. Glutathione permeability of CFTR.

    PubMed

    Linsdell, P; Hanrahan, J W

    1998-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) forms an ion channel that is permeable both to Cl- and to larger organic anions. Here we show, using macroscopic current recording from excised membrane patches, that the anionic antioxidant tripeptide glutathione is permeant in the CFTR channel. This permeability may account for the high concentrations of glutathione that have been measured in the surface fluid that coats airway epithelial cells. Furthermore, loss of this pathway for glutathione transport may contribute to the reduced levels of glutathione observed in airway surface fluid of cystic fibrosis patients, which has been suggested to contribute to the oxidative stress observed in the lung in cystic fibrosis. We suggest that release of glutathione into airway surface fluid may be a novel function of CFTR. PMID:9688865

  12. Glutathione in cyanobacteria

    NASA Technical Reports Server (NTRS)

    Bermudes, D.

    1985-01-01

    The effects of light and O2 on glutathione production were determined. Results of light and dark studies under normal and reduced oxygen tensions were compared to determine the effect of reduction in oxygen tension on glutathione levels. The growth rate of Anacystis nidulans and concurrent production of glutathione is presented. The generation of time of Anacystis nidulans was approximately 12 hours. Results of light and dark incubation of Aphanothece halophytica dominated planktonic microbial community from Pond 4 and Anacystis nidulans under high and low oxygen tension is also presented. It appears that light grown Anacystis nidulans cells have equal amounts of glutathione while dark grown cells produce more glutathione in the presence of increased O2.

  13. Mutagenicity of the Cysteine S-Conjugate Sulfoxides of Trichloroethylene and Tetrachloroethylene in the Ames Test

    PubMed Central

    Irving, Roy M.; Elfarra, Adnan A.

    2013-01-01

    The nephrotoxicity and nephrocarcinogenicity of trichloroethylene (TCE) and tetrachloroethylene (PCE) are believed to be mediated primarily through the cysteine S-conjugate β-lyase-dependent bioactivation of the corresponding cysteine S-conjugate metabolites S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), respectively. DCVC and TCVC have previously been demonstrated to be mutagenic by the Ames Salmonella mutagenicity assay, and reduction in mutagenicity was observed upon treatment with the β-lyase inhibitor aminooxyacetic acid (AOAA). Because DCVC and TCVC can also be bioactivated through sulfoxidation to yield the potent nephrotoxicants S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS) and S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide (TCVCS), respectively, the mutagenic potential of these two sulfoxides was investigated using the Ames S. typhimuriumTA100 mutagenicity assay. The results show both DCVCS and TCVCS were mutagenic, and TCVCS exhibited 3-fold higher mutagenicity than DCVCS. However, DCVCS and TCVCS mutagenic activity was approximately 700-fold and 30-fold lower than DCVC and TCVC, respectively. DCVC and DCVCS appeared to induce toxicity in TA100, as evidenced by increased microcolony formation and decreased mutant frequency above threshold concentrations. TCVC and TCVCS were not toxic in TA100. The toxic effects of DCVC limited the sensitivity of TA100 to DCVC mutagenic effects and rendered it difficult to investigate the effects of AOAA on DCVC mutagenic activity. Collectively, these results suggest that DCVCS and TCVCS exerted a definite but weak mutagenicity in the TA100 strain. Therefore, despite their potent nephrotoxicity, DCVCS and TCVCS are not likely to play a major role in DCVC or TCVC mutagenicity in this strain. PMID:23416178

  14. New cysteine-S-conjugate precursors of volatile sulfur compounds in bell peppers (Capsicum annuum L. cultivar).

    PubMed

    Starkenmann, Christian; Niclass, Yvan

    2011-04-13

    The objective of this study was to verify whether the volatile organic sulfur compounds recently discovered in bell pepper (Capsicum annuum, L. cultivars), such as the mercapto-ketones: 4-sulfanyl-2-heptanone and 2-sulfanyl-4-heptanone, the mercapto-alcohols: 4-sulfanyl-2-heptanol and 2-sulfanyl-4-heptanol, and heptane-2,4-dithiol, originate from their corresponding cysteine-S-conjugates. Analysis of aqueous extracts of red and green bell pepper by ultraperformance liquid chromatography-mass spectrometry with electrospray ionization in the positive mode (UPLC-MS ESI(+)) displayed masses corresponding to the expected cysteine-S-conjugates. To confirm this observation, four cysteine-S-conjugates were prepared as authentic samples: S-(3-hydroxy-1-methylhexyl)-L-cysteine, S-(3-hydroxy-1-propylbutyl)-L-cysteine, S-(3-oxo-1-propylbutyl)-L-cysteine, and (2R,2'R)-3,3'-(4-hydroxyheptane-2,6-diyl)bis(sulfanediyl) bis(2-aminopropanoic acid). By comparison with the fragmentation patterns and retention times of synthetic mixtures of cysteine-S-conjugate diastereoisomers, the natural occurrence of cysteine conjugates was confirmed in bell peppers. In addition, the cysteine-S-conjugates from red and green bell pepper extracts were concentrated by ion exchange chromatography and the fractions incubated with a β-lyase (apotryptophanase). The liberated thiols were concentrated by affinity chromatography, and their occurrence, detected by gas chromatography-mass spectrometry, confirmed our predictions. Moreover, 3-sulfanyl-1-hexanol was also detected and the occurrence of S-(1(2-hydroxyethyl)butyl)-L-cysteine confirmed. A quantitative estimation based on external calibration curves, established by UPLC-MS ESI(+) in selected reaction monitoring mode, showed that cysteine-S-conjugates were present at concentrations in the range of 1 to 100 μg/kg (±20%). PMID:21375341

  15. Unravelling glutathione conjugate catabolism in Saccharomyces cerevisiae: the role of glutathione/dipeptide transporters and vacuolar function in the release of volatile sulfur compounds 3-mercaptohexan-1-ol and 4-mercapto-4-methylpentan-2-one.

    PubMed

    Cordente, Antonio G; Capone, Dimitra L; Curtin, Chris D

    2015-11-01

    Sulfur-containing aroma compounds are key contributors to the flavour of a diverse range of foods and beverages, such as wine. The tropical fruit characters of Sauvignon Blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3-MH), its acetate ester 3-mercaptohexyl acetate (3-MHA), and 4-mercapto-4-methylpentan-2-one (4-MMP). These aromatic thiols are not detectable in grape juice to any significant extent but are released by yeast during alcoholic fermentation. While the processes involved in the release of 3-MH and 4-MMP from their cysteinylated precursors have been studied extensively, degradation pathways for glutathione S-conjugates (GSH-3-MH and GSH-4-MMP) have not. In this study, a candidate gene approach was taken, focusing on genes known to play a role in glutathione and glutathione-S-conjugate turnover in Saccharomyces cerevisiae. Our results confirm the role of Opt1p as the major transporter responsible for uptake of GSH-3-MH and GSH-4-MMP, and identify vacuolar Ecm38p as a key determinant of 3-MH release from GSH-3-MH. ECM38 was unimportant, on the other hand, for release of 4-MMP, and abolition of vacuolar biogenesis caused an increase in the amount of 4-MMP released. The alternative cytosolic glutathione degradation pathway was not involved in release of either thiol from their glutathionylated precursors. Finally, cycling of GSH-3-MH and/or its breakdown intermediates between the cytosol and the vacuole or extracellular space was implicated in modulation of 3-MH formation. Together, these results provide new targets for development of yeast strains that optimize release of these potent volatile sulfur compounds, and further our understanding of the processes involved in glutathione-S-conjugate turnover. PMID:26227410

  16. Formation of cysteine-S-conjugates in the Maillard reaction of cysteine and xylose.

    PubMed

    Cerny, Christoph; Guntz-Dubini, Renée

    2013-11-15

    Cysteine-S-conjugates (CS-conjugates) occur in foods derived from plant sources like grape, passion fruit, onion, garlic, bell pepper and hops. During eating CS-conjugates are degraded into aroma-active thiols by β-lyases that originate from oral microflora. The present study provides evidence for the formation of the CS-conjugates S-furfuryl-l-cysteine (FFT-S-Cys) and S-(2-methyl-3-furyl)-l-cysteine (MFT-S-Cys) in the Maillard reaction of xylose with cysteine at 100°C for 2h. The CS-conjugates were isolated using cationic exchange and reversed-phase chromatography and identified by (1)H NMR, (13)C NMR and LC-MS(2). Spectra and LC retention times matched those of authentic standards. To the best of our knowledge, this is the first time that CS-conjugates are described as Maillard reaction products. Furfuryl alcohol (FFA) is proposed as an intermediate which undergoes a nucleophilic substitution with cysteine. Both FFT-S-Cys and MFT-S-Cys are odourless but produce strong aroma when tasted in aqueous solutions, supposedly induced by β -lyases from the oral microflora. The perceived aromas resemble those of the corresponding aroma-active thiols 2-furfurylthiol (FFT) and 2-methyl-3-furanthiol (MFT) which smell coffee-like and meaty, respectively. PMID:23790889

  17. Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate.

    PubMed

    Homolya, László; Váradi, András; Sarkadi, Balázs

    2003-01-01

    Many endogenous or xenobiotic lipophilic substances are eliminated from the cells by the sequence of oxidation, conjugation to an anionic group (glutathione, glucuronate or sulfate) and transport across the plasma membrane into the extracellular space. The latter step is mediated by integral membrane glycoproteins belonging to the superfamily of ATP-Binding Cassette (ABC) transporters. A subfamily, referred as ABCC, includes the famous/infamous cystic fibrosis transmembrane regulator (CFTR), the sulfonylurea receptors (SUR 1 and 2), and the multidrug resistance-associated proteins (MRPs). The name of the MRPs refers to their potential role in clinical multidrug resistance, a phenomenon that hinders the effective chemotherapy of tumors. The MRPs that have been functionally characterized so far share the property of ATP-dependent export pumps for conjugates with glutathione (GSH), glucuronate or sulfate. MRP1 and MRP2 are also mediating the cotransport of unconjugated amphiphilic compounds together with free GSH. MRP3 preferentially transports glucuronides but not glutathione S-conjugates or free GSH. MRP1 and MRP2 also contribute to the control of the intracellular glutathione disulfide (GSSG) level. Although these proteins are low affinity GSSG transporters, they can play essential role in response to oxidative stress when the activity of GSSG reductase becomes rate limiting. The human MRP4, MRP5 and MRP6 have only partially been characterized. However, it has been revealed that MRP4 can function as an efflux pump for cyclic nucleotides and nucleoside analogues, used as anti-HIV drugs. MRP5 also transports GSH conjugates, nucleoside analogues, and possibly heavy metal complexes. Transport of glutathione S-conjugates mediated by MRP6, the mutation of which causes pseudoxantoma elasticum, has recently been shown. In summary, numerous members of the multidrug resistance-associated protein family serve as export pumps that prevent the accumulation of anionic conjugates

  18. Cysteine S-conjugate β-lyases: Important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents

    PubMed Central

    Cooper, Arthur J. L.; Krasnikov, Boris F.; Niatsetskaya, Zoya V.; Pinto, John T.; Callery, Patrick S.; Villar, Maria T.; Artigues, Antonio; Bruschi, Sam A.

    2010-01-01

    Summary Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously [Cooper and Pinto, 2006]. Here we focus on more recent findings regarding: 1) the identification of enzymes associated with high-Mr cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; 2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); 3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; 4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and 5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated. PMID:20306345

  19. A Mitochondria-Targeted Nitroxide/Hemigramicidin S Conjugate Protects Mouse Embryonic Cells Against Gamma Irradiation

    SciTech Connect

    Jiang Jianfei; Belikova, Natalia A.; Hoye, Adam T.; Zhao Qing; Epperly, Michael W.; Greenberger, Joel S.; Wipf, Peter; Kagan, Valerian E.

    2008-03-01

    Purpose: To evaluate the in vitro radioprotective effect of the mitochondria-targeted hemigramicidin S-conjugated 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (hemi-GS-TEMPO) 5-125 in {gamma}-irradiated mouse embryonic cells and adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells BEAS-2B and explore the mechanisms involved in its radioprotective effect. Methods and Materials: Cells were incubated with 5-125 before (10 minutes) or after (1 hour) {gamma}-irradiation. Superoxide generation was determined by using dihydroethidium assay, and lipid oxidation was quantitated by using a fluorescence high-performance liquid chromatography-based Amplex Red assay. Apoptosis was characterized by evaluating the accumulation of cytochrome c in the cytosol and externalization of phosphatidylserine on the cell surface. Cell survival was measured by means of a clonogenic assay. Results: Treatment (before and after irradiation) of cells with 5-125 at low concentrations (5, 10, and 20 {mu}M) effectively suppressed {gamma}-irradiation-induced superoxide generation, cardiolipin oxidation, and delayed irradiation-induced apoptosis, evaluated by using cytochrome c release and phosphatidylserine externalization. Importantly, treatment with 5-125 increased the clonogenic survival rate of {gamma}-irradiated cells. In addition, 5-125 enhanced and prolonged {gamma}-irradiation-induced G{sub 2}/M phase arrest. Conclusions: Radioprotection/mitigation by hemi-GS-TEMPO likely is caused by its ability to act as an electron scavenger and prevent superoxide generation, attenuate cardiolipin oxidation in mitochondria, and hence prevent the release of proapoptotic factors from mitochondria. Other mechanisms, including cell-cycle arrest at the G{sub 2}/M phase, may contribute to the protection.

  20. Plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology

    PubMed Central

    Ballatori, Nazzareno; Krance, Suzanne M.; Marchan, Rosemarie; Hammond, Christine L.

    2009-01-01

    Reduced glutathione (GSH) is critical for many cellular processes, and both its intracellular and extracellular concentrations are tightly regulated. Intracellular GSH levels are regulated by two main mechanisms: by adjusting the rates of synthesis and of export from cells. Some of the proteins responsible for GSH export from mammalian cells have recently been identified, and there is increasing evidence that these GSH exporters are multispecific and multifunctional, regulating a number of key biological processes. In particular, the multidrug resistance-associated proteins (Mrp/Abcc) appear to mediate GSH export and homeostasis. The Mrp proteins mediate not only GSH efflux, but they also export oxidized glutathione derivatives (e.g., glutathione disulfide (GSSG), S-nitrosoglutathione (GS-NO), and glutathione-metal complexes), as well as other glutathione S-conjugates. The ability to export both GSH and oxidized derivatives of GSH, endows these transporters with the capacity to directly regulate the cellular thiol-redox status, and therefore the ability to influence many key signaling and biochemical pathways. Among the many processes that are influenced by the GSH transporters are apoptosis, cell proliferation, and cell differentiation. This report summarizes the evidence that Mrps contribute to the regulation of cellular GSH levels and the thiol redox state, and thus to the many biochemical processes that are influenced by this tripeptide. PMID:18786560

  1. Glutathione and mitochondria

    PubMed Central

    Ribas, Vicent; García-Ruiz, Carmen; Fernández-Checa, José C.

    2014-01-01

    Glutathione (GSH) is the main non-protein thiol in cells whose functions are dependent on the redox-active thiol of its cysteine moiety that serves as a cofactor for a number of antioxidant and detoxifying enzymes. While synthesized exclusively in the cytosol from its constituent amino acids, GSH is distributed in different compartments, including mitochondria where its concentration in the matrix equals that of the cytosol. This feature and its negative charge at physiological pH imply the existence of specific carriers to import GSH from the cytosol to the mitochondrial matrix, where it plays a key role in defense against respiration-induced reactive oxygen species and in the detoxification of lipid hydroperoxides and electrophiles. Moreover, as mitochondria play a central strategic role in the activation and mode of cell death, mitochondrial GSH has been shown to critically regulate the level of sensitization to secondary hits that induce mitochondrial membrane permeabilization and release of proteins confined in the intermembrane space that once in the cytosol engage the molecular machinery of cell death. In this review, we summarize recent data on the regulation of mitochondrial GSH and its role in cell death and prevalent human diseases, such as cancer, fatty liver disease, and Alzheimer’s disease. PMID:25024695

  2. Characterization of thyroidal glutathione reductase

    SciTech Connect

    Raasch, R.J.

    1989-01-01

    Glutathione levels were determined in bovine and rat thyroid tissue by enzymatic conjugation with 1-chloro-2,4-dinitrobenzene using glutathione S-transferase. Bovine thyroid tissue contained 1.31 {+-} 0.04 mM reduced glutathione (GSH) and 0.14 {+-} 0.02 mM oxidized glutathione (GSSG). In the rat, the concentration of GSH was 2.50 {+-} 0.05 mM while GSSG was 0.21 {+-} 0.03 mM. Glutathione reductase (GR) was purified from bovine thyroid to electrophoretic homogeneity by ion exchange, affinity and molecular exclusion chromatography. A molecular weight range of 102-109 kDa and subunit size of 55 kDa were determined for GR. Thyroidal GR was shown to be a favoprotein with one FAD per subunit. The Michaelis constants of bovine thyroidal GR were determined to be 21.8 {mu}M for NADPH and 58.8 {mu}M for GSSG. The effect of thyroid stimulating hormone (TSH) and thyroxine (T{sub 4}) on in vivo levels of GR and glucose 6-phosphate dehydrogenase were determined in rat thyroid homogenates. Both enzymes were stimulated by TSH treatment and markedly reduced following T{sub 4} treatment. Lysosomal hydrolysis of ({sup 125}I)-labeled and unlabeled thyroglobulin was examined using size exclusion HPLC.

  3. Cross-functionalities of Bacillus deacetylases involved in bacillithiol biosynthesis and bacillithiol-S-conjugate detoxification pathways.

    PubMed

    Fang, Zhong; Roberts, Alexandra A; Weidman, Karissa; Sharma, Sunil V; Claiborne, Al; Hamilton, Christopher J; Dos Santos, Patricia C

    2013-09-01

    BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the acetyl group from N-acetylglucosamine malate to generate glucosamine malate. In Bacillus anthracis, BA1557 has been identified as the N-acetylglucosamine malate deacetylase (BshB); however, a high content of bacillithiol (~70%) was still observed in the B. anthracis ∆BA1557 strain. Genomic analysis led to the proposal that another deacetylase could exhibit cross-functionality in bacillithiol biosynthesis. In the present study, BA1557, its paralogue BA3888 and orthologous Bacillus cereus enzymes BC1534 and BC3461 have been characterized for their deacetylase activity towards N-acetylglucosamine malate, thus providing biochemical evidence for this proposal. In addition, the involvement of deacetylase enzymes is also expected in bacillithiol-detoxifying pathways through formation of S-mercapturic adducts. The kinetic analysis of bacillithiol-S-bimane conjugate favours the involvement of BA3888 as the B. anthracis bacillithiol-S-conjugate amidase (Bca). The high degree of specificity of this group of enzymes for its physiological substrate, along with their similar pH-activity profile and Zn²⁺-dependent catalytic acid-base reaction provides further evidence for their cross-functionalities. PMID:23758290

  4. Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase.

    PubMed

    Wang, Dezheng; Wang, Cheng; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-01-01

    Glutathione (GSH) is an important bioactive substance applied widely in pharmaceutical and food industries. Due to the strong product inhibition in the GSH biosynthetic pathway, high levels of intracellular content, yield and productivity of GSH are difficult to achieve. Recently, a novel bifunctional GSH synthetase was identified to be less sensitive to GSH. A recombinant Escherichia coli strain expressing gshF encoding the bifunctional glutathione synthetase of Streptococcus thermophilus was constructed for GSH production. In this study, efficient GSH production using this engineered strain was investigated. The cultivation process was optimized by controlling dissolved oxygen (DO), amino acid addition and glucose feeding. 36.8 mM (11.3 g/L) GSH were formed at a productivity of 2.06 mM/h when the amino acid precursors (75 mM each) were added and glucose was supplied as the sole carbon and energy source. PMID:26586402

  5. Dysregulation of Glutathione Homeostasis in Neurodegenerative Diseases

    PubMed Central

    Johnson, William M.; Wilson-Delfosse, Amy L.; Mieyal, John. J.

    2012-01-01

    Dysregulation of glutathione homeostasis and alterations in glutathione-dependent enzyme activities are increasingly implicated in the induction and progression of neurodegenerative diseases, including Alzheimer’s, Parkinson’s and Huntington’s diseases, amyotrophic lateral sclerosis, and Friedreich’s ataxia. In this review background is provided on the steady-state synthesis, regulation, and transport of glutathione, with primary focus on the brain. A brief overview is presented on the distinct but vital roles of glutathione in cellular maintenance and survival, and on the functions of key glutathione-dependent enzymes. Major contributors to initiation and progression of neurodegenerative diseases are considered, including oxidative stress, protein misfolding, and protein aggregation. In each case examples of key regulatory mechanisms are identified that are sensitive to changes in glutathione redox status and/or in the activities of glutathione-dependent enzymes. Mechanisms of dysregulation of glutathione and/or glutathione-dependent enzymes are discussed that are implicated in pathogenesis of each neurodegenerative disease. Limitations in information or interpretation are identified, and possible avenues for further research are described with an aim to elucidating novel targets for therapeutic interventions. The pros and cons of administration of N-acetylcysteine or glutathione as therapeutic agents for neurodegenerative diseases, as well as the potential utility of serum glutathione as a biomarker, are critically evaluated. PMID:23201762

  6. Changes of reduced glutathion, glutathion reductase, and glutathione peroxidase after radiation in guinea pigs

    SciTech Connect

    Erden, M.; Bor, N.M.

    1984-04-01

    In this series of experiments the protective action of reduced glutathion due to ionizing radiation has been studied. In the experimental group 18 guinea pigs were exposed to successive radiations of 150 rad 3 or 4 days apart. Total dose given amounted to 750 rad which is the LD50 for guinea pigs. Blood samples were taken 30 min after each exposure. The control series were sham radiated but otherwise treated identically. The cells of the removed blood samples were separated by centrifugation and were subjected to the reduced glutathion stability test. GSSGR, GPer, and LDH enzyme activities were also measured of which the latter served as a marked enzyme. It was found that LDH did not show any alteration after radiation. The reduced glutathion stability test showed a consistent but minor reduction (P greater than 0.05), in the experimental group. GSSGR enzyme activity on the other hand was reduced significantly (from 176.48 +/- 11.32 to 41.34 +/- 1.17 IU/ml of packed erythrocytes, P less than 0.001) in the same group. GPer activity showed a consistent but minor elevation during the early phase of the experimental group. It was later increased significantly beginning after 600 rad total radiation on the fourth session (P less than 0.050).

  7. Emerging regulatory paradigms in glutathione metabolism.

    PubMed

    Liu, Yilin; Hyde, Annastasia S; Simpson, Melanie A; Barycki, Joseph J

    2014-01-01

    One of the hallmarks of cancer is the ability to generate and withstand unusual levels of oxidative stress. In part, this property of tumor cells is conferred by elevation of the cellular redox buffer glutathione. Though enzymes of the glutathione synthesis and salvage pathways have been characterized for several decades, we still lack a comprehensive understanding of their independent and coordinate regulatory mechanisms. Recent studies have further revealed that overall central metabolic pathways are frequently altered in various tumor types, resulting in significant increases in biosynthetic capacity and feeding into glutathione synthesis. In this review, we will discuss the enzymes and pathways affecting glutathione flux in cancer and summarize current models for regulating cellular glutathione through both de novo synthesis and efficient salvage. In addition, we examine the integration of glutathione metabolism with other altered fates of intermediary metabolites and highlight remaining questions about molecular details of the accepted regulatory modes. PMID:24974179

  8. Emerging regulatory paradigms in glutathione metabolism

    PubMed Central

    Liu, Yilin; Hyde, Annastasia S.; Simpson, Melanie A.; Barycki, Joseph J.

    2015-01-01

    One of the hallmarks of cancer is the ability to generate and withstand unusual levels of oxidative stress. In part, this property of tumor cells is conferred by elevation of the cellular redox buffer glutathione. Though enzymes of the glutathione synthesis and salvage pathways have been characterized for several decades, we still lack a comprehensive understanding of their independent and coordinate regulatory mechanisms. Recent studies have further revealed that overall central metabolic pathways are frequently altered in various tumor types, resulting in significant increases in biosynthetic capacity, and feeding into glutathione synthesis. In this review, we will discuss the enzymes and pathways affecting glutathione flux in cancer, and summarize current models for regulating cellular glutathione through both de novo synthesis and efficient salvage. In addition, we examine the integration of glutathione metabolism with other altered fates of intermediary metabolites, and highlight remaining questions about molecular details of the accepted regulatory modes. PMID:24974179

  9. Roles for glutathione transferases in antioxidant recycling

    PubMed Central

    Dixon, David P; Steel, Patrick G

    2011-01-01

    Uniquely among the plant glutathione transferases, two classes possess a catalytic cysteine capable of performing glutathione-dependent reductions. These are the dehydroascorbate reductases (DHARs) and the lambda-class glutathione transferases (GSTLs). Using immobilized GSTLs probed with crude plant extracts we have identified flavonols as high affinity ligands and subsequently demonstrated a novel glutathione-dependent role for these enzymes in recycling oxidized quercetin. By comparing the activities of DHARs and GSTLs we now propose a unified catalytic mechanism that suggests oxidized anthocyanidins and tocopherols may be alternative polyphenolic substrates of GSTLs. PMID:21778824

  10. Expression of glutathione, glutathione peroxidase and glutathione S-transferase pi in canine mammary tumors

    PubMed Central

    2014-01-01

    Background Glutathione (GSH) is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase (GSH-Px) and glutathione S transferase pi (GSTpi), it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. This study evaluated the expression of GSH and the GSH-Px and GSTpi enzymes by immunohistochemistry in 30 canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. In an in vitro study, the expression of the genes glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS) that synthesize GSH and GSH-Px gene were verified by qPCR and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy. Results The immunohistochemical expression of GSH, GSH-Px and GSTpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death. The results showed that high immunoexpression of GSH was correlated to the absence of tumor ulceration and was present in dogs without metastasis (P < 0.05). There was significant correlation of survival with the increase of GSH (P < 0.05). The expression of the GSH-Px and GSTpi enzymes showed no statistically significant correlation with the analyzed variables (p > 0.05). The analysis of the relative expression of genes responsible for the synthesis of GSH (GCLC and GSS) and GSH-Px by quantitative PCR was done with cultured cells of 10 tumor fragments from dogs with mammary tumors. The culture cells showed a decrease in GCLC and GSS expression when compared with no treated cells (P < 0.05). High GSH immunoexpression was associated with better clinical outcomes. Conclusion Therefore, high expression of the GSH seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. The in

  11. Genetics Home Reference: glutathione synthetase deficiency

    MedlinePlus

    ... PubMed Njålsson R. Glutathione synthetase deficiency. Cell Mol Life Sci. 2005 Sep;62(17):1938-45. Review. Citation on PubMed Ristoff E, Larsson A. Inborn errors in the metabolism of glutathione. Orphanet J Rare Dis. 2007 Mar 30;2:16. Review. Citation on PubMed or ...

  12. Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

    PubMed

    Pinto, John T; Krasnikov, Boris F; Alcutt, Steven; Jones, Melanie E; Dorai, Thambi; Villar, Maria T; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J L

    2014-11-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. PMID:25231977

  13. Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine*

    PubMed Central

    Pinto, John T.; Krasnikov, Boris F.; Alcutt, Steven; Jones, Melanie E.; Dorai, Thambi; Villar, Maria T.; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J. L.

    2014-01-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. PMID:25231977

  14. Selenium, glutathione peroxidase and other selenoproteins

    SciTech Connect

    Wilhelmsen, E.C.

    1983-01-01

    Selenium, as essential trace element, has long been associated with protein. The essentiality of selenium is partially understood as glutathione peroxidase contains an essential selenocysteine. Glutathione peroxidase has been purified from many tissues including rat liver. An estimated molecular weight of 105,000 was obtained for glutathione peroxidase by comparison to standards. A subunit size of 26,000 was obtained by SDS-gel electrophoresis. Glutathione peroxidase is not the only selenoprotein in the rat. In seven rat tissues examined, there were many different subunit sizes and change groups representing between 9 and 23 selenoproteins. Selenocysteine in glutathione peroxidase accounts for ca. 36% of the selenium in the rat. The mode of synthesis of glutathione peroxidase and the other selenoproteins is not understood. Glutathione peroxidase is strongly and reversibly inhibited by mercaptocarboxylic acids and other mercaptans, including some used as slow-acting drugs for the symtomatic treatment of rheumatoid arthritis. The mechanism and chemistry of this inhibition is discussed. This inhibition may provide a link between selenium and arthritis.

  15. The biological functions of glutathione revisited in arabidopsis transgenic plants with altered glutathione levels.

    PubMed

    Xiang, C; Werner, B L; Christensen, E M; Oliver, D J

    2001-06-01

    A functional analysis of the role of glutathione in protecting plants from environmental stress was undertaken by studying Arabidopsis that had been genetically modified to have altered glutathione levels. The steady-state glutathione concentration in Arabidopsis plants was modified by expressing the cDNA for gamma-glutamyl-cysteine synthetase (GSH1) in both the sense and antisense orientation. The resulting plants had glutathione levels that ranged between 3% and 200% of the level in wild-type plants. Arabidopsis plants with low glutathione levels were hypersensitive to Cd due to the limited capacity of these plants to make phytochelatins. Plants with the lowest levels of reduced glutathione (10% of wild type) were sensitive to as little as 5 microM Cd, whereas those with 50% wild-type levels required higher Cd concentrations to inhibit growth. Elevating glutathione levels did not increase metal resistance. It is interesting that the plants with low glutathione levels were also less able to accumulate anthocyanins supporting a role for glutathione S-transferases for anthocyanin formation or for the vacuolar localization and therefore accumulation of these compounds. Plants with less than 5% of wild-type glutathione levels were smaller and more sensitive to environmental stress but otherwise grew normally. PMID:11402187

  16. Antioxidant action of glutathione and the ascorbic acid/glutathione pair in a model white wine.

    PubMed

    Sonni, Francesca; Clark, Andrew C; Prenzler, Paul D; Riponi, Claudio; Scollary, Geoffrey R

    2011-04-27

    Glutathione was assessed individually, and in combination with ascorbic acid, for its ability to act as an antioxidant with respect to color development in an oxidizing model white wine system. Glutathione was utilized at concentrations normally found in wine (30 mg/L), as well as at concentrations 20-fold higher (860 mg/L), the latter to afford ascorbic acid (500 mg/L) to glutathione ratios of 1:1. The model wine systems were stored at 45 °C without sulfur dioxide and at saturated oxygen levels, thereby in conditions highly conducive to oxidation. Under these conditions the results demonstrated the higher concentration of glutathione could initially provide protection against oxidative coloration, but eventually induced color formation. In the period during which glutathione offered a protective effect, the production of xanthylium cation pigment precursors and o-quinone-derived phenolic compounds was limited. When glutathione induced coloration, polymeric pigments were formed, but these were different from those found in model wine solutions without glutathione. In the presence of ascorbic acid, high concentrations of glutathione were able to delay the decay in ascorbic acid and inhibit the reaction of ascorbic acid degradation products with the wine flavanol compound (+)-catechin. However, on depletion, the glutathione again induced the production of a range of different polymeric pigments. These results highlight new mechanisms through which glutathione can offer both protection and spoilage during the oxidative coloration of a model wine. PMID:21384873

  17. The antioxidant master glutathione and periodontal health

    PubMed Central

    Bains, Vivek Kumar; Bains, Rhythm

    2015-01-01

    Glutathione, considered to be the master antioxidant (AO), is the most-important redox regulator that controls inflammatory processes, and thus damage to the periodontium. Periodontitis patients have reduced total AO capacity in whole saliva, and lower concentrations of reduced glutathione (GSH) in serum and gingival crevicular fluid, and periodontal therapy restores the redox balance. Therapeutic considerations for the adjunctive use of glutathione in management of periodontitis, in limiting the tissue damage associated with oxidative stress, and enhancing wound healing cannot be underestimated, but need to be evaluated further through multi-centered randomized controlled trials. PMID:26604952

  18. Glutathione cycle in stable chronic obstructive pulmonary disease.

    PubMed

    Biljak, Vanja Radisić; Rumora, Lada; Cepelak, Ivana; Pancirov, Dolores; Popović-Grle, Sanja; Sorić, Jasna; Grubisić, Tihana Zanić

    2010-08-01

    Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidant/antioxidant imbalance. Glutathione is the most abundant cellular low-molecular weight thiol and the glutathione redox cycle is the fundamental component of the cellular antioxidant defence system. Concentration of total glutathione and catalytic activities of glutathione peroxidase and glutathione reductase were determined in peripheral blood of patients (n = 109) and healthy subjects (n = 51). Concentration of total glutathione in patients was not changed in comparison to healthy controls. However, we found statistically significant difference between patients with moderate and severe disease stages. Glutathione reductase activity was increased, while glutathione proxidase activity was decreased in the patients with COPD, when compared to healthy controls. We found no significant difference in glutathione peroxidase and glutathione reductase activities between stages. Patients who smoked had lower concentration of total glutathione compared with former smokers and never-smoking patients. Lung function parameters were inversely associated with glutathione level. Evidence is presented for differential modulation of glutathione peroxidase and glutathione reductase activities in peripheral blood of patients with stable COPD. We suppose that in addition to glutathione biosynthesis, glutathione reductase-dependent regulation of the glutathione redox state is vital for protection against oxidative stress. PMID:20648694

  19. Effects of fraxetin on glutathione redox status.

    PubMed

    Martín-Aragón, S; Benedí, J M; Villar, A M

    1997-01-01

    We have evaluated the effects of an oral treatment of mice with fraxetin (25 mg/kg for 30 days) on the glutathione system (GSH, GSSG, and GSSG/GSH ratio as stress index), glutathione reductase (GR) and glutathione peroxidase (GPx) in liver supernatants from male C57BL/6J mice (18-month old). A significant antioxidant effect in vivo was found under this treatment by a decrease in the GSSG/GSH ratio and an increased activity of GR compared with the control mice. GSSG rate and GSSG/GSH ratio were correlated with the decline of GPx++ activity. Our results of increased GR activity could be considered as a supercompensation in glutathione redox status that involves a decrease in the accumulation of GSSG, as well as, in GSSG/GSH ratio. Finally, we suggest that this possible mechanism of supercompensation could lead to an enhancement in the average life span. PMID:9162171

  20. Quantification of cysteine S-conjugate of 3-sulfanylhexan-1-ol in must and wine of petite arvine vine by stable isotope dilution analysis.

    PubMed

    Luisier, Jean-Luc; Buettner, Hermann; Völker, Sebastian; Rausis, Thierry; Frey, Urban

    2008-05-14

    Making use of a convenient synthetic approach to prepare the deuterated S-3-(hexan-1-ol)-cysteine by a Michael addition reaction, an analytical method was developed to measure the presence of the cysteine S-conjugate, precursor of 3-sulfanylhexan-1-ol (3-mercaptohexan-1-ol), in must and wine from Petite Arvine vine. The method uses a stable isotope dilution assay with a suitable one-step sample preparation and HPLC-MS detection. The method has limits of detection and quantification of 3 and 10 microg/L, respectively. A correlation between the increase of the precursor concentration and the increase of the degree of rot has been established. PMID:18416552

  1. Transport of Glutathione Diethyl Ester Into Human Cells

    NASA Astrophysics Data System (ADS)

    Levy, Ellen J.; Anderson, Mary E.; Meister, Alton

    1993-10-01

    Glutathione monoesters in which the carboxyl group of the glycine residue is esterified were previously found, in contrast to glutathione itself, to be effectively transported into various types of cells and to be converted intracellularly into glutathione. Glutathione monoesters are thus useful for prevention of oxidative stress, certain toxicities, and for treatment of glutathione deficiency. Glutathione diethyl ester is rapidly split to the glutathione monoethyl ester by mouse plasma glutathione diester α-esterase activity. Thus, as expected, glutathione mono- and diesters have similar effects on cellular glutathione levels in mice. However, human plasma lacks glutathione diester α-esterase thus, it became of interest to compare the transport properties of glutathione mono- and diesters in human cells. We found that human cells (erythrocytes, peripheral blood mononuclear cells, fibroblasts, ovarian tumor cells, and purified T cells) transport glutathione diethyl ester much more effectively than the corresponding monoethyl (glycyl) ester. Human cells rapidly convert glutathione diethyl ester to the monoester, whose intracellular levels rise to levels that are significantly higher than levels found after application of the monoester to the cells. High levels of the monoester provide the cells with a means of producing glutathione over a period of time. We conclude that glutathione diethyl ester is highly effective as a delivery agent for glutathione monoester, and thus for glutathione, in human cells and therefore could serve to decrease oxidative stress and toxicity. Hamster (and certain other animals) also lack plasma glutathione diester α-esterase and therefore would be suitable animal models. Previously reported toxicity of certain glutathione ester preparations appears to reflect the presence of impurities rather than effects of the esters.

  2. Hepatobiliary transport of glutathione and glutathione conjugate in rats with hereditary hyperbilirubinemia.

    PubMed Central

    Elferink, R P; Ottenhoff, R; Liefting, W; de Haan, J; Jansen, P L

    1989-01-01

    TR- mutant rats have an autosomal recessive mutation that is expressed as a severely impaired hepatobiliary secretion of organic anions like bilirubin-(di)glucuronide and dibromosulphthalein (DBSP). In this paper, the hepatobiliary transport of glutathione and a glutathione conjugate was studied in normal Wistar rats and TR- rats. It was shown that glutathione is virtually absent from the bile of TR- rats. In the isolated, perfused liver the secretion of glutathione and the glutathione conjugate, dinitrophenyl-glutathione (GS-DNP), from hepatocyte to bile is severely impaired, whereas the sinusoidal secretion from liver to blood is not affected. The secretion of GS-DNP was also studied in isolated hepatocytes. The secretion of GS-DNP from cells isolated from TR- rat liver was significantly slower than from normal hepatocytes. Efflux of GS-DNP was a saturable process with respect to intracellular GS-DNP concentration: Vmax and Km for efflux from TR- cells was 498 nmol/min.g dry wt and 3.3 mM, respectively, as compared with 1514 nmol/min.g dry wt and 0.92 mM in normal hepatocytes. These results suggest that the canalicular transport system for glutathione and glutathione conjugates is severely impaired in TR- rats, whereas sinusoidal efflux is unaffected. Because the defect also comes to expression in isolated hepatocytes, efflux of GS-DNP from normal hepatocytes must predominantly be mediated by the canalicular transport mechanism, which is deficient in TR- rats. PMID:2760197

  3. Glutathione Metabolism and Parkinson’s Disease

    PubMed Central

    Smeyne, Michelle

    2013-01-01

    It has been established that oxidative stress, defined as the condition when the sum of free radicals in a cell exceeds the antioxidant capacity of the cell, contributes to the pathogenesis of Parkinson’s disease. Glutathione is a ubiquitous thiol tripeptide that acts alone, or in concert with enzymes within cells to reduce superoxide radicals, hydroxyl radicals and peroxynitrites. In this review, we examine the synthesis, metabolism and functional interactions of glutathione, and discuss how this relates to protection of dopaminergic neurons from oxidative damage and its therapeutic potential in Parkinson’s disease. PMID:23665395

  4. Role of glutathione metabolism and glutathione-related antioxidant defense systems in hypertension.

    PubMed

    Robaczewska, J; Kedziora-Kornatowska, K; Kozakiewicz, M; Zary-Sikorska, E; Pawluk, H; Pawliszak, W; Kedziora, J

    2016-06-01

    The risk of developing chronic hypertension increases with age. Among others factors, increased oxidative stress is a well-recognized etiological factor for the development of hypertension. The co-occurrence of oxidative stress and hypertension may occur as a consequence of a decrease in antioxidant defense system activity or elevated reactive oxygen species generation. Glutathione is a major intracellular thiol-disulfide redox buffer that serves as a cofactor for many antioxidant enzymes. Glutathione-related parameters are altered in hypertension, suggesting that there is an association between the glutathione-related redox system and hypertension. In this review, we provide mechanistic explanations for how glutathione maintains blood pressure. More specifically, we discuss glutathione's role in combating oxidative stress and maintaining nitric oxide bioavailability via the formation of nitrosothiols and nitrosohemoglobin. Although impaired vasodilator responses are observed in S-nitrosothiol-deficient red blood cells, this potential hypertensive mechanism is currently overlooked in the literature. Here we fill in this gap by discussing the role of glutathione in nitric oxide metabolism and controlling blood pressure. We conclude that disturbances in glutathione metabolism might explain age-dependent increases in blood pressure. PMID:27511994

  5. Glutathione metabolism and its implications for health.

    PubMed

    Wu, Guoyao; Fang, Yun-Zhong; Yang, Sheng; Lupton, Joanne R; Turner, Nancy D

    2004-03-01

    Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is the most abundant low-molecular-weight thiol, and GSH/glutathione disulfide is the major redox couple in animal cells. The synthesis of GSH from glutamate, cysteine, and glycine is catalyzed sequentially by two cytosolic enzymes, gamma-glutamylcysteine synthetase and GSH synthetase. Compelling evidence shows that GSH synthesis is regulated primarily by gamma-glutamylcysteine synthetase activity, cysteine availability, and GSH feedback inhibition. Animal and human studies demonstrate that adequate protein nutrition is crucial for the maintenance of GSH homeostasis. In addition, enteral or parenteral cystine, methionine, N-acetyl-cysteine, and L-2-oxothiazolidine-4-carboxylate are effective precursors of cysteine for tissue GSH synthesis. Glutathione plays important roles in antioxidant defense, nutrient metabolism, and regulation of cellular events (including gene expression, DNA and protein synthesis, cell proliferation and apoptosis, signal transduction, cytokine production and immune response, and protein glutathionylation). Glutathione deficiency contributes to oxidative stress, which plays a key role in aging and the pathogenesis of many diseases (including kwashiorkor, seizure, Alzheimer's disease, Parkinson's disease, liver disease, cystic fibrosis, sickle cell anemia, HIV, AIDS, cancer, heart attack, stroke, and diabetes). New knowledge of the nutritional regulation of GSH metabolism is critical for the development of effective strategies to improve health and to treat these diseases. PMID:14988435

  6. Five Decades with Glutathione and the GSTome

    PubMed Central

    Mannervik, Bengt

    2012-01-01

    Uncle Folke inspired me to become a biochemist by demonstrating electrophoresis experiments on butterfly hemolymph in his kitchen. Glutathione became the subject for my undergraduate project in 1964 and has remained a focal point in my research owing to its multifarious roles in the cell. Since the 1960s, the multiple forms of glutathione transferase (GST), the GSTome, were isolated and characterized, some of which were discovered in our laboratory. Products of oxidative processes were found to be natural GST substrates. Examples of toxic compounds against which particular GSTs provide protection include 4-hydroxynonenal and ortho-quinones, with possible links to the etiology of Alzheimer and Parkinson diseases and other degenerative conditions. The role of thioltransferase and glutathione reductase in the cellular reduction of disulfides and other oxidized forms of thiols was clarified. Glyoxalase I catalyzes still another glutathione-dependent detoxication reaction. The unusual steady-state kinetics of this zinc-containing enzyme initiated model discrimination by regression analysis. Functional properties of the enzymes have been altered by stochastic mutations based on DNA shuffling and rationally tailored by structure-based redesign. We found it useful to represent promiscuous enzymes by vectors or points in multidimensional substrate-activity space and visualize them by multivariate analysis. Adopting the concept “molecular quasi-species,” we describe clusters of functionally related enzyme variants that may emerge in natural as well as directed evolution. PMID:22247548

  7. Glutathione system in young spontaneously hypertensive rats.

    PubMed

    Lee, S K; Arunkumar, Sundaram; Sirajudeen, K N S; Singh, H J

    2010-12-01

    Glutathione (GSH) forms a part of the antioxidant system that plays a vital role in preventing oxidative stress, and an imbalance in the oxidant/antioxidant system has been linked to the pathogenesis of hypertension. The aim of this study was to investigate the status of the GSH system in the kidney of spontaneously hypertensive rats (SHR). Components of the GSH system, including glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), and total GSH content, were measured in the kidneys of 4, 6, 8, 12, and 16 weeks old SHR and Wistar-Kyoto (WKY) rats. Systolic blood pressure of SHR was significantly higher from the age of 6 weeks onwards compared with age-matched WKY rats. GPx activity in the SHR was significantly lower from the age of 8 weeks onwards when compared to that in age-matched WKY rats. No significant differences were evident in the GPx-1 protein abundance, and its relative mRNA levels, GR, GST activity, and total GSH content between SHR and age-matched WKY rats. The lower GPx activity suggests of an impairment of the GSH system in the SHR, which might be due to an abnormality in its protein rather than non-availability of a cofactor. Its role in the development of hypertension in SHR however remains unclear. PMID:20680541

  8. Glutathione and gamma-glutamyl transferases are involved in the formation of cadmium-glutathione complex.

    PubMed

    Adamis, Paula Daniela Braga; Mannarino, Sérgio Cantú; Eleutherio, Elis Cristina Araújo

    2009-05-01

    In a wild-type strain of Saccharomyces cerevisiae, cadmium induces the activities of both gamma-glutamyl transferase (gamma-GT) and glutathione transferase 2 (Gtt2). However, Gtt2 activity did not increase under gamma-GT or Ycf1 deficiencies, suggesting that the accumulation of glutathione-cadmium in the cytosol inhibits Gtt2. On the other hand, the balance between the cytoplasmic and vacuolar level of glutathione seems to regulate gamma-GT activity, since this enzyme was not activated in a gtt2 strain. Taken together, these results suggest that gamma-GT and Gtt2 work together to remove cadmium from the cytoplasm, a crucial mechanism for metal detoxification that is dependent on glutathione. PMID:19345220

  9. Enzyme-catalysed conjugations of glutathione with unsaturated compounds

    PubMed Central

    Boyland, E.; Chasseaud, L. F.

    1967-01-01

    1. Rat-liver supernatant catalyses the reaction of diethyl maleate with glutathione. 2. Evidence is presented that the enzyme involved is different from the known glutathione-conjugating enzymes, glutathione S-alkyltransferase, S-aryltransferase and S-epoxidetransferase. 3. Rat-liver supernatant catalyses the reaction of a number of other αβ-unsaturated compounds, including aldehydes, ketones, lactones, nitriles and nitro compounds, with glutathione: separate enzymes may be responsible for these reactions. PMID:6035529

  10. An enzyme catalysing the conjugation of epoxides with glutathione

    PubMed Central

    Boyland, E.; Williams, K.

    1965-01-01

    1. Liver supernatant preparations from rats and ferrets catalyse the conjugation of some epoxides with glutathione. The enzyme involved might be called `glutathione S-epoxidetransferase', as it is different from glutathione S-aryltransferase, the enzyme catalysing the conjugation of 1,2-dichloro-4-nitrobenzene, 4-nitro-pyridine N-oxide and other cyclic compounds with glutathione and from the enzyme catalysing the conjugation of iodomethane and glutathione. 2. The enzyme does not catalyse the reaction with cysteine. It is not inactivated by dialysis but is unstable at pH 5·0. 3. The role of the enzyme in metabolism of foreign compounds is discussed. PMID:14342229

  11. Effect of glutathione addition in sparkling wine.

    PubMed

    Webber, Vanessa; Dutra, Sandra Valduga; Spinelli, Fernanda Rodrigues; Marcon, Ângela Rossi; Carnieli, Gilberto João; Vanderlinde, Regina

    2014-09-15

    This study aims to evaluate the effect of the addition of glutathione (GSH) on secondary aromas and on the phenolic compounds of sparkling wine elaborated by traditional method. It was added 10 and 20 mg L(-1) of GSH to must and to base wine. The determination of aroma compounds was performed by gas chromatography. Phenolic compounds and glutathione content were analyzed by high performance liquid chromatography. Sparkling wines with addition of GSH to must showed lower levels of total phenolic compounds and hydroxycinnamic acids. Furthermore, the sparkling wine with addition of GSH to must showed higher levels of 2-phenylethanol, 3-methyl-1-butanol and diethyl succinate, and lower concentrations of ethyl decanoate, octanoic and decanoic acids. The GSH addition to the must show a greater influence on sparkling wine than to base wine, however GSH addition to base wine seems retain higher SO2 free levels. The concentration of GSH added showed no significant difference. PMID:24767072

  12. Glutathione and redox signaling in substance abuse

    PubMed Central

    Uys, Joachim D.; Mulholland, Patrick J.; Townsend, Danyelle M.

    2015-01-01

    Throughout the last couple decades, the cause and consequences of substance abuse has expanded to identify the underlying neurobiological signaling mechanisms associated with addictive behavior. Chronic use of drugs, such as cocaine, methamphetamine and alcohol leads to the formation of oxidative or nitrosative stress (ROS/RNS) and changes in glutathione and redox homeostasis. Of importance, redox-sensitive post-translational modifications on cysteine residues, such as S-glutathionylation and S-nitrosylation could impact on the structure and function of addiction related signaling proteins. In this commentary, we evaluate the role of glutathione and redox signaling in cocaine-, methamphetamine- and alcohol addiction and conclude by discussing the possibility of targeting redox pathways for the therapeutic intervention of these substance abuse disorders. PMID:25027386

  13. Glutathione and redox signaling in substance abuse.

    PubMed

    Uys, Joachim D; Mulholland, Patrick J; Townsend, Danyelle M

    2014-07-01

    Throughout the last couple decades, the cause and consequences of substance abuse has expanded to identify the underlying neurobiological signaling mechanisms associated with addictive behavior. Chronic use of drugs, such as cocaine, methamphetamine and alcohol leads to the formation of oxidative or nitrosative stress (ROS/RNS) and changes in glutathione and redox homeostasis. Of importance, redox-sensitive post-translational modifications on cysteine residues, such as S-glutathionylation and S-nitrosylation could impact on the structure and function of addiction related signaling proteins. In this commentary, we evaluate the role of glutathione and redox signaling in cocaine-, methamphetamine- and alcohol addiction and conclude by discussing the possibility of targeting redox pathways for the therapeutic intervention of these substance abuse disorders. PMID:25027386

  14. Effect of Glutathione on Phytochelatin Synthesis in Tomato Cells 1

    PubMed Central

    Mendum, Mary Lou; Gupta, Subhash C.; Goldsbrough, Peter B.

    1990-01-01

    Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably [γ-Glu-Cys]2-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little [35S]cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione. PMID:16667492

  15. A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thioredoxin for growth.

    PubMed Central

    Muller, E G

    1996-01-01

    A glutathione reductase null mutant of Saccharomyces cerevisiae was isolated in a synthetic lethal genetic screen for mutations which confer a requirement for thioredoxin. Yeast mutants that lack glutathione reductase (glr1 delta) accumulate high levels of oxidized glutathione and have a twofold increase in total glutathione. The disulfide form of glutathione increases 200-fold and represents 63% of the total glutathione in a glr1 delta mutant compared with only 6% in wild type. High levels of oxidized glutathione are also observed in a trx1 delta, trx2 delta double mutant (22% of total), in a glr1 delta, trx1 delta double mutant (71% of total), and in a glr1 delta, trx2 delta double mutant (69% of total). Despite the exceptionally high ratio of oxidized/reduced glutathione, the glr1 delta mutant grows with a normal cell cycle. However, either one of the two thioredoxins is essential for growth. Cells lacking both thioredoxins and glutathione reductase are not viable under aerobic conditions and grow poorly anaerobically. In addition, the glr1 delta mutant shows increased sensitivity to the thiol oxidant diamide. The sensitivity to diamide was suppressed by deletion of the TRX2 gene. The genetic analysis of thioredoxin and glutathione reductase in yeast runs counter to previous studies in Escherichia coli and for the first time links thioredoxin with the redox state of glutathione in vivo. Images PMID:8930901

  16. Glutathione is a Physiologic Reservoir of Neuronal Glutamate

    PubMed Central

    Koga, Minori; Serritella, Anthony V.; Messmer, Marcus M.; Hayashi-Takagi, Akiko; Hester, Lynda D.; Snyder, Solomon H.; Sawa, Akira; Sedlak, Thomas W.

    2013-01-01

    Glutamate, the principal excitatory neurotransmitter of the brain, participates in a multitude of physiologic and pathologic processes, including learning and memory. Glutathione, a tripeptide composed of the amino acids glutamate, cysteine, and glycine, serves important cofactor roles in antioxidant defense and drug detoxification, but glutathione deficits occur in multiple neuropsychiatric disorders. Glutathione synthesis and metabolism are governed by a cycle of enzymes, the γ-glutamyl cycle, which can achieve intracellular glutathione concentrations of 1-10 millimolar. Because of the considerable quantity of brain glutathione and its rapid turnover, we hypothesized that glutathione may serve as a reservoir of neural glutamate. We quantified glutamate in HT22 hippocampal neurons, PC12 cells and primary cortical neurons after treatment with molecular inhibitors targeting three different enzymes of the glutathione metabolic cycle. Inhibiting 5-oxoprolinase and γ-glutamyl transferase, enzymes that liberate glutamate from glutathione, leads to decreases in glutamate. In contrast, inhibition of γ-glutamyl cysteine ligase, which uses glutamate to synthesize glutathione, results in substantial glutamate accumulation. Increased glutamate levels following inhibition of glutathione synthesis temporally precede later effects upon oxidative stress. PMID:21539809

  17. Effects of concentrated drinking water injection on glutathione and glutathione-dependent enzymes in liver of Cyprinus carpio L.

    PubMed

    Elia, Antonia Concetta; Fanetti, Alessia; Dörr, Ambrosius Josef Martin; Taticchi, Maria I

    2008-06-01

    Two drinking water production plants located in North Italy, collecting water from the River Po (Plants 1 and 2) were chosen for this study. Water samples were collected before and after the disinfection process and at two points along the piping system. Water samples were concentrated by the solid-phase extraction system and injected intraperitoneally into specimens of Cyprinus carpio. The concentration of water samples was 3 l/equiv. In order to assess the effects of the water samples on carp liver, total glutathione and glutathione-dependent enzymes, such as glutathione S-transferase, glutathione peroxidase, glutathione reductase and glyoxalase I, were measured following this treatment for 6 days at two experimental times (3 and 6 days). Both water plant-treated carp showed a general increase of the enzymatic activities of glutathione S-transferase, and glutathione reductase which might be employed as potential biomarkers of oxidative stress induced by disinfected river water. Plant 1-treated carp showed higher glyoxalase I and glutathione levels and lower glutathione peroxidase activity. A depleted level of total glutathione and of glyoxalase I for specimens of water plant 2 (for both experimental times), without correlation with the distances in the pipeline, suggests that river plant water can also lead to potentially adverse effects on selected biochemical parameters in C. carpio. PMID:18457861

  18. Deficient Glutathione in the Pathophysiology of Mycotoxin-Related Illness

    PubMed Central

    Guilford, Frederick T.; Hope, Janette

    2014-01-01

    Evidence for the role of oxidative stress in the pathophysiology of mycotoxin-related illness is increasing. The glutathione antioxidant and detoxification systems play a major role in the antioxidant function of cells. Exposure to mycotoxins in humans requires the production of glutathione on an “as needed” basis. Research suggests that mycotoxins can decrease the formation of glutathione due to decreased gene expression of the enzymes needed to form glutathione. Mycotoxin-related compromise of glutathione production can result in an excess of oxidative stress that leads to tissue damage and systemic illness. The review discusses the mechanisms by which mycotoxin-related deficiency of glutathione may lead to both acute and chronic illnesses. PMID:24517907

  19. Deficient glutathione in the pathophysiology of mycotoxin-related illness.

    PubMed

    Guilford, Frederick T; Hope, Janette

    2014-02-01

    Evidence for the role of oxidative stress in the pathophysiology of mycotoxin-related illness is increasing. The glutathione antioxidant and detoxification systems play a major role in the antioxidant function of cells. Exposure to mycotoxins in humans requires the production of glutathione on an "as needed" basis. Research suggests that mycotoxins can decrease the formation of glutathione due to decreased gene expression of the enzymes needed to form glutathione. Mycotoxin-related compromise of glutathione production can result in an excess of oxidative stress that leads to tissue damage and systemic illness. The review discusses the mechanisms by which mycotoxin-related deficiency of glutathione may lead to both acute and chronic illnesses. PMID:24517907

  20. The regulation of gelation of Phloem exudate from cucurbita fruit by dilution, glutathione, and glutathione reductase.

    PubMed

    Alosi, M C; Melroy, D L; Park, R B

    1988-04-01

    The average glutathione equivalent concentration in phloem exudate collected from squash fruit (Cucurbita moschata [Duchesne] Poir. var Butternut) and pumpkin fruit (Cucurbita pepo [L.] var Jack-o-lattern) was 1.02 and 0.60 millimolar, respectively. Glutathione reductase (EC 1.6.4.2) activity in phloem exudate from squash and pumpkin fruit averaged 0.48 and 1.74 micromole NADPH oxidized per minute per milliliter, respectively. Protein concentrations in fruit phloem exudates averaged 67 milligrams per milliliter for squash and 57 milligrams per milliliter for pumpkin. The phloem-specific P-proteins account for most of the protein content of exudate. Pure exudate from fruit does not gel for hours or days, but when diluted with neutral or alkaline aqueous solutions, exudate gels rapidly. Exudate solutions undergo biphasic pH changes with dilution. We suggest that P-protein undergoes conformational change upon dilution, exposing titratable groups and sulfhydryl residues. Oxidation of the latter forms the intermolecular disulfide bridges of the gel. The gelation of diluted exudate is regulated by factors (oxygen, pH, glutathione, NADPH) which affect the maintenance of reduced sulfhydryl residues and the activity of glutathione reductase. While these factors may also act in vivo to regulate redox conditions in phloem, their relationship to hypothetical sol/gel transitions or motile and nonmotile phases in the transport conduit is unknown. PMID:16666036

  1. Detection of glutathione transferase activity on polyacrylamide gels.

    PubMed

    Ricci, G; Lo Bello, M; Caccuri, A M; Galiazzo, F; Federici, G

    1984-12-01

    A simple and sensitive assay for glutathione transferase activity on polyacrylamide gel is described. The method is based on the fast reduction of nitroblue tetrazolium salt by glutathione. Blue insoluble formazan colors the gel except in the glutathione transferase area. The stable and defined colorless zone is still detectable with 0.005 unit enzyme. This technique has been successfully applied with enzyme preparations of human heart and other tissues. PMID:6532239

  2. [The different aspects of the biological role of glutathione].

    PubMed

    Bilska, Anna; Kryczyk, Agata; Włodek, Lidia

    2007-01-01

    Glutathione plays a key role in maintaining a physiological balance between prooxidants and antioxidants, crucial for the life and death of a cell. Glutathione occurs in the human body in several redox forms, of which reduced glutathione (GSH), oxidized glutathione (GSSG), S-nitrosoglutathione (GSNO), and mixed disulfides of glutathione with proteins are the most important. There is a clear relationship between the levels of different redox forms of glutathione and the regulation of cellular metabolism in a broad sense. Therefore, each of these forms of glutathione can be beneficial or harmful to the organism depending on the cell type and its metabolic status. In such a situation, elevation of GSH level can constitute a very important factor aiding treatment. A rise in GSH level is beneficial in all pathological states, accompanied by lowered GSH content, while a lowering of GSH level is an indication to induce short-term immunosuppression required in organ transplantation and in tumor cells to selectively increase their sensitivity to chemo- and radiotherapy. GSH itself cannot be used as a therapeutic since it is not transported through plasma membranes. Cysteine, an amino acid which limits glutathione biosynthesis, also cannot be used in therapy due to its high neurotoxicity. For this reason, there is currently an intensive search for possibilities of modulating cellular glutathione and cysteine levels, and this problem can be the subject of interdisciplinary studies combining such scientific fields as biology, pharmacology, toxicology, and clinical medicine. PMID:17679914

  3. Cell free glutathione synthesizing activity of mercury resistant bacteria

    SciTech Connect

    Gachhui, R.; Pahan, K.; Ray, S., R.; Chaudhuri, J.; Mandal, A. )

    1991-03-01

    Reduced glutathione (GSH) is present in all living cells and is known to have a generalized role in protecting the cells from heavy metal toxicity. Depletion of both GSH and glutathione reductase (GR) level upon treatment with mercuric chloride (HgCl{sub 2}) is reported in various organs of rat. However, the effect of HgCl{sub 2} on glutathione level in bacterial system is not known. In the present communication, the authors report the results of their investigation on the glutathione status in mercury resistant bacterial cells exposed to HgCl{sub 2}.

  4. Measurement of glutathione in activated sludges.

    PubMed

    Dziurla, M A; Leroy, P; Strünkmann, G W; Salhi, M; Lee, D U; Camacho, P; Heinz, V; Müller, J A; Paul, E; Ginestet, Ph; Audic, J M; Block, J C

    2004-01-01

    Thermal, electric, mechanical or oxidative stress seem a promising way to reduce the production of excess activated sludge during biological wastewater treatment. However, the adaptation and the resistance of the sludge microbial ecosystem to stress conditions is a major question as it may definitively limit the effect of some treatments. Defence mechanisms developed by aerobic organisms, in particular, in response to oxidative stress involve various antioxidant activities and compounds such as glutathione. An HPLC method was developed for measuring reduced and total glutathione (GSH and GSHt) in perchloric acid sludge extracts. The method was sensitive, highly specific and validated for linearity, precision and recovery. Considering the extraction yield and the oxidation of GSH during extract storage, the measured GSH concentration was estimated to represent 60% of the GSH content from activated sludges. GSHt ranged from 0.32 to 3.34micromolg(-1) volatile solids and the GSH/GSHt ratio ranged from 32% to 91%. Measurements performed on sludges stressed in precise conditions selected to reach a reduction of sludge production showed a decrease of GSH and GSHt concentrations with thermal, mechanical, electric and ozone stress. PMID:14630122

  5. Manipulation of glutathione metabolism in transgenic plants.

    PubMed

    Creissen, G; Broadbent, P; Stevens, R; Wellburn, A R; Mullineaux, P

    1996-05-01

    There is clear potential for the genetic manipulation of key enzymes involved in stress metabolism in transgenic plants. However, the data emerging so far from such experiments are equivocal. The detailed analysis of stress responses in progeny of primary transgenics, coupled with comparisons with control transgenic plants that do not contain the GR transgene, allows us to take into account the possible variation in response to stress associated with regeneration of plants from tissue culture. The picture that is now beginning to emerge with respect to the role of GR in stress protection is that, although there are clearly benefits to be had from overexpression of the enzymes, there is no direct correlation between enzyme levels and stress tolerance. It may be that overexpression of the cytosolic isoform (gor2) will prove to be of greater benefit. Furthermore, the types of stresses to which transgenic plants have been exposed in order to assess the consequences of oxidative stress tolerance cannot reproduce those that will experienced in field conditions. Only when plants with higher GR levels and increased glutathione synthesis capacity are grown in field trials will it be possible to make a full assessment of the benefits of engineering plants with altered glutathione metabolism. PMID:8736785

  6. Plasma cysteine, cystine, and glutathione in cirrhosis.

    PubMed

    Chawla, R K; Lewis, F W; Kutner, M H; Bate, D M; Roy, R G; Rudman, D

    1984-10-01

    Plasma contains three forms of cyst(e)ine: cysteine, cystine, and protein-bound cysteine. The former is a thiol and the latter two are disulfides. The levels of all three types of cyst(e)ine, as well as the cysteinyl tripeptide glutathione, were measured in the plasma of 14 normal and 10 cirrhotic individuals. All subjects ate mixed foods. Some cirrhotic patients were studied during nasogastric hyperalimentation with Vivonex (Norwich Eaton Pharmaceuticals, Norwich, N.Y.) as well as during total parenteral nutrition with FreAmine III (American McGaw, Irvine, Calif.); neither formula contains cyst(e)ine. Regardless of the nature of the diet, cirrhotic patients had significantly subnormal values for cysteine, glutathione, and albumin. In addition, the following significant changes were found to be diet-dependent: (a) elevated methionine during Vivonex, (b) subnormal taurine during mixed foods and total parenteral nutrition, (c) depressed protein-bound cysteine during total parenteral nutrition, (d) depressed cyst(e)ine thiol/disulfide ratio during mixed foods, and (e) depressed total thiol during Vivonex and total parenteral nutrition. The data indicate multiple abnormalities in sulfur metabolism in cirrhosis. PMID:6468868

  7. Correction of glutathione deficiency in the lower respiratory tract of HIV seropositive individuals by glutathione aerosol treatment.

    PubMed Central

    Holroyd, K. J.; Buhl, R.; Borok, Z.; Roum, J. H.; Bokser, A. D.; Grimes, G. J.; Czerski, D.; Cantin, A. M.; Crystal, R. G.

    1993-01-01

    BACKGROUND--Concentrations of glutathione, a ubiquitous tripeptide with immune enhancing and antioxidant properties, are decreased in the blood and lung epithelial lining fluid of human immunodeficiency virus (HIV) seropositive individuals. Since the lung is the most common site of infection in those who progress to AIDS it is rational to consider whether it is possible to safely augment glutathione levels in the epithelial lining fluid of HIV seropositive individuals, thus potentially improving local host defence. METHODS--Purified reduced glutathione was delivered by aerosol to HIV seropositive individuals (n = 14) and the glutathione levels in lung epithelial lining fluid were compared before and at one, two, and three hours after aerosol administration. RESULTS--Before treatment total glutathione concentrations in the epithelial lining fluid were approximately 60% of controls. After three days of twice daily doses each of 600 mg reduced glutathione, total glutathione levels in the epithelial lining fluid increased and remained in the normal range for at least three hours after treatment. Strikingly, even though > 95% of the glutathione in the aerosol was in its reduced form, the percentage of oxidised glutathione in epithelial lining fluid increased from 5% before treatment to about 40% three hours after treatment, probably reflecting the use of glutathione as an antioxidant in vivo. No adverse effects were observed. CONCLUSIONS--It is feasible and safe to use aerosolised reduced glutathione to augment the deficient glutathione levels of the lower respiratory tract of HIV seropositive individuals. It is rational to evaluate further the efficacy of this tripeptide in improving host defence in HIV seropositive individuals. PMID:8256245

  8. Nomenclature for mammalian soluble glutathione transferases.

    PubMed

    Mannervik, Bengt; Board, Philip G; Hayes, John D; Listowsky, Irving; Pearson, William R

    2005-01-01

    The nomenclature for human soluble glutathione transferases (GSTs) is extended to include new members of the GST superfamily that have been discovered, sequenced, and shown to be expressed. The GST nomenclature is based on primary structure similarities and the division of GSTs into classes of more closely related sequences. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, etc., abbreviated in Roman capitals: A, M, P, and so on. (The Greek characters should not be used.) Class members are distinguished by Arabic numerals and the native dimeric protein structures are named according to their subunit composition (e.g., GST A1-2 is the enzyme composed of subunits 1 and 2 in the Alpha class). Soluble GSTs from other mammalian species can be classified in the same manner as the human enzymes, and this chapter presents the application of the nomenclature to the rat and mouse GSTs. PMID:16399376

  9. Quantification of Glutathione in Caenorhabditis elegans

    PubMed Central

    Caito, Samuel W.; Aschner, Michael

    2015-01-01

    Glutathione (GSH) is the most abundant intracellular thiol with diverse functions from redox signaling, xenobiotic detoxification, and apoptosis. The quantification of GSH is an important measure for redox capacity and oxidative stress. This protocol quantifies total GSH from Caenorhabditis elegans, an emerging model organism for toxicology studies. GSH is measured using the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) cycling method originally created for cell and tissue samples but optimized for whole worm extracts. DTNB reacts with GSH to from a 5′-thio-2-nitrobenzoic acid (TNB) chromophore with maximum absorbance of 412 nm. This method is both rapid and sensitive, making it ideal for studies involving a large number of transgenic nematode strains. PMID:26309452

  10. Glutathione Redox System in β-Thalassemia/Hb E Patients

    PubMed Central

    Tangjaidee, Thongchai; Hatairaktham, Suneerat; Charoensakdi, Ratiya; Panichkul, Narumol; Siritanaratkul, Noppadol; Fucharoen, Suthat

    2013-01-01

    β-thalassemia/Hb E is known to cause oxidative stress induced by iron overload. The glutathione system is the major endogenous antioxidant that protects animal cells from oxidative damage. This study aimed to determine the effect of disease state and splenectomy on redox status expressed by whole blood glutathione (GSH)/glutathione disulfide (GSSG) and also to evaluate glutathione-related responses to oxidation in β-thalassemia/Hb E patients. Twenty-seven normal subjects and 25 β-thalassemia/Hb E patients were recruited and blood was collected. The GSH/GSSG ratio, activities of glutathione-related enzymes, hematological parameters, and serum ferritin levels were determined in individuals. Patients had high iron-induced oxidative stress, shown as significantly increased serum ferritin, a decreased GSH/GSSG ratio, and increased activities of glutathione-related enzymes. Splenectomy increased serum ferritin levels and decreased GSH levels concomitant with unchanged glutathione-related enzyme activities. The redox ratio had a positive correlation with hemoglobin levels and negative correlation with levels of serum ferritin. The glutathione system may be the body's first-line defense used against oxidative stress and to maintain redox homeostasis in thalassemic patients based on the significant correlations between the GSH/GSSH ratio and degree of anemia or body iron stores. PMID:24223032

  11. Quantitation of protein S-glutathionylation by liquid chromatograph-tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein-glutathione mixed disulfides (PSSG) are commonly quantified by the reduction of the disulfide and detection of the resultant glutathione species. This met...

  12. Protective role of intracellular glutathione against ethanol-induced damage in cultured rat gastric mucosal cells

    SciTech Connect

    Mutoh, H.; Hiraishi, H.; Ota, S.; Yoshida, H.; Ivey, K.J.; Terano, A.; Sugimoto, T. )

    1990-06-01

    This study investigated whether intracellular glutathione is cytoprotective against ethanol-induced injury to cultured rat gastric mucosal cells in vitro. Secondly, it investigated whether reduced glutathione or oxidized glutathione is responsible for this cytoprotection. Cytolysis was quantified by measuring 51Cr release from prelabeled cells. Concentrations of ethanol greater than 12% caused cell damage and increased 51Cr release in a dose-dependent and time-related fashion. When a substrate for glutathione synthesis, N-acetyl-L-cysteine, was provided to cultured cells for 4 h before challenge with ethanol, cytolysis was significantly decreased corresponding with an increase in cellular glutathione content. Pretreatment with diethyl maleate, which depletes reduced glutathione without forming oxidized glutathione, potentiated ethanol-induced cell damage in a dose-dependent manner with the decrease of cellular glutathione content. The administration of tert-butyl hydroperoxide (which is specifically reduced by glutathione peroxidase to generate oxidized glutathione from reduced glutathione) or diamide (which nonenzymatically oxidizes reduced glutathione to oxidized glutathione) enhanced ethanol injury. We conclude that in cultured gastric mucosal cells, (a) intracellular glutathione maintains integrity of gastric mucosal cells against ethanol in vitro; and (b) reduced glutathione rather than oxidized glutathione is responsible for this cytoprotection. We postulate that the presence of reduced glutathione is essential to allow glutathione peroxidase to catalyze the ethanol-generated toxic oxygen radical, hydrogen peroxide.

  13. Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

    PubMed

    Okada, Kazushi; Fukui, Masayuki; Zhu, Bao-Ting

    2016-08-26

    Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity. PMID:27317486

  14. Fluorescein-labeled glutathione to study protein S-glutathionylation.

    PubMed

    Landino, Lisa M; Brown, Carolyn M; Edson, Carolyn A; Gilbert, Laura J; Grega-Larson, Nathan; Wirth, Anna Jean; Lane, Kelly C

    2010-07-01

    Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol-containing proteins to yield fluorescent proteins. PMID:20156418

  15. Saving the zone of stasis: is glutathione effective?

    PubMed

    Zor, Fatih; Ozturk, Serdar; Deveci, Mustafa; Karacalioglu, Ozgur; Sengezer, Mustafa

    2005-12-01

    One of the main subjects that burn researches are focused on is saving the zone of stasis. There are many molecules that are used for this purpose, but all have their drawbacks. Glutathione is one of the major buffer molecules of the cells and is known to increase the thermo-resistance of the cells. In this study, the effect of the systemic glutathione on the zone of stasis was evaluated. The results showed that glutathione is an effective molecule for saving the zone of stasis. It is well-known cheap, and easy to use. PMID:16278048

  16. The Genetic Architecture of Murine Glutathione Transferases

    PubMed Central

    Lu, Lu; Pandey, Ashutosh K.; Houseal, M. Trevor; Mulligan, Megan K.

    2016-01-01

    Glutathione S-transferase (GST) genes play a protective role against oxidative stress and may influence disease risk and drug pharmacokinetics. In this study, massive multiscalar trait profiling across a large population of mice derived from a cross between C57BL/6J (B6) and DBA2/J (D2)—the BXD family—was combined with linkage and bioinformatic analyses to characterize mechanisms controlling GST expression and to identify downstream consequences of this variation. Similar to humans, mice show a wide range in expression of GST family members. Variation in the expression of Gsta4, Gstt2, Gstz1, Gsto1, and Mgst3 is modulated by local expression QTLs (eQTLs) in several tissues. Higher expression of Gsto1 in brain and liver of BXD strains is strongly associated (P < 0.01) with inheritance of the B6 parental allele whereas higher expression of Gsta4 and Mgst3 in brain and liver, and Gstt2 and Gstz1 in brain is strongly associated with inheritance of the D2 parental allele. Allele-specific assays confirmed that expression of Gsto1, Gsta4, and Mgst3 are modulated by sequence variants within or near each gene locus. We exploited this endogenous variation to identify coexpression networks and downstream targets in mouse and human. Through a combined systems genetics approach, we provide new insight into the biological role of naturally occurring variants in GST genes. PMID:26829228

  17. The content of glutathione and glutathione S-transferases and the glutathione peroxidase activity in rat liver nuclei determined by a non-aqueous technique of cell fractionation.

    PubMed Central

    Soboll, S; Gründel, S; Harris, J; Kolb-Bachofen, V; Ketterer, B; Sies, H

    1995-01-01

    Hepatocellular nuclei require glutathione, glutathione S-transferases (GSTs) and Se-dependent glutathione peroxidase (GPx) for intranuclear protection against damage from electrophiles or products of active oxygen. Data so far available from the literature on nuclei isolated in aqueous systems range from glutathione, GSTs and GPx either being absent altogether to being present in quantities in excess of those in the cytoplasm. This paper describes a small-scale preparation of a nuclear fraction from rat liver by a non-aqueous technique, designed to retain nuclear water-soluble molecules in situ, since low-molecular-mass compounds can diffuse freely into other compartments during aqueous separation. This non-aqueous procedure shows the nucleus to contain glutathione at 8.4 mM and soluble GSTs at 38 micrograms/mg of protein, the enrichment over the homogenate being 1.2-1.4-fold. Se-dependent GPx activity was also present in the nucleus (56 m-units/mg), although with slightly lower activity than in the homogenate (0.7-fold). Images Figure 1 PMID:7487946

  18. Chronic Arsenic Exposure and Blood Glutathione and Glutathione Disulfide Concentrations in Bangladeshi Adults

    PubMed Central

    Hall, Megan N.; Niedzwiecki, Megan; Liu, Xinhua; Harper, Kristin N.; Alam, Shafiul; Slavkovich, Vesna; Ilievski, Vesna; Levy, Diane; Siddique, Abu B.; Parvez, Faruque; Mey, Jacob L.; van Geen, Alexander; Graziano, Joseph

    2013-01-01

    Background: In vitro and rodent studies have shown that arsenic (As) exposure can deplete glutathione (GSH) and induce oxidative stress. GSH is the primary intracellular antioxidant; it donates an electron to reactive oxygen species, thus producing glutathione disulfide (GSSG). Cysteine (Cys) and cystine (CySS) are the predominant thiol/disulfide redox couple found in human plasma. Arsenic, GSH, and Cys are linked in several ways: a) GSH is synthesized via the transsulfuration pathway, and Cys is the rate-limiting substrate; b) intermediates of the methionine cycle regulate both the transsulfuration pathway and As methylation; c) GSH serves as the electron donor for reduction of arsenate to arsenite; and d) As has a high affinity for sulfhydryl groups and therefore binds to GSH and Cys. Objectives: We tested the hypothesis that As exposure is associated with decreases in GSH and Cys and increases in GSSG and CySS (i.e., a more oxidized environment). Methods: For this cross-sectional study, the Folate and Oxidative Stress Study, we recruited a total of 378 participants from each of five water As concentration categories: < 10 (n = 76), 10–100 (n = 104), 101–200 (n = 86), 201–300 (n = 67), and > 300 µg/L (n = 45). Concentrations of GSH, GSSG, Cys, and CySS were measured using HPLC. Results: An interquartile range (IQR) increase in water As was negatively associated with blood GSH (mean change, –25.4 µmol/L; 95% CI: –45.3, –5.31) and plasma CySS (mean change, –3.00 µmol/L; 95% CI: –4.61, –1.40). We observed similar associations with urine and blood As. There were no significant associations between As exposure and blood GSSG or plasma Cys. Conclusions: The observed associations are consistent with the hypothesis that As may influence concentrations of GSH and other nonprotein sulfhydryls through binding and irreversible loss in bile and/or possibly in urine. Citation: Hall MN, Niedzwiecki M, Liu X, Harper KN, Alam S, Slavkovich V, Ilievski V, Levy

  19. Hemoglobin-catalyzed fluorometric method for the determination of glutathione

    NASA Astrophysics Data System (ADS)

    Wang, Ruiqiang; Tang, Lin; Li, Hua; Wang, Yi; Gou, Rong; Guo, Yuanyuan; Fang, Yudong; Chen, Fengmei

    2016-01-01

    A new spectrofluorometric method for the determination of glutathione based on the reaction catalyzed by hemoglobin was reported. The reaction product gave a highly fluorescent intensity with the excitation and emission wavelengths of 320.0 nm and 413.0 nm, respectively. The optimum experimental conditions were investigated. Results showed that low concentration glutathione enhanced the fluorescence intensity significantly. The line ranges were 1.0 × 10-6-1.0 × 10-5 mol L-1 of glutathione and 6.0 × 10-10 mol L-1-1.0 × 10-8 mol L-1, respectively. The detection limit was calculated to be 1.1 × 10-11 mol L-1. The recovery test by the standard addition method gave values in the range of 90.78%-102.20%. This method was used for the determination of glutathione in synthetic and real samples with satisfactory results.

  20. Nanofiltration concentration of extracellular glutathione produced by engineered Saccharomyces cerevisiae.

    PubMed

    Sasaki, Kengo; Hara, Kiyotaka Y; Kawaguchi, Hideo; Sazuka, Takashi; Ogino, Chiaki; Kondo, Akihiko

    2016-01-01

    This study aimed to optimize extracellular glutathione production by a Saccharomyces cerevisiae engineered strain and to concentrate the extracellular glutathione by membrane separation processes, including ultrafiltration (UF) and nanofiltration (NF). Synthetic defined (SD) medium containing 20 g L(-1) glucose was fermented for 48 h; the fermentation liquid was passed through an UF membrane to remove macromolecules. Glutathione in this permeate was concentrated for 48 h to 545.1 ± 33.6 mg L(-1) using the NF membrane; this was a significantly higher concentration than that obtained with yeast extract peptone dextrose (YPD) medium following 96 h NF concentration (217.9 ± 57.4 mg L(-1)). This higher glutathione concentration results from lower cellular growth in SD medium (final OD600 = 6.9 ± 0.1) than in YPD medium (final OD600 = 11.0 ± 0.6) and thus higher production of extracellular glutathione (16.0 ± 1.3 compared to 9.2 ± 2.1 mg L(-1) in YPD medium, respectively). Similar fermentation and membrane processing of sweet sorghum juice containing 20 g L(-1) total sugars provided 240.3 ± 60.6 mg L(-1) glutathione. Increased extracellular production of glutathione by this engineered strain in SD medium and subsequent UF permeation and NF concentration in shortend time may help realize industrial recovery of extracellular glutathione. PMID:26105794

  1. Amodiaquine failure associated with erythrocytic glutathione in Plasmodium falciparum malaria

    PubMed Central

    Zuluaga, Lina; Pabón, Adriana; López, Carlos; Ochoa, Aleida; Blair, Silvia

    2007-01-01

    Objective To establish the relationship between production of glutathione and the therapeutic response to amodiaquine (AQ) monotherapy in Plasmodium falciparum non-complicated malaria patients. Methodology Therapeutic response to AQ was evaluated in 32 patients with falciparum malaria in two townships of Antioquia, Colombia, and followed-up for 28 days. For every patient, total glutathione and enzymatic activity (glutathione reductase, GR, and γ-glutamylcysteine synthetase, γ-GCS) were determined in parasitized erythrocytes, non-infected erythrocytes and free parasites, on the starting day (day zero, before ingestion of AQ) and on the day of failure (in case of occurrence). Results There was found an AQ failure of 31.25%. Independent of the therapeutic response, on the starting day and on the day of failure, lower total glutathione concentration and higher GR activities in parasitized erythrocytes were found, compared with non-infected erythrocytes (p < 0.003). In addition, only on the day of failure, γ-GCS activity of parasitized erythrocytes was higher, compared with that of healthy erythrocytes (p = 0.01). Parasitized and non-parasitized erythrocytes in therapeutic failure patients (TF) had higher total glutathione on the starting day compared with those of adequate clinical response (ACR) (p < 0.02). Parasitized erythrocytes of TF patients showed lower total glutathione on the failure day, compared with starting day (p = 0.017). No differences was seen in the GR and γ-GCS activities by compartment, neither between the two therapeutic response groups nor between the two treatment days. Conclusion This study is a first approach to explaining P. falciparum therapeutic failure in humans through differences in glutathione metabolism in TF and ACR patients. These results suggest a role for glutathione in the therapeutic failure to antimalarials. PMID:17451604

  2. Evaluation of the antibiotic properties of glutathione.

    PubMed

    Schairer, David O; Chouake, Jason S; Kutner, Allison J; Makdisi, Joy; Nosanchuk, Josh D; Friedman, Adam J

    2013-11-01

    Skin and soft tissue infections (SSTIs) are growing in prevalence in both the outpatient and inpatient settings and are some of the most common diseases seen by dermatologists, who are often the first point of care for these patients. Microbial resistance to antibiotics continues to rise as more virulent strains evolve, and strains predominantly found in the hospital setting are now being seen in the community. Therefore, innovative approaches to combat this trend are needed. Glutathione (GSH) is a well-described and established antioxidant. It participates in detoxification of xenobiotics, regulation of cellular growth, modulation of immune response, and maintenance of the thiol status of proteins and cellular cysteine levels. GSH is also known to have a regulatory effect on immune cells and even inherent antibacterial properties have been reported. To this end, the value of GSH as an antibiotic was evaluated by growing methicillin resistant S. aureus, E. coli, K. pneumoniae and P. aeruginosa strains isolated from human skin and soft tissue infection in the presence of GSH. At a physiologic concentration of 10 mM, GSH had no effect on bacterial growth. At concentrations above 50 mM, which created acidic conditions (pH < 4), bacterial growth was completely inhibited. When adjusted to physiologic pH, GSH exhibited a bacteriostatic effect in a concentration-dependent manner. Additionally, the cytotoxicity of GSH was evaluated in a murine cell line. GSH was relatively non-toxic to murine macrophages, even at the highest concentration tested (160 mM). These results suggest the potential utility of GSH for the prevention and/or as adjunctive treatment of infection, most significantly in disease states associated with GSH deficiency. PMID:24196336

  3. Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase-Glutathione Interaction.

    PubMed

    Brenke, Jara K; Salmina, Elena S; Ringelstetter, Larissa; Dornauer, Scarlett; Kuzikov, Maria; Rothenaigner, Ina; Schorpp, Kenji; Giehler, Fabian; Gopalakrishnan, Jay; Kieser, Arnd; Gul, Sheraz; Tetko, Igor V; Hadian, Kamyar

    2016-07-01

    In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni(2+)-NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti-GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results. PMID:27044684

  4. Allyl isothiocyanate depletes glutathione and upregulates expression of glutathione S-transferases in Arabidopsis thaliana

    PubMed Central

    Øverby, Anders; Stokland, Ragni A.; Åsberg, Signe E.; Sporsheim, Bjørnar; Bones, Atle M.

    2015-01-01

    Allyl isothiocyanate (AITC) is a phytochemical associated with plant defense in plants from the Brassicaceae family. AITC has long been recognized as a countermeasure against external threats, but recent reports suggest that AITC is also involved in the onset of defense-related mechanisms such as the regulation of stomatal aperture. However, the underlying cellular modes of action in plants remain scarcely investigated. Here we report evidence of an AITC-induced depletion of glutathione (GSH) and the effect on gene expression of the detoxification enzyme family glutathione S-transferases (GSTs) in Arabidopsis thaliana. Treatment of A. thaliana wild-type with AITC resulted in a time- and dose-dependent depletion of cellular GSH. AITC-exposure of mutant lines vtc1 and pad2-1 with elevated and reduced GSH-levels, displayed enhanced and decreased AITC-tolerance, respectively. AITC-exposure also led to increased ROS-levels in the roots and loss of chlorophyll which are symptoms of oxidative stress. Following exposure to AITC, we found that GSH rapidly recovered to the same level as in the control plant, suggesting an effective route for replenishment of GSH or a rapid detoxification of AITC. Transcriptional analysis of genes encoding GSTs showed an upregulation in response to AITC. These findings demonstrate cellular effects by AITC involving a reversible depletion of the GSH-pool, induced oxidative stress, and elevated expression of GST-encoding genes. PMID:25954298

  5. Changes in biosynthesis and metabolism of glutathione upon ochratoxin A stress in Arabidopsis thaliana.

    PubMed

    Wang, Yan; Zhao, Weiwei; Hao, Junran; Xu, Wentao; Luo, Yunbo; Wu, Weihong; Yang, Zhuojun; Liang, Zhihong; Huang, Kunlun

    2014-06-01

    Ochratoxin A (OTA) is one of the most toxic mycotoxins, which is toxic to plants and simulates oxidative stress. Glutathione is an important antioxidant in plants and is closely associated with detoxification in cells. We have previously shown that OTA exposure induces obvious expression differences in genes associated with glutathione metabolism. To characterize glutathione metabolism and understand its role in OTA phytotoxicity, we observed the accumulation of GSH in the detached leaves of Arabidopsis thaliana under OTA treatment. OTA stimulated a defense response through enhancing glutathione-S-transferase, glutathione peroxidase, glutathione reductase activities, and the transcript levels of these enzymes were increased to maintain the total glutathione content. Moreover, the level of oxidized glutathione (GSSG) was increased and the ascorbate-glutathione cycle fluctuated in response to OTA. The depletion of glutathione using buthionine sulfoximine (BSO, inhibitor of glutamate-cysteine ligase) had no profound effect on OTA toxicity, as glutathione was regenerated through the ascorbate-glutathione cycle to maintain the total glutathione content. The ROS, MDA and GSH accumulation was significantly affected in the mutant gsh1, gr1 and gpx2 after treatment with OTA, which indicated that glutathione metabolism is directly involved in the oxidative stress response of Arabidopsis thaliana subjected to OTA. In conclusion, date demonstrate that glutathione-associated metabolism is closely related with OTA stress and glutathione play a role in resistance of Arabidopsis subjected to OTA. PMID:24662377

  6. EFFECT OF EXOGENOUS GLUTATHIONE, GLUTATHIONE REDUCTASE, CHLORINE DIOXIDE, AND CHLORITE ON OSMOTIC FRAGILITY OF RAT BLOOD IN VITRO

    EPA Science Inventory

    Chlorine dioxide (ClO2), chlorite (ClO2(-1)), and chlorate (ClO3(-1)) in drinking water decreased blood glutathione and RBC osmotic fragility in vivo. The osmotic fragility and glutathione content were also studied in rat blood treated with ClO2, ClO2(-1), ClO3(-1) in vitro. RBC ...

  7. Methylmercury alters glutathione homeostasis by inhibiting glutaredoxin 1 and enhancing glutathione biosynthesis in cultured human astrocytoma cells.

    PubMed

    Robitaille, Stephan; Mailloux, Ryan J; Chan, Hing Man

    2016-08-10

    Methylmercury (MeHg) is a neurotoxin that binds strongly to thiol residues on protein and low molecular weight molecules like reduced glutathione (GSH). The mechanism of its effects on GSH homeostasis particularly at environmentally relevant low doses is not fully known. We hypothesized that exposure to MeHg would lead to a depletion of reduced glutathione (GSH) and an accumulation of glutathione disulfide (GSSG) leading to alterations in S-glutathionylation of proteins. Our results showed exposure to low concentrations of MeHg (1μM) did not significantly alter GSH levels but increased GSSG levels by ∼12-fold. This effect was associated with a significant increase in total cellular glutathione content and a decrease in GSH/GSSG. Immunoblot analyses revealed that proteins involved in glutathione synthesis were upregulated accounting for the increase in cellular glutathione. This was associated an increase in cellular Nrf2 protein levels which is required to induce the expression of antioxidant genes in response to cellular stress. Intriguingly, we noted that a key enzyme involved in reversing protein S-glutathionylation and maintaining glutathione homeostasis, glutaredoxin-1 (Grx1), was inhibited by ∼50%. MeHg treatment also increased the S-glutathionylation of a high molecular weight protein. This observation is consistent with the inhibition of Grx1 and elevated H2O2 production however; contrary to our original hypothesis we found few S-glutathionylated proteins in the astrocytoma cells. Collectively, MeHg affects multiple arms of glutathione homeostasis ranging from pool management to protein S-glutathionylation and Grx1 activity. PMID:27180086

  8. The mitochondrial dicarboxylate and 2-oxoglutarate carriers do not transport glutathione

    PubMed Central

    Booty, Lee M.; King, Martin S.; Thangaratnarajah, Chancievan; Majd, Homa; James, Andrew M.; Kunji, Edmund R.S.; Murphy, Michael P.

    2015-01-01

    Glutathione carries out vital protective roles within mitochondria, but is synthesised in the cytosol. Previous studies have suggested that the mitochondrial dicarboxylate and 2-oxoglutarate carriers were responsible for glutathione uptake. We set out to characterise the putative glutathione transport by using fused membrane vesicles of Lactococcus lactis overexpressing the dicarboxylate and 2-oxoglutarate carriers. Although transport of the canonical substrates could be measured readily, an excess of glutathione did not compete for substrate uptake nor could transport of glutathione be measured directly. Thus these mitochondrial carriers do not transport glutathione and the identity of the mitochondrial glutathione transporter remains unknown. PMID:25637873

  9. The mitochondrial dicarboxylate and 2-oxoglutarate carriers do not transport glutathione.

    PubMed

    Booty, Lee M; King, Martin S; Thangaratnarajah, Chancievan; Majd, Homa; James, Andrew M; Kunji, Edmund R S; Murphy, Michael P

    2015-02-27

    Glutathione carries out vital protective roles within mitochondria, but is synthesised in the cytosol. Previous studies have suggested that the mitochondrial dicarboxylate and 2-oxoglutarate carriers were responsible for glutathione uptake. We set out to characterise the putative glutathione transport by using fused membrane vesicles of Lactococcus lactis overexpressing the dicarboxylate and 2-oxoglutarate carriers. Although transport of the canonical substrates could be measured readily, an excess of glutathione did not compete for substrate uptake nor could transport of glutathione be measured directly. Thus these mitochondrial carriers do not transport glutathione and the identity of the mitochondrial glutathione transporter remains unknown. PMID:25637873

  10. Cadmium(II) complex formation with glutathione.

    PubMed

    Mah, Vicky; Jalilehvand, Farideh

    2010-03-01

    Complex formation between heavy metal ions and glutathione (GSH) is considered as the initial step in many detoxification processes in living organisms. In this study the structure and coordination between the cadmium(II) ion and GSH were investigated in aqueous solutions (pH 7.5 and 11.0) and in the solid state, using a combination of spectroscopic techniques. The similarity of the Cd K-edge and L(3)-edge X-ray absorption spectra of the solid compound [Cd(GS)(GSH)]ClO(4).3H(2)O, precipitating at pH 3.0, with the previously studied cysteine compound {Cd(HCys)(2).H(2)O}(2).H(3)O(+).ClO(4) (-) corresponds to Cd(S-GS)(3)O (dominating) and Cd(S-GS)(4) four-coordination within oligomeric complexes with mean bond distances of 2.51 +/- 0.02 A for Cd-S and 2.24 +/- 0.04 A for Cd-O. For cadmium(II) solutions (C (Cd(II)) approximately 0.05 M) at pH 7.5 with moderate excess of GSH (C (GSH)/C (Cd(II)) = 3.0-5.0), a mix of Cd(S-GS)(3)O (dominating) and Cd(S-GS)(4) species is consistent with the broad (113)Cd NMR resonances in the range 632-658 ppm. In alkaline solutions (pH 11.0 and C (GSH)/C (Cd(II)) = 2.0 or 3.0), two distinct peaks at 322 and 674 ppm are obtained. The first peak indicates six-coordinated mononuclear and dinuclear complexes with CdS(2)N(2)(N/O)(2) and CdSN(3)O(2) coordination in fast exchange, whereas the second corresponds to Cd(S-GS)(4) sites. At high ligand excess the tetrathiolate complex, Cd(S-GS)(4), characterized by a sharp delta((113)Cd) NMR signal at 677 ppm, predominates. The average Cd-S distance, obtained from the X-ray absorption spectra, varied within a narrow range, 2.49-2.53 A, for all solutions (pH 7.5 and 11.0) regardless of the coordination geometry. PMID:20035360

  11. Lead(II) complex formation with glutathione.

    PubMed

    Mah, Vicky; Jalilehvand, Farideh

    2012-06-01

    A structural investigation of complexes formed between the Pb(2+) ion and glutathione (GSH, denoted AH(3) in its triprotonated form), the most abundant nonprotein thiol in biological systems, was carried out for a series of aqueous solutions at pH 8.5 and C(Pb(2+)) = 10 mM and in the solid state. The Pb L(III)-edge extended X-ray absorption fine structure (EXAFS) oscillation for a solid compound with the empirical formula [Pb(AH(2))]ClO(4) was modeled with one Pb-S and two short Pb-O bond distances at 2.64 ± 0.04 and 2.28 ± 0.04 Å, respectively. In addition, Pb···Pb interactions at 4.15 ± 0.05 Å indicate dimeric species in a network where the thiolate group forms an asymmetrical bridge between two Pb(2+) ions. In aqueous solution at the mole ratio GSH/Pb(II) = 2.0 (C(Pb(2+)) = 10 mM, pH 8.5), lead(II) complexes with two thiolate ligands form, characterized by a ligand-to-metal charge-transfer band (LMCT) S(-) → Pb(2+) at 317 nm in the UV-vis spectrum and mean Pb-S and Pb-(N/O) bond distances of 2.65 ± 0.04 and 2.51 ± 0.04 Å, respectively, from a Pb L(III)-edge EXAFS spectrum. For solutions with higher mole ratios, GSH/Pb(II) ≥ 3.0, electrospray ionization mass spectroscopy spectra identified a triglutathionyllead(II) complex, for which Pb L(III)-edge EXAFS spectroscopy shows a mean Pb-S distance of 2.65 ± 0.04 Å in PbS(3) coordination, (207)Pb NMR spectroscopy displays a chemical shift of 2793 ppm, and in the UV-vis spectrum, an S(-) → Pb(2+) LMCT band appears at 335 nm. The complex persists at high excess of GSH and also at ∼25 K in frozen glycerol (33%)/water glasses for GSH/Pb(II) mole ratios from 4.0 to 10 (C(Pb(2+)) = 10 mM) measured by Pb L(III)-edge EXAFS spectroscopy. PMID:22594853

  12. Do glutathione levels decline in aging human brain?

    PubMed

    Tong, Junchao; Fitzmaurice, Paul S; Moszczynska, Anna; Mattina, Katie; Ang, Lee-Cyn; Boileau, Isabelle; Furukawa, Yoshiaki; Sailasuta, Napapon; Kish, Stephen J

    2016-04-01

    For the past 60 years a major theory of "aging" is that age-related damage is largely caused by excessive uncompensated oxidative stress. The ubiquitous tripeptide glutathione is a major antioxidant defense mechanism against reactive free radicals and has also served as a marker of changes in oxidative stress. Some (albeit conflicting) animal data suggest a loss of glutathione in brain senescence, which might compromise the ability of the aging brain to meet the demands of oxidative stress. Our objective was to establish whether advancing age is associated with glutathione deficiency in human brain. We measured reduced glutathione (GSH) levels in multiple regions of autopsied brain of normal subjects (n=74) aged one day to 99 years. Brain GSH levels during the infancy/teenage years were generally similar to those in the oldest examined adult group (76-99 years). During adulthood (23-99 years) GSH levels remained either stable (occipital cortex) or increased (caudate nucleus, frontal and cerebellar cortices). To the extent that GSH levels represent glutathione antioxidant capacity, our postmortem data suggest that human brain aging is not associated with declining glutathione status. We suggest that aged healthy human brains can maintain antioxidant capacity related to glutathione and that an age-related increase in GSH levels in some brain regions might possibly be a compensatory response to increased oxidative stress. Since our findings, although suggestive, suffer from the generic limitations of all postmortem brain studies, we also suggest the need for "replication" investigations employing the new (1)H MRS imaging procedures in living human brain. PMID:26845616

  13. Glutathione deficiency down-regulates hepatic lipogenesis in rats

    PubMed Central

    2010-01-01

    Background Oxidative stress is supposed to increase lipid accumulation by stimulation of hepatic lipogenesis at transcriptional level. This study was performed to investigate the role of glutathione in the regulation of this process. For that purpose, male rats were treated with buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase, for 7 days and compared with untreated control rats. Results BSO treatment caused a significant reduction of total glutathione in liver (-70%), which was attributable to diminished levels of reduced glutathione (GSH, -71%). Glutathione-deficient rats had lower triglyceride concentrations in their livers than the control rats (-23%), whereas the circulating triglycerides and the cholesterol concentrations in plasma and liver were not different between the two groups of rats. Livers of glutathione-deficient rats had lower mRNA abundance of sterol regulatory element-binding protein (SREBP)-1c (-47%), Spot (S)14 (-29%) and diacylglycerol acyltransferase 2 (DGAT-2, -27%) and a lower enzyme activity of fatty acid synthase (FAS, -26%) than livers of the control rats. Glutathione-deficient rats had also a lower hepatic activity of the redox-sensitive protein-tyrosine phosphatase (PTP)1B, and a higher concentration of irreversible oxidized PTP1B than control rats. No differences were observed in protein expression of total PTP1B and the mature mRNA encoding active XBP1s, a key regulator of unfolded protein and ER stress response. Conclusion This study shows that glutathione deficiency lowers hepatic triglyceride concentrations via influencing lipogenesis. The reduced activity of PTP1B and the higher concentration of irreversible oxidized PTP1B could be, at least in part, responsible for this effect. PMID:20482862

  14. Role of glutathione and glutathione S-transferases in the metabolism of busulfan

    SciTech Connect

    Marchand, D.H.

    1987-01-01

    Busulfan (1,4-dimethanesulfonate butanediol) is a bifunctional alkylating agent used in the treatment of chronic myelogenous leukemia. The major urinary metabolite of busulfan in mammals is 3-hydroxytetrahydrothiophene-1,1-dioxide (3-HOTHT). Previous metabolic studies with /sup 35/S-busulfan indicated that the sulfur in 3-HOTHT results from the reaction of busulfan with endogenous thiols. These studies also found that mixing L-cysteine with busulfan at pH 8.0 produced a tetrahydrothiophenium-cysteine conjugate (THT-cys). Treatment of THT-cys with sodium hydroxide produced tetrahydrothiophene (THT). Administration of THT-cyc or THT to rats resulted in the appearance of 3-HOTHT in rat urine. Glutathione (GSH) is the major non-protein thiol in cells. As part of the present studies, the reaction between GSH and busulfan was examined.

  15. Hepatic glutathione and glutathione S-transferase in selenium deficiency and toxicity in the chick

    SciTech Connect

    Kim, Y. S.

    1989-01-01

    First, the hepatic activity of GSH-T{sub CDNB} was increased only under conditions of severe oxidative stress produced by combined Se- and vitamin E (VE)-deficiency, indicating that VE also affects GSH metabolism. Second, the incorporation of {sup 35}S-methionine into GSH and protein was about 4- and 2-fold higher, respectively, in Se- and VE-deficient chick hepatocytes as compared to controls. Third, chicks injected with the glutathione peroxidase (SeGSHpx) inhibitor, aurothioglucose (AuTG), showed increase hepatic GSH-T{sub CDNB} activity and plasma GSH concentration regardless of their Se status. Fourth, the effect of ascorbic acid (AA), on GSH metabolism was studied. Chicks fed 1000 ppm AA showed decreased hepatic GSH concentration compared to chicks fed no AA in a Se- and VE-deficient diet. Fifth, chicks fed excess Se showed increase hepatic activity of GSH-T{sub CDNB} and GSH concentration regardless of VE status.

  16. Activity of glutathione peroxidase, glutathione reductase, and lipid peroxidation in erythrocytes in workers exposed to lead.

    PubMed

    Kasperczyk, Slawomir; Kasperczyk, Aleksandra; Ostalowska, Alina; Dziwisz, Maria; Birkner, Ewa

    2004-01-01

    The aim of this study was to estimate the activity of glutathione peroxidase (GPx), glutathione reductase (GR), and malondialdehyde (MDA) in erythrocytes in healthy male employees of zinc and lead steelworks who were occupationally exposed to lead over a long period of time (about 15 yr). Workers were divided into two subgroups: the first included employees with low exposure to lead (LL) (n=75) with blood lead level PbB=25-40 microg/dL and the second with high exposure to lead (HL) (n=62) with PbB over 40 microg/dL. Administration workers (n=35) with normal levels of PbB and zinc protoporphyrin in blood (ZPP) in blood were the control group. The activity of GPx significantly increased in LL when compared to the control group (p<0.001) and decreased when compared to the HL group (p=0.036). There were no significant changes in activity of GR in the study population. MDA erythrocyte concentration significantly increased in the HL group compared to the control (p=0.014) and to the LL group (p=0.024). For the people with low exposure to lead (PbB=25-40 microg/dL), the increase of activity of GPx by about 79% in erythrocytes prevented lipid peroxidation and it appears to be the adaptive mechanism against the toxic effect of lead. People with high exposure to lead (with PbB over 40 microg/dL) have shown an increase in MDA concentration in erythrocytes by about 91%, which seems to have resulted from reduced activity of GPx and the lack of increase in activity of GR in blood red cells. PMID:15621928

  17. Voltammetric detection of glutathione: an adsorptive stripping voltammetry approach.

    PubMed

    Areias, Madalena C C; Shimizu, Kenichi; Compton, Richard G

    2016-05-10

    A simple, sensitive, and rapid detection of glutathione by cyclic voltammetry using a bare glassy carbon electrode is reported in which glutathione forms a 1 : 1 complex compound with copper(ii) ions. This complex compound is adsorbed onto the electrode surface and undergoes electrochemical oxidation at a characteristic oxidation potential of ca. -0.20 V vs. the standard mercury/mercurous sulphate reference electrode, which is used to detect the glutathione concentration. The linear dynamic range is obtained for a glutathione concentration from 1 μM to 12.5 μM, and the sensitivity is found to be 0.1 ± 0.002 μA μM(-1). A low limit of detection (n = 3) of 0.14 μM and a precision of 1.8% are achieved using a simple, unmodified electrode. The robustness of the present methodology is demonstrated by the successful quantitative analysis of glutathione in the presence of cysteine. PMID:27074944

  18. Glutathione is required for efficient production of infectious picornavirus virions

    SciTech Connect

    Smith, Allen D. . E-mail: smitha@ba.ars.usda.gov; Dawson, Harry . E-mail: dawsonh@ba.ars.usda.gov

    2006-09-30

    Glutathione is an intracellular reducing agent that helps maintain the redox potential of the cell and is important for immune function. The drug L-buthionine sulfoximine (BSO) selectively inhibits glutathione synthesis. Glutathione has been reported to block replication of HIV, HSV-1, and influenza virus, whereas cells treated with BSO exhibit increased replication of Sendai virus. Pre-treatment of HeLa cell monolayers with BSO inhibited replication of CVB3, CVB4, and HRV14 with viral titers reduced by approximately 6, 5, and 3 log{sub 1}, respectively. The addition of glutathione ethyl ester, but not dithiothreitol or 2-mercaptoethanol, to the culture medium reversed the inhibitory effect of BSO. Viral RNA and protein synthesis were not inhibited by BSO treatment. Fractionation of lysates from CVB3-infected BSO-treated cells on cesium chloride and sucrose gradients revealed that empty capsids but not mature virions were being produced. The levels of the 5S and 14S assembly intermediates, however, were not affected by BSO treatment. These results demonstrate that glutathione is important for production of mature infectious picornavirus virions.

  19. Thioltransferase activity of bovine lens glutathione S-transferase.

    PubMed Central

    Dal Monte, M; Cecconi, I; Buono, F; Vilardo, P G; Del Corso, A; Mura, U

    1998-01-01

    A Mu-class glutathione S-transferase purified to electrophoretic homogeneity from bovine lens displayed thioltransferase activity, catalysing the transthiolation reaction between GSH and hydroxyethyldisulphide. The thiol-transfer reaction is composed of two steps, the formation of GSSG occurring through the generation of an intermediate mixed disulphide between GSH and the target disulphide. Unlike glutaredoxin, which is only able to catalyse the second step of the transthiolation process, glutathioneS-transferase catalyses both steps of the reaction. Data are presented showing that bovine lens glutathione S-transferase and rat liver glutaredoxin, which was used as a thioltransferase enzyme model, can operate in synergy to catalyse the GSH-dependent reduction of hydroxyethyldisulphide. PMID:9693102

  20. Glutathione Homeostasis and Functions: Potential Targets for Medical Interventions

    PubMed Central

    Lushchak, Volodymyr I.

    2012-01-01

    Glutathione (GSH) is a tripeptide, which has many biological roles including protection against reactive oxygen and nitrogen species. The primary goal of this paper is to characterize the principal mechanisms of the protective role of GSH against reactive species and electrophiles. The ancillary goals are to provide up-to-date knowledge of GSH biosynthesis, hydrolysis, and utilization; intracellular compartmentalization and interorgan transfer; elimination of endogenously produced toxicants; involvement in metal homeostasis; glutathione-related enzymes and their regulation; glutathionylation of sulfhydryls. Individual sections are devoted to the relationships between GSH homeostasis and pathologies as well as to developed research tools and pharmacological approaches to manipulating GSH levels. Special attention is paid to compounds mainly of a natural origin (phytochemicals) which affect GSH-related processes. The paper provides starting points for development of novel tools and provides a hypothesis for investigation of the physiology and biochemistry of glutathione with a focus on human and animal health. PMID:22500213

  1. Characterization of recombinant glutathione reductase from the psychrophilic Antarctic bacterium Colwellia psychrerythraea.

    PubMed

    Ji, Mikyoung; Barnwell, Callie V; Grunden, Amy M

    2015-07-01

    Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat/K m at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems. PMID:26101017

  2. Recurrent Isolated Neonatal Hemolytic Anemia: Think About Glutathione Synthetase Deficiency.

    PubMed

    Signolet, Isabelle; Chenouard, Rachel; Oca, Florine; Barth, Magalie; Reynier, Pascal; Denis, Marie-Christine; Simard, Gilles

    2016-09-01

    Hemolytic anemia (HA) of the newborn should be considered in cases of rapidly developing, severe, or persistent hyperbilirubinemia. Several causes of corpuscular hemolysis have been described, among which red blood cell enzyme defects are of particular concern. We report a rare case of red blood cell enzyme defect in a male infant, who presented during his first months of life with recurrent and isolated neonatal hemolysis. All main causes were ruled out. At 6.5 months of age, the patient presented with gastroenteritis requiring hospitalization; fortuitously, urine organic acid chromatography revealed a large peak of 5-oxoproline. Before the association between HA and 5-oxoprolinuria was noted, glutathione synthetase deficiency was suspected and confirmed by a low glutathione synthetase concentration and a collapse of glutathione synthetase activity in erythrocytes. Moreover, molecular diagnosis revealed 2 mutations in the glutathione synthetase gene: a previously reported missense mutation (c.[656A>G]; p.[Asp219Gly]) and a mutation not yet described in the binding site of the enzyme (c.[902T>C]; p.[Leu301Pro]). However, 15 days later, a control sample revealed no signs of 5-oxoprolinuria and the clinical history discovered administration of acetaminophen in the 48 hours before hospitalization. Thus, in this patient, acetaminophen exposure allowed the diagnosis of a mild form of glutathione synthetase deficiency, characterized by isolated HA. Early diagnosis is important because treatment with bicarbonate, vitamins C and E, and elimination of trigger factors are recommended to improve long-term outcomes. Glutathione synthetase deficiency should be screened for in cases of unexplained newborn HA. PMID:27581854

  3. Studies on glutathione S-alkyltransferase of the rat

    PubMed Central

    Johnson, M. K.

    1966-01-01

    1. A rat-liver enzyme catalysing the S-alkylation of glutathione by iodomethane and various other alkyl compounds has been identified and partially purified; its stability, specificity and response to inhibitors and activators and to changes in reaction pH have been studied. 2. The enzyme is distinct from glutathione S-aryltransferase, but both enzymes respond similarly to various inhibitors. 3. A similar enzyme has been found in the kidney and adrenal of rat and in the liver and kidney of numerous species. 4. The identity and the physiological role of the enzyme are discussed. PMID:5938663

  4. Effects of Elevated Cytosolic Glutathione Reductase Activity on the Cellular Glutathione Pool and Photosynthesis in Leaves under Normal and Stress Conditions.

    PubMed

    Foyer, C; Lelandais, M; Galap, C; Kunert, K J

    1991-11-01

    Tobacco (Nicotiana tabacum var Samsun) was transformed using the bacterial gor gene coding for the enzyme glutathione reductase. Transgenic plants were selected by their kanamycin resistence and expression of the bacterial gor gene. After separation by isoelectric focusing techniques, leaf extracts from transgenic plants having both native and bacterial glutathione reductase activity gave, in addition to the six bands of the native enzyme, two further closely running isoenzymes. These additional bands originating from the expression of the bacterial gor gene were nonchloroplastic. Leaves from transgenic plants had two- to 10-fold higher glutathione reductase activity than non-transgenic controls. The amount of extractable glutathione reductase activity obtained in transgenic plants was dependent on leaf age and the conditions to which leaves were exposed. Both light and exposure to methylviologen increased leaf glutathione reductase activity. Elevated levels of cytosolic glutathione reductase activity in transgenic plants had no effect on the amount or reduction state of the reduced glutathione/oxidized glutathione pool under optimal conditions or oxidative conditions induced by methylviologen. The glutathione pool was unaltered despite the oxidation-dependent loss of CO(2) assimilation and oxidation of enzymes involved in photosynthesis. However, the reduction state of the ascorbate pool was greater in transgenic plants relative to nontransgenic controls following illumination of methylviologen-treated leaf discs. Therefore, we conclude that in the natural state glutathione reductase is present in tobacco at levels above those required for maximal operation of the ascorbate-glutathione pathway. PMID:16668524

  5. Analysis of glutathione and glutathione disulfide in whole cells and mitochondria by postcolumn derivatization high-performance liquid chromatography with ortho-phthalaldehyde.

    PubMed

    Lenton, K J; Therriault, H; Wagner, J R

    1999-10-01

    A method is described for the detection of glutathione (GSH) and glutathione disulfide (GSSG) based on a HPLC postcolumn reaction with ortho-phthalaldehyde (OPT) at pH 12 followed by fluorescence detection. Although similar methods have been reported, the high pH of the postcolumn reaction adds considerable selectivity and sensitivity to the measurement of GSH and glutathione disulfide. The limit of detection approaches 100 fmol, which is sufficient to detect whole-cell glutathione disulfide in 10,000 cells or mitochondrial glutathione disulfide in 20 million cells. Using this method, glutathione and glutathione disulfide were measured in human lymphocytes, granulocytes, and cultured Jurkat T cells, as well as in the corresponding samples of mitochondria. The percentage of glutathione disulfide to total glutathione in whole-cell extracts was approximately 1%. In contrast, the percentage was relatively high in mitochondria, with the mitochondria of granulocytes having the highest (25%) followed by those of lymphocytes (15%) and finally by cultured Jurkat T cells (9%). This method extends the analysis of glutathione and glutathione disulfide to mitochondria obtained from a relatively small number of cells. PMID:10527505

  6. Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures.

    PubMed

    Giustarini, Daniela; Galvagni, Federico; Tesei, Anna; Farolfi, Alberto; Zanoni, Michele; Pignatta, Sara; Milzani, Aldo; Marone, Ilaria M; Dalle-Donne, Isabella; Nassini, Romina; Rossi, Ranieri

    2015-12-01

    The analysis of the global thiol-disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported. Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3-5 and 10-20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15-30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol-disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0-200 μM concentration range). Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000

  7. Involvement of glutathione and glutathione metabolizing enzymes in human colorectal cancer cell lines and tissues.

    PubMed

    Kim, Areum Daseul; Zhang, Rui; Han, Xia; Kang, Kyoung Ah; Piao, Mei Jing; Maeng, Young Hee; Chang, Weon Young; Hyun, Jin Won

    2015-09-01

    Reduced glutathione (GSH) is an abundant tripeptide present in the majority of cell types. GSH is highly reactive and is often conjugated to other molecules, via its sulfhydryl moiety. GSH is synthesized from glutamic acid, cysteine, and glycine via two sequential ATP‑consuming steps, which are catalyzed by glutamate cysteine ligase (GCL) and GSH synthetase (GSS). However, the role of GSH in cancer remains to be elucidated. The present study aimed to determine the levels of GSH and GSH synthetic enzymes in human colorectal cancer. The mRNA and protein expression levels of GSH, the catalytic subunit of GCL (GCLC) and GSS were significantly higher in the following five colon cancer cell lines: Caco‑2, SNU‑407, SNU‑1033, HCT‑116, and HT‑29, as compared with the normal colon cell line, FHC. Similarly, in 9 out of 15 patients with colon cancer, GSH expression levels were higher in tumor tissue, as compared with adjacent normal tissue. In addition, the protein expression levels of GCLC and GSS were higher in the tumor tissue of 8 out of 15, and 10 out of 15 patients with colon cancer respectively, as compared with adjacent normal tissue. Immunohistochemical analyses confirmed that GCLC and GSS were expressed at higher levels in colon cancer tissue, as compared with normal mucosa. Since GSH and GSH metabolizing enzymes are present at elevated levels in colonic tumors, they may serve as clinically useful biomarkers of colon cancer, and/or targets for anti-colon cancer drugs. PMID:26059756

  8. Mechanism-based biomarker gene sets for glutathione depletion-related hepatotoxicity in rats

    SciTech Connect

    Gao Weihua; Mizukawa, Yumiko; Nakatsu, Noriyuki; Minowa, Yosuke; Yamada, Hiroshi; Ohno, Yasuo; Urushidani, Tetsuro

    2010-09-15

    Chemical-induced glutathione depletion is thought to be caused by two types of toxicological mechanisms: PHO-type glutathione depletion [glutathione conjugated with chemicals such as phorone (PHO) or diethyl maleate (DEM)], and BSO-type glutathione depletion [i.e., glutathione synthesis inhibited by chemicals such as L-buthionine-sulfoximine (BSO)]. In order to identify mechanism-based biomarker gene sets for glutathione depletion in rat liver, male SD rats were treated with various chemicals including PHO (40, 120 and 400 mg/kg), DEM (80, 240 and 800 mg/kg), BSO (150, 450 and 1500 mg/kg), and bromobenzene (BBZ, 10, 100 and 300 mg/kg). Liver samples were taken 3, 6, 9 and 24 h after administration and examined for hepatic glutathione content, physiological and pathological changes, and gene expression changes using Affymetrix GeneChip Arrays. To identify differentially expressed probe sets in response to glutathione depletion, we focused on the following two courses of events for the two types of mechanisms of glutathione depletion: a) gene expression changes occurring simultaneously in response to glutathione depletion, and b) gene expression changes after glutathione was depleted. The gene expression profiles of the identified probe sets for the two types of glutathione depletion differed markedly at times during and after glutathione depletion, whereas Srxn1 was markedly increased for both types as glutathione was depleted, suggesting that Srxn1 is a key molecule in oxidative stress related to glutathione. The extracted probe sets were refined and verified using various compounds including 13 additional positive or negative compounds, and they established two useful marker sets. One contained three probe sets (Akr7a3, Trib3 and Gstp1) that could detect conjugation-type glutathione depletors any time within 24 h after dosing, and the other contained 14 probe sets that could detect glutathione depletors by any mechanism. These two sets, with appropriate scoring

  9. Glutathione-related factors are not correlated with sensitivity of human tumour cells to actinomycin D.

    PubMed

    Zhang, K; Yang, E B; Zhao, Y N; Wong, K P; Mack, P

    2000-02-28

    Glutathione (GSH) contents and activities of glutathione S-transferases (GST), glutathione reductase (GSH-RD), glutathione peroxidase (GSHpx) and glutathione conjugate export pump (GS-X pump) were determined in eight human tumour cell lines with different sensitivities to melphalan, a substrate of glutathione conjugation, and actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms. Chang liver cells with highest GSH content and highest activities of GST, GSH-RD, GSHpx and GS-X pump were found to be most resistant to melphalan. Statistical analysis showed significant correlations between sensitivities of the human tumour cells to melphalan and the glutathione-related factors (r = 0.72-0.79; except for GST, r = 0.65, P = 0.08), while there were no significant correlations observed between sensitivities of the human tumour cells to actinomycin D and all the glutathione-related factors tested (r = -0.25-0.14). Significant correlations of the glutathione-related factors to resistance of human tumour cells to melphalan, a substrate of glutathione conjugation, but not to resistance of the human tumour cells to actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms suggested that glutathione-related mechanisms contribute to drug resistance by increased detoxification of the drugs involved. PMID:10737727

  10. Oxidation contributes to low glutathione in the airways of children with cystic fibrosis.

    PubMed

    Kettle, Anthony J; Turner, Rufus; Gangell, Catherine L; Harwood, D Timothy; Khalilova, Irada S; Chapman, Anna L; Winterbourn, Christine C; Sly, Peter D

    2014-07-01

    Glutathione is an important antioxidant in the lungs but its concentration is low in the airways of patients with cystic fibrosis. Whether this deficit occurs from an early age or how oxidative stress contributes to lowering glutathione is unknown. We measured glutathione, its oxidation products, myeloperoxidase, and biomarkers of hypochlorous acid in bronchoalveolar lavage from children with cystic fibrosis and disease controls using mass spectrometry and immunological techniques. The concentration of glutathione was lower in bronchoalveolar lavage from children with cystic fibrosis, whereas glutathione sulfonamide, a specific oxidation product of hypochlorous acid, was higher. Oxidised glutathione and glutathione sulfonamide correlated with myeloperoxidase and a biomarker of hypochlorous acid. The percentage of glutathione attached to proteins was higher in children with cystic fibrosis than controls. Pulmonary infections in cystic fibrosis resulted in lower levels of glutathione but higher levels of oxidised glutathione and glutathione sulfonamide in bronchoalveolar lavage. The concentration of glutathione is low in the airways of patients with cystic fibrosis from an early age. Increased oxidation of glutathione by hypochlorous acid and its attachment to proteins contribute to this deficiency. Therapies targeted against myeloperoxidase may boost antioxidant defence and slow the onset and progression of lung disease in cystic fibrosis. PMID:24659542

  11. Balneotherapy and platelet glutathione metabolism in type II diabetic patients

    NASA Astrophysics Data System (ADS)

    Ohtsuka, Yoshinori; Yabunaka, Noriyuki; Watanabe, Ichiro; Noro, Hiroshi; Agishi, Yuko

    1996-09-01

    Effects of balneotherapy on platelet glutathione metabolism were investigated in 12 type II (non-insulin-dependent) diabetic patients. Levels of the reduced form of glutathione (GSH) on admission were well correlated with those of fasting plasma glucose (FPG; r=0.692, P<0.02). After 4 weeks of balneotherapy, the mean level of GSH showed no changes; however, in well-controlled patients (FPG <150 mg/dl), the level increased ( P<0.01) and in poorly controlled patients (FPG >150 mg/dl), the value decreased ( P<0.05). There was a negative correlation between glutathione peroxidase (GPX) activities and the levels of FPG ( r=-0.430, P<0.05). After balneotherapy, the activity increased in 5 patients, decreased in 3 patients and showed no changes (alteration within ±3%) in all the other patients. From these findings in diabetic patients we concluded: (1) platelet GSH synthesis appeared to be induced in response to oxidative stress; (2) lowered GPX activities indicated that the antioxidative defense system was impaired; and (3) platelet glutathione metabolism was partially improved by 4 weeks balneotherapy, an effect thought to be dependent on the control status of plasma glucose levels. It is suggested that balneotherapy is beneficial for patients whose platelet antioxidative defense system is damaged, such as those with diabetes mellitus and coronary heart disease.

  12. Cystamine induces AIF-mediated apoptosis through glutathione depletion.

    PubMed

    Cho, Sung-Yup; Lee, Jin-Haeng; Ju, Mi-kyeong; Jeong, Eui Man; Kim, Hyo-Jun; Lim, Jisun; Lee, Seungun; Cho, Nam-Hyuk; Park, Hyun Ho; Choi, Kihang; Jeon, Ju-Hong; Kim, In-Gyu

    2015-03-01

    Cystamine and its reduced form cysteamine showed protective effects in various models of neurodegenerative disease, including Huntington's disease and Parkinson's disease. Other lines of evidence demonstrated the cytotoxic effect of cysteamine on duodenal mucosa leading to ulcer development. However, the mechanism for cystamine cytotoxicity remains poorly understood. Here, we report a new pathway in which cystamine induces apoptosis by targeting apoptosis-inducing factor (AIF). By screening of various cell lines, we observed that cystamine and cysteamine induce cell death in a cell type-specific manner. Comparison between cystamine-sensitive and cystamine-resistant cell lines revealed that cystamine cytotoxicity is not associated with unfolded protein response, reactive oxygen species generation and transglutaminase or caspase activity; rather, it is associated with the ability of cystamine to trigger AIF nuclear translocation. In cystamine-sensitive cells, cystamine suppresses the levels of intracellular glutathione by inhibiting γ-glutamylcysteine synthetase expression that triggers AIF translocation. Conversely, glutathione supplementation completely prevents cystamine-induced AIF translocation and apoptosis. In rats, cysteamine administration induces glutathione depletion and AIF translocation leading to apoptosis of duodenal epithelium. These results indicate that AIF translocation through glutathione depletion is the molecular mechanism of cystamine toxicity, and provide important implications for cystamine in the neurodegenerative disease therapeutics as well as in the regulation of AIF-mediated cell death. PMID:25549939

  13. 21 CFR 862.1365 - Glutathione test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Glutathione test system. 862.1365 Section 862.1365 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  14. 21 CFR 862.1365 - Glutathione test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Glutathione test system. 862.1365 Section 862.1365 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  15. 21 CFR 862.1365 - Glutathione test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione test system. 862.1365 Section 862.1365 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  16. 21 CFR 862.1365 - Glutathione test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Glutathione test system. 862.1365 Section 862.1365 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  17. 21 CFR 862.1365 - Glutathione test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Glutathione test system. 862.1365 Section 862.1365 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  18. METAL-INDUCED INHIBITION OF GLUTATHIONE S-TRANSFERASES

    EPA Science Inventory

    The glutathione S-transferases comprise a group of multi-functional enzymes involved in the biotransformation/detoxication of a broad spectrum of hydrophobic compounds bearing an electrophilic center. The enzymes facilitate the nucleophilic attack of the -SH group of reduced glut...

  19. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  20. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  1. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  2. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  3. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  4. REACTION OF BENZENE OXIDE WITH THIOLS INCLUDING GLUTATHIONE

    EPA Science Inventory

    This study accounts for the observations that the metabolism of benzene is dominated by the formation of phenol. As demonstrated here, the pathway leading to S-phenylmercapturic acid is necessarily minor on account of the low efficiency of benzene oxide capture by glutathione at ...

  5. Rational design of an organometallic glutathione transferase inhibitor

    SciTech Connect

    Ang, W.H.; Parker, L.J.; De Luca, A.; Juillerat-Jeanneret, L.; Morton, C.J.; LoBello, M.; Parker, M.W.; Dyson, P.J.

    2010-08-17

    A hybrid organic-inorganic (organometallic) inhibitor was designed to target glutathione transferases. The metal center is used to direct protein binding, while the organic moiety acts as the active-site inhibitor. The mechanism of inhibition was studied using a range of biophysical and biochemical methods.

  6. GLUTATHIONE S-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE

    EPA Science Inventory

    GLUTATHIONE s-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE. M K Ross1 and R A Pegram2. 1Curriculum in Toxicology, University of North Carolina at Chapel Hill; 2Experimental Toxicology Division, NHEERL/ORD, United States Environmental Protection Agency, Research Triangl...

  7. Gender differences in glutathione metabolism in Alzheimer's disease.

    PubMed

    Liu, Honglei; Harrell, Lindy E; Shenvi, Swapna; Hagen, Tory; Liu, Rui-Ming

    2005-03-15

    The mechanism underlying Alzheimer's disease (AD), an age-related neurodegenerative disease, is still an area of significant controversy. Oxidative damage of macromolecules has been suggested to play an important role in the development of AD; however, the underlying mechanism is still unclear. In this study, we showed that the concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, was decreased in red blood cells from male AD patients compared with age- and gender-matched controls. However, there was no difference in blood GSH concentration between the female patients and female controls. The decrease in GSH content in red blood cells from male AD patients was associated with reduced activities of glutamate cysteine ligase and glutathione synthase, the two enzymes involved in de novo GSH synthesis, with no change in the amount of oxidized glutathione or the activity of glutathione reductase, suggesting that a decreased de novo GSH synthetic capacity is responsible for the decline in GSH content in AD. These results showed for the first time that GSH metabolism was regulated differently in male and female AD patients. PMID:15693022

  8. Glutathione level after long-term occupational elemental mercury exposure

    SciTech Connect

    Kobal, Alfred Bogomir Prezelj, Marija; Horvat, Milena; Krsnik, Mladen; Gibicar, Darija; Osredkar, Josko

    2008-05-15

    Many in vitro and in vivo studies have elucidated the interaction of inorganic mercury (Hg) and glutathione. However, human studies are limited. In this study, we investigated the potential effects of remote long-term intermittent occupational elemental Hg vapour (Hg{sup o}) exposure on erythrocyte glutathione levels and some antioxidative enzyme activities in ex-mercury miners in the period after exposure. The study included 49 ex-mercury miners divided into subgroups of 28 still active, Hg{sup o}-not-exposed miners and 21 elderly retired miners, and 41 controls, age-matched to the miners subgroup. The control workers were taken from 'mercury-free works'. Reduced glutathione (GSH) and oxidized disulphide glutathione (GSSG) concentrations in haemolysed erythrocytes were determined by capillary electrophoresis, while total glutathione (total GSH) and the GSH/GSSG ratio were calculated from the determined values. Catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities in erythrocytes were measured using commercially available reagent kits, while urine Hg (U-Hg) concentrations were determined by cold vapour atomic absorption (CVAAS). No correlation of present U-Hg levels, GSH, GSSG, and antioxidative enzymes with remote occupational biological exposure indices were found. The mean CAT activity in miners and retired miners was significantly higher (p<0.05) than in the controls. No differences in mean GPx activity among the three groups were found, whereas the mean GR activity was significantly higher (p<0.05) in miners than in retired miners. The mean concentrations of GSH (mmol/g Hb) in miners (13.03{+-}3.71) were significantly higher (p<0.05) than in the control group (11.68{+-}2.66). No differences in mean total GSH, GSSG levels, and GSH/GSSG ratio between miners and controls were found. A positive correlation between GSSG and present U-Hg excretion (r=0.41, p=0.001) in the whole group of ex-mercury miners was observed. The

  9. Prolonged fasting increases glutathione biosynthesis in postweaned northern elephant seals

    PubMed Central

    Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Forman, Henry Jay; Crocker, Daniel E.; Ortiz, Rudy M.

    2011-01-01

    SUMMARY Northern elephant seals experience prolonged periods of absolute food and water deprivation (fasting) while breeding, molting or weaning. The postweaning fast in elephant seals is characterized by increases in the renin–angiotensin system, expression of the oxidant-producing protein Nox4, and NADPH oxidase activity; however, these increases are not correlated with increased oxidative damage or inflammation. Glutathione (GSH) is a potent reductant and a cofactor for glutathione peroxidases (GPx), glutathione-S transferases (GST) and 1-cys peroxiredoxin (PrxVI) and thus contributes to the removal of hydroperoxides, preventing oxidative damage. The effects of prolonged food deprivation on the GSH system are not well described in mammals. To test our hypothesis that GSH biosynthesis increases with fasting in postweaned elephant seals, we measured circulating and muscle GSH content at the early and late phases of the postweaning fast in elephant seals along with the activity/protein content of glutamate-cysteine ligase [GCL; catalytic (GCLc) and modulatory (GCLm) subunits], γ-glutamyl transpeptidase (GGT), glutathione disulphide reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), GST and PrxVI, as well as plasma changes in γ-glutamyl amino acids, glutamate and glutamine. GSH increased two- to four-fold with fasting along with a 40–50% increase in the content of GCLm and GCLc, a 75% increase in GGT activity, a two- to 2.5-fold increase in GR, G6PDH and GST activities and a 30% increase in PrxVI content. Plasma γ-glutamyl glutamine, γ-glutamyl isoleucine and γ-glutamyl methionine also increased with fasting whereas glutamate and glutamine decreased. Results indicate that GSH biosynthesis increases with fasting and that GSH contributes to counteracting hydroperoxide production, preventing oxidative damage in fasting seals. PMID:21430206

  10. CHARACTERIZATION OF DANSYLATED CYSTEINE, CYSTINE, GLUTATHIONE, AND GLUTATHIONE DISULFIDE BY NARROW BORE LIQUID CHROMATOGRAPHY - ELECTROSPRAY IONIZATION MASS SPECTROMETRY

    EPA Science Inventory

    A method using reversed phase high performance liquid chromtography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the dientity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and...

  11. Biotransformation of nitrosobenzene in the red cell and the role of glutathione.

    PubMed

    Eyer, P; Lierheimer, E

    1980-01-01

    1. In the red cell nitrosobenzene formed glutathione-sulphinanilide from reduced glutathione, and the corresponding sulphinanilide with the reactive cysteine residues of haemoglobin. 2. Glutathionesulphinanilide was reductively cleaved by an NADPH-linked reductase with formation of free analine half an equivalent of reduced glutathione and half of glutathione sulphinic acid. 3. About three quarters of the aniline produced from nitrosobenzene or phenylhydroxylamine was formed via this pathway within the red cell. PMID:6893778

  12. Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli.

    PubMed

    Goswami, Manish; Subramanian, Mahesh; Kumar, Ranjeet; Jass, Jana; Jawali, Narendra

    2016-07-01

    We have analyzed the contribution of different efflux components to glutathione-mediated abrogation of ciprofloxacin's activity in Escherichia coli and the underlying potential mechanism(s) behind this phenomenon. The results indicated that glutathione increased the total active efflux, thereby partially contributing to glutathione-mediated neutralization of ciprofloxacin's antibacterial action in E. coli However, the role of glutathione-mediated increased efflux becomes evident in the absence of a functional TolC-AcrAB efflux pump. PMID:27139480

  13. Blood glutathione status and activity of glutathione-metabolizing antioxidant enzymes in erythrocytes of young trotters in basic training.

    PubMed

    Janiak, M; Suska, M; Dudzińska, W; Skotnicka, E

    2010-04-01

    The aim of this study was to evaluate response of blood glutathione status and activity of glutathione-metabolizing antioxidant enzymes in erythrocytes of young trotters in basic training. Nine untrained trotters (aged 16-20 months) were exposed to a 4-month training program based on exercises at low-to-moderate intensity. The conditioning consisted of breaking the horses and running them on distances varying from 4 to 40 km a week. The workloads were increased on a 3-week basis. Exercise intensity was monitored by measuring heart rate and blood lactate. Blood samples were collected at rest, before (RES0) and after (RESt) the conditioning period; moreover, on the latter occasion (on day 112 of training), the blood was also taken immediately after the routine exercise (EXE0) and 60 min thereafter (EXE60). The whole blood samples were analysed for the concentration of reduced, oxidized and total glutathione (GSH, GSSG and TGSH, respectively), while the activities of glutathione peroxidase (GPX) and glutathione-disulfide reductase (GR) were determined in haemolysates. Additionally, the erythrocytic concentrations of oxidized nicotinamide adenine dinucleotide (NAD(+)) and its phosphate (NADP(+)) were measured. All investigated parameters except NAD(+) and reduced/oxidized glutathione ratio (GSH/GSSG) changed during the training period. Following the effortm GPX, NADP(+) and GSH/GSSG were significantly lower (p < 0.05, p < 0.01, p < 0.001, respectively) while GSSG was markedly higher than at rest (RESt). The drop in NADP(+), low GSH/GSSG and high GSSG concentration were sustained at EXE60. Glutathione-disulfide reductase activity was higher after the workout but only at EXE60 the increase in activity was significant. Despite the activities of the GSH-GSSG cycle, enzymes were considerably higher after the training period, the elevated concentration of GSSG and significantly lower GSH/GSSG ratio in the post-exercise measurements suggest that production of reactive oxygen

  14. Lead concentration and the level of glutathione, glutathione S-transferase, reductase and peroxidase in the blood of some occupational workers from Irbid City, Jordan.

    PubMed

    Hunaiti, A; Soud, M; Khalil, A

    1995-08-18

    Blood samples were collected from 263 lead-exposed suspected males living in Irbid area in the northern part of Jordan. The blood lead concentrations in the samples were determined by atomic absorption and were related to the type of work performed by the workers. The blood lead concentration was higher in metal casters, 41.6, and radiator welders, 32,8 micrograms/dl, compared to non-suspected lead-exposed university students, 5.7 micrograms/dl. Workers such as mechanics, bus drivers, car painters and gas station workers showed slightly higher but not significant blood lead. The blood glutathione content and the activities of glutathione reductase, glutathione peroxidase and glutathione S-transferase were also determined in non-suspected subjects and in those with occupational exposure to lead. With increasing blood lead concentration, glutathione content decreases as well as the activities of the glutathione utilizing enzymes. PMID:7569882

  15. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    PubMed

    Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

    2015-09-01

    Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. PMID:26091838

  16. Hepatic glutathione content in patients with alcoholic and non alcoholic liver diseases

    SciTech Connect

    Altomare, E.; Vendemiale, G.; Albano, O.

    1988-01-01

    Reduced and oxidized hepatic glutathione was evaluated during alcoholic and non alcoholic liver injury. We studied 35 chronic alcoholics, 20 patients with non alcoholic liver diseases, 15 control subjects. Hepatic glutathione was measured in liver biopsies and correlated with histology and laboratory tests. Alcoholic and non alcoholic patients exhibited a significant decrease of hepatic glutathione compared to control subjects. Oxidized glutathione was significantly higher in the two groups of patients compared to controls. The decreased hepatic glutathione level in patients with alcoholic and non alcoholic liver diseases may represent a contributing factor of liver injury and may enhance the risk of toxicity in these patients.

  17. Glutathione S-transferase class {pi} polymorphism in baboons

    SciTech Connect

    Aivaliotis, M.J.; Cantu, T.; Gilligan, R.

    1995-02-01

    Glutathione S-transferase (GST) comprises a family of isozymes with broad substrate specificities. One or more GST isozymes are present in most animal tissues and function in several detoxification pathways through the conjugation of reduced glutathione with various electrophiles, thereby reducing their potential toxicity. Four soluble GST isozymes encoded by genes on different chromosomes have been identified in humans. The acidic class pi GST, GSTP (previously designated GST-3), is widely distributed in adult tissues and appears to be the only GST isozyme present in leukocytes and placenta. Previously reported electrophoretic analyses of erythrocyte and leukocyte extracts revealed single bands of activity, which differed slightly in mobility between the two cell types, or under other conditions, a two-banded pattern. To our knowledge, no genetically determined polymorphisms have previously been reported in GSTP from any species. We now report a polymorphism of GSTP in baboon leukocytes, and present family data that verifies autosomal codominant inheritance. 14 refs., 2 figs., 1 tab.

  18. Delayed Cardiomyopathy in Dystrophin Deficient mdx Mice Relies on Intrinsic Glutathione Resource

    PubMed Central

    Khouzami, Lara; Bourin, Marie-Claude; Christov, Christo; Damy, Thibaud; Escoubet, Brigitte; Caramelle, Philippe; Perier, Magali; Wahbi, Karim; Meune, Christophe; Pavoine, Catherine; Pecker, Françoise

    2010-01-01

    Oxidative stress contributes to the pathogenesis of Duchenne muscular dystrophy (DMD). Although they have been a model for DMD, mdx mice exhibit slowly developing cardiomyopathy. We hypothesized that disease process was delayed owing to the development of an adaptive mechanism against oxidative stress, involving glutathione synthesis. At 15 to 20 weeks of age, mdx mice displayed a 33% increase in blood glutathione levels compared with age-matched C57BL/6 mice. In contrast, cardiac glutathione content was similar in mdx and C57BL/6 mice as a result of the balanced increased expression of glutamate cysteine ligase catalytic and regulatory subunits ensuring glutathione synthesis in the mdx mouse heart, as well as increased glutathione peroxidase-1 using glutathione. Oral administration from 10 weeks of age of the glutamate cysteine ligase inhibitor, l-buthionine(S,R)-sulfoximine (BSO, 5 mmol/L), led to a 33% and 50% drop in blood and cardiac glutathione, respectively, in 15- to 20-week-old mdx mice. Moreover, 20-week-old BSO-treated mdx mice displayed left ventricular hypertrophy associated with diastolic dysfunction, discontinuities in β-dystroglycan expression, micronecrosis and microangiopathic injuries. Examination of the glutathione status in four DMD patients showed that three displayed systemic glutathione deficiency as well. In conclusion, low glutathione resource hastens the onset of cardiomyopathy linked to a defect in dystrophin in mdx mice. This is relevant to the glutathione deficiency that DMD patients may suffer. PMID:20696779

  19. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network.

    PubMed

    Couto, Narciso; Wood, Jennifer; Barber, Jill

    2016-06-01

    In this review article we examine the role of glutathione reductase in the regulation, modulation and maintenance of cellular redox homoeostasis. Glutathione reductase is responsible for maintaining the supply of reduced glutathione; one of the most abundant reducing thiols in the majority of cells. In its reduced form, glutathione plays key roles in the cellular control of reactive oxygen species. Reactive oxygen species act as intracellular and extracellular signalling molecules and complex cross talk between levels of reactive oxygen species, levels of oxidised and reduced glutathione and other thiols, and antioxidant enzymes such as glutathione reductase determine the most suitable conditions for redox control within a cell or for activation of programmed cell death. Additionally, we discuss the translation and expression of glutathione reductase in a number of organisms including yeast and humans. In yeast and human cells, a single gene expresses more than one form of glutathione reductase, destined for residence in the cytoplasm or for translocation to different organelles; in plants, however, two genes encoding this protein have been described. In general, insects and kinetoplastids (a group of protozoa, including Plasmodia and Trypanosoma) do not express glutathione reductase or glutathione biosynthetic enzymes. Instead, they express either the thioredoxin system or the trypanothione system. The thioredoxin system is also present in organisms that have the glutathione system and there may be overlapping functions with cross-talk between the two systems. Finally we evaluate therapeutic targets to overcome oxidative stress associated cellular disorders. PMID:26923386

  20. Glutamine: a precursor of glutathione and its effect on liver

    PubMed Central

    Yu, Jian-Chun; Jiang, Zhu-Ming; Li, De-Min

    1999-01-01

    AIM To investigate the relationship between alanyl-glutamine (ALA-GLN) and glutathione (GSH) biosynthesis in hepatic protection. METHODS Twenty male Wistar rats were randomly divided into two groups: one receiving standard parenteral nutrition (STD) and the other supplemented with or without ALA-GLN for 7 days. The blood and liver tissue samples were examined after 5-fluorouracil (5-FU) was injected peritoneally. RESULTS The concentration measurements were significantly highe r in ALA-GLN group than in STD group in serum GLN (687 μmol/ L ± 50 μmol/L vs 505 μmol/L ± 39 μmol/L,P < 0.05), serum GSH (14 μmol/L ± 5 μmol/L vs 7 μmol/L ± 3 μmol/L, P < 0.01) and in liver GSH content (6.9 μmol/g ± 2.5 μmol/g vs 4.4 μmol/ g ± 1.6 μmol/g liver tissue, P < 0.05). Rats in ALA-GLN group had lesser elevations in hepatic enzymes after 5-FU administration. CONCLUSION The supplemented nutrition ALA-GLN can protect the liver function through increasing the glutathione biosynthesis and pre-serving the glutathione stores in hepatic tissue. PMID:11819414

  1. Glutathione conjugation of chlorambucil: measurement and modulation by plant polyphenols.

    PubMed

    Zhang, K; Wong, K P

    1997-07-15

    Chlorambucil (CMB), an anticancer drug, was cytotoxic at concentrations of 5-20 microM to human colon adenocarcinoma cells. It inhibited [14C]thymidine uptake in a dose-dependent manner. Both effects were potentiated by simultaneous exposure of the cells to 10 microM plant polyphenols. In an attempt to explain the possible mechanism of action of the polyphenols in relation to these observations, an HPLC-radiometric method was developed to measure the conjugation of CMB with glutathione in these cells and to monitor the export of monochloromonoglutathionyl CMB (MG-CMB), its main glutathione conjugate. At micromolar concentrations, five polyphenols, namely quercetin, butein, tannic acid, 2'-hydroxychalcone and morin, inhibited the efflux of CMB significantly; an inhibition of 40% was observed with 10 microM quercetin. The glutathione S-transferase (GST) activity of the cancer cells, measured with 1-chloro-2,4-dinitrobenzene, was also inhibited by the polyphenols. Their combined action on GST and on the efflux of MG-CMB conjugate could provide an enhanced positive modulation of sensitivity of the tumour cells to CMB. PMID:9230122

  2. Maintaining good hearing: calorie restriction, Sirt3, and glutathione.

    PubMed

    Han, Chul; Someya, Shinichi

    2013-10-01

    Reducing calorie intake extends the lifespan of a variety of experimental models and delays progression of age-related hearing loss (AHL). AHL is a common feature of aging and is characterized by age-related decline of hearing associated with loss of sensory hair cells, spiral ganglion neurons, and/or stria vascularis degeneration in the cochlea. Sirtuins are a family of NAD(+)-dependent enzymes that regulate lifespan in lower organisms and have emerged as broad regulators of cellular fate. Our recent study indicated that mitochondrial Sirt3, a member of the sirtuin family, mediates the anti-aging effects of calorie restriction (CR) on AHL in mice. Interestingly, we also found that weight loss alone may not be sufficient for maintaining normal hearing. How does CR slow the progression of AHL through regulation of Sirt3? Here we review the evidence that during CR, Sirt3 slows the progression of AHL by promoting the glutathione-mediated mitochondrial antioxidant defense system in mice. A significant reduction in food consumption in one's daily life may not be a desirable and realistic option for most people. Therefore, identification/discovery of compounds that induce the activation of SIRT3 or glutathione reductase, or that increase mitochondrial glutathione levels has potential for maintaining good hearing through mimicking the anti-aging effects of CR in human inner ear cells. PMID:23454634

  3. Benzene oxide is a substrate for glutathione S-transferases.

    PubMed

    Zarth, Adam T; Murphy, Sharon E; Hecht, Stephen S

    2015-12-01

    Benzene is a known human carcinogen which must be activated to benzene oxide (BO) to exert its carcinogenic potential. BO can be detoxified in vivo by reaction with glutathione and excretion in the urine as S-phenylmercapturic acid. This process may be catalyzed by glutathione S-transferases (GSTs), but kinetic data for this reaction have not been published. Therefore, we incubated GSTA1, GSTT1, GSTM1, and GSTP1 with glutathione and BO and quantified the formation of S-phenylglutathione. Kinetic parameters were determined for GSTT1 and GSTP1. At 37 °C, the putative Km and Vmax values for GSTT1 were 420 μM and 450 fmol/s, respectively, while those for GSTP1 were 3600 μM and 3100 fmol/s. GSTA1 and GSTM1 did not exhibit sufficient activity for determination of kinetic parameters. We conclude that GSTT1 is a critical enzyme in the detoxification of BO and that GSTP1 may also play an important role, while GSTA1 and GSTM1 seem to be less important. PMID:26554337

  4. Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

    PubMed Central

    Patzewitz, Eva-Maria; Wong, Eleanor H; Müller, Sylke

    2012-01-01

    Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG. PMID:22151036

  5. Application of superparamagnetic microspheres for affinity adsorption and purification of glutathione

    NASA Astrophysics Data System (ADS)

    Wang, Qiang; Guan, Yueping; Yang, Mingzhu

    2012-10-01

    The superparamagnetic poly-(MA-DVB) microspheres with micron size were synthesized by the modified suspension polymerization method. Adsorption of glutathione by magnetic poly-(MA-DVB) microspheres with IDA-copper was investigated. The effect of solution pH value, affinity adsorption and desorption of glutathione was studied. The results showed that the optimum pH value for glutathione adsorption was found at pH=3.5, the maximum capacity for glutathione of magnetic poly-(MA-DVB) microspheres was estimated at 42.4 mg/g by fitting the experimental data to the Langmuir equation. The adsorption equilibrium of glutathione was obtained in about 10 min and the adsorbed glutathione was desorbed from the magnetic microspheres in about 30 min using NaCl buffer solution. The magnetic microspheres could be repeatedly utilized for the affinity adsorption of glutathione.

  6. Effects of cold stress on glutathione and related enzymes in rat erythrocytes

    NASA Astrophysics Data System (ADS)

    Ohno, Hideki; Kondo, Takahito; Fujiwara, Yutaka; Tagami, Sei-Ichi; Kuroshima, Akihiro; Kawakami, Yoshikazu

    1991-06-01

    Effects of acute and chronic cold stress on glutathione and related enzymes in rat erythrocytes were investigated. Blood from both cold-acclimated (CA) and cold-adapted (CG) rats had significantly lower concentrations of glutathione than blood from control animals. Superoxide dismutase activity was increased significantly in CA rats and tended to rise in CG rats. Activity of glutathione peroxidase in erythrocytes was inconsistent in that it tended to increase in CA rats but decreased significantly in CG rats. The results may imply that CG rats suffered deleterious effects of hydrogen peroxide. On the other hand, there were marked decreases in glutathione peroxidase and glutathione reductase activities in acutely cold-exposed rats in conjunction with unchanged levels of glutathione. In all treatments the state of riboflavin metabolism was estimated to be adequate, since no increases were observed in the erythrocyte glutathione reductase activity coefficient.

  7. Effect of glutathione on phytochelatin synthesis in tomato cells. [Lycopersicon esculentum

    SciTech Connect

    Mendum, M.L.; Gupta, S.C.; Goldsbrough, P.B. )

    1990-06-01

    Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably ({gamma}-Glu-Cys){sub 2}-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little ({sup 35}S)cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione.

  8. Glutathione redox cycle dysregulation in Huntington's disease knock-in striatal cells.

    PubMed

    Ribeiro, Márcio; Rosenstock, Tatiana R; Cunha-Oliveira, Teresa; Ferreira, Ildete L; Oliveira, Catarina R; Rego, A Cristina

    2012-11-15

    Huntington's disease (HD) is a CAG repeat disorder affecting the HD gene, which encodes for huntingtin (Htt) and is characterized by prominent cell death in the striatum. Oxidative stress was previously implicated in HD neurodegeneration, but the role of the major endogenous antioxidant system, the glutathione redox cycle, has been less studied following expression of full-length mutant Htt (FL-mHtt). Thus, in this work we analyzed the glutathione system in striatal cells derived from HD knock-in mice expressing mutant Htt versus wild-type cells. Mutant cells showed increased intracellular reactive oxygen species (ROS) and caspase-3 activity, which were significantly prevented following treatment with glutathione ethyl ester. Interestingly, mutant cells exhibited an increase in intracellular levels of both reduced and oxidized forms of glutathione, and enhanced activities of glutathione peroxidase (GPx) and glutathione reductase (GRed). Furthermore, glutathione-S-transferase (GST) and γ-glutamyl transpeptidase (γ-GT) activities were also increased in mutant cells. Nevertheless, glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) activities and levels of GCL catalytic subunit were decreased in cells expressing FL-mHtt, highly suggesting decreased de novo synthesis of glutathione. Enhanced intracellular total glutathione, despite decreased synthesis, could be explained by decreased extracellular glutathione in mutant cells. This occurred concomitantly with decreased mRNA expression levels and activity of the multidrug resistance protein 1 (Mrp1), a transport protein that mediates cellular export of glutathione disulfide and glutathione conjugates. Additionally, inhibition of Mrp1 enhanced intracellular GSH in wild-type cells only. These data suggest that FL-mHtt affects the export of glutathione by decreasing the expression of Mrp1. Data further suggest that boosting of GSH-related antioxidant defense mechanisms induced by FL-mHtt is insufficient to

  9. Effect of ovariectomy and sex hormone replacement on glutathione and glutathione-related enzymes in rat hepatocarcinogenesis.

    PubMed

    Hambali, Z; Ngah, W Z; Wahid, S A; Kadir, K A

    1995-01-01

    The effects of ovariectomy and hormone replacement in control and carcinogen treated female rats were investigated by measuring whole blood and liver glutathione (WGSH, HGSH), glutathione S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GRx) and histological evaluation. Hepatocarcinogenesis was induced by diethylnitrosamine and 2-acetylaminofluorene. In control rats not receiving carcinogen, ovariectomy significantly increased the GST and GRx activities. Replacement with either estrogen or progesterone reduced the GST activities to below intact female values whereas replacement of both hormones together brought the GST activities to that of intact females. GRx activities were brought to intact female values by replacement with estrogen or progesterone, either singly or in combination. Neither ovariectomy nor sex hormone/s replacement influenced the levels of WGSH, HGSH and GPx activities. Carcinogen administration to intact rats increased all the parameters measured. Ovariectomized rats treated with carcinogen showed lower GPx and GRx activities at 2 mths. However, replacement with either progesterone or combined estrogen and progesterone increased GPx and GRx activities to original values. On the other hand GST and GPx activities in ovariectomized rats which had carcinogen treatment were lower than intact rats after 5 mths. Replacement with hormones either singly or both brought GST and GPx activities up to intact rat levels receiving carcinogen. The levels of WGSH, HGSH and GRx activities (5 mths) in carcinogen treated rats were not influenced by ovariectomy and/or hormone/s replacement. The results from this study suggested that ovariectomy reduced the severity of hepatocarcinogenesis which was restored by sex hormone/s replacement. PMID:7603748

  10. Synthesis and glutathione S-transferase structure-affinity relationships of nonpeptide and peptidase-stable glutathione analogues.

    PubMed

    Klotz, P; Slaoui-Hasnaoui, A; Banères, J L; Duckert, J F; Rossi, J C; Kerbal, A

    1998-06-18

    A series of nonpeptidic glutathione analogues where the peptide bonds were replaced by simple carbon-carbon bonds or isosteric E double bonds were prepared. The optimal length for the two alkyl chains on either side of the mercaptomethyl group was evaluated using structure-affinity relationships. Affinities of the analogues 14a-f, 23, and 25 were evaluated for a recombinant GST enzyme using a new affinity chromatography method previously developed in our laboratory. Analysis of these analogues gives an additional understanding for GST affinity requirements: (a) the carbon skeleton must conserve that of glutathione since analogue 14a showed the best affinity (IC50 = 5.2 microM); (b) the GST G site is not able to accommodate a chain length elongation of one methylene group (no affinity for analogues 14c,f); (c) a one-methylene group chain length reduction is tolerated, much more for the "Glu side" (14d, IC50 = 10.1 microM) than for the "Gly side" (14b, IC50 = 1800 microM); (d) the mercaptomethyl group must remain at position 5 as shown from the null affinity of the 6-mercaptomethyl analogue 14e; (e) the additional peptide isosteric E double bond (25) or hydroxyl derivative (23) in 14e did not help to retrieve affinity. This work reveals useful information for the design of new selective nonpeptidic and peptidase-stable glutathione analogues. PMID:9632361

  11. [Individual and joint stress of lead and mercury on growth, glutathione and glutathione-related enzymes of Scenedesmus quadricauda].

    PubMed

    Li, Yan; Zhu, Lin; Liu, Shuo

    2009-01-01

    To understand the toxicity mechanisms of mixed heavy metals on aquatic plant, indicators of algea growth rate,content of reduced glutathione (GSH), activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx) of green algae, Scenedesmus quadricauda were measured to analyze the individual and joint toxic effects of lead and mercury. The results show that the 96h EC50 of algae growth inhibition by lead [Pb(NO3)2] and mercury (HgCl2) are 0.6789 mg/L and 0.1401 mg/L respectively. After 12 h individual and joint lead and mercury exposure, the content of GSH in alga cells is decreased to about 70% of the level of the control, and keeps a steady level with the increase of the exposure concentration. The GST activities are increased to a peak in lower concentration groups and then decrease with the increase of the exposure concentration. Indeed,the higher concentration of lead and mercury combined-poisoning can inhibit the activities of GST significantly, with 13.04% inhibitory rate. The activity of GPx is almost suppressed continuously with the increase of the exposure concentration, and the lowest activity is only 38.77% of the control. The toxic action of the mixture of Pb and Hg on growth inhibition,GSH content,activities of GST and activities of GPx for Scenedesmus quadricauda are addition. PMID:19353889

  12. Plastid-Localized Glutathione Reductase2–Regulated Glutathione Redox Status Is Essential for Arabidopsis Root Apical Meristem Maintenance[C][W

    PubMed Central

    Yu, Xin; Pasternak, Taras; Eiblmeier, Monika; Ditengou, Franck; Kochersperger, Philip; Sun, Jiaqiang; Wang, Hui; Rennenberg, Heinz; Teale, William; Paponov, Ivan; Zhou, Wenkun; Li, Chuanyou; Li, Xugang; Palme, Klaus

    2013-01-01

    Glutathione is involved in thiol redox signaling and acts as a major redox buffer against reactive oxygen species, helping to maintain a reducing environment in vivo. Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) into reduced glutathione (GSH). The Arabidopsis thaliana genome encodes two GRs: GR1 and GR2. Whereas the cytosolic/peroxisomal GR1 is not crucial for plant development, we show here that the plastid-localized GR2 is essential for root growth and root apical meristem (RAM) maintenance. We identify a GR2 mutant, miao, that displays strong inhibition of root growth and severe defects in the RAM, with GR activity being reduced to ∼50%. miao accumulates high levels of GSSG and exhibits increased glutathione oxidation. The exogenous application of GSH or the thiol-reducing agent DTT can rescue the root phenotype of miao, demonstrating that the RAM defects in miao are triggered by glutathione oxidation. Our in silico analysis of public microarray data shows that auxin and glutathione redox signaling generally act independently at the transcriptional level. We propose that glutathione redox status is essential for RAM maintenance through both auxin/PLETHORA (PLT)-dependent and auxin/PLT-independent redox signaling pathways. PMID:24249834

  13. Targeting aberrant glutathione metabolism to eradicate human acute myelogenous leukemia cells.

    PubMed

    Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P; Balys, Marlene; Ashton, John M; Neering, Sarah J; Lagadinou, Eleni D; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L; O'Dwyer, Kristen M; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K; Munger, Joshua; Crooks, Peter A; Becker, Michael W; Jordan, Craig T

    2013-11-22

    The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

  14. Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

    PubMed Central

    Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

    2013-01-01

    The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

  15. Response of glutathione in mussels (Mytilus) exposed to common environmental contaminants

    SciTech Connect

    Inouye, L.S.; Casillas, E.

    1995-12-31

    Mussels (Mytilus sp.) were exposed to PCBs, a mixture of polycyclic aromatic hydrocarbons (PAHs), or extracts of contaminated sediments to determine the response of glutathione content in gill and digestive gland to chemicals contaminants. In addition, a field transplant investigation was conducted to determine if the differences observed in tissue glutathione levels of mussels from reference and contaminated sites were due to the presence of chemical contaminants rather than to population differences in basal glutathione concentrations. Exposure to PCBs or to sediment extracts from a contaminated site resulted in a decrease in glutathione content in the digestive gland, but an increase in glutathione content in gills. In contrast, no alterations in tissue glutathione were observed after exposure to PAHs. Transplant investigation results were consistent with those from the contaminated sediment extract exposure. Glutathione content in digestive glands was higher in mussels from a reference site compared to that found in mussels from the contaminated site, while the opposite trend was found in gill glutathione content of the same mussels. Eight weeks after being transplanted from the reference site to the contaminated site or alternatively from a contaminated site to a reference site, glutathione levels in the gland tissues matched those found in mussels native to the site to which they were transplanted. Although gill glutathione content was significantly different from that found at the site of origin, it did not match levels found in mussels native to the site to which they had been transplanted.

  16. Exposure to cadmium changes the content of glutathione in maize seedlings

    SciTech Connect

    Rauser, W.E.

    1987-04-01

    Glutathione may be involved in the biosynthesis of Cd-binding peptides known as phytochelatins. Five-day old maize seedlings in hydroponic culture were exposed to 3 ..mu..M CdSO/sub 4/ for 2, 6 and 12 hours and 1, 2 and 3 days. Total glutathione (glutathione + glutathione disulfide) in roots and shoots was measured enzymatically. Exposure to Cd for 12 hours or longer reduced root elongation growth. Shoots contained more glutathione than did roots. Within 2 hours of exposure to Cd the glutathione content declined by 47% of control and stayed low for a day. Shoot glutathione decreased gradually and less markedly (by 34% in 24 hours). Following the decline in the first day the glutathione per seedling increased with time in the presence of Cd. Cadmium-binding peptide in roots increased during the recovery phase. If glutathione is a substrate for Cd-binding peptide synthesis, such a use accounts for only part of the decline in root glutathione observed during the first day.

  17. Unveiling the roles of the glutathione redox system in vivo by analyzing genetically modified mice

    PubMed Central

    Fujii, Junichi; Ito, Jun-itsu; Zhang, Xuhong; Kurahashi, Toshihiro

    2011-01-01

    Redox status affects various cellular activities, such as proliferation, differentiation, and death. Recent studies suggest pivotal roles of reactive oxygen species not only in pathogenesis under oxidative insult but also in intracellular signal transduction. Glutathione is present in several millimolar concentrations in the cytoplasm and has multiple roles in the regulation of cellular homeostasis. Two enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, constitute the de novo synthesis machinery, while glutathione reductase is involved in the recycling of oxidized glutathione. Multidrug resistant proteins and some other transporters are responsible for exporting oxidized glutathione, glutathione conjugates, and S-nitrosoglutathione. In addition to antioxidation, glutathione is more positively involved in cellular activity via its sulfhydryl moiety of a molecule. Animals in which genes responsible for glutathione metabolism are genetically modified can be used as beneficial and reliable models to elucidate roles of glutathione in vivo. This review article overviews recent progress in works related to genetically modified rodents and advances in the elucidation of glutathione-mediated reactions. PMID:21980221

  18. A Western diet induced NAFLD in LDLR(-/)(-) mice is associated with reduced hepatic glutathione synthesis.

    PubMed

    Li, Ling; Zhang, Guo-Fang; Lee, Kwangwon; Lopez, Rocio; Previs, Stephen F; Willard, Belinda; McCullough, Arthur; Kasumov, Takhar

    2016-07-01

    Oxidative stress plays a key role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Glutathione is the major anti-oxidant involved in cellular oxidative defense, however there are currently no simple non-invasive methods for assessing hepatic glutathione metabolism in patients with NAFLD. As a primary source of plasma glutathione, liver plays an important role in interorgan glutathione homeostasis. In this study, we have tested the hypothesis that measurements of plasma glutathione turnover could be used to assess the hepatic glutathione metabolism in LDLR(-/)(-) mice, a mouse model of diet-induced NAFLD. Mice were fed a standard low fat diet (LFD) or a high fat diet containing cholesterol (a Western type diet (WD)). The kinetics of hepatic and plasma glutathione were quantified using the (2)H2O metabolic labeling approach. Our results show that a WD leads to reduced fractional synthesis rates (FSR) of hepatic (25%/h in LFD vs. 18%/h in WD, P<0.05) and plasma glutathione (43%/h in LFD vs. 21%/h in WD, P<0.05), without any significant effect on their absolute production rates (PRs). WD-induced concordant changes in both hepatic and plasma glutathione turnover suggest that the plasma glutathione turnover measurements could be used to assess hepatic glutathione metabolism. The safety, simplicity, and low cost of the (2)H2O-based glutathione turnover approach suggest that this method has the potential for non-invasive probing of hepatic glutathione metabolism in patients with NAFLD and other diseases. PMID:27036364

  19. Glutathione Reductase Is Essential for Host Defense against Bacterial Infection

    PubMed Central

    Yan, Jing; Ralston, Melissa M.; Meng, Xiaomei; Bongiovanni, Kathleen D.; Jones, Amanda L.; Benndorf, Rainer; Nelin, Leif D.; Frazier, W. Joshua; Rogers, Lynette K.; Smith, Charles V.; Liu, Yusen

    2013-01-01

    Glutathione reductase (Gsr)1 catalyzes the reduction of glutathione disulfide to glutathione, a major cellular antioxidant. We have recently shown that Gsr is essential for host defense against the Gram-negative bacteria Escherichia coli in a mouse model of sepsis. While we have demonstrated that Gsr is required for sustaining the oxidative burst and the development of neutrophil extracellular traps, the role of Gsr in other phagocytic functions remains unclear. It is also unclear whether Gsr-deficient mice exhibit host defense defects against Gram-positive bacteria. In the present study, we characterized the effects of Gsr deficiency on the innate immune responses to a Gram-positive bacterium, group B Streptococcus, and to the Gram-negative bacterial cell wall component lipopolysaccharide (LPS). We found that like, E. coli, group B Streptococcus resulted in a substantially more robust cytokine response and a markedly higher morbidity and mortality in Gsr-deficient mice than in wildtype mice. The increased morbidity and mortality were associated with greater bacterial burden in the Gsr-deficient mice. Interestingly, Gsr-deficient mice did not exhibit a greater sensitivity to LPS than did wildtype mice. Analysis of the neutrophils of Gsr-deficient mice revealed impaired phagocytosis. In response to thioglycollate stimulation, Gsr-deficient mice mobilized far fewer phagocytes, including neutrophils, macrophages, and eosinophils, into their peritoneal cavities than did wildtype mice. The defective phagocyte mobilization is associated with profound oxidation and aggregation of ascitic proteins, particularly albumin. Our results indicate that the oxidative defense mechanism mediated by Gsr is required for an effective innate immune response against bacteria, likely by preventing phagocyte dysfunction due to oxidative damage. PMID:23623936

  20. Preclinical Pharmacokinetic Analysis of NOV-002, a Glutathione Disulfide Mimetic

    PubMed Central

    Uys, Joachim D.; Manevich, Yefim; DeVane, Lindsay C.; He, Lin; Garret, Tracy E.; Pazoles, Christopher J.; Tew, Kenneth D.; Townsend, Danyelle M.

    2010-01-01

    Summary NOV-002 is a glutathione disulfide (GSSG) mimetic that is in Phase III clinical trials for the treatment of advanced non-small cell lung cancer and other oncology indications. GSSG is reduced by glutathione reductase (GR) to form glutathione (GSH), thereby maintaining redox homeostasis. The purpose of the study was to report the pharmacokinetic properties of NOV-002 and evaluate the effect that NOV-002 elicits in redox homeostasis. The pharmacokinetic analysis and tissue distribution of NOV-002 and GSH was evaluated in mice following a dose of 250 mg/kg, i.p. The redox potential and total protein thiol status was calculated. Here we show that NOV-002 is a substrate for GR and that GSH is a primary metabolite. Nonlinear pharmacokinetic modeling predicted that the estimated absorption and elimination rate constants correspond to a half-life of ~13 mins with an AUC of 1.18 μg.h/ml, a Cmax of 2.16 μg/ml and a volume of distribution of 42.61 L/kg. In addition, measurement of the redox potential and total protein thiol status indicated the generation of a transient oxidative signal in the plasma compartment after administration of NOV-002. These results indicate that NOV-002 exerts kinetic and dynamic effects in mice consistent with the GSSG component as the active pharmacological constituent of the drug. A longer-lasting decrease in total plasma free thiol content was also seen, suggesting that the oxidative effect of the GSSG from NOV-002 was impacting redox homeostasis. PMID:20359856

  1. Preclinical pharmacokinetic analysis of NOV-002, a glutathione disulfide mimetic.

    PubMed

    Uys, J D; Manevich, Y; Devane, L C; He, L; Garret, T E; Pazoles, C J; Tew, K D; Townsend, D M

    2010-09-01

    NOV-002 is a glutathione disulfide (GSSG) mimetic that is the subject of clinical investigation in oncology indications. GSSG is reduced by glutathione reductase (GR) to form glutathione (GSH), thereby maintaining redox homeostasis. The purpose of the study was to report the pharmacokinetic properties of NOV-002 and evaluate the effect that NOV-002 elicits in redox homeostasis. The pharmacokinetic analysis and tissue distribution of NOV-002 and GSH was evaluated in mice following a dose of 250 mg/kg, i.p. The redox potential and total protein thiol status was calculated. Here we show that NOV-002 is a substrate for GR and that GSH is a primary metabolite. Non-linear pharmacokinetic modeling predicted that the estimated absorption and elimination rate constants correspond to a half-life of approximately 13 min with an AUC of 1.18 μgh/mL, a C(max) of 2.16 μg/ml and a volume of distribution of 42.61 L/kg. In addition, measurement of the redox potential and total protein thiol status indicated the generation of a transient oxidative signal in the plasma compartment after administration of NOV-002. These results indicate that NOV-002 exerts kinetic and dynamic effects in mice consistent with the GSSG component as the active pharmacological constituent of the drug. A longer-lasting decrease in total plasma free thiol content was also seen, suggesting that the oxidative effect of the GSSG from NOV-002 was impacting redox homeostasis. PMID:20359856

  2. The glutathione system: a new drug target in neuroimmune disorders.

    PubMed

    Morris, Gerwyn; Anderson, George; Dean, Olivia; Berk, Michael; Galecki, Piotr; Martin-Subero, Marta; Maes, Michael

    2014-12-01

    Glutathione (GSH) has a crucial role in cellular signaling and antioxidant defenses either by reacting directly with reactive oxygen or nitrogen species or by acting as an essential cofactor for GSH S-transferases and glutathione peroxidases. GSH acting in concert with its dependent enzymes, known as the glutathione system, is responsible for the detoxification of reactive oxygen and nitrogen species (ROS/RNS) and electrophiles produced by xenobiotics. Adequate levels of GSH are essential for the optimal functioning of the immune system in general and T cell activation and differentiation in particular. GSH is a ubiquitous regulator of the cell cycle per se. GSH also has crucial functions in the brain as an antioxidant, neuromodulator, neurotransmitter, and enabler of neuron survival. Depletion of GSH leads to exacerbation of damage by oxidative and nitrosative stress; hypernitrosylation; increased levels of proinflammatory mediators and inflammatory potential; dysfunctions of intracellular signaling networks, e.g., p53, nuclear factor-κB, and Janus kinases; decreased cell proliferation and DNA synthesis; inactivation of complex I of the electron transport chain; activation of cytochrome c and the apoptotic machinery; blockade of the methionine cycle; and compromised epigenetic regulation of gene expression. As such, GSH depletion has marked consequences for the homeostatic control of the immune system, oxidative and nitrosative stress (O&NS) pathways, regulation of energy production, and mitochondrial survival as well. GSH depletion and concomitant increase in O&NS and mitochondrial dysfunctions play a role in the pathophysiology of diverse neuroimmune disorders, including depression, myalgic encephalomyelitis/chronic fatigue syndrome and Parkinson's disease, suggesting that depleted GSH is an integral part of these diseases. Therapeutical interventions that aim to increase GSH concentrations in vivo include N-acetyl cysteine; Nrf-2 activation via hyperbaric

  3. Glutathione: new roles in redox signaling for an old antioxidant.

    PubMed

    Aquilano, Katia; Baldelli, Sara; Ciriolo, Maria R

    2014-01-01

    The physiological roles played by the tripeptide glutathione have greatly advanced over the past decades superimposing the research on free radicals, oxidative stress and, more recently, redox signaling. In particular, GSH is involved in nutrient metabolism, antioxidant defense, and regulation of cellular metabolic functions ranging from gene expression, DNA and protein synthesis to signal transduction, cell proliferation and apoptosis. This review will be focused on the role of GSH in cell signaling by analysing the more recent advancements about its capability to modulate nitroxidative stress, autophagy, and viral infection. PMID:25206336

  4. Glutathione: new roles in redox signaling for an old antioxidant

    PubMed Central

    Aquilano, Katia; Baldelli, Sara; Ciriolo, Maria R.

    2014-01-01

    The physiological roles played by the tripeptide glutathione have greatly advanced over the past decades superimposing the research on free radicals, oxidative stress and, more recently, redox signaling. In particular, GSH is involved in nutrient metabolism, antioxidant defense, and regulation of cellular metabolic functions ranging from gene expression, DNA and protein synthesis to signal transduction, cell proliferation and apoptosis. This review will be focused on the role of GSH in cell signaling by analysing the more recent advancements about its capability to modulate nitroxidative stress, autophagy, and viral infection. PMID:25206336

  5. The comprehensive acid-base characterization of glutathione

    NASA Astrophysics Data System (ADS)

    Mirzahosseini, Arash; Somlyay, Máté; Noszál, Béla

    2015-02-01

    Glutathione in its thiol (GSH) and disulfide (GSSG) forms, and 4 related compounds were studied by 1H NMR-pH titrations and a case-tailored evaluation method. The resulting acid-base properties are quantified in terms of 128 microscopic protonation constants; the first complete set of such parameters for this vitally important pair of compounds. The concomitant 12 interactivity parameters were also determined. Since biological redox systems are regularly compared to the GSH-GSSG pair, the eight microscopic thiolate basicities determined this way are exclusive means for assessing subtle redox parameters in a wide pH range.

  6. Chemical reactions of As complexation by glutathione: an XAFS study

    NASA Astrophysics Data System (ADS)

    Franco, M. W.; Vasconcelos, I. F.; Modolo, L. V.; Barbosa, F. A. R.

    2016-05-01

    In this study, the chemical reactions between As(III) and As(V) with glutathione, which is a target compound in As biochemistry due to its primordial role in As immobilization and intracellular reduction, in various molar ratios were investigated using As K-edge XAFS spectroscopy. Results showed a gradual substitution of As-O bonds in the coordination of aqueous As(III) and As(V) for three As-S bonds in the As+GSH complex. Moreover, the data showed reduction of As(V) to As(III) prior or concomitant to the As+GSH complex formation.

  7. S-(4-bromo-2,3-dioxobutyl)glutathione: A new affinity label for the 4-4 isoenzyme of rat liver glutathione S-transferase

    SciTech Connect

    Katusz, R.M.; Colman, R.F. )

    1991-11-26

    S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200 {mu}M) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25C results in a time-dependent inactivation of the enzyme. The k{sub obs} exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000 {mu}M. Modified enzyme, prepared by incubating glutathione S-transferase with ({sup 3}H)S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH{sub 4}, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified. These results suggest that S-BDB-G functions as an affinity label at or near the active site of glutathione S-transferase and that modification of one site per enzyme subunit causes inactivation. It is proposed that the new compound, S-(4-bromo-2,3-dioxobutyl)glutathione, may have general applicability as an affinity label of other enzymes with glutathione binding sites.

  8. Interaction of glutathione with bovine serum albumin: Spectroscopy and molecular docking.

    PubMed

    Jahanban-Esfahlan, Ali; Panahi-Azar, Vahid

    2016-07-01

    This study aims to investigate the interaction between glutathione and bovine serum albumin (BSA) using ultraviolet-visible (UV-vis) absorption, fluorescence spectroscopies under simulated physiological conditions (pH 7.4) and molecular docking methods. The results of fluorescence spectroscopy indicated that the fluorescence intensity of BSA was decreased considerably upon the addition of glutathione through a static quenching mechanism. The fluorescence quenching obtained was related to the formation of BSA-glutathione complex. The values of KSV, Ka and Kb for the glutathione and BSA interaction were in the order of 10(5). The thermodynamic parameters including enthalpy change (ΔH), entropy change (ΔS) and also Gibb's free energy (ΔG) were determined using Van't Hoff equation. These values showed that hydrogen bonding and van der Waals forces were the main interactions in the binding of glutathione to BSA and the stabilization of the complex. Also, the interaction of glutathione and BSA was spontaneous. The effects of glutathione on the BSA conformation were determined using UV-vis spectroscopy. Moreover, glutathione was docked in BSA using ArgusLab as a molecular docking program. It was recognized that glutathione binds within the sub-domain IIA pocket in domain II of BSA. PMID:26920314

  9. Interaction of glutathione and ascorbic acid in guinea pig lungs exposed to nitrogen dioxide

    SciTech Connect

    Leung, H.-W.; Morrow, P.E.

    1981-01-01

    The interaction of two important water-soluble antioxidants, glutathione and ascorbic acid, was studied. The perfused guinea pig lung was found to contain about twice as much reduced glutathione as ascorbic acid. Nitrogen dioxide exposure decreased the levels of the two antioxidants both in vitro and in vivo. Ascorbic acid concentration was lowered to a greater extent than glutathione. The pulmonary ascorbic acid level was identical in both control and glutathione-deficient guinea pigs exposed to nitrogen dioxide, suggesting that there was little interaction between the two antioxidants in the lungs during oxidant stress.

  10. Glutathione depletion due to copper-induced phytochelatin synthesis causes oxidative stress in Silene cucubalus

    SciTech Connect

    Ric De Vos, C.H.; Vonk, M.J.; Vooijs, R.; Schat, H. )

    1992-03-01

    The relation between loss of glutathione due to metal-induced phytochelatin synthesis and oxidative stress was studied in the roots of copper-sensitive and tolerant Silene cucubalus (L.) Wib., resistant to 1 and 40 micromolar Cu, respectively. The amount of nonprotein sulfhydryl compounds other then glutathione was taken as a measure of phytochelatins. At a supply of 20 micromolar Cu, which is toxic for sensitive plants only, phytochelatin synthesis and loss of total glutathione were observed only in sensitive plants within 6 h of exposure. When the plants were exposed to a range of copper concentrations for 3 d, a marked production of phytochelatins in sensitive plants was already observed at 0.5 micromolar Cu, whereas the production in tolerant plants was negligible at 40 micromolar or lower. The highest production in tolerant plants was only 40% of that in sensitive plants. In both varieties, the synthesis of phytochelatins was coupled to a loss of glutathione. Copper at toxic concentrations caused oxidative stress, as was evidenced by both the accumulation of lipid peroxidation products and a shift in the glutathione redox couple to a more oxidized state. Depletion of glutathione by pretreatment with buthionine sulfoximine significantly increased the oxidative damage by copper. At a comparably low glutathione level, cadmium had no effect on either lipid peroxidation or the glutathione redox couple in buthionine sulfoximine-treated plants. These results indicate that copper may specifically cause oxidative stress by depletion of the antioxidant glutathione due to phytochelatin synthesis.

  11. Molecularly imprinted solid-phase extraction of glutathione from urine samples.

    PubMed

    Song, Renyuan; Hu, Xiaoling; Guan, Ping; Li, Ji; Zhao, Na; Wang, Qiaoli

    2014-11-01

    Molecularly imprinted polymer (MIP) particles for glutathione were synthesized through iniferter-controlled living radical precipitation polymerization (IRPP) under ultraviolet radiation at ambient temperature. Static adsorption, solid-phase extraction, and high-performance liquid chromatography were carried out to evaluate the adsorption properties and selective recognition characteristics of the polymers for glutathione and its structural analogs. The obtained IRPP-MIP particles exhibited a regularly spherical shape, rapid binding kinetics, high imprinting factor, and high selectivity compared with the MIP particles prepared using traditional free-radical precipitation polymerization. The selective separation and enrichment of glutathione from the mixture of glycyl-glycine and glutathione disulfide could be achieved on the IRPP-MIP cartridge. The recoveries of glutathione, glycyl-glycine, and glutathione disulfide were 95.6% ± 3.65%, 29.5% ± 1.26%, and 49.9% ± 1.71%, respectively. The detection limit (S/N=3) of glutathione was 0.5 mg·L(-1). The relative standard deviations (RSDs) for 10 replicate detections of 50 mg·L(-1) of glutathione were 5.76%, and the linear range of the calibration curve was 0.5 mg·L(-1) to 200 mg·L(-1) under optimized conditions. The proposed approach was successfully applied to determine glutathione in spiked human urine samples with recoveries of 90.24% to 96.20% and RSDs of 0.48% to 5.67%. PMID:25280681

  12. Physical exercise intensity can be related to plasma glutathione levels.

    PubMed

    Gambelunghe, C; Rossi, R; Micheletti, A; Mariucci, G; Rufini, S

    2001-03-01

    The aim of the present study was to examine the effect of different kinds of physical exercise on plasma glutathione levels. Male Wistar rats were randomly divided into four groups: In walking group (W; n=6), rats were trained to walk 0.8 m/min for 45 min; slow running group (SR; n=6) were trained to run 4 m/min for 45 min; fast running group (FR; n=6) ran 8 m/min for 60 min and control rats (C; n=6) remained in their home cages. All animals were sacrificed after exercise and the levels of reduced glutathione (GSH) in plasma samples determined by high performance liquid chromatography (HPLC) with a fluorescent detector. Compared to controls, exercise did not change GSH plasma levels of the W group. A tendency to decrease blood GSH was observed in plasma samples of the SR group and in the FR group, physical exercise resulted in a dramatic decrease in GSH plasma levels. These data suggest that during light physical exercise there is a low production of reactive oxygen species (ROS) with a low request for antioxidant defence such as oxidation of GSH. The dramatic decrease observed in GSH levels in FR rats would indicate the presence of oxidative stress able to modify blood antioxidant profiles. Our results suggest that GSH plays a central antioxidant role in blood during intensive physical exercise and that its modifications are closely related to exercise intensity. PMID:11519887

  13. Physical exercise intensity can be related to plasma glutathione levels.

    PubMed

    Gambelunghe, C; Rossi, R; Micheletti, A; Mariucci, G; Rufini, S

    2001-03-01

    The aim of the present study was to examine the effect of different kinds of physical exercise on plasma glutathione levels. Male Wistar rats were randomly divided into four groups: In walking group (W; n=6), rats were trained to walk 0.8 m/min for 45 min; slow running group (SR; n=6) were trained to run 4 m/min for 45 min; fast running group (FR; n=6) ran 8m/min for 60 min and control rats (C; n=6) remained in their home cages. All animals were sacrificed after exercise and the levels of reduced glutathione (GSH) in plasma samples determined by high performance liquid chromatography (HPLC) with a fluorescent detector. Compared to controls, exercise did not change GSH plasma levels of the W group. A tendency to decrease blood GSH was observed in plasma samples of the SR group and in the FR group, physical exercise resulted in a dramatic decrease in GSH plasma levels. These data suggest that during light physical exercise there is a low production of reactive oxygen species (ROS) with a low request for antioxidant defence such as oxidation of GSH. The dramatic decrease observed in GSH levels in FR rats would indicate the presence of oxidative stress able to modify blood antioxidant profiles. Our results suggest that GSH plays a central antioxidant role in blood during intensive physical exercise and that its modifications are closely related to exercise intensity. PMID:11579999

  14. Bioavailability Study of an Innovative Orobuccal Formulation of Glutathione

    PubMed Central

    Buonocore, Daniela; Grosini, Matteo; Giardina, Silvana; Michelotti, Angela; Carrabetta, Mariaelena; Seneci, Antonio; Verri, Manuela; Dossena, Maurizia; Marzatico, Fulvio

    2016-01-01

    Alteration of the ubiquitous thiol tripeptide glutathione (GSH) is involved in oxidative stress, which plays a role in ageing; consequently, GSH is closely related to this process characterized by progressive decline in the efficiency of physiological function and increased susceptibility to disease. When circulating GSH decreases, oral administration might be considered a therapeutic benefit. Unfortunately, due to the hydrolysis of the tripeptide by intestinal γ-glutamyltransferase, dietary glutathione is not a major determinant for its increase. Aim of this work was to evaluate improvement of GSH systemic availability testing, in vitro and in vivo, an optimized orobuccal fast-slow release formulation tablet containing pure stabilized GSH. In vitro evaluation of the penetration capability of the innovative GSH-release formulation showed that GSH was well absorbed by the reconstructed oral epithelium and its absorption has features of time-dependence. In addition, in vivo results, obtained from 15 healthy volunteers, were in favor of GSH level improvement in blood showing fast (after 30 and 60 minutes) absorption through oral mucosa. In conclusion, the intake of GSH formulated through optimized orobuccal fast-slow release tablets gave positive results in raising GSH blood concentration. PMID:26649136

  15. Glutathione and γ-glutamylcysteine in hydrogen peroxide detoxification.

    PubMed

    Quintana-Cabrera, Ruben; Bolaños, Juan P

    2013-01-01

    Hydrogen peroxide (H2O2) is an important regulator of cell redox status and signaling pathways. However, if produced in excess, it can trigger oxidative damage, which can be counteracted by the antioxidant systems. Amongst these, the glutathione (GSH) precursor, γ-glutamylcysteine (γGC), has recently been shown to detoxify H2O2 in a glutathione peroxidase-1 (GPx1)-dependent fashion. To analyze how both γGC and GSH reduce H2O2, we have taken advantage of a colorimetric assay that allows simple and reliable quantification of H2O2 in the micromolar range. Whereas most assays rely on coupled enzymatic reactions, this method determines the formation of a ferric thiocyanate derivative after direct Fe(2+) oxidation by H2O2. Here, we detail the procedure and considerations to determine H2O2 reduction by both γGC and GSH, either from cell samples or in vitro reactions with purified enzymes from GSH metabolism. PMID:23830629

  16. Glutathione plays a role in the chick intestinal calcium absorption.

    PubMed

    Tolosa de Talamoni, N; Marchionatti, A; Baudino, V; Alisio, A

    1996-10-01

    DL-buthionine-S,R-sulfoximine (BSO) administration to vitamin D-deficient chicks treated with cholecalciferol produces a rapid decrease in the Ca2+ transfer from lumen-to-plasma and in the intestinal glutathione content. This response was reversed by addition of glutathione monoester to the intestinal sac. Variables related to the Ca2+ homeostasis such as plasma Ca and P, and intestinal calbindin D28k were not modified by BSO given to vitamin D-deficient chicks treated with cholecalciferol. Intestinal alkaline phosphatase activity, on the contrary, was highly reduced by BSO in vitamin D-deficient chicks treated with vitamin D3. This effect showed time and dose-dependency. Although the mechanism/s of action of BSO on the intestinal Ca absorption is unknown, it is quite possible that thiol groups of protein involved in the Ca2+ transport are affected by the GSH depletion and/or by block of the antioxidant ability of vitamin D3. Thus, reactive oxygen compounds would be increased and, therefore, the Ca2+ movement from lumen to plasma decreases. PMID:8916550

  17. Glutathione transferase mimics: micellar catalysis of an enzymic reaction.

    PubMed Central

    Lindkvist, B; Weinander, R; Engman, L; Koetse, M; Engberts, J B; Morgenstern, R

    1997-01-01

    Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as alpha,beta-unsaturated carbonyls. The nucleophilic aromatic substitution reaction is enhanced by the following surfactants in descending order: poly(dimethyldiallylammonium - co - dodecylmethyldiallylammonium) bromide (86/14) >>cetyltrimethylammonium bromide>zwittergent 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate)>zwittergent+ ++ 3-14 (n-tetradecyl-N,N-dimethyl - 3 - ammonio -1 - propanesulphonate) approximately N,N - dimethyl - laurylamine N-oxide>N,N-dimethyloctylamine N-oxide. The most efficient catalyst studied is a polymeric material that incorporates surfactant properties (n-dodecylmethyldiallylammonium bromide) and opens up possibilities for engineering sequences of reactions on a polymeric support. Michael addition to alpha,beta-unsaturated carbonyls is exemplified by a model substance, trans-4-phenylbut-3-en-2-one, and a toxic compound that is formed during oxidative stress, 4-hydroxy-2-undecenal. The latter compound is conjugated with the highest efficiency of those tested. Micellar catalysts can thus be viewed as simple models for the glutathione transferases highlighting the influence of a positive electrostatic field and a non-specific hydrophobic binding site, pertaining to two catalytic aspects, namely thiolate anion stabilization and solvent shielding. PMID:9173899

  18. Glutathione synthesis and homeostasis in isolated type II alveolar cells

    SciTech Connect

    Saito, K.; Warshaw, J.B.; Prough, R.A.

    1986-03-05

    After isolation of Type II cells from neonatal rat lung, the glutathione (GSH) levels in these cells were greatly depressed. The total glutathione content could be increased 5-fold within 12-24 h by incubating the cells in media containing sulfur amino acids. Similarly, the activity of ..gamma..-glutamyltranspeptidase was low immediately after isolation, but was increased 2-fold during the first 24 h culture. Addition of either GSH or GSSG to the culture media increased the GSH content of Type II cells 2-2.5-fold. Buthionine sulfoximine and NaF prevented this replenishment of GSH during 24 h culture. When the rates of de novo synthesis of GSH and GSSG from /sup 35/S-cysteine were measured, the amounts of newly formed GSH decreased to 80% in the presence of GSH or GSSG. This suggests that exogenous GSH/GSSG can be taken up by the Type II cells to replenish the intracellular pool of GSH. Methionine was not as effective as cysteine in the synthesis of GSH. These results suggest that GSH levels in the isolated Type II cell can be maintained by de novo synthesis or uptake of exogenous GSH. Most of the GSH synthesized from cysteine, however, was excreted into the media of the cultured cells indicative of a potential role for the type II cell in export of the non-protein thiol.

  19. Renal Clearance and Degradation of Glutathione-Coated Copper Nanoparticles

    PubMed Central

    Yang, Shengyang; Sun, Shasha; Zhou, Chen; Hao, Guiyang; Liu, Jinbin; Ramezani, Saleh; Yu, Mengxiao; Sun, Xiankai; Zheng, Jie

    2016-01-01

    Degradation of inorganic nanoparticles (NPs) into small molecular complexes is often observed in the physiological environment; however, how this process influences renal clearance of inorganic NPs is largely unknown. By systematically comparing renal clearance of degradable luminescent glutathione coated copper NPs (GS-CuNPs) and their dissociated products, Cu(II)-glutathione disulfide (GSSG) complexes (Cu(II)-GSSG), we found that GS-CuNPs were eliminated through the urinary system surprisingly faster and accumulated in the liver much less than their smaller dissociation counterparts. With assistance of radiochemistry and positron emission tomography (PET) imaging, we found that the observed “nano size” effect in enhancing renal clearance is attributed to the fact that GS-CuNPs are more resistant to serum protein adsorption than Cu(II)-GSSG. In addition, since dissociation of GS-CuNPs follows zero-order chemical kinetics, their renal clearance and biodistribution also depend on initial injection doses and their dissociation processes. Quantitative understanding of size effect and other factors involved in renal clearance and biodistribution of degradable inorganic NPs will lay down a foundation for further development of renal-clearable inorganic NPs with minimized nanotoxicity. PMID:25674666

  20. Interspecific variation for thermal dependence of glutathione reductase in sainfoin.

    PubMed

    Kidambi, S P; Mahan, J R; Matches, A G

    1990-05-01

    Understanding the biochemical and physiological consequences of species variation would expedite improvement in agronomically useful genotypes of sainfoin (Onobrychis spp.) Information on variation among sainfoin species is lacking on thermal dependence of glutathione reductase (B.C. 1.6.4.2.), which plays an important role in the protection of plants from both high and low temperature stresses by preventing harmful oxidation of enzymes and membranes. Our objective was to investigate the interspecific variation for thermal dependency of glutathione reductase in sainfoin. Large variation among species was found for: (i) the minimum apparent Km (0.4-2.5 μM NADPH), (ii) the temperature at which the minimum apparent Km was observed (15°-5°C), and (iii) the thermal kinetic windows (2°-30°C width) over a 15°-45°C temperature gradient. In general, tetraploid species had narrower (≤17°C) thermal kinetic windows than did diploid species (∼30°C), with one exception among the diploids. Within the tetraploid species, the cultivars of O. viciifolia had a broader thermal kinetic window (≥7°C) than the plant introduction (PI 212241, >2 °C) itself. PMID:24226572

  1. Increased efflux of glutathione conjugate in acutely diabetic cardiomyocytes.

    PubMed

    Ghosh, Sanjoy; Ting, Simon; Lau, Howard; Pulinilkunnil, Thomas; An, Ding; Qi, Dake; Abrahani, Mohammed A; Rodrigues, Brian

    2004-10-01

    In diabetes, cell death and resultant cardiomyopathy have been linked to oxidative stress and depletion of antioxidants like glutathione (GSH). Although the de novo synthesis and recycling of GSH have been extensively studied in the chronically diabetic heart, their contribution in modulating cardiac oxidative stress in acute diabetes has been largely ignored. Additionally, the possible contribution of cellular efflux in regulating GSH levels during diabetes is unknown. We used streptozotocin to make Wistar rats acutely diabetic and after 4 days examined the different processes that regulate cardiac GSH. Reduction in myocyte GSH in diabetic rats was accompanied by increased oxidative stress, excessive reactive oxygen species, and an elevated apoptotic cell death. The effect on GSH was not associated with any change in either synthesis or recycling, as both gamma-glutamylcysteine synthetase gene expression (responsible for bio syn thesis) and glutathione reductase activity (involved with GSH recycling) remained unchanged. However, gene expression of multidrug resistance protein 1, a transporter implicated in effluxing GSH during oxidative stress, was elevated. GSH conjugate efflux mediated by multidrug resistance protein 1 also increased in diabetic cardiomyocytes, an effect that was blocked using MK-571, a specific inhibitor of this transporter. As MK-571 also decreased oxidative stress in diabetic cardiomyocytes, an important role can be proposed for this transporter in GSH and reactive oxygen species homeostasis in the acutely diabetic heart. PMID:15573148

  2. Fasciola gigantica thioredoxin glutathione reductase: Biochemical properties and structural modeling.

    PubMed

    Gupta, Ankita; Kesherwani, Manish; Velmurugan, Devadasan; Tripathi, Timir

    2016-08-01

    Platyhelminth thioredoxin glutathione reductase (TGR) is a multifunctional enzyme that crosstalk between the conventional thioredoxin (Trx) and glutathione (GSH) system. It has been validated as a potential drug target in blood flukes. In the present study, we have performed a biochemical study on Fasciola gigantica TGR with substrates DTNB and GSSG. The Michaelis constant (Km) with DTNB was found to be 4.34±0.12μM while it was 61.15±1.50μM with GSSG. The kinetic results were compared with the TGR activities of other helminths. FgTGR showed typical hysteretic behavior with GSSG as other TGRs. We also described a homology-based structure of FgTGR. The cofactors (NADPH and FAD) and substrates (GSSG and DTNB) were docked, and two possible binding sites for substrates were identified in a single chain. The substrates were found to bind more favorably in the second site of TrxR domains. We also presented the first report on binding interaction of DTNB with a TGR. DTNB forms H-bond with His204 and Arg450 of chain A, Sec597, and Gly598 from chain B, salt-bridge with Lys124, and numerous other hydrophobic interactions. Helminth TGR represents an important enzyme in the redox and antioxidant system; hence, its inhibition can be used as an effective strategy against liver flukes. PMID:27112978

  3. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

    PubMed

    Wongsantichon, Jantana; Robinson, Robert C; Ketterman, Albert J

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  4. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

    PubMed Central

    Wongsantichon, Jantana; Robinson, Robert C.; Ketterman, Albert J.

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  5. Dairy intake is associated with brain glutathione concentration in older adults123

    PubMed Central

    Lee, Phil; Denney, Douglas R; Spaeth, Kendra; Nast, Olivia; Ptomey, Lauren; Roth, Alexandra K; Lierman, Jo Ann; Sullivan, Debra K

    2015-01-01

    Background: A reduction in key antioxidants such as glutathione has been noted in brain tissue undergoing oxidative stress in aging and neurodegeneration. To date, no dietary factor has been linked to a higher glutathione concentration. However, in an earlier pilot study, we showed evidence of a positive association between cerebral glutathione and dairy intake. Objective: We tested the hypothesis that dairy food consumption is associated with cerebral glutathione concentrations in older adults. Design: In this observational study, we measured cerebral glutathione concentrations in 60 healthy subjects (mean ± SD age: 68.7 ± 6.2 y) whose routine dairy intakes varied. Glutathione concentrations were measured by using a unique, noninvasive magnetic resonance chemical shift imaging technique at 3 T and compared with dairy intakes reported in 7-d food records. Results: Glutathione concentrations in the frontal [Spearman's rank-order correlation (rs) = 0.39, P = 0.013], parietal (rs = 0.50, P = 0.001), and frontoparietal regions (rs = 0.47, P = 0.003) were correlated with average daily dairy servings. In particular, glutathione concentrations in all 3 regions were positively correlated with milk servings (P ≤ 0.013), and those in the parietal region were also correlated with cheese servings (P = 0.015) and calcium intake (P = 0.039). Dairy intake was related to sex, fat-free mass, and daily intakes of energy, protein, and carbohydrates. However, when these factors were controlled through a partial correlation, correlations between glutathione concentrations and dairy and milk servings remained significant. Conclusions: Higher cerebral glutathione concentrations were associated with greater dairy consumption in older adults. One possible explanation for this association is that dairy foods may serve as a good source of substrates for glutathione synthesis in the human brain. PMID:25646325

  6. [35S]-LABELING OF THE SALMONELLA TYPHIMURIUM GLUTATHIONE POOL TO ASSESS GLUTATHIONE-MEDIATED DNA BINDING BY 1,2-DIBROMOETHANE

    EPA Science Inventory

    Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures. In general, glutathione (GSH) conjugation serves as a detoxification m...

  7. Glutathione peroxidase 4 prevents necroptosis in mouse erythroid precursors

    PubMed Central

    Canli, Özge; Alankuş, Yasemin B.; Grootjans, Sasker; Vegi, Naidu; Hültner, Lothar; Hoppe, Philipp S.; Schroeder, Timm; Vandenabeele, Peter; Bornkamm, Georg W.

    2016-01-01

    Maintaining cellular redox balance is vital for cell survival and tissue homoeostasis because imbalanced production of reactive oxygen species (ROS) may lead to oxidative stress and cell death. The antioxidant enzyme glutathione peroxidase 4 (Gpx4) is a key regulator of oxidative stress–induced cell death. We show that mice with deletion of Gpx4 in hematopoietic cells develop anemia and that Gpx4 is essential for preventing receptor-interacting protein 3 (RIP3)-dependent necroptosis in erythroid precursor cells. Absence of Gpx4 leads to functional inactivation of caspase 8 by glutathionylation, resulting in necroptosis, which occurs independently of tumor necrosis factor α activation. Although genetic ablation of Rip3 normalizes reticulocyte maturation and prevents anemia, ROS accumulation and lipid peroxidation in Gpx4-deficient cells remain high. Our results demonstrate that ROS and lipid hydroperoxides function as not-yet-recognized unconventional upstream signaling activators of RIP3-dependent necroptosis. PMID:26463424

  8. Oxidative stress and glutathione in TGF-β-mediated fibrogenesis

    PubMed Central

    Liu, R-M.; Gaston Pravia, K. A.

    2009-01-01

    Transforming growth factor beta (TGF-β) is the most potent and ubiquitous profibrogenic cytokine and its expression is increased in almost all the fibrotic diseases and in experimental fibrosis models. TGF-β increases ROS production and decreases the concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, in various types of cells, which mediates many of TGF-β’s fibrogenic effects. A decreased GSH concentration is also observed in human fibrotic diseases and in experimental fibrosis models. Although the biological significance of GSH depletion in the development of fibrosis remains obscure, GSH and N-acetylcysteine (NAC), a precursor of GSH, have been used in clinics for the treatment of fibrotic diseases. This review summarizes recent findings in the field to address the potential mechanism whereby oxidative stress mediates TGF-β’s fibrogenesis and the potential therapeutic values of antioxidant treatment in fibrotic diseases. PMID:19800967

  9. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    PubMed

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  10. Fumarate induces redox-dependent senescence by modifying glutathione metabolism

    PubMed Central

    Zheng, Liang; Cardaci, Simone; Jerby, Livnat; MacKenzie, Elaine D.; Sciacovelli, Marco; Johnson, T. Isaac; Gaude, Edoardo; King, Ayala; Leach, Joshua D. G.; Edrada-Ebel, RuAngelie; Hedley, Ann; Morrice, Nicholas A.; Kalna, Gabriela; Blyth, Karen; Ruppin, Eytan; Frezza, Christian; Gottlieb, Eyal

    2015-01-01

    Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers. PMID:25613188

  11. Glutathione in metastases: From mechanisms to clinical applications.

    PubMed

    Estrela, José M; Ortega, Angel; Mena, Salvador; Sirerol, J Antoni; Obrador, Elena

    2016-08-01

    Metastatic spread, not primary tumors, is the leading cause of cancer death. Glutathione (γ-glutamyl-cysteinyl-glycine, GSH) is particularly relevant in cancer cells as it is involved in regulating carcinogenic mechanisms, growth and dissemination, and multidrug and radiation resistance. Upon interaction of metastatic cells with the vascular endothelium, a high percentage of metastatic cells with high GSH levels survive the combined nitrosative and oxidative stresses elicited by the vascular endothelium. GSH release from different organs, mainly the liver, and its interorgan transport through the blood circulation to metastatic foci, promote their growth. This review focuses on the relationship among GSH and different key mechanisms that facilitate metastatic cell survival and growth, i.e. adaptive responses to stress, cell death evasion and utilization of physiological neuroendocrine mechanisms. Different strategies that are aimed at sensitizing metastases to cancer therapy by depleting metastatic cell GSH are analyzed. PMID:26754151

  12. Studies on the Glutathione-Dependent Formaldehyde-Activating Enzyme from Paracoccus denitrificans

    PubMed Central

    Hopkinson, Richard J.; Leung, Ivanhoe K. H.; Smart, Tristan J.; Rose, Nathan R.; Henry, Luc; Claridge, Timothy D. W.; Schofield, Christopher J.

    2015-01-01

    Formaldehyde is a toxin and carcinogen that is both an environmental pollutant and an endogenous metabolite. Formaldehyde metabolism, which is probably essential for all aerobic cells, likely proceeds via multiple mechanisms, including via a glutathione-dependent pathway that is widely conserved in bacteria, plants and animals. However, it is unclear whether the first step in the glutathione-dependent pathway (i.e. formation of S-hydroxymethylglutathione (HMG)) is enzyme-catalysed. We report studies on glutathione-dependent formaldehyde-activating enzyme (GFA) from Paracoccus denitrificans, which has been proposed to catalyse HMG formation from glutathione and formaldehyde on the basis of studies using NMR exchange spectroscopy (EXSY). Although we were able to replicate the EXSY results, time course experiments unexpectedly imply that GFA does not catalyse HMG formation under standard conditions. However, GFA was observed to bind glutathione using NMR and mass spectrometry. Overall, the results reveal that GFA binds glutathione but does not directly catalyse HMG formation under standard conditions. Thus, it is possible that GFA acts as a glutathione carrier that acts to co-localise glutathione and formaldehyde in a cellular context. PMID:26675168

  13. Enhancement of Combined Umami and Salty Taste by Glutathione in the Human Tongue and Brain.

    PubMed

    Goto, Tazuko K; Yeung, Andy Wai Kan; Tanabe, Hiroki C; Ito, Yuki; Jung, Han-Sung; Ninomiya, Yuzo

    2016-09-01

    Glutathione, a natural substance, acts on calcium receptors on the tongue and is known to enhance basic taste sensations. However, the effects of glutathione on brain activity associated with taste sensation on the tongue have not been determined under standardized taste delivery conditions. In this study, we investigated the sensory effect of glutathione on taste with no effect of the smell when glutathione added to a combined umami and salty taste stimulus. Twenty-six volunteers (12 women and 14 men; age 19-27 years) performed a sensory evaluation of taste of a solution of monosodium L-glutamate and sodium chloride, with and without glutathione. The addition of glutathione changed taste qualities and significantly increased taste intensity ratings under standardized taste delivery conditions (P < 0.001). Functional magnetic resonance imaging showed that glutathione itself elicited significant activation in the left ventral insula. These results are the first to demonstrate the enhancing effect of glutathione as reflected by brain data while tasting an umami and salty mixture. PMID:27353260

  14. Glutathione peroxidase 4 (Gpx4) and ferroptosis: what's so special about it?

    PubMed Central

    Conrad, Marcus; Friedmann Angeli, José Pedro

    2015-01-01

    The system XC−/glutathione/glutathione peroxidase 4 (Gpx4) axis pivotally controls ferroptosis, a recently described form of regulated non-apoptotic cell death. Compelling evidence has established that this route of cell death is not only of high relevance for triggering cancer cell death, but also proves to be amenable for therapeutic intervention to halt ischemia/reperfusion-related diseases. PMID:27308484

  15. METABOLISM OF 1,1- AND 1,3- DICHLOROPROPENE: A MECHANISM OF BIOACTIVATION BY GLUTATHIONE

    EPA Science Inventory

    Glutathione transferases (GST) catalyze the reaction of glutathione (GSH) with haloalkenes via a nucleophilic vinylic substitution mechanism (SNV reaction). The source water contaminants 1,1-dichloropropene and 1,3-dichloropropene, which are under scrutiny by the U.S.EPA, were...

  16. Glutathione and GSH-dependent enzymes in bronchoalveolar lavage fluid cells in response to ozone

    SciTech Connect

    Boehme, D.S.; Hotchkiss, J.A.; Henderson, R.F. )

    1992-02-01

    The purpose of this study was to determine if in vivo ozone exposure results in elevations in the levels of glutathione and glutathione-dependent enzymes in cells derived from bronchoalveolar lavage fluid (BALF). Our hypothesis was that, as part of a defense mechanism against oxygen toxicity, such cells would have increased levels of glutathione (GSH) in response to an oxidant stress. Female F344/N rats were exposed to 0.8 ppm ozone, 6 hr/day, for 1, 3, or 7 days, after which cells were collected by lung lavage. The GSH and GSH-peroxidase activity per milligram of protein in the cellular fraction, both necessary for reducing cellular peroxides, were elevated after 3 days of ozone exposure. After 7 days of exposure, cellular GSH had returned to control values, but the activity of glutathione reductase, the enzyme that reduces oxidized glutathione to GSH, was increased. Extracellular GSH concentration and glutathione reductase activity in BALF were also increased after 7 days of exposure. The total glutathione equivalents (GSH and GSSG, both cellular and extracellular) in BALF increased throughout the 7-day exposure, with GSH increasing first in the cells, and then in the extracellular fluid. This study demonstrated that the glutathione anti-oxidant system of BALF cells is stimulated by exposure to ozone. This response may serve to protect cells from the toxic effects of oxidant stress.

  17. Glutathione peroxidase-1 protects from CD95-induced apoptosis.

    PubMed

    Gouaze, Valerie; Andrieu-Abadie, Nathalie; Cuvillier, Olivier; Malagarie-Cazenave, Sophie; Frisach, Marie-Francoise; Mirault, Marc-Edouard; Levade, Thierry

    2002-11-01

    Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis. PMID:12221075

  18. Radiosensitization of mouse skin by oxygen and depletion of glutathione

    SciTech Connect

    Stevens, G.; Joiner, M.; Joiner, B.

    1995-09-30

    To determine the oxygen enhancement ratio (OER) and shape of the oxygen sensitization curve of mouse foot skin, the extent to which glutathione (GSH) depletion radiosensitized skin, and the dependence of such sensitization on the ambient oxygen tension. Carbogen caused the greatest radiosensitization of skin, with a reproducible enhancement of 2.2 relative to the anoxic response. The OER of 2.2 is lower than other reports for mouse skin. This may indicate that the extremes of oxygenation were not produced, although there was no direct evidence for this. Depletion of GSH caused minimal radiosensitization when skin was irradiated under anoxic or well-oxygenated conditions. Radiosensitization by GSH depletion was maximal at intermediate oxygen tensions of 10-21% O{sub 2} in the ambient gas. Increasing the extent of GSH depletion led to increasing radiosensitization, with sensitization enhancement ratios of 1.2 and 1.1, respectively, for extensive and intermediated levels of GSH depletion. In mice exposed to 100% O{sub 2}, a significant component of skin radiosensitivity was due to diffusion of oxygen directly through the skin. Pentobarbitone anesthesia radiosensitized skin in mice exposed to 100% O{sub 2} by a factor of 1.2, but did not further sensitize skin in mice exposed to carbogen. Glutathione levels and the local oxygen tension at the time of irradiation were important determinants of mouse foot skin radiosensitivity. The extent to which GSH levels altered the radiosensitivity of skin was critically dependent on the local oxygen tension. These results have significant implications for potential clinical applications of GSH depletion. 53 refs., 7 figs., 2 tabs.

  19. Analysis of Arabidopsis glutathione-transferases in yeast.

    PubMed

    Krajewski, Matthias P; Kanawati, Basem; Fekete, Agnes; Kowalski, Natalie; Schmitt-Kopplin, Philippe; Grill, Erwin

    2013-07-01

    The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as herbicides, extensive redundancy within this large gene family impedes a functional analysis in planta. In this study, a GST-deficient yeast strain was established as a system for analyzing plant GSTs that allows screening for GST substrates and identifying substrate preferences within the plant GST family. To this end, five yeast genes encoding GSTs and GST-related proteins were simultaneously disrupted. The resulting yeast quintuple mutant showed a strongly reduced conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). Consistently, the quintuple mutant was hypersensitive to CDNB, and this phenotype was complemented by the inducible expression of Arabidopsis GSTs. The conjugating activity of the plant GSTs was assessed by in vitro enzymatic assays and via analysis of exposed yeast cells. The formation of glutathione adducts with dinitrobenzene was unequivocally verified by stable isotope labeling and subsequent accurate ultrahigh-resolution mass spectrometry (ICR-FTMS). Analysis of Arabidopsis GSTs encompassing six clades and 42 members demonstrated functional expression in yeast by using CDNB and NBD-Cl as model substrates. Subsequently, the established yeast system was explored for its potential to screen the Arabidopsis GST family for conjugation of the fungicide anilazine. Thirty Arabidopsis GSTs were identified that conferred increased levels of glutathionylated anilazine. Efficient anilazine conjugation was observed in the presence of the phi, tau, and theta clade GSTs including AtGSTF2, AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF10, and AtGSTT2, none of which had previously been known to contribute to fungicide detoxification. ICR-FTMS analysis of yeast extracts allowed the simultaneous detection and

  20. Glutathione dysregulation and the etiology and progression of human diseases

    PubMed Central

    Ballatori, Nazzareno; Krance, Suzanne M.; Notenboom, Sylvia; Shi, Shujie; Tieu, Kim; Hammond, Christine L.

    2009-01-01

    Glutathione (GSH) plays an important role in a multitude of cellular processes, including cell differentiation, proliferation, and apoptosis, and as a result, disturbances in GSH homeostasis are implicated in the etiology and/or progression of a number of human diseases, including cancer, diseases of aging, cystic fibrosis, and cardiovascular, inflammatory, immune, metabolic, and neurodegenerative diseases. Because of GSH’s pleiotropic effects on cell functions, it has been quite difficult to define the role of GSH in the onset and/or the expression of human diseases, although significant progress is being made. GSH levels, turnover rates and/or oxidation state can be compromised by inherited or aquired defects in the enzymes, transporters, signaling molecules, or transcription factors that are involved in its homeostasis, or from exposure to reactive chemicals or metabolic intermediates. GSH deficiency or a decrease in the GSH/glutathione disulfide (GSSG) ratio manifests itself largely through an increased susceptibility to oxidative stress, and the resulting damage is thought to be involved in diseases such as cancer, Parkinson’s disease, and Alzheimer’s disease. In addition, imbalances in GSH levels affect immune system function, and are thought to play a role in the aging process. Just as low intracellular GSH levels decrease cellular antioxidant capacity, elevated GSH levels generally increase antioxidant capacity and resistance to oxidative stress, and this is observed in many cancer cells. The higher GSH levels in some tumor cells are also typically associated with higher levels of GSH-related enzymes and transporters. Although neither the mechanism nor the implications of these changes are well defined, the high GSH content makes cancer cells chemoresistant, which is a major factor that limits drug treatment. The present report highlights and integrates the growing connections between imbalances in GSH homeostasis and a multitude of human diseases

  1. Glutathione S-transferase activity and glutathione S-transferase mu expression in subjects with risk for colorectal cancer.

    PubMed

    Szarka, C E; Pfeiffer, G R; Hum, S T; Everley, L C; Balshem, A M; Moore, D F; Litwin, S; Goosenberg, E B; Frucht, H; Engstrom, P F

    1995-07-01

    The glutathione S-transferases (alpha, mu, and pi), a family of Phase II detoxication enzymes, play a critical role in protecting the colon mucosa by catalyzing the conjugation of dietary carcinogens with glutathione. We investigated the efficacy of using the glutathione S-transferase (GST) activity of blood lymphocytes and GST-mu expression as biomarkers of risk for colorectal cancer. GST activity was measured in the blood lymphocytes of control individuals (n = 67) and in the blood lymphocytes (n = 60) and colon tissue (n = 34) of individuals at increased risk for colon cancer. Total GST activity was determined spectrophotometrically with the use of 1-chloro-2,4-dinitrobenzene as a substrate. The ability to express the um subclass of GST was determined with the use of an ELISA. Although interindividual variability in the GST activity of blood lymphocytes was greater than 8-fold (range, 16.7-146.8 nmol/min/mg), the GST activity of blood lymphocytes and colon tissue within an individual was constant over time and was unrelated to sex, age, or race. The GST activity of blood lymphocytes from high-risk individuals was significantly lower than that of blood lymphocytes from control individuals (P < or = 0.004). No association was observed between the frequency of GST-mu phenotype and risk for colorectal cancer. Blood lymphocytes from high-risk individuals unable to express GST-mu had lower levels of GST activity than did those from control subjects with the GST-mu null phenotype; however, this difference was significant in male subjects only (P < or = 0.006). Analysis of paired samples of blood lymphocytes and colon tissue indicated a strong correlation between the GST activity of the two tissue types (Spearman's rank correlation, r = 0.87; P < or = 0.0001). The GST activity of blood lymphocytes may be used to identify high-risk individuals with decreased protection from this Phase II detoxication enzyme who may benefit from clinical trials evaluating GST modulators

  2. Glutathione prevents ethanol induced gastric mucosal damage and depletion of sulfhydryl compounds in humans.

    PubMed Central

    Loguercio, C; Taranto, D; Beneduce, F; del Vecchio Blanco, C; de Vincentiis, A; Nardi, G; Romano, M

    1993-01-01

    Whether parenteral administration of reduced glutathione prevented ethanol induced damage to and depletion of sulfhydryl compounds in the human gastric mucosa was investigated. Ten healthy volunteers underwent endoscopy on three separate occasions. Gastric mucosal damage was induced by spraying 80% ethanol on to the gastric mucosa through the biopsy channel of the endoscope. The gastric mucosal score, total sulfhydryls, glutathione, and cysteine were evaluated in basal conditions and after ethanol administration with and without pretreatment with parenteral glutathione. Glutathione significantly decreased the extent of ethanol induced macroscopic injury to the mucosa of the gastric body and antrum. Glutathione's protective effect is associated with appreciable inhibition of ethanol induced depletion of gastric sulfhydryl compounds. This is the first report of protection against ethanol induced gastric mucosal damage by a sulfhydryl containing agent in humans. PMID:8432465

  3. Storage of Heparinised Canine Whole Blood for the Measurement of Glutathione Peroxidase Activity.

    PubMed

    van Zelst, Mariëlle; Hesta, Myriam; Gray, Kerry; Janssens, Geert P J

    2016-08-01

    Glutathione peroxidase activity is used as a biomarker of selenium status in dogs. Freshly collected blood samples are usually measured, due to the lack of knowledge on the effect of storing the samples. This study investigated if the analysis of glutathione peroxidase activity in whole blood collected from dogs was affected by storage of between 5 and 164 days. Results indicated that glutathione peroxidase activity was more variable in the freshly analysed samples compared to the stored samples. Although the mean differences between fresh and stored samples were not always equal to zero, this is thought to be caused by the variability of reagent preparation rather than by storage, as no consistent increase or decrease in glutathione peroxidase activity was found. Therefore, it can be concluded that heparinised dog blood samples can be successfully stored up to 164 days before analysis of glutathione peroxidase activity. PMID:26701335

  4. Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori.

    PubMed

    Yamamoto, Kohji; Yamada, Naotaka

    2016-01-01

    The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription-polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides. PMID:27440377

  5. Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori

    PubMed Central

    Yamamoto, Kohji; Yamada, Naotaka

    2016-01-01

    The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription–polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides. PMID:27440377

  6. Effect of methylmercury on the activity of glutathione peroxidase in rat liver

    SciTech Connect

    Hirota, Y.

    1986-09-01

    The effect of methylmercury on the activity of glutathione peroxidase in rat liver was studied in vivo. A daily dose of 10mg methylmercuric chloride/kg body weight was administered subcutaneously to 15 male Wistar rats for 10 days, and the glutathione peroxidase activity in the liver was measured to compare with the control activity. A marked decrease was observed in the glutathione peroxidase activity in the experimental animals, which measured as low as 40% in comparison to that in the control animals. It can be speculated that the inhibition of glutathione peroxidase activity plays a significant role in the development of mercury toxicity and that the protective effect of selenium and vitamin E on the mercury intoxication might be partly due to preserving the glutathione peroxidase activity in the antioxidative defense mechanisms.

  7. Dynamics of glutathione regulation in Schistosoma mansoni: correlations with the acute effects of oltipraz

    SciTech Connect

    Morrison, D.D.

    1984-01-01

    Glutathione is present in adult Schistosoma mansoni (0.336 +/- 0.012 nmol/mg protein) at significantly lower levels than uninfected host tissues (1.051 +/- 0.013 nmol/mg protein, liver; 0.627 +/- 0.013 nmol/mg protein, kidney). Host hepatic glutathione levels decline significantly during the course of infection, while renal cortical glutathione levels are unaffected. Of the enzymes regulating glutathione utilization, glutathione reductase in the male parasite exhibits a specific activity of 10.3 +/- 4.2 nmol/mg protein, 15% of hepatic values. The apparent glutathione S-transferase activity was 26 +/- 7 ..mu..mol conjugate formed/min/mg protein with p-nitrobenzyl chloride as substrate (13% of hepatic values) and 526 +/- 18 ..mu..mol conjugate formed/min/mg protein with 1-chloro-2,4-dinitrobenzene as substrate (43% of hepatic values). Male schistosomes exhibited negligible glutathione peroxidase activity. Oltipraz, an antischistosomal compound, effected a significant depletion of parasite and host glutathione levels within 1 h of exposure in vivo and in vitro (at 250 mg/kg and 10 ..mu..M, respectively). Host tissue glutathionine levels returned to, or above, control levels by 6 h after oltipraz administration, while parasite glutathione levels remained significantly depressed. Uptake of (/sup 35/S) cysteine or (/sup 35/S) cystine by schistosomes was inhibited by oltipraz. However, the drug did not alter the relative distribution of label once incorporated into the parasite, indicating that the enzymes of glutathione synthesis were not directly inhibited.

  8. Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

    PubMed

    Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

    2005-01-01

    The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase. PMID:15619514

  9. Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis.

    PubMed

    Yang, Jianhua; Li, Wei; Wang, Dezheng; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-07-01

    Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, γ-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production. PMID:26996628

  10. Glutathione, glutathione S-transferases, and related redox enzymes in Adriamycin-resistant cell lines with a multidrug resistant phenotype.

    PubMed

    Schisselbauer, J C; Crescimanno, M; D'Alessandro, N; Clapper, M; Toulmond, S; Tapiero, H; Tew, K D

    1989-01-01

    Friend erythroleukemia cells (FLC) selected by exposure to Adriamycin (doxorubicin) express an approximate 2.5-fold (ARN1) or 13-fold (ARN2) resistance to the drug with various degrees of cross-resistance to other anthracyclines, vinca alkaloids, and epipodophyllotoxins. Because the redox cycling of the quinone moiety of Adriamycin is known to produce oxidative stress, however, an analysis of glutathione (GSH) and related enzyme systems was undertaken in the wild-type and selected resistant cells. In ARN1 and ARN2, superoxide dismutase (SOD) and catalase activities were slightly decreased, intracellular GSH and GSH reductase were essentially unchanged, and total GSH peroxidase, glutathione S-transferase (GST), and DT-diaphorase activities were slightly elevated. In each case there was no stoichiometric relationship between degree of resistance and level of activity. GST isozymes were purified from each cell line by HPLC GSH affinity column chromatography. Two-dimensional gel electrophoresis and western blot immunoreactivity against a battery of GST isozyme polyclonal antibodies determined that both the resistant and sensitive cells expressed isozymes of the alpha, pi, and mu classes (alternative murine nomenclature: M1, M2, M3). Of significance, both ARN1 and ARN2 cell lines expressed a unique alpha subunit which was absent from the parent FLC cell line. This isozyme presumably accounted for the increased GSH peroxidase activity (cumene hydroperoxide as substrate) found in ARN1 and ARN2 and may play a role in the small incremental resistance to melphalan found for both resistant lines. Expression of the isozyme was not stoichiometric with respect to degree of resistance. The presence of this isozyme may contribute to the resistant phenotype or may be the consequence of a more general cellular response to oxidative stress. PMID:2639724

  11. Viral load is associated with abnormal serum levels of micronutrients and glutathione and glutathione-dependent enzymes in genotype 3 HCV patients

    PubMed Central

    Razzaq, Zarish; Malik, Arif

    2014-01-01

    Background Oxidative stress in hepatitis C patients has been linked to hepatitis C virus. We verified this assumption in HCV genotype 3 patients by detecting the relationship between viral load and certain specific oxidative stress markers like Cu, Mn, Fe, Se, Zn and glutathione and glutathione-dependent enzymes. Method Subjects (n = 200, average age 24 years) with quantitative HCV RNA polymerase chain reaction-proven genotype 3 hepatitis C were simultaneously evaluated. Cu, Mn, Fe, Se and Zn serum levels were by using atomic absorption spectrophotometer. Internationally accepted methods were used for viral load quantification of glutathione, GR and Gpx serum levels. Result There was a significant correlation between HCV viral load and studied parameters. With the increase of viral load from mild group (200,000–1,000,000 copies/ml) to severe group (5,000,000–25,000,000 copies/ml) the serum levels of Cu, Mn, Zn, and Fe and glutathione reductase were found to be abnormally high. However, in severe viral load group serum concentration of Se and glutathione was less than the healthy controls. Conclusion As a significant correlation was detected between the study parameters in genotype 3 HCV patients, it is concluded that the studied micronutrients and glutathione and glutathione-dependent enzymes are the biomolecular targets of HCV to induce oxidative stress. General significance Constant monitoring and regulation of the recommended biomolecular targets of HCV can improve the plight of more than 170 million patients suffering from hepatitis C virus around the globe. PMID:26674880

  12. Purification and Biochemical Characterization of Glutathione S-Transferase from Down Syndrome and Normal Children Erythrocytes: A Comparative Study

    ERIC Educational Resources Information Center

    Hamed, Ragaa R.; Maharem, Tahany M.; Abdel-Meguid, Nagwa; Sabry, Gilane M.; Abdalla, Abdel-Monem; Guneidy, Rasha A.

    2011-01-01

    Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was…

  13. Unbalanced activation of glutathione metabolic pathways suggests potential involvement in plant defense against the gall midge mayetiola destructor in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutathione, a thiol tripeptide of '-glutamylcysteinylglycine, exists abundantly in nearly all organisms. Glutathione participates in various physiological processes involved in redox reactions by serving as an electron donor/acceptor. In this study, we found that the abundance of total glutathion...

  14. Protective role of glutathione reductase in paraquat induced neurotoxicity.

    PubMed

    Djukic, Mirjana M; Jovanovic, Marina D; Ninkovic, Milica; Stevanovic, Ivana; Ilic, Katarina; Curcic, Marijana; Vekic, Jelena

    2012-08-30

    Paraquat (PQ), a widely used herbicide is a well-known free radical producing agent. The mechanistic pathways of PQ neurotoxicity were examined by assessing oxidative/nitrosative stress markers. Focus was on the role of glutathione (GSH) cycle and to examine whether the pre-treatment with enzyme glutathione reductase (GR) could protect the vulnerable brain regions (VBRs) against harmful oxidative effect of PQ. The study was conducted on Wistar rats, randomly divided in five groups: intact-control group, (n = 8) and four experimental groups (n = 24). All tested compounds were administered intrastriatally (i.s.) in one single dose. The following parameters of oxidative status were measured in the striatum, hippocampus and cortex, at 30 min, 24 h and 7 days post treatment: superoxide anion radical (O₂·⁻), nitrate (NO₃⁻), malondialdehyde (MDA), superoxide dismutase (SOD), total GSH (tGSH) and its oxidized, disulfide form (GSSG) and glutathione peroxidase (GPx). Results obtained from the intact and the sham operated groups were not statistically different, confirming that invasive i.s. route of administration would not influence the reliability of results. Also, similar pattern of changes were observed between ipsi- and contra- lateral side of examined VBRs, indicating rapid spatial spreading of oxidative stress. Mortality of the animals (10%), within 24h, along with symptoms of Parkinsonism, after awakening from anesthesia for 2-3 h, were observed in the PQ group, only. Increased levels of O₂·⁻, NO₃⁻ and MDA, increased ratio of GSSG/GSH and considerably high activity of GPx were measured at 30 min after the treatment. Cytotoxic effect of PQ was documented by drastic drop of all measured parameters and extremely high peak of the ratio GSSG/GSH at 24th hrs after the PQ i.s. injection. In the GR+PQ group, markedly low activity of GPx and low content of NO₃⁻ (in striatum and cortex) were measured during whole experiment, while increase value was

  15. Effect of fish oil on glutathione redox system in multiple sclerosis

    PubMed Central

    Sorto-Gomez, Tania E; Ortiz, Genaro G; Pacheco-Moises, Fermín P; Torres-Sanchez, Erandis D; Ramirez-Ramirez, Viridiana; Macias-Islas, Miguel A; de la Rosa, Alfredo Celis; Velázquez-Brizuela, Irma E

    2016-01-01

    Multiple sclerosis (MS) is a chronic, inflammatory and autoimmune disease of the central nervous system. Dysregulation of glutathione homeostasis and alterations in glutathione-dependent enzyme activities are implicated in the induction and progression of MS. Evidence suggests that Omega-3 polyunsaturated fatty acids (PUFAs) have anti-inflammatory, antioxidant and neuroprotective effects. The aim of the present work was to evaluate the effect of fish oil on the activity of glutathione reductase (GR), content of reduced and oxidized glutathione, and GSH/GSSG ratio in MS. 50 patients with relapsing-remitting MS were enrolled. The experimental group received orally 4 g/day of fish oil for 12 months. Fish oil supplementation resulted in a significant increase in n-3 fatty acids and a decrease n-6 fatty acids. No differences in glutathione reductase activity, content of reduced and oxidized glutathione, and GSH/GSSG ratio were found. Conclusion: Glutathione reductase activity was not significantly different between the groups; however, fish oil supplementation resulted in smaller increase in GR compared with control group, suggesting a possible effect on antioxidant defence mechanisms. PMID:27335704

  16. The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

    PubMed Central

    Markovic, Jelena; Mora, Nancy J.; Broseta, Ana M.; Gimeno, Amparo; de-la-Concepción, Noelia; Viña, José; Pallardó, Federico V.

    2009-01-01

    Background Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate. Principal Findings We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation. Conclusions Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle. PMID:19641610

  17. Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

    PubMed Central

    Freitas, Hercules R.; Ferraz, Gabriel; Ferreira, Gustavo C.; Ribeiro-Resende, Victor T.; Chiarini, Luciana B.; do Nascimento, José Luiz M.; Matos Oliveira, Karen Renata H.; Pereira, Tiago de Lima; Ferreira, Leonardo G. B.; Kubrusly, Regina C.; Faria, Robson X.

    2016-01-01

    Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1–10mM) showed that 5–10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit. PMID:27078878

  18. Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells.

    PubMed

    Freitas, Hercules R; Ferraz, Gabriel; Ferreira, Gustavo C; Ribeiro-Resende, Victor T; Chiarini, Luciana B; do Nascimento, José Luiz M; Matos Oliveira, Karen Renata H; Pereira, Tiago de Lima; Ferreira, Leonardo G B; Kubrusly, Regina C; Faria, Robson X; Herculano, Anderson Manoel; Reis, Ricardo A de Melo

    2016-01-01

    Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10 mM) showed that 5-10 mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50 mM KCl (labeled as βIII tubulin positive cells). BBG 100 nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70 μM and MK-801 20 μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5 mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit. PMID:27078878

  19. Glutathione Preservation during Storage of Rat Lenses in Optisol-GS and Castor Oil

    PubMed Central

    Holm, Thomas; Brøgger-Jensen, Martin Rocho; Johnson, Leif; Kessel, Line

    2013-01-01

    Background Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations. Methodology/Principal Findings Rat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree. Conclusions/Significance It was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours. PMID:24260265

  20. Plasma glutathione turnover in the rat: effect of fasting and buthionine sulfoximine.

    PubMed

    Hum, S; Robitaille, L; Hoffer, L J

    1991-05-01

    Hepatic glutathione (GSH) plays an important role in the detoxification of reactive molecular intermediates. Because of evidence that the intrahepatic turnover of glutathione in the rat may be largely accounted for by efflux from hepatocytes into the general circulation, the quantitation of plasma GSH turnover in vivo could provide a noninvasive index of hepatic glutathione metabolism. We developed a method to estimate plasma glutathione turnover and clearance in the intact, anesthetized rat using a 30-min unprimed, continuous infusion of 35S-labelled GSH. A steady state of free plasma glutathione specific radioactivity was achieved within 10 min, as determined by high-pressure liquid chromatography with fluorometric detection after precolumn derivatization of the plasma samples with monobromobimane. The method was tested after two treatments known to alter hepatic GSH metabolism: 90 min after intraperitoneal injection of 4 mmol/kg buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, and after a 48-h fast. Liver glutathione concentration (mean +/- SEM) was 5.00 +/- 0.53 mumol/g wet weight in control rats. It decreased to 3.10 +/- 0.35 mumol/g wet weight after BSO injection and to 3.36 +/- 0.14 mumol/g wet weight after fasting (both p less than 0.05). Plasma glutathione turnover was 63.0 +/- 7.46 nmol.min-1.100 g-1 body weight in control rats, 35.0 +/- 2.92 nmol.min-1.g-1 body weight in BSO-treated rats, and 41.7 +/- 2.28 nmol.min-1.g-1 body weight after fasting (both p less than 0.05), thus reflecting the hepatic alterations. This approach might prove useful in the noninvasive assessment of liver glutathione status. PMID:1863907

  1. 1-3-A Resolution Structure of Human Glutathione S-Transferase With S-Hexyl Glutathione Bound Reveals Possible Extended Ligandin Binding Site

    SciTech Connect

    Trong, I.Le; Stenkamp, R.E.; Ibarra, C.; Atkins, W.M.; Adman, E.T.

    2005-08-22

    Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathione binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.

  2. Glyoxalase 1 and glutathione reductase 1 regulate anxiety in mice.

    PubMed

    Hovatta, Iiris; Tennant, Richard S; Helton, Robert; Marr, Robert A; Singer, Oded; Redwine, Jeffrey M; Ellison, Julie A; Schadt, Eric E; Verma, Inder M; Lockhart, David J; Barlow, Carrolee

    2005-12-01

    Anxiety and fear are normal emotional responses to threatening situations. In human anxiety disorders--such as panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, social phobia, specific phobias and generalized anxiety disorder--these responses are exaggerated. The molecular mechanisms involved in the regulation of normal and pathological anxiety are mostly unknown. However, the availability of different inbred strains of mice offers an excellent model system in which to study the genetics of certain behavioural phenotypes. Here we report, using a combination of behavioural analysis of six inbred mouse strains with quantitative gene expression profiling of several brain regions, the identification of 17 genes with expression patterns that correlate with anxiety-like behavioural phenotypes. To determine if two of the genes, glyoxalase 1 and glutathione reductase 1, have a causal role in the genesis of anxiety, we performed genetic manipulation using lentivirus-mediated gene transfer. Local overexpression of these genes in the mouse brain resulted in increased anxiety-like behaviour, while local inhibition of glyoxalase 1 expression by RNA interference decreased the anxiety-like behaviour. Both of these genes are involved in oxidative stress metabolism, linking this pathway with anxiety-related behaviour. PMID:16244648

  3. Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase.

    PubMed

    Sun, Qi-An; Su, Dan; Novoselov, Sergey V; Carlson, Bradley A; Hatfield, Dolph L; Gladyshev, Vadim N

    2005-11-01

    Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes. PMID:16262253

  4. Catalytic Cycle of Human Glutathione Reductase Near 1 Å Resolution

    SciTech Connect

    Berkholz, Donald S.; Faber, H. Richard; Savvides, Savvas N.; Karplus, P. Andrew

    2008-09-08

    Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 {angstrom} that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity -- a net deviation of 53 deg. across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.

  5. Glutathione Decrement Drives Thermogenic Program In Adipose Cells.

    PubMed

    Lettieri Barbato, Daniele; Tatulli, Giuseppe; Maria Cannata, Stefano; Bernardini, Sergio; Aquilano, Katia; Ciriolo, Maria R

    2015-01-01

    Adipose tissue metabolically adapts to external stimuli. We demonstrate that the induction of the thermogenic program in white adipocytes, through cold exposure in mice or in vitro adrenergic stimulation, is accompanied by a decrease in the intracellular content of glutathione (GSH). Moreover, the treatment with a GSH depleting agent, buthionine sulfoximine (BSO), recapitulates the effect of cold exposure resulting in the induction of thermogenic program. In particular, BSO treatment leads to enhanced uncoupling respiration as demonstrated by increased expression of thermogenic genes (e.g. Ucp1, Ppargc1a), augmented oxygen consumption and decreased mitochondrial transmembrane potential. Buffering GSH decrement by pre-treatment with GSH ester prevents the up-regulation of typical markers of uncoupling respiration. We demonstrate that FoxO1 activation is responsible for the conversion of white adipocytes into a brown phenotype as the "browning" effects of BSO are completely abrogated in cells down-regulating FoxO1. In mice, the BSO-mediated up-regulation of uncoupling genes results in weight loss that is at least in part ascribed to adipose tissue mass reduction. The induction of thermogenic program has been largely proposed to counteract obesity-related diseases. Based on these findings, we propose GSH as a novel therapeutic target to increase energy expenditure in adipocytes. PMID:26260892

  6. New role for leucyl aminopeptidase in glutathione turnover.

    PubMed Central

    Cappiello, Mario; Lazzarotti, Alessandra; Buono, Francesca; Scaloni, Andrea; D'Ambrosio, Chiara; Amodeo, Pietro; Méndez, Blanca L; Pelosi, Paolo; Del Corso, Antonella; Mura, Umberto

    2004-01-01

    A manganese-dependent cysteinyl-glycine hydrolysing activity has been purified to electrophoretic homogeneity from bovine lens. The characterization of the purified enzyme (molecular mass of the native protein, molecular mass of the subunit and extensive primary structure analysis) allowed the unequivocal attribution of the cysteinyl-glycine hydrolysing activity, which is usually associated with alanyl aminopeptidase (EC 3.4.11.2) or membrane-bound dipeptidase (EC 3.4.13.19), to LAP (leucyl aminopeptidase; EC 3.4.11.1). Analysis of the pH dependence of Cys-Gly hydrolysis catalysed by LAP, supported by a molecular modelling approach to the enzyme-substrate conformation, gave insights into the ability of the enzyme to recognize Cys-Gly as a substrate. Due to the effectiveness of LAP in hydrolysing Cys-Gly (K(m)=0.57 mM, kcat=6.0x10(3) min(-1) at pH 7.4 and 25 degrees C) with respect to other dipeptide substrates, a new role for this enzyme in glutathione turnover is proposed. PMID:14583094

  7. MITOCHONDRIAL GLUTATHIONE: FEATURES, REGULATION AND ROLE IN DISEASE

    PubMed Central

    Marí, Montserrat; Morales, Albert; Colell, Anna; García-Ruiz, Carmen; Kaplowitz, Neil; Fernández-Checa, José C

    2012-01-01

    BACKGROUND Mitochondria are the powerhouse of mammalian cells and the main source of reactive oxygen species (ROS) associated with oxygen consumption. In addition, they also play a strategic role in controlling the fate of cells through regulation of death pathways. Mitochondrial ROS production fulfills a signaling role through regulation of redox pathways, but also contributes to mitochondrial damage in a number of pathological states. SCOPE OF REVIEW Mitochondria are exposed to the constant generation of oxidant species, and yet the organelle remains functional due to the existence of an armamentarium of antioxidant defense systems aimed to repair oxidative damage, of which mitochondrial glutathione (mGSH) is of particular relevance. Thus, the aim of the review is to cover the regulation of mGSH and its role in disease. MAJOR CONCLUSIONS Cumulating evidence over recent years has demonstrated the essential role for mGSH in mitochondrial physiology and disease. Despite its high concentration in the mitochondrial matrix, mitochondria lack the enzymes to synthesize GSH de novo, so that mGSH originates from cytosolic GSH via transport through specific mitochondrial carriers, which exhibit sensitivity to membrane dynamics. Depletion of mGSH sensitizes cells to stimuli leading to oxidative stress such as TNF, hypoxia or amyloid β-peptide, thereby contributing to disease pathogenesis. GENERAL SIGNIFICANCE Understanding the regulation of mGSH may provide novel insights to disease pathogenesis and toxicity and the opportunity to design therapeutic targets of intervention in cell death susceptibility and disease. PMID:23123815

  8. Oral glutathione supplementation drastically reduces Helicobacter-induced gastric pathologies

    PubMed Central

    De Bruyne, Ellen; Ducatelle, Richard; Foss, Dennis; Sanchez, Margaret; Joosten, Myrthe; Zhang, Guangzhi; Smet, Annemieke; Pasmans, Frank; Haesebrouck, Freddy; Flahou, Bram

    2016-01-01

    Helicobacter (H.) suis causes gastric pathologies in both pigs and humans. Very little is known on the metabolism of this bacterium and its impact on the host. In this study, we have revealed the importance of the glutamate-generating metabolism, as shown by a complete depletion of glutamine (Gln) in the medium during H. suis culture. Besides Gln, H. suis can also convert glutathione (GSH) to glutamate, and both reactions are catalyzed by the H. suis γ-glutamyltranspeptidase (GGT). Both for H. pylori and H. suis, it has been hypothesized that the degradation of Gln and GSH may lead to a deficiency for the host, possibly initiating or promoting several pathologies. Therefore the in vivo effect of oral supplementation with Gln and GSH was assessed. Oral supplementation with Gln was shown to temper H. suis induced gastritis and epithelial (hyper)proliferation in Mongolian gerbils. Astonishingly, supplementation of the feed with GSH, another GGT substrate, resulted in inflammation and epithelial proliferation levels returning to baseline levels of uninfected controls. This indicates that Gln and GSH supplementation may help reducing tissue damage caused by Helicobacter infection in both humans and pigs, highlighting their potential as a supportive therapy during and after Helicobacter eradication therapy. PMID:26833404

  9. Inactivation of Anopheles gambiae Glutathione Transferase ε2 by Epiphyllocoumarin

    PubMed Central

    Marimo, Patience; Hayeshi, Rose; Mukanganyama, Stanley

    2016-01-01

    Glutathione transferases (GSTs) are part of a major family of detoxifying enzymes that can catalyze the reductive dehydrochlorination of dichlorodiphenyltrichloroethane (DDT). The delta and epsilon classes of insect GSTs have been implicated in conferring resistance to this insecticide. In this study, the inactivation of Anopheles gambiae GSTε2 by epiphyllocoumarin (Tral 1) was investigated. Recombinant AgGSTε2 was expressed in Escherichia coli cells containing a pET3a-AGSTε2 plasmid and purified by affinity chromatography. Tral 1 was shown to inactivate GSTε2 both in a time-dependent manner and in a concentration-dependent manner. The half-life of GSTε2 in the presence of 25 μM ethacrynic acid (ETA) was 22 minutes and with Tral 1 was 30 minutes, indicating that Tral 1 was not as efficient as ETA as an inactivator. The inactivation parameters kinact and KI were found to be 0.020 ± 0.001 min−1 and 7.5 ± 2.1 μM, respectively, after 90 minutes of incubation. Inactivation of GSTε2 by Tral 1 implies that Tral 1 covalently binds to this enzyme in vitro and would be expected to exhibit time-dependent effects on the enzyme in vivo. Tral 1, therefore, would produce irreversible effects when used together with dichlorodiphenyltrichloroethane (DDT) in malaria control programmes where resistance is mediated by GSTs. PMID:26925266

  10. Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

    PubMed Central

    Bellomo, G; Vairetti, M; Stivala, L; Mirabelli, F; Richelmi, P; Orrenius, S

    1992-01-01

    The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury. Images PMID:1584774

  11. The evolution of glutathione metabolism in phototrophic microorganisms

    NASA Technical Reports Server (NTRS)

    Fahey, Robert C.; Buschbacher, Ralph M.; Newton, Gerald L.

    1988-01-01

    The low molecular weight thiol composition of a variety of phototropic microorganisms is examined in order to ascertain how evolution of glutathione (GSH) production is related to the evolution of oxygenic photosynthesis. Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols (RSH) to fluorescent derivatives (RSmB) which were analyzed by high performance liquid chromatography (HPLC). Significant levels of GSH were not found in green sulfur bacteria. Substantial levels were present in purple bacteria, cyanobacteria, and eukaryotic algae. Other thiols measured included cysteine, gamma-glutamylcysteine, thiosulfate, coenzyme A, and sulfide. Many of the organisms also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability which was quenched by treatment with 2-pyridyl disulfide or 5,5 prime-bisdithio - (2-nitrobenzoic acid) prior to reaction with mBBr. These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of reactive disulfides. The distribution of GSH in phototropic eubacteria indicates that GSH synthesis evolved at or around the time that oxygenic photosynthesis evolved.

  12. Regional modulation of the response to glutathione in Hydra vulgaris.

    PubMed

    Pierobon, Paola

    2015-07-01

    In the presence of prey, or upon exposure to reduced glutathione (GSH), Hydra polyps open a mouth to ingest the captured prey and close it after feeding; at rest the mouth is not evident. In previous papers we have shown that GABA, glycine and NMDA modulate the mechanisms of mouth closure through ligand-gated-ion-channel receptors that are similar to their mammalian analogues in terms of biochemical and pharmacological properties. In order to study the regional distribution of these receptors, we have applied the GSH assay to polyps amputated at different levels of the body column. The response to 1-10 µmol l(-1) GSH of polyps lacking either peduncle and foot or the entire body columns (heads) was not different from control, whole animals. In the presence of GABA or muscimol, duration of the response was significantly decreased in heads; the decrease was suppressed by the GABA antagonists gabazine and bicuculline. By contrast, in animals lacking peduncle and foot, duration of the response did not vary upon GABA administration. Conversely, in the presence of glycine, duration of the response in heads preparations was similar to control, whereas in footless polyps, it was significantly reduced. The decrease was mimicked by the glycine agonists taurine and β-alanine, and counteracted by strychnine. These results suggest a regional distribution of receptors to GABA and glycine in the neuromuscular circuitry modulating the feeding behaviour. PMID:25987735

  13. Measuring glutathione-induced feeding response in hydra.

    PubMed

    Kulkarni, Ram; Galande, Sanjeev

    2014-01-01

    Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra. PMID:25490534

  14. Glutathione modulation in cancer treatment: will it work

    SciTech Connect

    Mitchell, J.B.; Cook, J.A.; DeGraff, W.; Glatstein, E.; Russo, A.

    1989-05-01

    Glutathione (GSH) assumes a pivotal role in numerous cellular functions including bioreductive reactions, maintenance of enzyme activity, amino acid transport, protection from harmful oxidative species, and detoxification of xenobiotics. The importance of GSH in modifying the cellular response to several anti-cancer treatment modalities has become better appreciated with the introduction of agents which can either decrease or elevate GSH levels in cells and tissues. In general, GSH depletion has been demonstrated to further enhance the cytotoxicity of several chemotherapy drugs and nitroimidazole hypoxic cell radiosensitizers. Conversely, GSH elevation affords varying degrees of protection. Whether or not GSH modulating agents will be useful as an adjuvant to selected cancer treatment modalities will depend on whether differential levels of GSH can be achieved in tumor versus normal tissues. Accurate GSH measurements in tumor and normal tissues will be required to adequately use and interpret the results of clinical studies where GSH modulating agents are employed. Precise tumor GSH measurements pose a considerable challenge due to the complicated cellular makeup of tumors.44 references.

  15. Role of glutathione transport processes in kidney function

    SciTech Connect

    Lash, Lawrence H. . E-mail: l.h.lash@wayne.edu

    2005-05-01

    The kidneys are highly dependent on an adequate supply of glutathione (GSH) to maintain normal function. This is due, in part, to high rates of aerobic metabolism, particularly in the proximal tubules. Additionally, the kidneys are potentially exposed to high concentrations of oxidants and reactive electrophiles. Renal cellular concentrations of GSH are maintained by both intracellular synthesis and transport from outside the cell. Although function of specific carriers has not been definitively demonstrated, it is likely that multiple carriers are responsible for plasma membrane transport of GSH. Data suggest that the organic anion transporters OAT1 and OAT3 and the sodium-dicarboxylate 2 exchanger (SDCT2 or NaDC3) mediate uptake across the basolateral plasma membrane (BLM) and that the organic anion transporting polypeptide OATP1 and at least one of the multidrug resistance proteins mediate efflux across the brush-border plasma membrane (BBM). BLM transport may be used pharmacologically to provide renal proximal tubular cells with exogenous GSH to protect against oxidative stress whereas BBM transport functions physiologically in turnover of cellular GSH. The mitochondrial GSH pool is derived from cytoplasmic GSH by transport into the mitochondrial matrix and is mediated by the dicarboxylate and 2-oxoglutarate exchangers. Maintenance of the mitochondrial GSH pool is critical for cellular and mitochondrial redox homeostasis and is important in determining susceptibility to chemically induced apoptosis. Hence, membrane transport processes are critical to regulation of renal cellular and subcellular GSH pools and are determinants of susceptibility to cytotoxicity induced by oxidants and electrophiles.

  16. Hepatic mitochondrial glutathione: transport and role in disease and toxicity

    SciTech Connect

    Fernandez-Checa, Jose C. . E-mail: checa229@yahoo.com; Kaplowitz, Neil . E-mail: kaplowitz@hsc.usc.edu

    2005-05-01

    Synthesized in the cytosol of cells, a fraction of cytosolic glutathione (GSH) is then transported into the mitochondrial matrix where it reaches a high concentration and plays a critical role in defending mitochondria against oxidants and electrophiles. Evidence mainly from kidney and liver mitochondria indicated that the dicarboxylate and the 2-oxoglutarate carriers contribute to the transport of GSH across the mitochondrial inner membrane. However, differential features between kidney and liver mitochondrial GSH (mGSH) transport seem to suggest the existence of additional carriers the identity of which remains to be established. One of the characteristic features of the hepatic mitochondrial transport of GSH is its regulation by membrane fluidity. Conditions leading to increased cholesterol deposition in the mitochondrial inner membrane such as in alcohol-induced liver injury decrease membrane fluidity and impair the mitochondrial transport of GSH. Depletion of mitochondrial GSH by alcohol is believed to contribute to the sensitization of the liver to alcohol-induced injury through tumor necrosis factor (TNF)-mediated hepatocellular death. Through control of mitochondrial electron transport chain-generated oxidants, mitochondrial GSH modulates cell death and hence its regulation may be a key target to influence disease progression and drug-induced cell death.

  17. Atherogenic diets exacerbate colitis in mice deficient in glutathione peroxidase

    PubMed Central

    Gao, Qiang; Esworthy, R. Steven; Kim, Byung-Wook; Synold, Timothy W.; Smith, David D.; Chu, Fong-Fong

    2010-01-01

    The pro-inflammatory effect of high-fat diet has been observed beyond the cardiovascular system, but there is little evidence to support its role in triggering inflammatory bowel disease. GPx1/2-double knockout (DKO) mice deficient in two intracellular glutathione peroxidases, GPx1 and GPx2, on a C57BL/6 (B6) background, have mild ileocolitis on a conventional chow. We fed B6 DKO mice two atherogenic diets to test the dietary effect on atherosclerosis and ileocolitis. Both atherogenic diets have high cholesterol, the Chol+/CA diet has cholic acid (CA) and the Chol+ diet has no CA. The Chol+/CA diet induced severe colitis, but not ileitis, in the DKO mice compared with Chol+ and a Chol- control diet. On the Chol+/CA diet, the wild-type (WT) mice had similar levels of aortic lesions and hypercholesterolemia as DKO mice did, but had no intestinal pathology. The diet-associated inflammatory responses in the DKO mice included increase of colonic pro-inflammatory serum amyloid A 3 expression, plasma lipopolysaccharide and TNF-α levels. The Chol+/CA diet has lowered the expression of unfolded protein response genes, ATF6, CHOP, unspliced XbpU and Grp78/Bip, in WT and DKO mice on the Chol- diet. Thus, we conclude that cholesterol diet weakens colon unfolded protein response, which can aggravate spontaneous colitis leading to gut barrier breakdown. GPx has no impact on atherosclerosis without ultra-hypercholesterolemia. PMID:20848490

  18. Serine incorporation into the selenocysteine moiety of glutathione peroxidase

    SciTech Connect

    Sunde, R.A.; Evenson, J.K.

    1987-01-15

    The selenium in mammalian glutathione peroxidase is present as a selenocysteine ((Se)Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the (Se)Cys moiety, we perfused isolated rat liver with /sup 14/C- or /sup 3/H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the (Se)Cys in GSH peroxidase to carboxymethylselenocysteine ((Se)Cys(Cm)), and determined the amino acid specific activity. Perfusion with (/sup 14/C)cystine resulted in (/sup 14/C)cystine incorporation into GSH peroxidase without labeling (Se)Cys(Cm), indicating that cysteine is not a direct precursor for (Se)Cys. (/sup 14/C)Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and (Se)Cys(Cm) in purified GSH peroxidase, whereas (3-3H)serine perfusion only labeled serine and (Se)Cys(Cm), thus demonstrating that the (Se)Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and (Se)Cys(Cm) strongly suggest that the precursor pool of serine used for (Se) Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.

  19. Glutathione Decrement Drives Thermogenic Program In Adipose Cells

    PubMed Central

    Lettieri Barbato, Daniele; Tatulli, Giuseppe; Maria Cannata, Stefano; Bernardini, Sergio; Aquilano, Katia; Ciriolo, Maria R.

    2015-01-01

    Adipose tissue metabolically adapts to external stimuli. We demonstrate that the induction of the thermogenic program in white adipocytes, through cold exposure in mice or in vitro adrenergic stimulation, is accompanied by a decrease in the intracellular content of glutathione (GSH). Moreover, the treatment with a GSH depleting agent, buthionine sulfoximine (BSO), recapitulates the effect of cold exposure resulting in the induction of thermogenic program. In particular, BSO treatment leads to enhanced uncoupling respiration as demonstrated by increased expression of thermogenic genes (e.g. Ucp1, Ppargc1a), augmented oxygen consumption and decreased mitochondrial transmembrane potential. Buffering GSH decrement by pre-treatment with GSH ester prevents the up-regulation of typical markers of uncoupling respiration. We demonstrate that FoxO1 activation is responsible for the conversion of white adipocytes into a brown phenotype as the “browning” effects of BSO are completely abrogated in cells down-regulating FoxO1. In mice, the BSO-mediated up-regulation of uncoupling genes results in weight loss that is at least in part ascribed to adipose tissue mass reduction. The induction of thermogenic program has been largely proposed to counteract obesity-related diseases. Based on these findings, we propose GSH as a novel therapeutic target to increase energy expenditure in adipocytes. PMID:26260892

  20. Preparation of N-tBoc L-glutathione dimethyl and di-tert-butyl esters: versatile synthetic building blocks.

    PubMed

    Falck, J R; Sangras, Bhavani; Capdevila, Jorge H

    2007-01-15

    The title l-glutathione derivatives, containing acid- and base-labile esters, respectively, were obtained in good overall yields. N-(t)Boc l-glutathione dimethyl ester was prepared via Fischer esterification of l-glutathione disulfide (GSSG) using HCl in dry methanol, protection of the amine with (t)Boc(2)O, and tributylphosphine cleavage of the disulfide in wet isopropanol. Alternatively, Fischer esterification and (t)Boc-protection of l-glutathione (GSH) also furnished N-(t)Boc glutathione dimethyl ester accompanied by a small amount of S-(t)Boc that was removed chromatographically. The di-tert-butyl ester was obtained by S-palmitoylation of GSH in TFA as solvent, N-(t)Boc-protection, esterification using (t)BuOH mediated by diisopropylcarbodiimide/copper(I) chloride, and saponification of the thioester. These l-glutathione derivatives are versatile synthetic building blocks for the preparation of S-glutathione adducts. PMID:17070060

  1. A novel glutathione-S transferase immunosensor based on horseradish peroxidase and double-layer gold nanoparticles.

    PubMed

    Lu, Dingqiang; Lu, Fuping; Pang, Guangchang

    2016-06-01

    GSTs, a biotransformation enzyme group, can perform metabolism, drug transfer and detoxification functions. Rapid detection of the GSTs with more sensitive approaches is of great importance. In the current study, a novel double-layer gold nanoparticles-electrochemical immunosensor electrode (DGN-EIE) immobilized with Glutathione S-Transferase (GST) antibody derived from Balb/c mice was developed. To increase the fixed quantity of antibodies and electrochemical signal, an electrochemical biosensing signal amplification system was utilized with gold nanoparticles-thionine-chitosan absorbing horseradish peroxidase (HRP). In addition, transmission electron microscope (TEM) was used to characterize the nanogold solution. To evaluate the quality of DGN-EIE, the amperometric I-t curve method was applied to determine the GST in PBS. The results showed that the response current had a good linear correlation with the GST concentration ranged from 0.1-10(4) pg/mL. The lowest detection limit was found at 0.03 pg/mL(S/N = 3). The linear equation was deduced as △I/% = 7.386lgC + 22.36 (R(2) = 0.998). Moreover, it was validated with high sensitivity and reproducibility. Apparently, DGN-EIE may be a very useful tool for monitoring the GST. PMID:27220630

  2. A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

    PubMed Central

    Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

    2010-01-01

    Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control. PMID:20824165

  3. Cruciferous vegetables and glutathione: their effects on colon mucosal glutathione level and colon tumor development in rats induced by DMH.

    PubMed

    Chen, M F; Chen, L T; Boyce, H W

    1995-01-01

    The effect of a diet containing 10-40% lyophilized cabbage or broccoli as cruciferous vegetable or 10-40% lyophilized potato as noncruciferous vegetable fed for 14 days on the colon mucosal glutathione (GSH) level was studied in male rats. The GSH levels of the duodenum mucosa and the liver were also measured. Cabbage and broccoli enhanced the colon and duodenum mucosal GSH levels in a dose-related manner; potato had no effect. All three vegetables had no effect on the liver GSH level. The effect of GSH on colon tumorigenesis induced by 1,2-dimethylhydrazine (DMH) was also examined in rats. Male Sprague-Dawley rats were injected with DMH (20 mg/kg body wt) weekly for 20 weeks. DMH lowered the colon mucosal GSH level. GSH (100 mg/day/rat) dissolved in the drinking water and given to rats during and after DMH injections had little or no effect on tumor incidence and total number of colon tumors. Tumors were larger in rats that received GSH than in those that received water. This study shows that the colon mucosal GSH level can be enhanced by feeding rats a diet high in cabbage or broccoli and that GSH added to the drinking water did not affect DMH-induced colon tumorigenesis under the experimental conditions used. PMID:7739917

  4. Efficacy of free glutathione and niosomal glutathione in the treatment of acetaminophen-induced hepatotoxicity in cats

    PubMed Central

    Vulcano, L.A. Denzoin; Confalonieri, O.; Franci, R.; Tapia, M.O.; Soraci, A.L.

    2013-01-01

    Acetaminophen (APAP) administration results in hepatotoxicity and hematotoxicity in cats. The response to three different treatments against APAP poisoning was evaluated. Free glutathione (GSH) (200mg/kg), niosomal GSH (14 mg/kg) and free amino acids (180 mg/kg of N-acetylcysteine and 280 mg/kg of methionine) were administered to cats that were intoxicated with APAP (a single dose of 150 mg/kg, p.o.). Serum concentration of alanine aminotransferase (ALT) along with serum, liver and erythrocyte concentration of GSH and methemoglobin percentage were measured before and 4, 24 and 72 hours after APAP administration. Free GSH (200 mg/kg) and niosomal GSH (14 mg/kg) were effective in reducing hepatotoxicity and hematotoxicity in cats intoxicated with a dose of 150 mg/kg APAP. We conclude that both types of treatments can protect the liver and haemoglobin against oxidative stress in APAP intoxicated cats. Furthermore, our results showed that treatment with niosomal GSH represents an effective therapeutic approach for APAP poisoning. PMID:26623313

  5. Nineteen-year follow-up of a patient with severe glutathione synthetase deficiency.

    PubMed

    Atwal, Paldeep S; Medina, Casey R; Burrage, Lindsay C; Sutton, V Reid

    2016-07-01

    Glutathione synthetase deficiency is a rare autosomal recessive disorder resulting in low levels of glutathione and an increased susceptibility to oxidative stress. Patients with glutathione synthetase deficiency typically present in the neonatal period with hemolytic anemia, metabolic acidosis and neurological impairment. Lifelong treatment with antioxidants has been recommended in an attempt to prevent morbidity and mortality associated with the disorder. Here, we present a 19-year-old female who was diagnosed with glutathione synthetase deficiency shortly after birth and who has been closely followed in our metabolic clinic. Despite an initial severe presentation, she has had normal intellectual development and few complications of her disorder with a treatment regimen that includes polycitra (citric acid, potassium citrate and sodium citrate), vitamin C, vitamin E and selenium. PMID:26984560

  6. High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

    NASA Technical Reports Server (NTRS)

    Abukhalaf, Imad K.; Silvestrov, Natalia A.; Menter, Julian M.; von Deutsch, Daniel A.; Bayorh, Mohamed A.; Socci, Robin R.; Ganafa, Agaba A.

    2002-01-01

    A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

  7. Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

    SciTech Connect

    Hernandez-Montes, Eva; Pollard, Susan E.; Vauzour, David; Jofre-Montseny, Laia; Rota, Cristina; Rimbach, Gerald; Weinberg, Peter D.; Spencer, Jeremy P.E. . E-mail: j.p.e.spencer@reading.ac.uk

    2006-08-04

    Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of {gamma}-glutamylcysteine synthetase-heavy subunit ({gamma}-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis.

  8. Magnetically separable nanoferrite-anchored glutathione: Aqueous homocoupling of arylboronic acids under microwave irradiation

    EPA Science Inventory

    A highly active, stable and magnetically separable glutathione based organocatalyst provided good to excellent yields to symmetric biaryls in the homocoupling of arylboronic acids under microwave irradiation. Symmetrical biaryl motifs are present in a wide range of natural p...

  9. Measuring protein-bound glutathioine (PSSG): Critical correction for cytosolic glutathione species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Protein glutathionylation is gaining recognition as an important posttranslational protein modification. The common first step in measuring protein glutathionylation is the denaturation and precipitation of protein away from soluble, millimolar quantities of glutathione (GSH) and glut...

  10. A turn-on fluorescent sensor for the discrimination of cystein from homocystein and glutathione.

    PubMed

    Niu, Li-Ya; Guan, Ying-Shi; Chen, Yu-Zhe; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng

    2013-02-14

    We report a turn-on fluorescent sensor based on nitrothiophenolate boron dipyrromethene (BODIPY) derivatives for the discrimination of cystein (Cys) from homocystein (Hcy) and glutathione (GSH). The sensor was applied for detection of Cys in living cells. PMID:23295243

  11. Cadmium-induced synthesis of HSP70 and a role of glutathione in Euglena gracilis.

    PubMed

    Watanabe, Masumi; Suzuki, Tetsuya

    2004-01-01

    The effect of cadmium-induced oxidative stress, with or without glutathione supplementation, was investigated in the single cell eukaryotic phytoflagellate, Euglena gracilis strains Z and its achlorophyllous mutant SMZ as experimental models. Both these strains actively synthesize thiols to prevent or resist cadmium toxicity. The content of glutathione, as a representative antioxidant, was also examined in both strains. Exposure to cadmium induced heat-shock protein 70 (HSP70) synthesis in both strains of E. gracilis. Glutathione supplementation also induced HSP70. Overall, these results indicate that glutathione was closely linked to the induction of stress-related proteins. The sensitivity to cadmium-stress was higher in strain Z than SMZ. The results suggest that chloroplasts may have a role in the regulation of HSP70 expression. The relationship between HSP70 and GSH levels is still far from understood, and further research may shed light upon their up-regulation in the presence of Cd. PMID:15720831

  12. Lectin-Gated, Mesoporous, Photofunctionalized Glyconanoparticles for Glutathione-Responsive Drug Delivery

    PubMed Central

    Zhou, Juan; Hao, Nanjing; De Zoyza, Thareendra; Yan, Mingdi

    2015-01-01

    A stimuli-responsive drug delivery system based on fluorescent, lectin-gated, mesoporous glyconanoparticles has been developed and evaluated in normal- and cancer lung epithelial cells. The gating process proved efficient, exhibiting good sealing properties in the absence of the glutathione redox trigger, avoiding premature release in normal cells. In the presence of higher levels of glutathione in cancer cells, the lectin gate was rapidly opened and the anticancer drug released. PMID:25989158

  13. Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

    PubMed Central

    Schraufstätter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

    1985-01-01

    Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells. PMID:3840176

  14. Nitro-fatty Acid Reaction with Glutathione and Cysteine

    PubMed Central

    Baker, Laura M. S.; Baker, Paul R. S.; Golin-Bisello, Franca; Schopfer, Francisco J.; Fink, Mitchell; Woodcock, Steven R.; Branchaud, Bruce P.; Radi, Rafael; Freeman, Bruce A.

    2007-01-01

    Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M−1s−1 and 355 M−1s−1, respectively, at pH 7.4 and 37 °C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A2 and 15-deoxy-Δ12,14-prostaglandin J2. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pKa of target thiols increased. Increasing concentrations of the detergent octyl-β-D-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids. PMID:17720974

  15. Ascorbate and glutathione regulation in hibernating ground squirrels.

    PubMed

    Drew, K L; Osborne, P G; Frerichs, K U; Hu, Y; Koren, R E; Hallenbeck, J M; Rice, M E

    1999-12-18

    Ground squirrels withstand up to 90% reductions in cerebral blood flow during hibernation as well as rapid reperfusion upon periodic arousals from torpor. Metabolic suppression likely plays a primary adaptive role which allows hibernating species to tolerate such phenomena. However, several other aspects of hibernation physiology are also consistent with tolerance to dramatic fluctuations in cerebral blood flow, suggesting that multiple neuroprotective adaptations may work in concert during hibernation. The purpose of the present work was to study the dynamics of the low molecular weight antioxidants, ascorbate and glutathione (GSH), during hibernation. Alterations in concentrations of ascorbate during hibernation and arousal in two species of hibernating ground squirrels suggest that it could play a protective role during hibernation or arousal. Samples were collected during the hibernation season from arctic ground squirrels (AGS; Spermophilus parryii) and 13-lined ground squirrels (TLS; S. tridecemlineatus) during prolonged torpor and in squirrels that did not hibernate or had not been hibernating for several weeks. We determined antioxidant levels in plasma, cerebrospinal fluid (CSF), and in frontal cortex, hippocampus and cerebellum using high-performance liquid chromatography (HPLC). Plasma ascorbate concentrations increased dramatically (3-4-fold) in both species during hibernation and rapidly returned to prehibernation levels upon arousal. By contrast, plasma GSH concentrations fell slightly or remained stable during hibernation. Ascorbate levels in the CSF doubled in hibernating AGS (not determined in TLS), while brain ascorbate content fell slightly (10-15%) in both species. Substantial increases in plasma and CSF ascorbate concentrations suggest that this antioxidant could play a protective role during hibernation and reperfusion upon arousal from hibernation. PMID:10642822

  16. A supramolecular microgel glutathione peroxidase mimic with temperature responsive activity.

    PubMed

    Yin, Yanzhen; Jiao, Shufei; Lang, Chao; Liu, Junqiu

    2014-05-21

    Glutathione peroxidase (GPx) protects cells from oxidative damage by scavenging surplus reactive oxygen species (ROS). Commonly, an appropriate amount of ROS acts as a signal molecule in the metabolism. A smart artificial GPx exhibits adjustable catalytic activity, which can potentially reduce the amount of ROS to an appropriate degree and maintain its important physiological functions in metabolism. To construct an optimum and excellent smart artificial GPx, a novel supramolecular microgel artificial GPx (SM-Te) was prepared based on the supramolecular host-guest interaction employing the tellurium-containing guest molecule (ADA-Te-ADA) and the cyclodextrin-containing host block copolymer (poly(N-isopropylacrylamide)-b-[polyacrylamides-co-poly(6-o-(triethylene glycol monoacrylate ether)-β-cyclodextrin)], PPAM-CD) as building blocks. Subsequently, based on these building blocks, SM-Te was constructed and the formation of its self-assembled structure was confirmed by dynamic light scattering, NMR, SEM, TEM, etc. Typically, benefitting from the temperature responsive properties of the PNIPAM scaffold, SM-Te also exhibited similar temperature responsive behaviour. Importantly, the GPx catalytic rates of SM-Te displayed a noticeable temperature responsive characteristic. Moreover, SM-Te exhibited the typical saturation kinetics behaviour of a real enzyme catalyst. It was proved that the changes of the hydrophobic microenvironment and the pore size in the supramolecular microgel network of SM-Te played significant roles in altering the temperature responsive catalytic behaviour. The successful construction of SM-Te not only overcomes the insurmountable disadvantages existing in previous covalent bond crosslinked microgel artificial GPx but also bodes well for the development of novel intelligent antioxidant drugs. PMID:24652520

  17. Function of glutathione peroxidases in legume root nodules.

    PubMed

    Matamoros, Manuel A; Saiz, Ana; Peñuelas, Maria; Bustos-Sanmamed, Pilar; Mulet, Jose M; Barja, Maria V; Rouhier, Nicolas; Moore, Marten; James, Euan K; Dietz, Karl-Josef; Becana, Manuel

    2015-05-01

    Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function. PMID:25740929

  18. Function of glutathione peroxidases in legume root nodules

    PubMed Central

    Matamoros, Manuel A.; Saiz, Ana; Peñuelas, Maria; Bustos-Sanmamed, Pilar; Mulet, Jose M.; Barja, Maria V.; Rouhier, Nicolas; Moore, Marten; James, Euan K.; Dietz, Karl-Josef; Becana, Manuel

    2015-01-01

    Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function. PMID:25740929

  19. Glutathione and cellular redox control in epigenetic regulation.

    PubMed

    García-Giménez, José Luis; Ibañez-Cabellos, José Santiago; Seco-Cervera, Marta; Pallardó, Federico V

    2014-10-01

    Epigenetics is defined as the mitotically/meiotically heritable changes in gene expression that are not due to changes in the primary DNA sequence. Over recent years, growing evidence has suggested a link between redox metabolism and the control of epigenetic mechanisms. The effect of the redox control, oxidative stress, and glutathione (GSH) on the epigenetic mechanisms occur at different levels affecting DNA methylation, miRNAs expression, and histone post-translational modifications (PTMs). Furthermore, a number of redox PTMs are being described, so enriching the histone code. Pioneer works showed how oxidized GSH inhibits the activity of S-adenosyl methionine synthetase, MAT1A, a key enzyme involved in the synthesis of S-adenosyl methionine (SAM), which is used by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). Alteration in NAD /NADH ratio affects the activity of class III histone deacetylases (HDACs) and poly-ADP ribosyltransferases (PARPs). Furthermore, the iron redox state of the catalytic center of key enzymes influences the activity of HDACs and the activity of Tet methylcytosine dioxygenases (DNA demetylases) and JmjC histone demethylases. In this communication, we will show the intricate mechanisms that participate in the redox control of the epigenetic mechanisms. We specially focus our work in the characterization of new PTMs in histones, such as histone carbonylation and glutathionylation. Demonstrating how GSH influences the epigenetic mechanisms beyond a mere regulation of SAM levels. The mechanisms described in this communication place GSH and redox control in the landscape of the epigenetic regulation. The results shown underscore the relevant role that oxidative stress and GSH play as key factors in epigenetics, opening a new window for understating the underlying mechanisms that control cell differentiation, proliferation, development, and disease. PMID:26461333

  20. Measuring glutathione redox potential of HIV-1-infected macrophages.

    PubMed

    Bhaskar, Ashima; Munshi, MohamedHusen; Khan, Sohrab Zafar; Fatima, Sadaf; Arya, Rahul; Jameel, Shahid; Singh, Amit

    2015-01-01

    Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E(GSH); Grx1-roGFP2) and measured subcellular changes in E(GSH) during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E(GSH) (approximately -310 mV), active viral replication induces substantial oxidative stress (E(GSH) more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E(GSH) between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in E(GSH) is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E(GSH). Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV. PMID:25406321

  1. Characterization of thioredoxin glutathione reductase in Schiotosoma japonicum.

    PubMed

    Han, Yanhui; Zhang, Min; Hong, Yang; Zhu, Zhu; Li, Dong; Li, Xiangrui; Fu, Zhiqiang; Lin, Jiaojiao

    2012-09-01

    Schistosomiasis is one of the most prevalent and serious parasitic diseases in the world and remains an important public health problem in China. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, we cloned a cDNA encoding Schistosoma japonicum (S. japonicum) thioredoxin glutathione reductase (SjTGR) from the cDNA of 42-day-old adult worms. The open reading frame (ORF) of the gene was 1791 base pairs (bp) encoding a protein of 596 amino acids. SjTGR was subcloned into pET-32a (+) and expressed in Escherichia coli (E. coli) BL21 (DE3). The recombinant protein rSjTGR exhibited enzymatic activity of 5.13U/mg with DTNB as the substrate, and showed strong immunogenecity. Real-time PCR results indicated that SjTGR was expressed at a higher level in 35-day-old schistosome worms in transcript. We vaccinated BALB/c mice with rSjTGR in combination with MONTANIDE™ ISA 206 VG (ISA 206) and observed a 33.50% to 36.51% (P<0.01) decrease in the adult worm burden and a 33.73%to 43.44% (P<0.01) decrease in the number of eggs counted compared to the ISA 206 or blank control groups in two independent vaccination tests. ELISA analysis demonstrated that rSjTGR induced a high level of SjTGR-specific IgG, IgG1, and IgG 2a antibodies and induced elevated production of IFN-γ. This study provides the basis for further investigations into the biological function of SjTGR and further evaluation of the potential use of this molecule as a vaccine candidate or new drug target is warranted. PMID:22484130

  2. Measuring Glutathione Redox Potential of HIV-1-infected Macrophages*

    PubMed Central

    Bhaskar, Ashima; Munshi, MohamedHusen; Khan, Sohrab Zafar; Fatima, Sadaf; Arya, Rahul; Jameel, Shahid; Singh, Amit

    2015-01-01

    Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (EGSH; Grx1-roGFP2) and measured subcellular changes in EGSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial EGSH (approximately −310 mV), active viral replication induces substantial oxidative stress (EGSH more than −240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular EGSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in EGSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular EGSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV. PMID:25406321

  3. Exercise-induced oxidative stress: glutathione supplementation and deficiency.

    PubMed

    Sen, C K; Atalay, M; Hänninen, O

    1994-11-01

    Glutathione (GSH) plays a central role in coordinating the synergism between different lipid- and aqueous-phase antioxidants. We documented 1) how exogenous GSH and N-acetylcysteine (NAC) may affect exhaustive exercise-induced changes in tissue GSH status, lipid peroxides [thiobarbituric acid-reactive substances (TBARS)], and endurance and 2) the relative role of endogenous GSH in the circumvention of exercise-induced oxidative stress by using GSH-deficient [L-buthionine-(S,R)-sulfoximine (BSO)-treated] rats. Intraperitoneal injection of GSH remarkably increased plasma GSH; exogenous GSH per se was an ineffective delivery agent of GSH to tissues. Repeated administration of GSH (1 time/day for 3 days) increased blood and kidney total GSH [TGSH; GSH+oxidized GSH (GSSG)]. Neither GSH nor NAC influenced endurance to exhaustion. NAC decreased exercise-induced GSH oxidation in the lung and blood. BSO decreased TGSH pools in the liver, lung, blood, and plasma by approximately 50% and in skeletal muscle and heart by 80-90%. Compared with control, resting GSH-deficient rats had lower GSSG in the liver, red gastrocnemius muscle, heart, and blood; similar GSSG/TGSH ratios in the liver, heart, lung, blood, and plasma; higher GSSG/TGSH ratios in the skeletal muscle; and more TBARS in skeletal muscle, heart, and plasma. In contrast to control, exhaustive exercise of GSH-deficient rats did not decrease TGSH in the liver, muscle, or heart or increase TGSH of plasma; GSSG of muscle, blood, or plasma; or TBARS of plasma or muscle. GSH-deficient rats had approximately 50% reduced endurance, which suggests a critical role of endogenous GSH in the circumvention of exercise-induced oxidative stress and as a determinant of exercise performance. PMID:7868431

  4. The evolution of glutathione metabolism in phototrophic microorganisms

    NASA Technical Reports Server (NTRS)

    Fahey, R. C.; Buschbacher, R. M.; Newton, G. L.

    1987-01-01

    Of the many roles ascribed to glutathione (GSH) the one most clearly established is its role in the protection of higher eucaryotes against oxygen toxicity through destruction of thiol-reactive oxygen byproducts. If this is the primary function of GSH then GSH metabolism should have evolved during or after the evolution of oxygenic photosynthesis. That many bacteria do not produce GSH is consistent with this view. In the present study we have examined the low-molecular-weight thiol composition of a variety of phototrophic microorganisms to ascertain how evolution of GSH production is related to evolution of oxygenic photosynthesis. Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols to fluorescent derivatives, which were analyzed by high-pressure liquid chromatography. Significant levels of GSH were not found in the green bacteria (Chlorobium thiosulfatophilum and Chloroflexus aurantiacus). Substantial levels of GSH were present in the purple bacteria (Chromatium vinosum, Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodocyclus gelatinosa), the cyanobacteria [Anacystis nidulans, Microcoleus chthonoplastes S.G., Nostoc muscorum, Oscillatoria amphigranulata, Oscillatoria limnetica, Oscillatoria sp. (Stinky Spring, Utah), Oscillatoria terebriformis, Plectonema boryanum, and Synechococcus lividus], and eucaryotic algae (Chlorella pyrenoidsa, Chlorella vulgaris, Euglena gracilis, Scenedesmus obliquus, and Chlamydomonas reinhardtii). Other thiols measured included cysteine, gamma-glutamylcysteine, thiosulfate, coenzyme A, and sulfide; several unidentified thiols were also detected. Many of the organisms examined also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability that was quenched by treatment with 2-pyridyl disulfide or 5,5'-bisdithio-(2-nitrobenzoic acid) prior to reaction with mBBr. These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of

  5. Selection of antisense oligodeoxynucleotides against glutathione S-transferase Mu.

    PubMed Central

    't Hoen, Peter A C; Out, Ruud; Commandeur, Jan N M; Vermeulen, Nico P E; van Batenburg, F H D; Manoharan, Muthiah; van Berkel, Theo J C; Biessen, Erik A L; Bijsterbosch, Martin K

    2002-01-01

    The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression. PMID:12515389

  6. Beta-amyloidolysis and glutathione in Alzheimer’s disease

    PubMed Central

    Lasierra-Cirujeda, J; Coronel, P; Aza, MJ; Gimeno, M

    2013-01-01

    In this review, we hypothesized the importance of the interaction between the brain glutathione (GSH) system, the proteolytic tissue plasminogen activator (t-PA)/plasminogen/ plasmin system, regulated by plasminogen activator inhibitor (PAI-1), and neuroserpin in the pathogenesis of Alzheimer’s disease. The histopathological characteristic hallmark that gives personality to the diagnosis of Alzheimer’s disease is the accumulation of neurofibroid tangles located intracellularly in the brain, such as the protein tau and extracellular senile plaques made primarily of amyloidal substance. These formations of complex etiology are intimately related to GSH, brain protective antioxidants, and the proteolytic system, in which t-PA plays a key role. There is scientific evidence that suggests a relationship between aging, a number of neurodegenerative disorders, and the excessive production of reactive oxygen species and accompanying decreased brain proteolysis. The plasminogen system in the brain is an essential proteolytic mechanism that effectively degrades amyloid peptides (“beta-amyloidolysis”) through action of the plasmin, and this physiologic process may be considered to be a means of prevention of neurodegenerative disorders. In parallel to the decrease in GSH levels seen in aging, there is also a decrease in plasmin brain activity and a progressive decrease of t-PA activity, caused by a decrease in the expression of the t-PA together with an increase of the PAI-1 levels, which rise to an increment in the production of amyloid peptides and a lesser clearance of them. Better knowledge of the GSH mechanism and cerebral proteolysis will allow us to hypothesize about therapeutic practices. PMID:23650462

  7. Blood selenium concentrations and enzyme activities related to glutathione metabolism in wild emperor geese

    USGS Publications Warehouse

    Franson, J.C.; Hoffman, D.J.; Schmutz, J.A.

    2002-01-01

    In 1998, we collected blood samples from 63 emperor geese (Chen canagica) on their breeding grounds on the Yukon-Kuskokwim Delta (YKD) in western Alaska, USA. We studied the relationship between selenium concentrations in whole blood and the activities of glutathione peroxidase and glutathione reductase in plasma. Experimental studies have shown that plasma activities of these enzymes are useful biomarkers of selenium-induced oxidative stress, but little information is available on their relationship to selenium in the blood of wild birds. Adult female emperor geese incubating their eggs in mid-June had a higher mean concentration of selenium in their blood and a greater activity of glutathione peroxidase in their plasma than adult geese or goslings that were sampled during the adult flight feathera??molting period in late July and early August. Glutathione peroxidase activity was positively correlated with the concentration of selenium in the blood of emperor geese, and the rate of increase relative to selenium was greater in goslings than in adults. The activity of glutathione reductase was greatest in the plasma of goslings and was greater in molting adults than incubating females but was not significantly correlated with selenium in the blood of adults or goslings. Incubating female emperor geese had high selenium concentrations in their blood, accompanied by increased glutathione peroxidase activity consistent with early oxidative stress. These findings indicate that further study of the effects of selenium exposure, particularly on reproductive success, is warranted in this species.

  8. Blood selenium concentrations and enzyme activities related to glutathione metabolism in wild emperor geese

    USGS Publications Warehouse

    Franson, J.C.; Hoffman, D.J.; Schmutz, J.A.

    2002-01-01

    In 1998, we collected blood samples from 63 emperor geese (Chen canagica) on their breeding grounds on the Yukon-Kuskokwim Delta (YKD) in western Alaska, USA. We studied the relationship between selenium concentrations in whole blood and the activities of glutathione peroxidase and glutathione reductase in plasma. Experimental studies have shown that plasma activities of these enzymes are useful biomarkers of selenium-induced oxidative stress, but little information is available on their relationship to selenium in the blood of wild birds. Adult female emperor geese incubating their eggs in mid-June had a higher mean concentration of selenium in their blood and a greater activity of glutathione peroxidase in their plasma than adult geese or goslings that were sampled during the adult flight feathermolting period in late July and early August. Glutathione peroxidase activity was positively correlated with the concentration of selenium in the blood of emperor geese, and the rate of increase relative to selenium was greater in goslings than in adults. The activity of glutathione reductase was greatest in the plasma of goslings and was greater in molting adults than incubating females but was not significantly correlated with selenium in the blood of adults or goslings. Incubating female emperor geese had high selenium concentrations in their blood, accompanied by increased glutathione peroxidase activity consistent with early oxidative stress. These findings indicate that further study of the effects of selenium exposure, particularly on reproductive success, is warranted in this species.

  9. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants.

    PubMed

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1(CtoS) purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER.DOI: http://dx.doi.org/10.7554/eLife.03421.001. PMID:25073928

  10. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

    PubMed Central

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

  11. A Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Efficient Wound Repair

    PubMed Central

    Telorack, Michèle; Meyer, Michael; Ingold, Irina; Conrad, Marcus; Bloch, Wilhelm; Werner, Sabine

    2016-01-01

    The tripeptide glutathione is the most abundant cellular antioxidant with high medical relevance, and it is also required as a co-factor for various enzymes involved in the detoxification of reactive oxygen species and toxic compounds. However, its cell-type specific functions and its interaction with other cytoprotective molecules are largely unknown. Using a combination of mouse genetics, functional cell biology and pharmacology, we unraveled the function of glutathione in keratinocytes and its cross-talk with other antioxidant defense systems. Mice with keratinocyte-specific deficiency in glutamate cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis, showed a strong reduction in keratinocyte viability in vitro and in the skin in vivo. The cells died predominantly by apoptosis, but also showed features of ferroptosis and necroptosis. The increased cell death was associated with increased levels of reactive oxygen and nitrogen species, which caused DNA and mitochondrial damage. However, epidermal architecture, and even healing of excisional skin wounds were only mildly affected in the mutant mice. The cytoprotective transcription factor Nrf2 was strongly activated in glutathione-deficient keratinocytes, but additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione dependent. However, we show that deficiency in glutathione biosynthesis is efficiently compensated in keratinocytes by the cysteine/cystine and thioredoxin systems. Therefore, our study highlights a remarkable antioxidant capacity of the epidermis that ensures skin integrity and efficient wound healing. PMID:26808544

  12. Auranofin-induced oxidative stress causes redistribution of the glutathione pool in Taenia crassiceps cysticerci.

    PubMed

    Martínez-González, J J; Guevara-Flores, A; Rendón, J L; del Arenal, I P

    2015-05-01

    Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites. PMID:26024834

  13. A Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Efficient Wound Repair.

    PubMed

    Telorack, Michèle; Meyer, Michael; Ingold, Irina; Conrad, Marcus; Bloch, Wilhelm; Werner, Sabine

    2016-01-01

    The tripeptide glutathione is the most abundant cellular antioxidant with high medical relevance, and it is also required as a co-factor for various enzymes involved in the detoxification of reactive oxygen species and toxic compounds. However, its cell-type specific functions and its interaction with other cytoprotective molecules are largely unknown. Using a combination of mouse genetics, functional cell biology and pharmacology, we unraveled the function of glutathione in keratinocytes and its cross-talk with other antioxidant defense systems. Mice with keratinocyte-specific deficiency in glutamate cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis, showed a strong reduction in keratinocyte viability in vitro and in the skin in vivo. The cells died predominantly by apoptosis, but also showed features of ferroptosis and necroptosis. The increased cell death was associated with increased levels of reactive oxygen and nitrogen species, which caused DNA and mitochondrial damage. However, epidermal architecture, and even healing of excisional skin wounds were only mildly affected in the mutant mice. The cytoprotective transcription factor Nrf2 was strongly activated in glutathione-deficient keratinocytes, but additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione dependent. However, we show that deficiency in glutathione biosynthesis is efficiently compensated in keratinocytes by the cysteine/cystine and thioredoxin systems. Therefore, our study highlights a remarkable antioxidant capacity of the epidermis that ensures skin integrity and efficient wound healing. PMID:26808544

  14. Study of Linkage between Glutathione Pathway and the Antibiotic Resistance of Escherichia coli from Patients’ Swabs

    PubMed Central

    Kominkova, Marketa; Michalek, Petr; Cihalova, Kristyna; Guran, Roman; Cernei, Natalia; Nejdl, Lukas; Smerkova, Kristyna; Dostalova, Simona; Chudobova, Dagmar; Heger, Zbynek; Vesely, Radek; Gumulec, Jaromir; Kynicky, Jindrich; Xhaxhiu, Kledi; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

    2015-01-01

    In this work, we focused on the differences between bacterial cultures of E. coli obtained from swabs of infectious wounds of patients compared to laboratory E. coli. In addition, blocking of the protein responsible for the synthesis of glutathione (γ-glutamylcysteine synthase—GCL) using 10 mM buthionine sulfoximine was investigated. Each E. coli showed significant differences in resistance to antibiotics. According to the determined resistance, E. coli were divided into experimental groups based on a statistical evaluation of their properties as more resistant and more sensitive. These groups were also used for finding the differences in a dependence of the glutathione pathway on resistance to antibiotics. More sensitive E. coli showed the same kinetics of glutathione synthesis while blocking GCL (Km 0.1 µM), as compared to non-blocking. In addition, the most frequent mutations in genes of glutathione synthetase, glutathione peroxidase and glutathione reductase were observed in this group compared to laboratory E.coli. The group of “more resistant” E. coli exhibited differences in Km between 0.3 and 0.8 µM. The number of mutations compared to the laboratory E. coli was substantially lower compared to the other group. PMID:25837469

  15. Study of linkage between glutathione pathway and the antibiotic resistance of Escherichia coli from patients' swabs.

    PubMed

    Kominkova, Marketa; Michalek, Petr; Cihalova, Kristyna; Guran, Roman; Cernei, Natalia; Nejdl, Lukas; Smerkova, Kristyna; Dostalova, Simona; Chudobova, Dagmar; Heger, Zbynek; Vesely, Radek; Gumulec, Jaromir; Kynicky, Jindrich; Xhaxhiu, Kledi; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

    2015-01-01

    In this work, we focused on the differences between bacterial cultures of E. coli obtained from swabs of infectious wounds of patients compared to laboratory E. coli. In addition, blocking of the protein responsible for the synthesis of glutathione (γ-glutamylcysteine synthase-GCL) using 10 mM buthionine sulfoximine was investigated. Each E. coli showed significant differences in resistance to antibiotics. According to the determined resistance, E. coli were divided into experimental groups based on a statistical evaluation of their properties as more resistant and more sensitive. These groups were also used for finding the differences in a dependence of the glutathione pathway on resistance to antibiotics. More sensitive E. coli showed the same kinetics of glutathione synthesis while blocking GCL (Km 0.1 µM), as compared to non-blocking. In addition, the most frequent mutations in genes of glutathione synthetase, glutathione peroxidase and glutathione reductase were observed in this group compared to laboratory E.coli. The group of "more resistant" E. coli exhibited differences in Km between 0.3 and 0.8 µM. The number of mutations compared to the laboratory E. coli was substantially lower compared to the other group. PMID:25837469

  16. Transsulfuration Is a Significant Source of Sulfur for Glutathione Production in Human Mammary Epithelial Cells

    PubMed Central

    Belalcázar, Andrea D.; Frost, Leslie M.; Valentovic, Monica A.; Wilkinson, John

    2013-01-01

    The transsulfuration pathway, through which homocysteine from the methionine cycle provides sulfur for cystathionine formation, which may subsequently be used for glutathione synthesis, has not heretofore been identified as active in mammary cells. Primary human mammary epithelial cells (HMEC's) were labeled with S35-methionine for 24 hours following pretreatment with a vehicle control, the cysteine biosynthesis inhibitor propargylglycine or the gamma-glutamylcysteine synthesis inhibitor buthionine sulfoximine. Cell lysates were prepared and reacted with glutathione-S-transferase and the fluorescent labeling compound monochlorobimane to form a fluorescent glutathione-bimane conjugate. Comparison of fluorographic and autoradiographic images indicated that glutathione had incorporated S35-methionine demonstrating that functional transsulfuration occurs in mammary cells. Pathway inhibitors reduced incorporation by roughly 80%. Measurement of glutathione production in HMEC's treated with and without hydrogen peroxide and/or pathway inhibitors indicates that the transsulfuration pathway plays a significant role in providing cysteine for glutathione production both normally and under conditions of oxidant stress. PMID:24634789

  17. Glutathione biosynthesis is upregulated at the initiation of MYCN-driven neuroblastoma tumorigenesis.

    PubMed

    Carter, Daniel R; Sutton, Selina K; Pajic, Marina; Murray, Jayne; Sekyere, Eric O; Fletcher, Jamie; Beckers, Anneleen; De Preter, Katleen; Speleman, Frank; George, Rani E; Haber, Michelle; Norris, Murray D; Cheung, Belamy B; Marshall, Glenn M

    2016-06-01

    The MYCN gene is amplified and overexpressed in a large proportion of high stage neuroblastoma patients and has been identified as a key driver of tumorigenesis. However, the mechanism by which MYCN promotes tumor initiation is poorly understood. Here we conducted metabolic profiling of pre-malignant sympathetic ganglia and tumors derived from the TH-MYCN mouse model of neuroblastoma, compared to non-malignant ganglia from wildtype littermates. We found that metabolites involved in the biosynthesis of glutathione, the most abundant cellular antioxidant, were the most significantly upregulated metabolic pathway at tumor initiation, and progressively increased to meet the demands of tumorigenesis. A corresponding increase in the expression of genes involved in ribosomal biogenesis suggested that MYCN-driven transactivation of the protein biosynthetic machinery generated the necessary substrates to drive glutathione biosynthesis. Pre-malignant sympathetic ganglia from TH-MYCN mice had higher antioxidant capacity and required glutathione upregulation for cell survival, when compared to wildtype ganglia. Moreover, in vivo administration of inhibitors of glutathione biosynthesis significantly delayed tumorigenesis when administered prophylactically and potentiated the anticancer activity of cytotoxic chemotherapy against established tumors. Together these results identify enhanced glutathione biosynthesis as a selective metabolic adaptation required for initiation of MYCN-driven neuroblastoma, and suggest that glutathione-targeted agents may be used as a potential preventative strategy, or as an adjuvant to existing chemotherapies in established disease. PMID:26996379

  18. Structure of glutathione S-transferase 1 from the major human hookworm parasite Necator americanus (Na-GST-1) in complex with glutathione

    PubMed Central

    Asojo, Oluwatoyin A.; Ceccarelli, Christopher

    2014-01-01

    Glutathione S-transferase 1 from Necator americanus (Na-GST-1) is a vaccine candidate for hookworm infection that has a high affinity for heme and metal porphyrins. As part of attempts to clarify the mechanism of heme detoxification by hookworm GSTs, co-crystallization and soaking studies of Na-GST-1 with the heme-like molecules protoporphyrin IX disodium salt, hematin and zinc protoporphyrin were undertaken. While these studies did not yield the structure of the complex of Na-GST-1 with any of these molecules, co-crystallization experiments resulted in the first structures of the complex of Na-GST-1 with the substrate glutathione. The structures of the complex of Na-GST-1 with glutathione were solved from pathological crystalline aggregates comprising more than one crystal form. These first structures of the complex of Na-GST-1 with the substrate glutathione were solved by molecular replacement from data collected with a sealed-tube home source using the previously reported apo structure as the search model. PMID:25195885

  19. Formation of S-substituted glutathione adducts of styrene catalyzed by protaglandin H synthase: a possible new mechanism for the formation of glutathione conjugates

    SciTech Connect

    Stock, B.H.; Bend, J.R.; Eling, T.E.

    1986-03-01

    The prostaglandin hydroperoxidase (PHP)- and horseradish peroxidase (HRP)-dependent metabolism of styrene was examined. In the presence of arachidonic acid or hydrogen peroxide and glutathione (GSH), microsomes prepared from ram seminal vesicles catalyzed the formation of styrene-GSH adducts. Neither styrene 7,8-oxide nor styrene-GSH adducts were isolated by HPLC and shown by NMR and MS-MS mass spectrometry to be a mixture of (2R)- and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione. No (1R)- or (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione was formed. The addition of phenol or aminopyrine to incubation mixtures containing the appropriate cofactor and PHP or HRP, which greatly enhances the oxidation of GSH to a thiyl radical by these peroxidases, increased the amount of the two styrene-GSH adducts produced. The authors propose that the formation of (2R)- and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione is dependent on the initial oxidation of GSH to a thiyl radical by the peroxidases, and on the subsequent reaction of this thiyl radical with the terminal alkene carbon atom of styrene. This is an epoxide-independent mechanism for the formation of GSH conjugates of alkenes that can occur in the absence of cytochrome P-450-dependent monooxygenases.

  20. Crystallization and preliminary X-ray crystallographic analysis of Escherichia coli glutaredoxin 2 in complex with glutathione and of a cysteine-less variant without glutathione

    SciTech Connect

    Sheng, Ju; Ye, Jun; Rosen, Barry P.

    2007-04-01

    Glutaredoxin 2 from E. coli was cocrystallized with glutathione and data were collected to 1.60 Å. A mutant with the active-site residues Cys9 and Cys12 changed to serine was crystallized in the absence of glutathione and data were collected to 2.4 Å. Glutaredoxin 2 (Grx2) from Escherichia coli is larger in size than classical glutaredoxins. It is extremely efficient in the catalysis of reduced glutathione-dependent disulfide reduction. A complex of Grx2 with reduced glutathione (GSH) has been crystallized. Data were collected to 1.60 Å. The crystals belong to space group P3{sub 2}21, with one Grx2–GSH complex in the asymmetric unit. The unit-cell parameters are a = b = 50.10, c = 152.47 Å. A Grx2 mutant, C9S/C12S, which cannot form a disulfide bond with GSH was also crystallized. The crystals diffracted to 2.40 Å and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with one molecule in the asymmetric unit. The unit-cell parameters are a = 28.16, b = 78.65, c = 89.16 Å.

  1. Synthesis, characterization and cytotoxicity of glutathione- and PEG-glutathione-superparamagnetic iron oxide nanoparticles for nitric oxide delivery

    NASA Astrophysics Data System (ADS)

    Santos, M. C.; Seabra, A. B.; Pelegrino, M. T.; Haddad, P. S.

    2016-03-01

    Superparamagnetic iron oxide nanoparticles (SPIONs), with appropriate surface coatings, are commonly used for biomedical applications, such as drug delivery. For the successful application of SPIONs, it is necessary that the nanoparticles have well-defined morphological, structural and magnetic characteristics, in addition to high stability and biocompatibility in biological environments. The present work is focused on the synthesis and characterization of SPIONs, which were prepared using the co-precipitation method and have great potential for drug delivery. The surfaces of the SPIONs were functionalized with the tripeptide glutathione (GSH) and poly(ethylene glycol) (PEG) to form GSH-SPIONs and PEG-GSH-SPIONs. The structural, morphological, magnetic properties and the cytotoxicity of the obtained nanoparticles were characterized using different techniques. The results showed that the nanoparticles have a mean diameter of 10 nm in the solid state and are superparamagnetic at room temperature. No cytotoxicity was observed for either nanoparticle (up to 500 μg L-1) on mouse normal fibroblasts (3T3 cell line) or acute T cell leukemia (Jurkat cell line) after 24 h of incubation. Free thiol groups (SH) on the surfaces of GSH-SPIONs and PEG-GSH-SPIONs were nitrosated, leading to the formation of S-nitrosated SPIONs, which act as a nitric oxide (NO) donor. The amounts of NO released from GSNO-SPIONs and PEG-GSNO-SPIONs were (124.0 ± 1.0) μmol and (33.2 ± 5.1) μmol of NO per gram, respectively. This study highlights the successful capping of the SPION surfaces with antioxidant GSH and biocompatible PEG, which improved the dispersion and biocompatibility of the NPs in aqueous/biological environments, thereby enhancing the potential uses of SPIONs as drug delivery systems, such as a NO donor vehicle, in biomedical applications.

  2. Artificial elevation of glutathione affects symptom development in ZYMV-infected Cucurbita pepo L. plants.

    PubMed

    Zechmann, B; Zellnig, G; Urbanek-Krajnc, A; Müller, M

    2007-01-01

    Styrian oil pumpkin seedlings (Cucurbita pepo L. subsp. pepo var. styriaca GREB: .) were treated for 48 h with 1 mM OTC (L-2-oxothiazolidine-4-carboxylic acid) in order to artificially increase cellular glutathione content. They were inoculated with zucchini yellow mosaic virus (ZYMV) 10 days later. The effects of OTC treatment and ZYMV infection on glutathione levels were examined at the subcellular level by immunogold labeling of glutathione using a transmission electron microscope (TEM). These effects were further tested at the whole-tissue level by high performance liquid chromatography (HPLC). Such tests were carried out a) on roots, cotyledons and the first true leaves immediately after OTC treatment in order to analyze to which extent OTC increases glutathione levels in different cell compartments as well as in the whole organ; and b) in older and younger leaves and in roots three weeks after ZYMV inoculation in order to study how possible effects of OTC on symptom development would correlate with glutathione levels at the subcellular level and in the whole organ. Immunocytological and biochemical investigations revealed that, 48 h after OTC treatment, glutathione content had increased in all investigated organs, up to 144% in peroxisomes of cotyledons. Three weeks after ZYMV inoculation, glutathione labeling density had significantly increased within intact cells of infected leaves, up to 124% in the cytosol of younger leaves. Roots showed decreased amounts of glutathione in the TEM. Biochemical studies revealed that OTC treatment resulted in 41 and 51% higher glutathione content in older and younger ZYMV-infected leaves, respectively, in comparison to untreated and ZYMV-infected plants. Evaluation of symptom development at this point revealed that all untreated ZYMV-infected plants had symptoms, whereas only 42% of OTC-treated ZYMV-infected plants showed signs of symptoms. Quantification of ZYMV particles revealed that all organs of OTC-treated and ZYMV

  3. Significance of Polymorphisms and Expression of Enzyme-Encoding Genes Related to Glutathione in Hematopoietic Cancers and Solid Tumors

    PubMed Central

    Zmorzyński, Szymon; Świderska-Kołacz, Grażyna; Koczkodaj, Dorota; Filip, Agata Anna

    2015-01-01

    Antioxidant compounds such as glutathione and its enzymes have become the focus of attention of medical sciences. Glutathione, a specific tripeptide, is involved in many intercellular processes. The glutathione concentration is determined by the number of GAG repeats in gamma-glutamylcysteine synthetase. GAG polymorphisms are associated with an increased risk of schizophrenia, berylliosis, diabetes, lung cancer, and nasopharyngeal tumors. Cancer cells with high glutathione concentration are resistant to chemotherapy treatment. The oxidized form of glutathione is formed by glutathione peroxidases (GPXs). The changes in activity of GPX1, GPX2, and GPX3 isoforms may be associated with the development of cancers, for example, prostate cancer or even colon cancer. Detoxification of glutathione conjugates is possible due to activity of glutathione S-transferases (GSTs). Polymorphisms in GSTM1, GSTP1, and GSTO1 enzymes increase the risk of developing breast cancer and hepatocellular carcinoma. Gamma-glutamyl transpeptidases (GGTs) are responsible for glutathione degradation. Increased activity of GGT correlates with adverse prognosis in patients with breast cancer. Studies on genes encoding glutathione enzymes are continued in order to determine the correlation between DNA polymorphisms in cancer patients. PMID:26682223

  4. Carcinogen treatment increases glutathione hydrolysis by gamma-glutamyl transpeptidase.

    PubMed

    Conway, J G; Neptun, D A; Garvey, L K; Popp, J A

    1987-07-01

    The effect of carcinogen treatment on gamma-glutamyl transpeptidase (GGT)-mediated hydrolysis of GSH to glutamate and cysteinylglycine in the blood and bile compartments was investigated in livers perfused in situ. Treatment of rats with 40 p.p.m. diethylnitrosamine (DEN) in the drinking water or 0.02% 2-acetylaminofluorene (AAF) in the diet for 50-60 days increased GGT activity in liver homogenates by 100 and 800% respectively. Bile flow and the sum of glutamate and glutathione (GSH) efflux into the bile of perfused livers was not affected by carcinogen treatment. However, the ratio of GSH to glutamate in bile was 2.1, 1.1 and 0.2 in livers from control, DEN- and AAF-treated rats respectively. Pretreatment with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT125) decreased GGT activity in liver homogenates by about 85% and elevated the ratio of GSH to glutamate in the bile to 3.2 in all groups. Thus, the hydrolysis of GSH to glutamate in the bile of perfused livers correlated with the degree of induction of GGT by DEN and AAF treatments. Exogenous GSH (10 microM) infused into the portal vein of perfused livers from control, DEN- and AAF-treated rats was recovered completely in the effluent perfusate. Pretreatment with AT125 had no effect on the recovery of exogenous GSH in the effluent perfusate. Thus, metabolism of GSH in the blood space was not detected after short-term carcinogen treatment. To increase the possible hydrolysis of GSH in the perfusate, rats were treated for 130-180 days with DEN and GSH (60 microM) was infused into the hepatic artery of livers perfused simultaneously via the hepatic artery and portal vein. Only 50% of the infused GSH was recovered in the effluent perfusate of perfused livers from DEN-treated rats. In contrast, significantly more GSH (80-90%) was recovered from livers from control rats or DEN-treated rats that had received AT125 pretreatment. In addition AT125 pretreatment increased the basal rates of GSH

  5. Lung glutathione adaptive responses to cigarette smoke exposure

    PubMed Central

    2011-01-01

    Background Smoking tobacco is a leading cause of chronic obstructive pulmonary disease (COPD), but although the majority of COPD cases can be directly related to smoking, only a quarter of smokers actually develop the disease. A potential reason for the disparity between smoking and COPD may involve an individual's ability to mount a protective adaptive response to cigarette smoke (CS). Glutathione (GSH) is highly concentrated in the lung epithelial lining fluid (ELF) and protects against many inhaled oxidants. The changes in GSH that occur with CS are not well investigated; therefore the GSH adaptive response that occurs with a commonly utilized CS exposure was examined in mice. Methods Mice were exposed to CS for 5 h after which they were rested in filtered air for up to 16 h. GSH levels were measured in the ELF, bronchoalveolar lavage cells, plasma, and tissues. GSH synthesis was assessed by measuring γ-glutamylcysteine ligase (GCL) activity in lung and liver tissue. Results GSH levels in the ELF, plasma, and liver were decreased by as much as 50% during the 5 h CS exposure period whereas the lung GSH levels were unchanged. Next, the time course of rebound in GSH levels after the CS exposure was examined. CS exposure initially decreased ELF GSH levels by 50% but within 2 h GSH levels rebound to about 3 times basal levels and peaked at 16 h with a 6-fold increase and over repeat exposures were maintained at a 3-fold elevation for up to 2 months. Similar changes were observed in tissue GCL activity which is the rate limiting step in GSH synthesis. Furthermore, elevation in ELF GSH levels was not arbitrary since the CS induced GSH adaptive response after a 3d exposure period prevented GSH levels from dropping below basal levels. Conclusions CS exposures evoke a powerful GSH adaptive response in the lung and systemically. These data suggests there may be a sensor that sets the ELF GSH adaptive response to prevent GSH levels from dipping below basal levels. Factors

  6. Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione

    PubMed Central

    Kilanczyk, Ewa; Saraswat Ohri, Sujata; Whittemore, Scott R.

    2016-01-01

    The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10−8 M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter. PMID:27449129

  7. Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione.

    PubMed

    Kilanczyk, Ewa; Saraswat Ohri, Sujata; Whittemore, Scott R; Hetman, Michal

    2016-08-01

    The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10(-8) M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter. PMID:27449129

  8. Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ -ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation

    PubMed Central

    Jobe, Timothy O.; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A.; Mendoza-Cózatl, David G.; Schroeder, Julian I.

    2015-01-01

    Summary Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M2 seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione

  9. A mitochondria-targeted turn-on fluorescent probe for the detection of glutathione in living cells.

    PubMed

    Zhang, Jian; Bao, Xiaolong; Zhou, Junliang; Peng, Fangfang; Ren, Hang; Dong, Xiaochun; Zhao, Weili

    2016-11-15

    A novel turn-on red fluorescent BODIPY-based probe (Probe 1) for the detection of glutathione was developed. Such a probe carries a para-dinitrophenoxy benzyl pyridinium moiety at the meso position of a BODIPY dye as self-immolative linker. Probe 1 responds selectively to glutathione with the detection limit of 109nM over other amino acids, common metal ions, reactive oxygen species, reactive nitrogen species, and reactive sulfur species. A novel electrostatic interaction to modulate the SNAr attack of glutathione was believed to play significant role for the observed selective response to glutathione. The cleavage of dinitrophenyl ether by glutathione leads to the production of para-hydroxybenzyl moiety which is able to self-immolate through an intramolecular 1,4-elimination reaction to release the fluorescent BODIPY dye. The low toxic probe has been successfully used to detect mitochondrial glutathione in living cells. PMID:27176914

  10. Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity, and iron homeostasis of Beauveria bassiana.

    PubMed

    Zhang, Long-Bin; Tang, Li; Ying, Sheng-Hua; Feng, Ming-Guang

    2016-07-01

    Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for the thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized the Beauveria bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5, and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some other grx genes in normal cultures. Particularly, grx3 compensated the absence of grx1, grx2, grx5, or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most of other undeleted grx and glr genes in response to menadione. Consequently, the redox state was disturbed in Δglr more than in Δgrx3 but not in Δgrx1/2/5. Superoxide dismutases were more active in normal Δgrx1-3 cultures but less in Δgrx5 or Δglr response to menadione. Total catalase activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Δglr than of Δgrx3. Among the mutants, Δgrx3 showed slightly increased sensitivity to menadione or H2O2; Δglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing 1-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe(3+)-inclusive medium perhaps due to elevated transcripts of two Fe(3+) transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Δgrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other Grx homologues in the Glr-dependent redox system of the fungal entomopathogen. PMID:26969041

  11. Survival of Escherichia coli cells on solid copper surfaces is increased by glutathione.

    PubMed

    Große, Cornelia; Schleuder, Grit; Schmole, Christin; Nies, Dietrich H

    2014-11-01

    Bacteria are rapidly killed on solid copper surfaces, so this material could be useful to limit the spread of multiple-drug-resistant bacteria in hospitals. In Escherichia coli, the DNA-protecting Dps protein and the NADH:ubiquinone oxidoreductase II Ndh were not involved in tolerance to copper ions or survival on solid copper surfaces. Decreased copper tolerance under anaerobic growth conditions in the presence of ascorbate and with melibiose as the carbon source indicated that sodium-dependent symport systems may provide an import route for Cu(I) into the cytoplasm. Glutathione-free ΔcopA ΔgshA double mutants of E. coli were more rapidly inactivated on solid copper surfaces than glutathione-containing wild-type cells. Therefore, while DNA protection by Dps was not required, glutathione was needed to protect the cytoplasm and the DNA against damage mediated by solid copper surfaces, which may explain the differences in the molecular mechanisms of killing between glutathione-containing Gram-negative and glutathione-free Gram-positive bacteria. PMID:25192999

  12. PKLR promotes colorectal cancer liver colonization through induction of glutathione synthesis.

    PubMed

    Nguyen, Alexander; Loo, Jia Min; Mital, Rohit; Weinberg, Ethan M; Man, Fung Ying; Zeng, Zhaoshi; Paty, Philip B; Saltz, Leonard; Janjigian, Yelena Y; de Stanchina, Elisa; Tavazoie, Sohail F

    2016-02-01

    Colorectal cancer metastasis to the liver is a major cause of cancer-related death; however, the genes and pathways that govern this metastatic colonization event remain poorly characterized. Here, using a large-scale in vivo RNAi screen, we identified liver and red blood cell pyruvate kinase (PKLR) as a driver of metastatic liver colonization. PKLR expression was increased in liver metastases as well as in primary colorectal tumors of patients with metastatic disease. Evaluation of a murine liver colonization model revealed that PKLR promotes cell survival in the tumor core during conditions of high cell density and oxygen deprivation by increasing glutathione, the primary endogenous antioxidant. PKLR negatively regulated the glycolytic activity of PKM2, the major pyruvate kinase isoenzyme known to regulate cellular glutathione levels. Glutathione is critical for metastasis, and we determined that the rate-limiting enzyme of glutathione synthesis, GCLC, becomes overexpressed in patient liver metastases, promotes cell survival under hypoxic and cell-dense conditions, and mediates metastatic liver colonization. RNAi-mediated inhibition of glutathione synthesis impaired survival of multiple colon cancer cell lines, and pharmacological targeting of this metabolic pathway reduced colonization in a primary patient-derived xenograft model. Our findings highlight the impact of metabolic reprogramming within the niche as metastases progress and suggest clinical potential for targeting this pathway in colorectal cancer. PMID:26784545

  13. Chlortetracycline detoxification in maize via induction of glutathione S-transferases after antibiotic exposure.

    PubMed

    Farkas, Michael H; Berry, James O; Aga, Diana S

    2007-02-15

    Soil contamination with nonmetabolized antibiotics is an emerging environmental concern, especially on agricultural croplands that receive animal manure as fertilizer. In this study, phytotoxicity of chlortetracycline (CTC) antibiotics on pinto beans (Phaseolus vulgaris) and maize (Zea mays) was investigated under controlled conditions. When grown in CTC-treated soil, a significant increase in the activities of the plant stress proteins glutathione S-transferases (GST) and peroxidases (POX) were observed in maize plants, but not in pinto beans. In vitro conjugation reactions demonstrated that the induced GST in maize catalyzed the conjugation of glutathione (GSH) with CTC, producing stable conjugates that were structurally characterized using liquid chromatography/mass spectrometry. The antibiotic-induced GST produced CTC-glutathione conjugate at relative concentrations 2-fold higher than that produced by constitutively expressed GST extracted from untreated maize. On the other hand, GST extracted from pinto beans (both treated and untreated) did not efficiently catalyze glutathione conjugation with CTC. These results suggest that maize is able to detoxify chlortetracycline via the glutathione pathway, whereas pinto beans cannot. This may explain the observed stunted growth of pinto beans after antibiotic treatment. This study demonstrates the importance of plant uptake in determining the fate of antibiotics in soil and their potential phytotoxicity to susceptible plants. PMID:17593756

  14. Glutathione reductase gsr-1 is an essential gene required for Caenorhabditis elegans early embryonic development.

    PubMed

    Mora-Lorca, José Antonio; Sáenz-Narciso, Beatriz; Gaffney, Christopher J; Naranjo-Galindo, Francisco José; Pedrajas, José Rafael; Guerrero-Gómez, David; Dobrzynska, Agnieszka; Askjaer, Peter; Szewczyk, Nathaniel J; Cabello, Juan; Miranda-Vizuete, Antonio

    2016-07-01

    Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress, have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode. PMID:27117030

  15. PKLR promotes colorectal cancer liver colonization through induction of glutathione synthesis

    PubMed Central

    Nguyen, Alexander; Loo, Jia Min; Mital, Rohit; Weinberg, Ethan M.; Man, Fung Ying; Zeng, Zhaoshi; Paty, Philip B.; Saltz, Leonard; Janjigian, Yelena Y.; de Stanchina, Elisa; Tavazoie, Sohail F.

    2016-01-01

    Colorectal cancer metastasis to the liver is a major cause of cancer-related death; however, the genes and pathways that govern this metastatic colonization event remain poorly characterized. Here, using a large-scale in vivo RNAi screen, we identified liver and red blood cell pyruvate kinase (PKLR) as a driver of metastatic liver colonization. PKLR expression was increased in liver metastases as well as in primary colorectal tumors of patients with metastatic disease. Evaluation of a murine liver colonization model revealed that PKLR promotes cell survival in the tumor core during conditions of high cell density and oxygen deprivation by increasing glutathione, the primary endogenous antioxidant. PKLR negatively regulated the glycolytic activity of PKM2, the major pyruvate kinase isoenzyme known to regulate cellular glutathione levels. Glutathione is critical for metastasis, and we determined that the rate-limiting enzyme of glutathione synthesis, GCLC, becomes overexpressed in patient liver metastases, promotes cell survival under hypoxic and cell-dense conditions, and mediates metastatic liver colonization. RNAi-mediated inhibition of glutathione synthesis impaired survival of multiple colon cancer cell lines, and pharmacological targeting of this metabolic pathway reduced colonization in a primary patient-derived xenograft model. Our findings highlight the impact of metabolic reprogramming within the niche as metastases progress and suggest clinical potential for targeting this pathway in colorectal cancer. PMID:26784545

  16. Yeast mitochondrial glutathione is an essential antioxidant with mitochondrial thioredoxin providing a back-up system

    PubMed Central

    Gostimskaya, Irina; Grant, Chris M.

    2016-01-01

    Glutathione is an abundant, low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl moiety. Its role as the main determinant of thiol-redox control has been challenged such that it has been proposed to play a crucial role in iron–sulphur clusters maturation, and only a minor role in thiol redox regulation, predominantly as a back-up system for the cytoplasmic thioredoxin system. Here, we have tested the importance of mitochondrial glutathione in thiol-redox regulation. Glutathione reductase (Glr1) is an oxidoreductase which converts oxidized glutathione to its reduced form. Yeast Glr1 localizes to both the cytosol and mitochondria and we have used a Glr1M1L mutant that is constitutively localized to the cytosol to test the requirement for mitochondrial Glr1. We show that the loss of mitochondrial Glr1 specifically accounts for oxidant sensitivity of a glr1 mutant. Loss of mitochondrial Glr1 does not influence iron–sulphur cluster maturation and we have used targeted roGFP2 fluorescent probes to show that oxidant sensitivity is linked to an altered redox environment. Our data indicate mitochondrial glutathione is crucial for mitochondrial thiol-redox regulation, and the mitochondrial thioredoxin system provides a back-up system, but cannot bear the redox load of the mitochondria on its own. PMID:26898146

  17. In Vivo Magnetic Resonance Studies of Glycine and Glutathione Metabolism in a Rat Mammary Tumor

    PubMed Central

    Thelwall, Peter E.; Simpson, Nicholas E.; Rabbani, Zahid N.; Clark, M. Daniel; Pourdeyhimi, Roxana; Macdonald, Jeffrey M.; Blackband, Stephen J.; Gamcsik, Michael P.

    2011-01-01

    The metabolism of glycine into glutathione was monitored noninvasively in vivo in intact R3230Ac rat tumors by magnetic resonance imaging and spectroscopy. Metabolism was tracked by following the isotope label from intravenously infused [2-13C]-glycine into the glycinyl residue of glutathione. Signals from [2-13C]-glycine and γ-glutamylcysteinyl-[2-13C]-glycine (13C-glutathione) were detected by nonlocalized 13C spectroscopy as these resonances are distinct from background signals. In addition, using spectroscopic imaging methods, heterogeneity in the in vivo tumor distribution of glutathione was observed. In vivo spectroscopy also detected isotope incorporation from [2-13C]-glycine into both the 2- and 3-carbons of serine. Analyses of tumor tissue extracts show single and multiple label incorporation from [2-13C]-glycine into serine from metabolism through the serine hydroxymethyltransferase and glycine cleavage system pathways. Mass spectrometric analysis of extracts also shows that isotope-labeled serine is further metabolized via the transsulfuration pathway as the 13C-isotope labels appear in both the glycinyl- and the cysteinyl-residue of glutathione. Our studies demonstrate the use of magnetic resonance imaging and spectroscopy for monitoring tumor metabolic processes central to oxidative stress defense. PMID:21751272

  18. Yeast mitochondrial glutathione is an essential antioxidant with mitochondrial thioredoxin providing a back-up system.

    PubMed

    Gostimskaya, Irina; Grant, Chris M

    2016-05-01

    Glutathione is an abundant, low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl moiety. Its role as the main determinant of thiol-redox control has been challenged such that it has been proposed to play a crucial role in iron-sulphur clusters maturation, and only a minor role in thiol redox regulation, predominantly as a back-up system for the cytoplasmic thioredoxin system. Here, we have tested the importance of mitochondrial glutathione in thiol-redox regulation. Glutathione reductase (Glr1) is an oxidoreductase which converts oxidized glutathione to its reduced form. Yeast Glr1 localizes to both the cytosol and mitochondria and we have used a Glr1(M1L) mutant that is constitutively localized to the cytosol to test the requirement for mitochondrial Glr1. We show that the loss of mitochondrial Glr1 specifically accounts for oxidant sensitivity of a glr1 mutant. Loss of mitochondrial Glr1 does not influence iron-sulphur cluster maturation and we have used targeted roGFP2 fluorescent probes to show that oxidant sensitivity is linked to an altered redox environment. Our data indicate mitochondrial glutathione is crucial for mitochondrial thiol-redox regulation, and the mitochondrial thioredoxin system provides a back-up system, but cannot bear the redox load of the mitochondria on its own. PMID:26898146

  19. 1-Methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons

    SciTech Connect

    Drechsel, Derek A.; Liang, L.-P.; Patel, Manisha . E-mail: manisha.patel@uchsc.edu

    2007-05-01

    Decreased glutathione levels associated with increased oxidative stress are a hallmark of numerous neurodegenerative diseases, including Parkinson's disease. GSH is an important molecule that serves as an anti-oxidant and is also a major determinant of cellular redox environment. Previous studies have demonstrated that neurotoxins can cause changes in reduced and oxidized GSH levels; however, information regarding steady state levels remains unexplored. The goal of this study was to characterize changes in cellular GSH levels and its regulatory enzymes in a dopaminergic cell line (N27) following treatment with the Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP{sup +}). Cellular GSH levels were initially significantly decreased 12 h after treatment, but subsequently recovered to values greater than controls by 24 h. However, oxidized glutathione (GSSG) levels were increased 24 h following treatment, concomitant with a decrease in GSH/GSSG ratio prior to cell death. In accordance with these changes, ROS levels were also increased, confirming the presence of oxidative stress. Decreased enzymatic activities of glutathione reductase and glutamate-cysteine ligase by 20-25% were observed at early time points and partly account for changes in GSH levels after MPP{sup +} exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP{sup +} treatment was not associated with increased efflux of glutathione to the medium. These data further elucidate the mechanisms underlying GSH depletion in response to the Parkinsonian toxin, MPP{sup +}.

  20. Current status and emerging role of glutathione in food grade lactic acid bacteria

    PubMed Central

    2012-01-01

    Lactic acid bacteria (LAB) have taken centre stage in perspectives of modern fermented food industry and probiotic based therapeutics. These bacteria encounter various stress conditions during industrial processing or in the gastrointestinal environment. Such conditions are overcome by complex molecular assemblies capable of synthesizing and/or metabolizing molecules that play a specific role in stress adaptation. Thiols are important class of molecules which contribute towards stress management in cell. Glutathione, a low molecular weight thiol antioxidant distributed widely in eukaryotes and Gram negative organisms, is present sporadically in Gram positive bacteria. However, new insights on its occurrence and role in the latter group are coming to light. Some LAB and closely related Gram positive organisms are proposed to possess glutathione synthesis and/or utilization machinery. Also, supplementation of glutathione in food grade LAB is gaining attention for its role in stress protection and as a nutrient and sulfur source. Owing to the immense benefits of glutathione, its release by probiotic bacteria could also find important applications in health improvement. This review presents our current understanding about the status of glutathione and its role as an exogenously added molecule in food grade LAB and closely related organisms. PMID:22920585

  1. Purification and thermal dependence of glutathione reductase from two forage legume species.

    PubMed

    Kidambi, S P; Mahan, J R; Matches, A G

    1990-02-01

    Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from ;Cimarron' alfalfa and ;PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45 degrees C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25 degrees C), and (c) the thermal kinetic windows (6-19 degrees C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season. PMID:16667283

  2. Effect of transport on blood selenium and glutathione status in feeder lambs.

    PubMed

    Hall, J A; Bobe, G; Nixon, B K; Vorachek, W R; Hugejiletu; Nichols, T; Mosher, W D; Pirelli, G J

    2014-09-01

    Stress from transport may be linked to increased generation of reactive oxygen species, the removal of which requires reduced glutathione and selenium. The aim of this experiment was to examine the effect of transport on glutathione and Se status of feeder lambs. Recently weaned lambs (n = 40) were blocked by gender and BW on d 0 of the experiment and randomly assigned to 2 treatment groups: group 1, no transport and full access to feed and water (control), and group 2, 8-h road transport followed by another 16 h of feed deprivation (transport). After 24 h, both treatment groups were treated the same. All lambs were weighed, and blood samples were collected at 0, 8, 24, and 72 h and analyzed for whole-blood (WB) and serum Se concentrations, serum NEFA concentrations, and erythrocyte concentrations of glutathione. Transport of feeder lambs for 8 h followed by another 16 h of feed deprivation transiently (significant at 24 h but no longer different at 72 h) decreased BW and erythrocyte glutathione concentrations and increased serum NEFA and blood Se concentrations compared with control lambs. Our results suggest that 8 h of transport followed by another 16 h of feed deprivation results in fatty acid and Se mobilization from tissue stores with a coincident decrease in erythrocyte glutathione concentrations. PMID:25035242

  3. Survival of Escherichia coli Cells on Solid Copper Surfaces Is Increased by Glutathione

    PubMed Central

    Große, Cornelia; Schleuder, Grit; Schmole, Christin

    2014-01-01

    Bacteria are rapidly killed on solid copper surfaces, so this material could be useful to limit the spread of multiple-drug-resistant bacteria in hospitals. In Escherichia coli, the DNA-protecting Dps protein and the NADH:ubiquinone oxidoreductase II Ndh were not involved in tolerance to copper ions or survival on solid copper surfaces. Decreased copper tolerance under anaerobic growth conditions in the presence of ascorbate and with melibiose as the carbon source indicated that sodium-dependent symport systems may provide an import route for CuI into the cytoplasm. Glutathione-free ΔcopA ΔgshA double mutants of E. coli were more rapidly inactivated on solid copper surfaces than glutathione-containing wild-type cells. Therefore, while DNA protection by Dps was not required, glutathione was needed to protect the cytoplasm and the DNA against damage mediated by solid copper surfaces, which may explain the differences in the molecular mechanisms of killing between glutathione-containing Gram-negative and glutathione-free Gram-positive bacteria. PMID:25192999

  4. Developmental aspects of glutathione S-transferase B (ligandin) in rat liver.

    PubMed Central

    Hales, B F; Neims, A H

    1976-01-01

    The postnatal development in male Sprague-Dawley rats of hepatic glutathione S-transferase B (ligandin) in relation to the other glutathione S-transferases is described. The concentration of glutathione S-transferase B in 1-day-old male rats is about one-fifth of that in adult animals. The enzyme reaches adult concentrations 4-5 weeks later. When assessed by substrate specificity or immunologically, the proportion of transferase B relative to the other glutathione S-transferases is high during the first week after birth. At this age, 67.5% of the transferase activity towards 1-chloro-2,4-dinitrobenzene is immunoprecipitable by anti-(transferase B), compared with about 50% in adults and older pups. Between the second and the fifth postnatal week, the fraction of transferase B increases in parallel fashion with the other transferases in hepatic cytosol. Neither L-thyroxine nor cortisol induce a precocious increase in glutathione S-transferase activity. Phenobarbital did induce transferase activity towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene in both pups and adults. The extent of induction by phenobarbital was a function of basal activity during development such that the percentage stimulation remained constant from 5 days postnatally to adulthood. PMID:1008852

  5. Airway glutathione homeostasis is altered in children with severe asthma: Evidence for oxidant stress

    PubMed Central

    Fitzpatrick, Anne M.; Teague, W. Gerald; Holguin, Fernando; Yeh, Mary; Brown, Lou Ann S.

    2009-01-01

    Background Severe asthma is characterized by persistent airway inflammation and increased formation of reactive oxygen species. Objectives Glutathione (GSH) is an important antioxidant in the epithelial lining fluid (ELF). We hypothesized that airway GSH homeostasis was altered in children with severe asthma and was characterized by decreased GSH and increased glutathione disulfide (GSSG) concentrations. Methods Bronchoalveolar lavage was obtained from 65 children with severe asthma, including 35 children with baseline airway obstruction evidenced by FEV1 <80%. Control data were obtained from 6 children with psychogenic (habit) cough or vocal cord dysfunction undergoing diagnostic bronchoscopy and 35 healthy adult controls. GSH, GSSG, and other determinants of airway oxidative stress including glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), malondialdehyde, 8-isoprostane, and H2O2 were measured in the ELF. The ELF redox potential was calculated from GSH and GSSG by using the Nernst equation. Results: Compared with controls, subjects with severe asthma had lower airway GSH with increased GSSG despite no differences in GST, GR, and GPx activities between groups. This was accompanied by increased malondialdehyde, 8-isoprostane, and H2O2 concentrations in the ELF. GSH oxidation was most apparent in subjects with severe asthma with airway obstruction and was supported by an upward shift in the ELF GSH redox potential. Conclusion Children with severe asthma have increased biomarkers of oxidant stress in the ELF that are associated with increased formation of GSSG and a shift in the GSH redox potential toward the more oxidized state. PMID:19130935

  6. Selenium concentrations and enzyme activities of glutathione metabolism in wild long-tailed ducks and common eiders

    USGS Publications Warehouse

    Franson, J. Christian; Hoffman, David J.; Flint, Paul L.

    2011-01-01

    The relationships of selenium (Se) concentrations in whole blood with plasma activities of total glutathione peroxidase, Se-dependent glutathione peroxidase, and glutathione reductase were studied in long-tailed ducks (Clangula hyemalis) and common eiders (Somateria mollissima) sampled along the Beaufort Sea coast of Alaska, USA. Blood Se concentrations were >8 μg/g wet weight in both species. Linear regression revealed that the activities of total and Se-dependent glutathione peroxidase were significantly related to Se concentrations only in long-tailed ducks, raising the possibility that these birds were experiencing early oxidative stress.

  7. Mammalian proapoptotic factor ChaC1 and its homologues function as γ-glutamyl cyclotransferases acting specifically on glutathione.

    PubMed

    Kumar, Akhilesh; Tikoo, Shweta; Maity, Shuvadeep; Sengupta, Shantanu; Sengupta, Sagar; Kaur, Amandeep; Bachhawat, Anand Kumar

    2012-12-01

    ChaC1 is a mammalian proapoptic protein of unknown function induced during endoplasmic reticulum stress. We show using in vivo studies and novel in vitro assays that the ChaC family of proteins function as γ-glutamyl cyclotransferases acting specifically to degrade glutathione but not other γ-glutamyl peptides. The overexpression of these proteins (but not the catalytically dead E>Q mutants) led to glutathione depletion and enhanced apoptosis in yeast. The ChaC family is conversed across all phyla and represents a new pathway for glutathione degradation in living cells, and the first cytosolic pathway for glutathione degradation in mammalian cells. PMID:23070364

  8. The gene encoding human glutathione synthetase (GSS) maps to the long arm of chromosome 20 at band 11.2

    SciTech Connect

    Webb, G.C.; Vaska, V.L.; Ford, J.H.

    1995-12-10

    Two forms of glutathione synthetase deficiency have been described. While one form is mild, causing hemolytic anemia, the other more severe form causes 5-oxoprolinuria with secondary neurological involvement. Despite the existence of two deficiency phenotypes, Southern blots hybridized with a glutathione synthetase cDNA suggest that there is a single glutathione synthetase gene in the human genome. Analysis of somatic cell hybrids showed the human glutathione synthetase gene (GSS) to be located on chromosome 20, and this assignment has been refined to subband 20q11.2 using in situ hybridization. 16 refs., 2 figs.

  9. Regiospecificity of placental metabolism by cytochromes P450 and glutathione S-transferase.

    PubMed

    McRobie, D J; Glover, D D; Tracy, T S

    1996-01-01

    The placenta possesses the ability to metabolize numerous xenobiotics and endogenous steroids. However, it is unknown whether regional differences in these enzymatic reactions exist in the human placenta. To this end, we undertook a study of four regions of the placenta, the chorionic plate, maternal surface, placental margin and whole tissue, to assess the activities of cytochrome P450 1A1 and 19A1 (aromatase) and glutathione S-stransferase in these fractions. No differences in either P450 1A1 or glutathione S-transferase activities were noted among any of the placental fractions. However, with respect to P450 19A1 activity, the placental margin differed significantly from all other fractions (p < 0.05). This study demonstrates that whole tissue samples of the human placenta are adequate for placental cytochrome P450 and glutathione S-transferase metabolism studies. PMID:8938464

  10. A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

    NASA Astrophysics Data System (ADS)

    Lou, Xiaoding; Hong, Yuning; Chen, Sijie; Leung, Chris Wai Tung; Zhao, Na; Situ, Bo; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2014-03-01

    In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells.

  11. A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

    PubMed Central

    Lou, Xiaoding; Hong, Yuning; Chen, Sijie; Leung, Chris Wai Tung; Zhao, Na; Situ, Bo; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2014-01-01

    In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells. PMID:24603274

  12. Influence of gender and season on reduced glutathione concentration and energy reserves of Gammarus roeseli.

    PubMed

    Gismondi, Eric; Beisel, Jean-Nicolas; Cossu-Leguille, Carole

    2012-10-01

    As biomarkers are known to be influenced by biotic and abiotic factors (e.g. gender, temperature), we investigated over a one-year long sampling period, the influence of season and gender on reduced glutathione concentrations and its synthesis in the crustacean amphipod Gammarus roeseli. At the same time, we assessed energy reserves and malondialdehyde levels as toxic biomarker. Results have shown that, in both genders, reduced glutathione concentrations were inversely correlated to water temperature, and higher in females than in males whatever the season. Total lipid and glycogen contents were higher in females than in males, allowing females to have enough energy to assume the reproductive period and maintain high GSH concentrations for detoxification processes. Conversely, females have lower cell damages than males. These differences between genders could induce differential sensitivity in a contamination context, and thus affect the population. Females could resist better than males in contaminated environments, especially in spring when reduced glutathione concentration is the highest. PMID:22769238

  13. The effects of age on glutathione synthesis enzymes in lenses of Old World simians and prosimians.

    PubMed

    Rathbun, W B; Holleschau, A M

    1992-07-01

    The activities of gamma-glutamylcysteine synthetase and glutathione synthetase, the two enzymes required for glutathione synthesis, were determined as a function of age in lenses of three species of Old World higher primates: orangutan, pigtail monkey and olive baboon. These were compared to enzyme activities in lenses of two prosimians: mouse lemur and galago. gamma-Glutamylcysteine synthetase activity decreased as a function of age in all three Old World simians. The rate of decrease was greatest in the juvenile lenses. In contrast, the enzyme activity increased continuously with age in the galago lens. In the mouse lemur the enzyme activity increased per lens, but was constant when expressed as specific activity or as units per gram of lens. The loss of enzyme activity with age was limited to Old World higher primates apparently representing genetic change. Glutathione synthetase activity decreased logarithmically with age in the lenses of all five species. PMID:1355706

  14. Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12.

    PubMed

    Pacheco, Catarina C; Passos, João F; Castro, A Rita; Moradas-Ferreira, Pedro; De Marco, Paolo

    2008-03-01

    Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (DeltagshA and DeltagshB) and one respiration deficient mutant (DeltaubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the DeltaubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification. PMID:17968530

  15. Bioaccumulation of PCB-153 and effects on molecular biomarkers acetylcholinesterase, glutathione-S-transferase and glutathione peroxidase in Mytilus galloprovincialis mussels.

    PubMed

    Vidal-Liñán, Leticia; Bellas, Juan; Soriano, José Antonio; Concha-Graña, Estefanía; Muniategui, Soledad; Beiras, Ricardo

    2016-07-01

    In this study, PCB-153 bioaccumulation kinetics and concentration-response experiments were performed employing wild Mytilus galloprovincialis mussels. In addition, the activity of three enzymatic biomarkers: glutathione S-transferase (GST), glutathione peroxidase (GPx) and acetylcholinesterase (AChE), were measured in the mussel gills. The experimental data fitted well to an asymptotic accumulation model with a high bioconcentration factor (BCF) of 9324 L kg(-1) and a very limited depuration capacity, described by a low excretion rate coefficient (Kd = 0.083 d(-1)). This study reports by first time in mussels significant inhibition of GST activity and significant induction of GPx activity as a result of exposure to dissolved PCB-153. In contrast, AChE activity was unaffected at all concentrations and exposure times tested. The effects on both enzymes are time-dependent, which stresses the difficulties inherent to the use of these biomarkers in chemical pollution monitoring programs. PMID:27176625

  16. High Glutathione and Glutathione Peroxidase-2 Levels Mediate Cell-Type-Specific DNA Damage Protection in Human Induced Pluripotent Stem Cells

    PubMed Central

    Dannenmann, Benjamin; Lehle, Simon; Hildebrand, Dominic G.; Kübler, Ayline; Grondona, Paula; Schmid, Vera; Holzer, Katharina; Fröschl, Mirjam; Essmann, Frank; Rothfuss, Oliver; Schulze-Osthoff, Klaus

    2015-01-01

    Summary Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage. PMID:25937369

  17. High glutathione and glutathione peroxidase-2 levels mediate cell-type-specific DNA damage protection in human induced pluripotent stem cells.

    PubMed

    Dannenmann, Benjamin; Lehle, Simon; Hildebrand, Dominic G; Kübler, Ayline; Grondona, Paula; Schmid, Vera; Holzer, Katharina; Fröschl, Mirjam; Essmann, Frank; Rothfuss, Oliver; Schulze-Osthoff, Klaus

    2015-05-12

    Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage. PMID:25937369

  18. Glutathione peroxidase's reaction intermediate selenenic acid is stabilized by the protein microenvironment.

    PubMed

    Li, Fei; Liu, Jun; Rozovsky, Sharon

    2014-11-01

    Selenenic acids are highly reactive intermediates of selenoproteins' enzymatic reactions. Knowledge of how the protein environment protects and stabilizes them is fundamental not only to descriptions of selenoproteins' reactivity but also potentially for proteomics and therapeutics. However, selenenic acids are considered particularly short-lived and are not yet identified in wild-type selenoproteins. Here, we report trapping the selenenic acid in glutathione peroxidase, an antioxidant enzyme that efficiently eliminates hydroperoxides. It has long been thought that selenium-containing glutathione peroxidases form a selenenic acid intermediate. However, this putative species has eluded detection. Here, we report its identification. The selenenic acid in bovine glutathione peroxidase 1 was chemically trapped using dimedone, an alkylating agent specific to sulfenic and selenenic acids. The alkylation of the catalytic selenocysteine was verified by electrospray ionization mass spectrometry. In the presence of glutathione, the selenocysteine was not alkylated because the selenenic acid condenses faster with glutathione than the alkylation reaction. In the absence of thiols, the selenenic acid was surprisingly long-lived with 95% of the protein still able to react with dimedone 10 min after hydrogen peroxide was removed, indicating that the protein environment stabilizes the selenenic acid by shielding it from reactive groups in the protein. After 30 min, the selenocysteine was no longer modified but became accessible once the protein was exposed to reducing agents. This suggests that the selenenic acid reacted with a protein's amide or amine to form a selenylamide bond. Such a modification may play a role in protecting glutathione peroxidase׳' reactivity. PMID:25124921

  19. Targeting brain cells with glutathione-modulated nanoliposomes: in vitro and in vivo study

    PubMed Central

    Salem, Heba F; Ahmed, Sayed M; Hassaballah, Ashraf E; Omar, Mahmoud M

    2015-01-01

    Background The blood–brain barrier prevents many drug moieties from reaching the central nervous system. Therefore, glutathione-modulated nanoliposomes have been engineered to enhance the targeting of flucytosine to the brain. Methods Glutathione-modulated nanoliposomes were prepared by thin-film hydration technique and evaluated in the primary brain cells of rats. Lecithin, cholesterol, and span 65 were mixed at 1:1:1 molar ratio. The molar percentage of PEGylated glutathione varied from 0 mol% to 0.75 mol%. The cellular binding and the uptake of the targeted liposomes were both monitored by epifluorescent microscope and flow cytometry techniques. A biodistribution and a pharmacokinetic study of flucytosine and flucytosine-loaded glutathione–modulated liposomes was carried out to evaluate the in vivo brain-targeting efficiency. Results The size of glutathione-modulated nanoliposomes was <100 nm and the zeta potential was more than −65 mV. The cumulative release reached 70% for certain formulations. The cellular uptake increased as molar percent of glutathione increased to reach the maximum at 0.75 mol%. The uptake of the targeted liposomes by brain cells of the rats was three times greater than that of the nontargeted liposomes. An in vivo study showed that the relative efficiency was 2.632±0.089 and the concentration efficiency was 1.590±0.049, and also, the drug-targeting index was 3.670±0.824. Conclusion Overall, these results revealed that glutathione-PEGylated nanoliposomes enhance the effective delivery of flucytosine to brain and could become a promising new therapeutic option for the treatment of the brain infections. PMID:26229435

  20. Impact of glutathione supplementation of parenteral nutrition on hepatic methionine adenosyltransferase activity

    PubMed Central

    Elremaly, Wesam; Mohamed, Ibrahim; Rouleau, Thérèse; Lavoie, Jean-Claude

    2015-01-01

    Background The oxidation of the methionine adenosyltransferase (MAT) by the combined impact of peroxides contaminating parenteral nutrition (PN) and oxidized redox potential of glutathione is suspected to explain its inhibition observed in animals. A modification of MAT activity is suspected to be at origin of the PN-associated liver disease as observed in newborns. We hypothesized that the correction of redox potential of glutathione by adding glutathione in PN protects the MAT activity. Aim To investigate whether the addition of glutathione to PN can reverse the inhibition of MAT observed in animal on PN. Methods Three days old guinea pigs received through a jugular vein catheter 2 series of solutions. First with methionine supplement, (1) Sham (no infusion); (2) PN: amino acids, dextrose, lipids and vitamins; (3) PN-GSSG: PN+10 μM GSSG. Second without methionine, (4) D: dextrose; (5) D+180 μM ascorbylperoxide; (6) D+350 μM H2O2. Four days later, liver was sampled for determination of redox potential of glutathione and MAT activity in the presence or absence of 1 mM DTT. Data were compared by ANOVA, p<0.05. Results MAT activity was 45±4% lower in animal infused with PN and 23±7% with peroxides generated in PN. The inhibition by peroxides was associated with oxidized redox potential and was reversible by DTT. Correction of redox potential (PN+GSSG) or DTT was without effect on the inhibition of MAT by PN. The slope of the linear relation between MAT activity and redox potential was two fold lower in animal infused with PN than in others groups. Conclusion The present study suggests that prevention of peroxide generation in PN and/or correction of the redox potential by adding glutathione in PN are not sufficient, at least in newborn guinea pigs, to restore normal MAT activity. PMID:26722840

  1. Glutathione Peroxidase’s Reaction Intermediate Selenenic Acid is Stabilized by the Protein Microenvironment

    PubMed Central

    Li, Fei; Liu, Jun; Rozovsky, Sharon

    2014-01-01

    Selenenic acids are highly reactive intermediates of selenoproteins’ enzymatic reactions. Knowledge of how the protein environment protects and stabilizes them is fundamental not only to descriptions of selenoproteins’ reactivity but also potentially for proteomics and therapeutics. However, selenenic acids are considered particularly short-lived and were not yet identified in wild-type selenoproteins. Here, we report trapping the selenenic acid in glutathione peroxidase, an anti-oxidant enzyme that efficiently eliminates hydroperoxides. It has long been thought that selenium-containing glutathione peroxidases form a selenenic acid intermediate. However, this putative species has eluded detection. Here, we report its identification. The selenenic acid in bovine glutathione peroxidase 1 was chemically trapped using dimedone, an alkylating agent specific to sulfenic and selenenic acids. The alkylation of the catalytic selenocysteine was verified by electrospray ionization mass spectrometry. In the presence of glutathione, the selenocysteine was not alkylated because the selenenic acid condenses faster with glutathione than the alkylation reaction. In the absence of thiols, the selenenic acid was surprisingly long-lived with 95% of the protein still able to react with dimedone 10 min after hydrogen peroxide was removed, indicating that the protein environment stabilizes the selenenic acid by shielding it from reactive groups in the protein. After 30 min, the selenocysteine was no longer modified but became accessible once the protein was exposed to reducing agents. This suggests that the selenenic acid reacted with a protein’s amide or amine to form a selenylamide bond. Such a modification may play a role in protecting glutathione peroxidase’s reactivity. PMID:25124921

  2. The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats

    SciTech Connect

    Nishiya, Takayoshi Mori, Kazuhiko Hattori, Chiharu Kai, Kiyonori Kataoka, Hiroko Masubuchi, Noriko Jindo, Toshimasa Manabe, Sunao

    2008-10-15

    To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.

  3. Glutathione as a skin whitening agent: Facts, myths, evidence and controversies.

    PubMed

    Sonthalia, Sidharth; Daulatabad, Deepashree; Sarkar, Rashmi

    2016-01-01

    Glutathione is a low molecular weight thiol-tripeptide that plays a prominent role in maintaining intracellular redox balance. In addition to its remarkable antioxidant properties, the discovery of its antimelanogenic properties has led to its promotion as a skin-lightening agent. It is widely used for this indication in some ethnic populations. However, there is a dichotomy between evidence to support its efficacy and safety. The hype around its depigmentary properties may be a marketing gimmick of pharma-cosmeceutical companies. This review focuses on the various aspects of glutathione: its metabolism, mechanism of action and the scientific evidence to evaluate its efficacy as a systemic skin-lightening agent. Glutathione is present intracellularly in its reduced form and plays an important role in various physiological functions. Its skin-lightening effects result from direct as well as indirect inhibition of the tyrosinase enzyme and switching from eumelanin to phaeomelanin production. It is available in oral, parenteral and topical forms. Although the use of intravenous glutathione injections is popular, there is no evidence to prove its efficacy. In fact, the adverse effects caused by intravenous glutathione have led the Food and Drug Administration of Philippines to issue a public warning condemning its use for off-label indications such as skin lightening. Currently, there are three randomized controlled trials that support the skin-lightening effect and good safety profile of topical and oral glutathione. However, key questions such as the duration of treatment, longevity of skin-lightening effect and maintenance protocols remain unanswered. More randomized, double-blind, placebo-controlled trials with larger sample size, long-term follow-up and well-defined efficacy outcomes are warranted to establish the relevance of this molecule in disorders of hyperpigmentation and skin lightening. PMID:27088927

  4. Glutathione depletion sensitizes cisplatin- and temozolomide-resistant glioma cells in vitro and in vivo

    PubMed Central

    Rocha, C R R; Garcia, C C M; Vieira, D B; Quinet, A; de Andrade-Lima, L C; Munford, V; Belizário, J E; Menck, C F M

    2014-01-01

    Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3–7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating

  5. Glutathione depletion sensitizes cisplatin- and temozolomide-resistant glioma cells in vitro and in vivo.

    PubMed

    Rocha, C R R; Garcia, C C M; Vieira, D B; Quinet, A; de Andrade-Lima, L C; Munford, V; Belizário, J E; Menck, C F M

    2014-01-01

    Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3-7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating

  6. gamma-Glutamylcysteinylglutamic acid--a new homologue of glutathione in maize seedlings exposed to cadmium.

    PubMed

    Meuwly, P; Thibault, P; Rauser, W E

    1993-12-28

    Exposure of plants to Cd induces the appearance of several thiols based on glutathione and known as class III metallothioneins (or phytochelatins). A new tripeptide with the structure gamma-GluCysGlu accumulated in roots and shoots of Cd-exposed maize seedlings. This thiol was purified and identified by tandem mass spectrometry. The fragmentation pattern of the maize tripeptide was identical to that of the synthetic compound. Like glutathione, this new tripeptide may serve as a precursor for longer-chain peptides involved in metal detoxification through the formation of Cd-binding complexes. PMID:8282113

  7. Steady-state kinetics and chemical mechanism of octopus hepatopancreatic glutathione transferase.

    PubMed Central

    Tang, S S; Chang, G G

    1995-01-01

    The kinetic mechanism of glutathione S-transferase (GST) from Octopus vulgaris hepatopancreas was investigated by steady-state analysis. Initial-velocity studies showed an intersecting pattern, which suggests a sequential kinetic mechanism for the enzyme. Product-inhibition patterns by chloride and the conjugate product were all non-competitive with respect to glutathione or 1-chloro-2,4-dinitrobenzene (CDNB), which indicates that the octopus digestive gland GST conforms to a steady-state sequential random Bi Bi kinetic mechanism. Dead-end inhibition patterns indicate that ethacrynic acid ([2,3-dichloro-4-(2-methyl-enebutyryl) phenoxy]acetic acid) binds at the hydrophobic H-site, norophthalmic acid (gamma-glutamylalanylglycine) binds at the glutathione G-site, and glutathione-ethacrynate conjugate occupied both H- and G-sites of the enzyme. The chemical mechanism of the enzyme was examined by pH and kinetic solvent-isotope effects. At pH (and p2H) = 8.011, in which kcat. was independent of pH or p2H, the solvent isotope effects on V and V/KmGSH were near unity, in the range 1.069-1.175. An inverse isotope effect was observed for V/KmCDNB (0.597), presumably resulting from the hydrogen-bonding of enzyme-bound glutathione, which has pKa of 6.83 +/- 0.04, a value lower by 2.34 pH units than the pKa of glutathione in aqueous solution. This lowering of the pKa value for the sulphydryl group of the bound glutathione was presumably due to interaction with the active site Tyr7, which had a pKa value of 8.46 +/- 0.09 that was raised to 9.63 +/- 0.08 in the presence of glutathione thiolate. Subsequent chemical reaction involves attacking of thiolate anion at the electrophilic substrate with the formation of a negatively charged Meisenheimer complex, which is the rate-limiting step of the reaction. Images Scheme 2 PMID:7619078

  8. [Levels of glutathione and anaerobic glycolysis in the kidney and liver of rats treated with chloroethanol].

    PubMed

    Rinaudo, M T; Curto, M; Bruno, R

    1983-11-30

    Chloroethanol administration produces in rats a strong fall of glutathione levels in liver and kidney tissues. In liver, such a modification does not imply alterations in the levels of glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-biphosphate, lactate and pyruvate and in the glycolytic activity. In kidney, the glycolytic activity does not result modified, while it appears a reduction in levels of glucose, glucose-6-phosphate, fructose-1,6-biphosphate and triose phosphates and a remarkable increase of pyruvate. The administration of chloroethanol produces a strong fall of glutathione in the soluble and nuclear fractions of liver and in the soluble and mitochondrial fractions of kidney. PMID:6230087

  9. Bcl-2 is a novel interacting partner for the 2-oxoglutarate carrier and a key regulator of mitochondrial glutathione.

    PubMed

    Wilkins, Heather M; Marquardt, Kristin; Lash, Lawrence H; Linseman, Daniel A

    2012-01-15

    Despite making up only a minor fraction of the total cellular glutathione, recent studies indicate that the mitochondrial glutathione pool is essential for cell survival. Selective depletion of mitochondrial glutathione is sufficient to sensitize cells to mitochondrial oxidative stress (MOS) and intrinsic apoptosis. Glutathione is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. Therefore, regulation of mitochondrial glutathione transport is a key factor in maintaining the antioxidant status of mitochondria. Bcl-2 resides in the outer mitochondrial membrane where it acts as a central regulator of the intrinsic apoptotic cascade. In addition, Bcl-2 displays an antioxidant-like function that has been linked experimentally to the regulation of cellular glutathione content. We have previously demonstrated a novel interaction between recombinant Bcl-2 and reduced glutathione (GSH), which was antagonized by either Bcl-2 homology-3 domain (BH3) mimetics or a BH3-only protein, recombinant Bim. These previous findings prompted us to investigate if this novel Bcl-2/GSH interaction might play a role in regulating mitochondrial glutathione transport. Incubation of primary cultures of cerebellar granule neurons (CGNs) with the BH3 mimetic HA14-1 induced MOS and caused specific depletion of the mitochondrial glutathione pool. Bcl-2 was coimmunoprecipitated with GSH after chemical cross-linking in CGNs and this Bcl-2/GSH interaction was antagonized by preincubation with HA14-1. Moreover, both HA14-1 and recombinant Bim inhibited GSH transport into isolated rat brain mitochondria. To further investigate a possible link between Bcl-2 function and mitochondrial glutathione transport, we next examined if Bcl-2 associated with the 2-oxoglutarate carrier (OGC), an inner mitochondrial membrane protein known to transport glutathione in liver and kidney. After cotransfection of CHO cells, Bcl-2 was coimmunoprecipitated with OGC and this novel

  10. Bcl-2 is a novel interacting partner for the 2-oxoglutarate carrier and a key regulator of mitochondrial glutathione

    PubMed Central

    Wilkins, Heather M.; Marquardt, Kristin; Lash, Lawrence H.; Linseman, Daniel A.

    2011-01-01

    Despite making up only a minor fraction of the total cellular glutathione, recent studies indicate that the mitochondrial glutathione pool is essential for cell survival. Selective depletion of mitochondrial glutathione is sufficient to sensitize cells to mitochondrial oxidative stress (MOS)1 and intrinsic apoptosis. Glutathione is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. Therefore, regulation of mitochondrial glutathione transport is a key factor in maintaining the antioxidant status of mitochondria. Bcl-2 is resident in the outer mitochondrial membrane where it acts as a central regulator of the intrinsic apoptotic cascade. In addition, Bcl-2 displays an antioxidant-like function that has been linked experimentally to the regulation of cellular glutathione content. We have previously demonstrated a novel interaction between recombinant Bcl-2 and reduced glutathione (GSH) which was antagonized by either Bcl-2 homology-3 domain (BH3) mimetics or a BH3-only protein, recombinant Bim. These previous findings prompted us to investigate if this novel Bcl-2/GSH interaction might play a role in regulating mitochondrial glutathione transport. Incubation of primary cultures of cerebellar granule neurons (CGNs) with the BH3 mimetic, HA14-1, induced MOS and caused specific depletion of the mitochondrial glutathione pool. Bcl-2 was co-immunoprecipitated with GSH following chemical cross-linking in CGNs and this Bcl-2/GSH interaction was antagonized by pre-incubation with HA14-1. Moreover, both HA14-1 and recombinant Bim inhibited GSH transport into isolated rat brain mitochondria. To further investigate a possible link between Bcl-2 function and mitochondrial glutathione transport, we next examined if Bcl-2 associated with the 2-oxoglutarate carrier (OGC), an inner mitochondrial membrane protein known to transport glutathione in liver and kidney. Following co-transfection of CHO cells, Bcl-2 was co-immunoprecipitated with OGC

  11. Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The extensive similarities between helminth proteins and allergens are thought to contribute to helminth-driven allergic sensitization. We investigated the molecular and structural similarities between Bla g 5, a major glutathione-S transferase (GST) allergen of cockroaches, and the GST of Wucherer...

  12. Oxidative stress protection and glutathione metabolism in response to hydrogen peroxide and menadione in riboflavinogenic fungus Ashbya gossypii.

    PubMed

    Kavitha, S; Chandra, T S

    2014-11-01

    Ashbya gossypii is a plant pathogen and a natural overproducer of riboflavin and is used for industrial riboflavin production. A few literature reports depict a link between riboflavin overproduction and stress in this fungus. However, the stress protection mechanisms and glutathione metabolism are not much explored in A. gossypii. In the present study, an increase in the activity of catalase and superoxide dismutase was observed in response to hydrogen peroxide and menadione. The lipid peroxide and membrane lipid peroxide levels were increased by H2O2 and menadione, indicating oxidative damage. The glutathione metabolism was altered with a significant increase in oxidized glutathione (GSSG), glutathione peroxidase (GPX), glutathione S transferase (GST), and glutathione reductase (GR) and a decrease in reduced glutathione (GSH) levels in the presence of H2O2 and menadione. Expression of the genes involved in stress mechanism was analyzed in response to the stressors by semiquantitative RT-PCR. The messenger RNA (mRNA) levels of CTT1, SOD1, GSH1, YAP1, and RIB3 were increased by H2O2 and menadione, indicating the effect of stress at the transcriptional level. A preliminary bioinformatics study for the presence of stress response elements (STRE)/Yap response elements (YRE) depicted that the glutathione metabolic genes, stress genes, and the RIB genes hosted either STRE/YRE, which may enable induction of these genes during stress. PMID:25178419

  13. Oxidative-stress detoxification and signalling in cyanobacteria: the crucial glutathione synthesis pathway supports the production of ergothioneine and ophthalmate.

    PubMed

    Narainsamy, Kinsley; Farci, Sandrine; Braun, Emilie; Junot, Christophe; Cassier-Chauvat, Corinne; Chauvat, Franck

    2016-04-01

    Using genetics and metabolomics we investigated the synthesis (gshA and gshB genes) and catabolism (ggt) of the conserved antioxidant glutathione in the model cyanobacterium Synechocystis PCC6803. These three genes are crucial to Synechocystis, in agreement with the proposed invention of glutathione by ancient cyanobacteria to protect themselves against the toxicity of oxygen they produced through photosynthesis. Consistent with their indispensability, gshA and gshB also operate in the production of another antioxidant, ergothioneine, as well as of the glutathione analogues ophthalmate and norophthalmate. Furthermore, we show that glutathione, ophthalmate and norophthalmate are accumulated in cells stressed by glucose, and that the two glutathione-dependent glyoxalase enzymes operate in the protection against glucose and its catabolite methylglyoxal. These findings are interesting because ophthalmate and norophthalmate were observed only in mammals so far, where ophthalmate is regarded as a biomarker of glutathione depletion. Instead, our data suggest that ophthalmate and norophthalmate are stress-induced markers of cysteine depletion triggered by its accelerated incorporation into glutathione, to face its increased demand for detoxification purposes. Hence, Synechocystis is an attractive model for the analysis of the role of glutathione, ergothioneine, ophthalmate and norophthalmate, in signalling and detoxification of oxidants and metabolic by-products. PMID:26713511

  14. Protective effect of sesamol against 3-nitropropionic acid-induced cognitive dysfunction and altered glutathione redox balance in rats.

    PubMed

    Kumar, Puneet; Kalonia, Harikesh; Kumar, Anil

    2010-07-01

    Sesamol (SML) (Sesamum indicum, Linn, Pedaliaceae) has been used traditionally as a health supplement in India and other countries for a long time. It is a well-known antioxidant, currently being tried against several neurological disorders. The present study was designed to evaluate the potential of sesamol treatment against 3-nitropropionic acid (3-NP)-induced cognitive impairment and oxidative damage in striatal, cortex and hippocampal regions of the rat. The memory performance was assessed by Morris water maze and elevated plus maze paradigms. The oxidative damage was assessed by estimating the total glutathione, reduced glutathione, oxidized glutathione levels and glutathione redox ratio. Glutathione-S-transferase and lactate dehydrogenase enzymes were also measured in different brain areas. 3-NP significantly impaired memory performance as assessed in Morris water maze and elevated plus maze, which was significantly attenuated by sesamol (5, 10 and 20 mg/kg) pre-treatment. On the other hand, 3-NP significantly induced oxidative stress and depleted total glutathione, reduced glutathione, glutathione-S-transferase, lactate dehydrogenase enzyme levels and redox ratio in the striatum, cortex and hippocampal regions as compared to the vehicle-treated group. Sesamol pre-treatment restored oxidative defence possibly by its free radical scavenging activity as compared to the 3NP-treated group. The present study suggests that sesamol could be used as an effective agent in the management of Huntington's disease. PMID:20102363

  15. An energy-saving glutathione production method from low-temperature cooked rice using amylase-expressing Saccharomyces cerevisiae.

    PubMed

    Hara, Kiyotaka Y; Kim, Songhee; Kiriyama, Kentaro; Yoshida, Hideyo; Arai, Shogo; Ishii, Jun; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2012-05-01

    Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is industrially produced by fermentation using Saccharomyces cerevisiae. Before the glutathione fermentation process with S. cerevisiae, a glucose extraction process from starchy materials is required. This glucose extraction is usually carried out by converting starchy materials to starch using high-temperature cooking and subsequent hydrolysis by amylases to convert starch to glucose. In this study, to develop an energy-saving glutathione production process by reducing energy consumption during the cooking step, we efficiently produced glutathione from low-temperature cooked rice using amylase-expressing S. cerevisiae. The combination of the amylase-expressing yeast with low-temperature cooking is potentially applicable to a variety of energy-saving bio-production methods of chemicals from starchy bio-resources. PMID:22294378

  16. [Ascorbate-glutathione cycle enzymes activity in Zea mays leaves under salinity and treatment by adaptogenic compounds].

    PubMed

    Konturs'ka, O O; Palladina, T O

    2012-01-01

    The effect of different salinity levels and synthetic compounds treatments on ascorbate-glutathione cycle enzymes activity in maize leaves has been investigated. One-day seedlings exposition with 0.05 M NaCl increased ascorbate peroxidase activity, whereas 10-day exposition did not affect it. However the exposition with 0.1 M NaCl, which is extreme for maize, decreased ascorbate peroxidase activity in leaves during 10 days. On the other hand glutathione reductase activity in leaves increased under both salt concentrations. Seeds treatments with Methyure and Ivine increased ascorbate peroxidase activity in the leaves of seedlings under 0.1 M NaCl, but did not affect glutathione reductase activity as compared to the salt control. The results obtained have shown differences of ascorbate-glutathione cycle enzymes responses to salt exposition of seedlings and the effects of adaptogenic compounds on the ascorbate-glutathione cycle via ascorbate peroxidase activation. PMID:23387279

  17. Low levels of glutathione are sufficient for survival of keratinocytes after UV irradiation and for healing of mouse skin wounds.

    PubMed

    Telorack, Michèle; Abplanalp, Jeannette; Werner, Sabine

    2016-08-01

    Reduced levels of the cellular antioxidant glutathione are associated with premature skin aging, cancer and impaired wound healing, but the in vivo functions of glutathione in the skin remain largely unknown. Therefore, we analyzed mice lacking the modifier subunit of the glutamate cysteine ligase (Gclm), the enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Glutathione levels in the skin of these mice were reduced by 70 %. However, neither skin development and homeostasis, nor UVA- or UVB-induced apoptosis in the epidermis were affected. Histomorphometric analysis of excisional wounds did not reveal wound healing abnormalities in young Gclm-deficient mice, while the area of hyperproliferative epithelium as well as keratinocyte proliferation were affected in aged mice. These findings suggest that low levels of glutathione are sufficient for wound repair in young mice, but become rate-limiting upon aging. PMID:27262586

  18. Cysteine and glutathione concentrations in plasma and bronchoalveolar lavage fluid after treatment with N-acetylcysteine.

    PubMed Central

    Bridgeman, M. M.; Marsden, M.; MacNee, W.; Flenley, D. C.; Ryle, A. P.

    1991-01-01

    N-acetylcysteine (600 mg/day) was given to patients by mouth for five days before bronchoscopy and bronchoalveolar lavage to determine whether N-acetylcysteine could increase the concentrations of the antioxidant reduced glutathione in plasma and bronchoalveolar lavage fluid. Bronchoalveolar lavage was performed 1-3 hours (group 2, n = 9) and 16-20 hours (group 3, n = 10) after the last dose of N-acetylcysteine and the values were compared with those in a control group receiving no N-acetylcysteine (group 1, n = 8). N-acetylcysteine was not detected in plasma or lavage fluid. Plasma concentrations of cysteine, the main metabolite of N-acetylcysteine and a precursor of reduced glutathione, were greater in the groups receiving treatment (groups 2 and 3) than in group 1. Cysteine concentrations in lavage fluid were similar in the three groups. Concentrations of reduced glutathione were greater in both plasma and lavage fluid in group 2 than in group 1. These data suggest that N-acetylcysteine given by mouth is rapidly deacetylated to cysteine, with resulting increases in the concentrations of cysteine in plasma and of reduced glutathione in plasma and the airways, which thus temporarily increase the antioxidant capacity of the lung. Images PMID:1871695

  19. Glutathione depletion and acute exercise increase O-GlcNAc protein modification in rat skeletal muscle.

    PubMed

    Peternelj, Tina Tinkara; Marsh, Susan A; Strobel, Natalie A; Matsumoto, Aya; Briskey, David; Dalbo, Vincent J; Tucker, Patrick S; Coombes, Jeff S

    2015-02-01

    Post-translational modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) profoundly affects protein structure, function, and metabolism. Although many skeletal muscle proteins are O-GlcNAcylated, the modification has not been extensively studied in this tissue, especially in the context of exercise. This study investigated the effects of glutathione depletion and acute exercise on O-GlcNAc protein modification in rat skeletal muscle. Diethyl maleate (DEM) was used to deplete intracellular glutathione and rats were subjected to a treadmill run. White gastrocnemius and soleus muscles were analyzed for glutathione status, O-GlcNAc and O-GlcNAc transferase (OGT) protein levels, and mRNA expression of OGT, O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase. DEM and exercise both reduced intracellular glutathione and increased O-GlcNAc. DEM upregulated OGT protein expression. The effects of the interventions were significant 4 h after exercise (P < 0.05). The changes in the mRNA levels of O-GlcNAc enzymes were different in the two muscles, potentially resulting from different rates of oxidative stress and metabolic demands between the muscle types. These findings indicate that oxidative environment promotes O-GlcNAcylation in skeletal muscle and suggest an interrelationship between cellular redox state and O-GlcNAc protein modification. This could represent one mechanism underlying cellular adaptation to oxidative stress and health benefits of exercise. PMID:25416863

  20. Biomimetic 'Green' Synthesis of Nanomaterials Using Antioxidants-Vitamins, Glutathione and Polyphenols from Tea and Wine

    EPA Science Inventory

    The presentation summarizes our recent activity in chemical synthesis of nanomaterials via benign biomimetic ‘greener’ alternatives,1 such as the use antioxidants present in a variety of natural products, and ubiquitous glutathione in aqueous media.2 Vitamins B1, B2, C, and tea ...

  1. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

  2. A clickable glutathione approach for identification of protein glutathionylation in response to glucose metabolism.

    PubMed

    Samarasinghe, Kusal T G; Munkanatta Godage, Dhanushka N P; Zhou, Yani; Ndombera, Fidelis T; Weerapana, Eranthie; Ahn, Young-Hoon

    2016-07-19

    Glucose metabolism and mitochondrial function are closely interconnected with cellular redox-homeostasis. Although glucose starvation, which mimics ischemic conditions or insufficient vascularization, is known to perturb redox-homeostasis, global and individual protein glutathionylation in response to glucose metabolism or mitochondrial activity remains largely unknown. In this report, we use our clickable glutathione approach, which forms clickable glutathione (azido-glutathione) by using a mutant of glutathione synthetase (GS M4), for detection and identification of protein glutathionylation in response to glucose starvation. We found that protein glutathionylation is readily induced in HEK293 cells in response to low glucose concentrations when mitochondrial reactive oxygen species (ROS) are elevated in cells, and glucose is the major determinant for inducing reversible glutathionylation. Proteomic and biochemical analysis identified over 1300 proteins, including SMYD2, PP2Cα, and catalase. We further showed that PP2Cα is glutathionylated at C314 in a C-terminal domain, and PP2Cα C314 glutathionylation disrupts the interaction with mGluR3, an important glutamate receptor associated with synaptic plasticity. PMID:27216279

  3. [Selective N-heterylazimine inhibition of reactions catalyzed by rat liver glutathione transferase].

    PubMed

    Stulovskiĭ, A V; Voznyĭ, I V; Rozengart, E V; Suvorov, A A; Khovanskikh, A E

    1992-01-01

    Three reactions (nucleophile substitution, thiolysis and N-deoxygenation) catalyzed by rat liver glutathione transferase have been studied using several N-heterylazimine inhibitors. The inhibitors are sharply different in their effectiveness in the transferase reactions. Their efficiency depends on their structure. The mechanism which underlies the found regularities is suggested. PMID:1413125

  4. Changes in cysteine, glutathione and phytochelatins in roots of maize seedlings exposed to cadmium

    SciTech Connect

    Tukendorf, A. ); Rauser, W.E. )

    1990-05-01

    The effects of Cd on the contents of free cysteine, total glutathione and phytochelatin (PC) were measured in roots of intact maize seedlings. Crude extracts in 100 mM HCl were separated by reverse-phase HPLC and thiols quantitated by continuous addition of Ellman's reagent. Exposure to 3 {mu}M Cd for 15 min caused PCs to appear. The kinetics indicated that shorter PCs were substrates for longer PCs. Total glutathione levels declined with PC synthesis, free cysteine contents changed little. In the 1 cm apical region a high production of PCs occurred with a moderate loss of total glutathione. In the nature region (1-10 cm), PC content was 2.5 fold less than in species and total glutathione levels declined drastically. Exposure to 0.05 {mu}M Cd for 24 h induced PCs, more PCs appeared with greater Cd supplies. The roots produced PCs in excess of that required to chelate the Cd present, as if some PCs were compartmentalized or had not yet formed Cd-PC complexes.

  5. Noninvasive quantification of ascorbate and glutathione concentration in the elderly human brain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, Ascorbate (Asc) and glutathione (GSH) concentrations were non-invasively quantified using double edited 1H MRS at 4T in the occipital cortex of healthy young(age 18 – 22 years, N = 22) and elderly (age 70 – 89 years, N = 22) human subjects. Elderly subjects had a lower GSH concentrati...

  6. The role of glutathione detoxification pathway in MCLR-induced hepatotoxicity in SD rats.

    PubMed

    Li, Shangchun; Chen, Jun; Xie, Ping; Guo, Xiaochun; Fan, Huihui; Yu, Dezhao; Zeng, Cheng; Chen, Liang

    2015-12-01

    In the present study, we investigated the role of glutathione (GSH) and its related enzymes in Sprague Dawley (SD) rats subjected to microcystin-leucine-arginine (MCLR)-induced hepatotoxicity. SD rats were intraperitoneally (i.p.) injected with MCLR after pretreating with or without buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis. The depletion of GSH with BSO enhanced MCLR-induced oxidative stress, resulting in more severe liver damage and higher MCLR accumulation. Similarly, the contents of malondialdehyde (MDA), total GSH (T-GSH), oxidized GSH (GSSG) and GSH were significantly enhanced in BSO pretreated rats following MCLR treatment. The study showed that the transcription of GSH-related enzymes such as glutathione-S-transferase (GST), γ-glutamylcysteine synthetase (γ-GCS), glutathione reductase (GR) varied in different ways (expect for glutathione peroxidase (GPx), whose gene expression was induced in all treated groups) with or without BSO pretreatment before MCLR exposure, suggesting an adaptative response of GSH-related enzymes at transcription level to combat enhancement of oxidative stress induced by MCLR when pretreated with BSO. These data suggested the tissues with low GSH concentration are highly vulnerable to MCLR toxicity and GSH was critical for the detoxification in MCLR-induced hepatotoxicity in vivo. PMID:24964298

  7. Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

    SciTech Connect

    Noguchi, K.; Hattori, T.; Igarashi, T.; Ueno, K.; Satoh, T.; Kitagawa, H.; Hori, H.; Shibata, T.; Inayama, S.

    1985-08-19

    A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitromidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H/sub 2/O/sub 2/ as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H/sub 2/O/sub 2/ as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H/sub 2/O/sub 2/ as substrate. 26 references, 2 figures, 4 tables.

  8. The concentration of ascorbic acid and glutathione in 13 provenances of Acacia melanoxylon.

    PubMed

    Wujeska-Klause, Agnieszka; Bossinger, Gerd; Tausz, Michael

    2016-04-01

    Climate change can negatively affect sensitive tree species, affecting their acclimation and adaptation strategies. A common garden experiment provides an opportunity to test whether responses of trees from different provenances are genetically driven and if this response is related to factors at the site of origin. We hypothesized that antioxidative defence systems and leaf mass area ofAcacia melanoxylonR. Br. samples collected from different provenances will vary depending on local rainfall. Thirteen provenances ofA. melanoxylonoriginating from different rainfall habitats (500-2000 mm) were grown for 5 years in a common garden. For 2 years, phyllode samples were collected during winter and summer, for measurements of leaf mass area and concentrations of glutathione and ascorbic acid. Leaf mass area varied between seasons, years and provenances ofA. melanoxylon, and an increase was associated with decreasing rainfall at the site of origin. Ascorbic acid and glutathione concentrations varied between seasons, years (i.e., environmental factors) and among provenances ofA. melanoxylon In general, glutathione and ascorbic acid concentrations were higher in winter compared with summer. Ascorbic acid and glutathione were different among provenances, but this was not associated with rainfall at the site of origin. PMID:26960387

  9. Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system

    PubMed Central

    Baxter, Paul S.; Bell, Karen F.S.; Hasel, Philip; Kaindl, Angela M.; Fricker, Michael; Thomson, Derek; Cregan, Sean P.; Gillingwater, Thomas H.; Hardingham, Giles E.

    2015-01-01

    How the brain's antioxidant defenses adapt to changing demand is incompletely understood. Here we show that synaptic activity is coupled, via the NMDA receptor (NMDAR), to control of the glutathione antioxidant system. This tunes antioxidant capacity to reflect the elevated needs of an active neuron, guards against future increased demand and maintains redox balance in the brain. This control is mediated via a programme of gene expression changes that boosts the synthesis, recycling and utilization of glutathione, facilitating ROS detoxification and preventing Puma-dependent neuronal apoptosis. Of particular importance to the developing brain is the direct NMDAR-dependent transcriptional control of glutathione biosynthesis, disruption of which can lead to degeneration. Notably, these activity-dependent cell-autonomous mechanisms were found to cooperate with non-cell-autonomous Nrf2-driven support from astrocytes to maintain neuronal GSH levels in the face of oxidative insults. Thus, developmental NMDAR hypofunction and glutathione system deficits, separately implicated in several neurodevelopmental disorders, are mechanistically linked. PMID:25854456

  10. Ascorbic acid prevents acetaminophen-induced hepatotoxicity in mice by ameliorating glutathione recovery and autophagy.

    PubMed

    Kurahashi, Toshihiro; Lee, Jaeyong; Nabeshima, Atsunori; Homma, Takujiro; Kang, Eun Sil; Saito, Yuka; Yamada, Sohsuke; Nakayama, Toshiyuki; Yamada, Ken-Ichi; Miyata, Satoshi; Fujii, Junichi

    2016-08-15

    Aldehyde reductase (AKR1A) plays a role in the biosynthesis of ascorbic acid (AsA), and AKR1A-deficient mice produce about 10-15% of the AsA that is produced by wild-type mice. We found that acetaminophen (AAP) hepatotoxicity was aggravated in AKR1A-deficient mice. The pre-administration of AsA in the drinking water markedly ameliorated the AAP hepatotoxicity in the AKR1A-deficient mice. Treatment of the mice with AAP decreased both glutathione and AsA levels in the liver in the early phase after AAP administration, and an AsA deficiency delayed the recovery of the glutathione content in the healing phase. While in cysteine supply systems; a neutral amino acid transporter ASCT1, a cystine transporter xCT, enzymes for the transsulfuration pathway, and autophagy markers, were all elevated in the liver as the result of the AAP treatment, the AsA deficiency suppressed their induction. Thus, AsA appeared to exert a protective effect against AAP hepatotoxicity by ameliorating the supply of cysteine that is available for glutathione synthesis as a whole. Because some drugs produce reactive oxygen species, resulting in the consumption of glutathione during the metabolic process, the intake of sufficient amounts of AsA would be beneficial for protecting against the hepatic damage caused by such drugs. PMID:27288086

  11. Overexpression of Glutathione Synthetase in Indian Mustard Enhances Cadmium Accumulation and Tolerance1

    PubMed Central

    Liang Zhu, Yong; Pilon-Smits, Elizabeth A.H.; Jouanin, Lise; Terry, Norman

    1999-01-01

    An important pathway by which plants detoxify heavy metals is through sequestration with heavy-metal-binding peptides called phytochelatins or their precursor, glutathione. To identify limiting factors for heavy-metal accumulation and tolerance, and to develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, the Escherichia coli gshII gene encoding glutathione synthetase (GS) was overexpressed in the cytosol of Indian mustard (Brassica juncea). The transgenic GS plants accumulated significantly more Cd than the wild type: shoot Cd concentrations were up to 25% higher and total Cd accumulation per shoot was up to 3-fold higher. Moreover, the GS plants showed enhanced tolerance to Cd at both the seedling and mature-plant stages. Cd accumulation and tolerance were correlated with the gshII expression level. Cd-treated GS plants had higher concentrations of glutathione, phytochelatin, thiol, S, and Ca than wild-type plants. We conclude that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and phytochelatins, and that overexpression of GS offers a promising strategy for the production of plants with superior heavy-metal phytoremediation capacity. PMID:9880348

  12. Preliminary X-ray crystallographic analysis of glutathione transferase zeta 1 (GSTZ1a-1a)

    SciTech Connect

    Boone, Christopher D.; Zhong, Guo; Smeltz, Marci; James, Margaret O. McKenna, Robert

    2014-01-21

    Crystals of glutathione transferase zeta 1 were grown and shown to diffract X-rays to 3.1 Å resolution. They belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, β = 69.9, γ = 73.4°.

  13. DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1

    EPA Science Inventory


    DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1. R A Pegram1 and M K Ross2. 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC; 1Pharmacokinetics Branch, NHEERL, ORD, United States Environmental Protection Ag...

  14. Neuroprotective effects of a glutathione depletor in rat post-ischemic reperfusion brain damage.

    PubMed

    Giacomo, Claudia Di; Santangelo, Rosa; Sorrenti, Valeria; Volti, Giovanni L; Acquaviva, Rosaria

    2015-01-01

    The induction of heme oxygenase (HO), the rate-limiting enzyme in heme degradation, occurs as an adaptative response to oxidative stress and is consequent to decrease in cellular glutathione levels. Our previous studies demonstrated significant increase in survival rates of rats treated with glutathione depletors and submitted to transient cerebral ischemia. The aim of the present research was to test the effects of L-Buthionine sulfoximine (BSO), a glutathione depletor, during cerebral post-ischemic reperfusion. Cerebral ischemia was induced by bilateral clamping of common carotid arteries for 20 min. Each sample was used for glutathione ad lipid peroxidation level dosage and for evaluating the expression of heme oxygenase both after a single subcutaneous administration of BSO and without treatment. In the same experimental conditions, endothelial, inducible and neuronal Nitric Oxide Synthase (eNOS, iNOS and nNOS) and Dimethylarginine Dimethyl amine Hydrolases (DDAH-1 and DDAH-2) were also evaluated. Results obtained in the present study suggested that HO-1 over-expression may be implicated in the protective effect of BSO in post-ischemic reperfusion brain damage, although the involvement of other important stress mediators cannot be ruled out. PMID:25613502

  15. GLUTATHIONE S-TRANSFERASE THETA 1-1-DEPENDENT METABOLISM OF THE DISINFECTION BYPRODUCT BROMODICHLOROMETHANE

    EPA Science Inventory

    ABSTRACT
    Bromodichloromethane (BDCM), a prevalent drinking water disinfection by-product, was previously shown to be mutagenic in Salmonella expressing glutathione S-transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the...

  16. Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells.

    PubMed

    Mauzeroll, Janine; Bard, Allen J

    2004-05-25

    The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV-visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-microm-diameter electrode situated 10 microm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster. PMID:15148374

  17. Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells

    PubMed Central

    Mauzeroll, Janine; Bard, Allen J.

    2004-01-01

    The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV–visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-μm-diameter electrode situated 10 μm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster. PMID:15148374

  18. Effects of commercial selenium products on glutathione peroxidase activity and semen quality in stud boars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to determine how dietary supplementation of inorganic and organic selenium affects selenium concentration and glutathione peroxidase activity in blood and sperm of sexually mature stud boars. Twenty-four boars of the Large White, Landrace, Pietrain, and Duroc breeds of opt...

  19. Correlation between Glutathione Peroxidase Activity and Anthropometrical Parameters in Adolescents with Down Syndrome

    ERIC Educational Resources Information Center

    Ordonez, F. J.; Rosety-Rodriguez, M.

    2007-01-01

    Since we have recently found that regular exercise increased erythrocyte antioxidant enzyme activities such as glutathione peroxidase (GPX) in adolescents with Down syndrome, these programs may be recommended. This study was designed to assess the role of anthropometrical parameters as easy, economic and non-invasive biomarkers of GPX. Thirty-one…

  20. Identification and characterization of a selenium-dependent glutathione peroxidase in Setaria cervi

    SciTech Connect

    Singh, Anchal; Rathaur, Sushma . E-mail: sushmarathaur@yahoo.com

    2005-06-17

    Setaria cervi a bovine filarial parasite secretes selenium glutathione peroxidase during in vitro cultivation. A significant amount of enzyme activity was detected in the somatic extract of different developmental stages of the parasite. Among different stages, microfilariae showed a higher level of selenium glutathione peroxidase activity followed by males then females. However, when the activity was compared in excretory secretory products of these stages males showed higher activity than microfilariae and female worms. The enzyme was purified from female somatic extract using a combination of glutathione agarose and gel filtration chromatography, which migrated as a single band of molecular mass {approx}20 kDa. Selenium content of purified enzyme was estimated by atomic absorption spectroscopy and found to be 3.5 ng selenium/{mu}g of protein. Further, inhibition of enzyme activity by potassium cyanide suggested the presence of selenium at the active site of enzyme. This is the first report of identification of selenium glutathione peroxidase from any filarial parasite.

  1. A tyrosine-reactive irreversible inhibitor for glutathione S-transferase Pi (GSTP1).

    PubMed

    Crawford, L A; Weerapana, E

    2016-05-24

    Glutathione S-transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, we report LAS17, a dichlorotriazine-containing compound that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition. PMID:27113843

  2. Interaction between vitamin E and glutathione in rat brain: Effect of chronic ethanol administration.

    PubMed

    Marcus, S R; Chandrakala, M V; Nadiger, H A

    1998-12-01

    The protection against ethanol-induced lipid peroxidation is rendered by antioxidants such as vitamin E and glutathione (GSH) interacting with each other and also functioning independently. A study of the levels of GSH and activities of glutathione peroxidase (GP), glutathione reductase (GR) and glutathione transferase (GST) in the cerebral cortex (CC), cerebellum (CB) and brain stem (BS) of vitamin E-supplemented and -deficient rats subjected to ethanol administration for 30 days was carried out. Chronic ethanol administration to vitamin E-supplemented rats elevated GP, GR and GST activities in the three regions and GSH levels in the CB. Chronic ethanol administration to vitamin E-deficient rats elevated GR activity in the three regions and GP activity in the CC and CB, decreased GST activity in the CC and CB, but did not alter GSH levels compared with normal rats subjected to chronic ethanol administration. The results indicate that vitamin E helps to maintain GSH levels to combat increased peroxidation while its absence has a deleterious effect. PMID:24393672

  3. Characterization of affinity-purified isoforms of Acinetobacter calcoaceticus Y1 glutathione transferases.

    PubMed

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

  4. Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

    PubMed Central

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

  5. Covert hepatic encephalopathy: elevated total glutathione and absence of brain water content changes.

    PubMed

    Oeltzschner, Georg; Butz, Markus; Wickrath, Frithjof; Wittsack, Hans-Jörg; Schnitzler, Alfons

    2016-06-01

    Recent pathophysiological models suggest that oxidative stress and hyperammonemia lead to a mild brain oedema in hepatic encephalopathy (HE). Glutathione (GSx) is a major cellular antioxidant and known to be involved in the interception of both. The aim of this work was to study total glutathione levels in covert HE (minimal HE and HE grade 1) and to investigate their relationship with local brain water content, levels of glutamine (Gln), myo-inositol (mI), neurotransmitter levels, critical flicker frequency (CFF), and blood ammonia. Proton magnetic resonance spectroscopy ((1)H MRS) data were analysed from visual and sensorimotor cortices of thirty patients with covert HE and 16 age-matched healthy controls. Total glutathione levels (GSx/Cr) were quantified with respect to creatine. Furthermore, quantitative MRI brain water content measures were evaluated. Data were tested for links with the CFF and blood ammonia. GSx/Cr was elevated in the visual (mHE) and sensorimotor (mHE, HE 1) MRS volumes and correlated with blood ammonia levels (both P < 0.001). It was further linked to Gln/Cr and mI/Cr (P < 0.01 in visual, P < 0.001 in sensorimotor) and to GABA/Cr (P < 0.01 in visual). Visual GSx/Cr correlated with brain water content in the thalamus, nucleus caudatus, and visual cortex (P < 0.01). Brain water measures did neither show group effects nor correlations with CFF or blood ammonia. Elevated total glutathione levels in covert HE (< HE 2) correlate with blood ammonia and may be a regional-specific reaction to hyperammonemia and oxidative stress. Brain water content is locally linked to visual glutathione levels, but appears not to be associated with changes of clinical parameters. This might suggest that cerebral oedema is only marginally responsible for the symptoms of covert HE. PMID:26563124

  6. Biomolecularly capped uniformly sized nanocrystalline materials: glutathione-capped ZnS nanocrystals

    NASA Astrophysics Data System (ADS)

    Torres-Martínez, Claudia L.; Nguyen, Liem; Kho, Richard; Bae, Weon; Bozhilov, Krassimir; Klimov, Victor; Mehra, Rajesh K.

    1999-09-01

    Micro-organisms such as bacteria and yeasts form CdS to detoxify toxic cadmium ions. Frequently, CdS particles formed in yeasts and bacteria were found to be associated with specific biomolecules. It was later determined that these biomolecules were present at the surface of CdS. This coating caused a restriction in the growth of CdS particles and resulted in the formation of nanometre-sized semiconductors (NCs) that exhibited typical quantum confinement properties. Glutathione and related phytochelatin peptides were shown to be the biomolecules that capped CdS nanocrystallites synthesized by yeasts Candida glabrata and Schizosaccharomyces pombe. Although early studies showed the existence of specific biochemical pathways for the synthesis of biomolecularly capped CdS NCs, these NCs could be formed in vitro under appropriate conditions. We have recently shown that cysteine and cysteine-containing peptides such as glutathione and phytochelatins can be used in vitro to dictate the formation of discrete sizes of CdS and ZnS nanocrystals. We have evolved protocols for the synthesis of ZnS or CdS nanocrystals within a narrow size distribution range. These procedures involve three steps: (1) formation of metallo-complexes of cysteine or cysteine-containing peptides, (2) introduction of stoichiometric amounts of inorganic sulfide into the metallo-complexes to initiate the formation of nanocrystallites and finally (3) size-selective precipitation of NCs with ethanol in the presence of Na+. The resulting NCs were characterized by optical spectroscopy, high-resolution transmission electron microscopy (HRTEM), x-ray diffraction and electron diffraction. HRTEM showed that the diameter of the ZnS-glutathione nanocrystals was 3.45+/-0.5 nm. X-ray diffraction and electron diffraction analyses indicated ZnS-glutathione to be hexagonal. Photocatalytic studies suggest that glutathione-capped ZnS nanocrystals prepared by our procedure are highly efficient in degrading a test model

  7. Redox-sensitive YFP sensors for monitoring dynamic compartment-specific glutathione redox state.

    PubMed

    Banach-Latapy, Agata; He, Tiantian; Dardalhon, Michèle; Vernis, Laurence; Chanet, Roland; Huang, Meng-Er

    2013-12-01

    Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. Genetically encoded biosensors including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP) may provide an alternative way to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. This study describes the use of rxYFP sensors for investigating compartment-specific steady redox state and their dynamics in response to stress in human cells. RxYFP expressed in the cytosol, nucleus, or mitochondrial matrix of HeLa cells was responsive to the intracellular redox state changes induced by reducing as well as oxidizing agents. Compartment-targeted rxYFP sensors were able to detect different steady-state redox conditions among the cytosol, nucleus, and mitochondrial matrix. These sensors expressed in human epidermal keratinocytes HEK001 responded to stress induced by ultraviolet A radiation in a dose-dependent manner. Furthermore, rxYFP sensors were able to sense dynamic and compartment-specific redox changes caused by 100 μM hydrogen peroxide (H2O2). Mitochondrial matrix-targeted rxYFP displayed a greater dynamics of oxidation in response to a H2O2 challenge than the cytosol- and nucleus-targeted sensors, largely due to a more alkaline local pH environment. These observations support the view that mitochondrial glutathione redox state is maintained and regulated independently from that of the cytosol and nucleus. Taken together, our data show the robustness of the rxYFP sensors to measure compartmental redox changes in human cells. Complementary to existing redox sensors and conventional redox measurements, compartment-targeted rxYFP sensors provide a novel tool for examining mammalian cell redox homeostasis, permitting high-resolution readout of steady glutathione state and dynamics of redox changes. PMID:23891676

  8. Withanolide A prevents neurodegeneration by modulating hippocampal glutathione biosynthesis during hypoxia.

    PubMed

    Baitharu, Iswar; Jain, Vishal; Deep, Satya Narayan; Shroff, Sabita; Sahu, Jayanta Kumar; Naik, Pradeep Kumar; Ilavazhagan, Govindasamy

    2014-01-01

    Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner. PMID

  9. Effects of mercury and selenium on glutathione metabolism and oxidative stress in mallard ducks

    USGS Publications Warehouse

    Hoffman, D.J.; Heinz, G.H.

    1998-01-01

    Earlier studies reported on the toxicity and related oxidative stress of different forms of Se, including seleno-D,L-methionine, in mallards (Anas platyrhynchos). This study compares the effects of Se (seleno-D,L-methionine) and Hg (methylmercury chloride) separately and in combination. Mallard drakes received one of the following diets: untreated feed (controls), or feed containing 10 ppm Se, 10 ppm Hg, or 10 ppm Se in combination with 10 ppm Hg. After 10 weeks, blood, liver, and brain samples were collected for biochemical assays. The following clinical and biochemical alterations occurred in response to mercury exposure: hematocrit and hemoglobin concentrations decreased; activities of the enzymes glutathione (GSH) peroxidase (plasma and liver), glutathione-S-transferase (liver), and glucose-6-phosphate dehydrogenase (G-6-PDH) (liver and brain) decreased; hepatic oxidized glutathione (GSSG) concentration increased relative to reduced glutathione (GSH); and lipid peroxidation in the brain was evident as detected by increased thiobarbituric reactive substances (TBARS). Effects of Se alone included increased hepatic GSSG reductase activity and brain TBARS concentration. Se in combination with Hg partially or totally alleviated effects of Hg on GSH peroxidase, G-6-PDH, and GSSG. These findings are compared in relation to field observations for diving ducks and other aquatic birds. It is concluded that since both Hg and excess Se can affect thiol status, measurement of associated enzymes in conjunction with thiol status may be a useful bioindicator to discriminate between Hg and Se effects. The ability of Se to restore the activities of G-6-PDH, GSH peroxidase, and glutathione status involved in antioxidative defense mechanisms may be crucial to biological protection from the toxic effects of methyl mercury.

  10. Withanolide A Prevents Neurodegeneration by Modulating Hippocampal Glutathione Biosynthesis during Hypoxia

    PubMed Central

    Baitharu, Iswar; Jain, Vishal; Deep, Satya Narayan; Shroff, Sabita; Sahu, Jayanta Kumar; Naik, Pradeep Kumar; Ilavazhagan, Govindasamy

    2014-01-01

    Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)–related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner

  11. A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin.

    PubMed

    McLaughlin, John E; Bin-Umer, Mohamed Anwar; Widiez, Thomas; Finn, Daniel; McCormick, Susan; Tumer, Nilgun E

    2015-01-01

    Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione

  12. Glutathione: an intracellular and extracellular protective agent in Salmonella typhimurium and Escherichia coli

    SciTech Connect

    Owens, R.A.

    1986-01-01

    Levels of glutathione, were measured in several aerobically grown strains of Salmonella typhimurium and Escherichia coli. External accumulation of GSH was inhibited by 30 mM NaN/sub 3/. Thus, GSH export may be energy dependent. Greater than 50% of the glutathione detected in the media was in the reduced form. Since the oxidized glutathione in the media could be accounted for by oxidation during aerobic incubation as well as in sample processing, the glutathione was predominantly exported in the reduced form. Extracellular glutathione was detected in log phase cultures of 2 out of 2 E. coli strains and 6 of 8 Salmonella strains tested. Two-dimensional paper chromatography of supernatants from cultures labelled with Na/sub 2//sup 35/SO/sub 4/ confirmed the presence of GSH and revealed five other sulfur-containing compounds in the media of Salmonella and E. coli cultures. Since media from cultures of an E. coli GSH/sup -/ strain contained compounds with identical R/sub f/'s, the five unidentified compounds were not derivatives of GSH. The addition of 26 ..mu..M GSH to cultures of TA1534 partially protected the bacteria from the toxic effects of 54 ..mu..M N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When MNNG was preincubated with equimolar GSH, the mutagenicity of the MNNG was neutralized. The addition of micromolar GSH to cultures and E. coli GSH/sup -/ strain protected the cells from growth inhibition by micromolar concentrations of mercuric chloride, silver nitrate, cisplatin, cadmium chloride, and iodoacetamide. The data presented demonstrate that micromolar concentrations of external GSH can significantly shorten the recovery time of cells after exposure to toxic agents in the environment.

  13. Sulforaphane Restores Cellular Glutathione Levels and Reduces Chronic Periodontitis Neutrophil Hyperactivity In Vitro

    PubMed Central

    Dias, Irundika H. K.; Chapple, Ian L. C.; Milward, Mike; Grant, Melissa M.; Hill, Eric; Brown, James; Griffiths, Helen R.

    2013-01-01

    The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2. - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients’ neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2. - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. PMID:23826097

  14. A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin

    PubMed Central

    McLaughlin, John E.; Bin-Umer, Mohamed Anwar; Widiez, Thomas; Finn, Daniel; McCormick, Susan; Tumer, Nilgun E.

    2015-01-01

    Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione

  15. Glutathione and its related enzymes in the gonad of Nile Tilapia (Oreochromis niloticus).

    PubMed

    Hamed, R R; Saleh, N S M; Shokeer, A; Guneidy, R A; Abdel-Ghany, S S

    2016-02-01

    Glutathione (GSH) concentration, the activity of its metabolizing enzymes, glutathione transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and the antioxidant enzyme catalase (CAT) in O. niloticus ovary and testis were examined. GSH concentration of O. niloticus testis exhibited high concentration (129 ± 21 nmol/g tissue) compared with GSH concentration (49.2 ± 8.3 nmol/g tissue) in the ovary. GST, GPx, GR, and CAT activities of O. niloticus testis exhibited high values compared with their corresponding values in ovary homogenates. However, protein concentration in ovary homogenates exhibited higher values (175 ± 40.6 mg) compared with testis homogenates (27.1 ± 3.7 mg). O. niloticus ovary was less effective in excretion of xenobiotices compared with the testis, where its function is mainly in increasing the protein content of the eggs; however, in O. niloticus testis, the glutathione cycle operated in accelerated way in the direction of reduced GSH production in order to protect the maturation stages in a save way. A simple reproducible procedure for the purification of GST from O. niloticus ovary was established. The enzymes proved to be homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its molecular weight was calculated to be 25.1 kDa. GST of O. niloticus ovary exhibited maximum activity at pH 7.5. The Michaelis-Menten constant (K(m)) of the purified ovary GST for GSH and CDNB was 0.076 mM and 1.0 mM, respectively. Cibacron blue was the most potent inhibitor of ovary GST activity (IC50 value, concentration of inhibitor that will give 50% inhibition, equal 0.002 μM). The specific activity of GST toward different electrophilic substrates was determined. GST activity toward benzyl isothiocyanate was the highest compared with phenethyl isothiocyanate and allyl isothiocyanate. PMID:26476660

  16. Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization.

    PubMed Central

    Prabhu, K S; Reddy, P V; Gumpricht, E; Hildenbrandt, G R; Scholz, R W; Sordillo, L M; Reddy, C C

    2001-01-01

    A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage. PMID:11716762

  17. Short-term effects of T-2 toxin exposure on some lipid peroxide and glutathione redox parameters of broiler chickens.

    PubMed

    Bócsai, A; Pelyhe, Cs; Zándoki, E; Ancsin, Zs; Szabó-Fodor, J; Erdélyi, M; Mézes, M; Balogh, K

    2016-06-01

    The purpose of this study was to investigate the short-term effects of T-2 toxin exposure (3.09 mg/kg feed) on lipid peroxidation and glutathione redox system of broiler chicken. A total of 54 Cobb 500 cockerels were randomly distributed to two experimental groups at 21 days of age. Samples (blood plasma, red blood cell, liver, kidney and spleen) were collected every 12 h during a 48-h period. The results showed that the initial phase of lipid peroxidation, as measured by conjugated dienes and trienes in the liver, was continuously, but not significantly higher in T-2 toxin-dosed birds than in control birds. The termination phase of lipid peroxidation, as measured by malondialdehyde, was significantly higher in liver and kidney as a result of T-2 toxin exposure at the end of the experimental period (48th hour). The glutathione redox system activated shortly after starting the T-2 toxin exposure, which is supported by the significantly higher concentration of reduced glutathione and glutathione peroxidase activity in blood plasma at 24 and 48 h, in liver at 12, 24 and 36 h, and in kidney and spleen at 24 h. These results suggest that T-2 toxin, or its metabolites, may be involved in the generation of reactive oxygen substances which causes an increase in lipid peroxidation, and consequently activates the glutathione redox system, namely synthesis of reduced glutathione and glutathione peroxidase. PMID:26412027

  18. Dorsal Anterior Cingulate Lactate and Glutathione Levels in Euthymic Bipolar I Disorder: 1H-MRS Study

    PubMed Central

    Pastorello, Bruno F.; Leite, Cláudia da Costa; Henning, Anke; Moreno, Ricardo A.; Garcia Otaduy, Maria Concepción

    2016-01-01

    Objective: Oxidative stress and mitochondrial dysfunction are 2 closely integrated processes implicated in the physiopathology of bipolar disorder. Advanced proton magnetic resonance spectroscopy techniques enable the measurement of levels of lactate, the main marker of mitochondrial dysfunction, and glutathione, the predominant brain antioxidant. The objective of this study was to measure brain lactate and glutathione levels in bipolar disorder and healthy controls. Methods: Eighty-eight individuals (50 bipolar disorder and 38 healthy controls) underwent 3T proton magnetic resonance spectroscopy in the dorsal anterior cingulate cortex (2x2x4.5cm3) using a 2-D JPRESS sequence. Lactate and glutathione were quantified using the ProFit software program. Results: Bipolar disorder patients had higher dorsal anterior cingulate cortex lactate levels compared with controls. Glutathione levels did not differ between euthymic bipolar disorder and controls. There was a positive correlation between lactate and glutathione levels specific to bipolar disorder. No influence of medications on metabolites was observed. Conclusion: This is the most extensive magnetic resonance spectroscopy study of lactate and glutathione in bipolar disorder to date, and results indicated that euthymic bipolar disorder patients had higher levels of lactate, which might be an indication of altered mitochondrial function. Moreover, lactate levels correlated with glutathione levels, indicating a compensatory mechanism regardless of bipolar disorder diagnosis. PMID:27207914

  19. Effect of selenium- and glutathione-enriched yeast supplementation on a combined atherosclerosis and diabetes hamster model.

    PubMed

    Agbor, Gabriel A; Vinson, Joe A; Patel, Shachi; Patel, Kunal; Scarpati, Jenyne; Shiner, Drew; Wardrop, Forbes; Tompkins, Thomas A

    2007-10-17

    Selenium has a central role in antioxidant pathways as a cofactor to glutathione peroxidase. The present study evaluated the effects of four different preparations of inactivated yeast containing various concentrations of selenium and glutathione on a combined atherosclerosis and diabetes hamster model. The hamsters were supplemented with the yeast products for three months. The enriched yeast with the highest selenium and glutathione levels reduced the weight loss induced by diabetes, inhibited an increase in plasma cholesterol and triglyceride caused by a high-cholesterol and high-fat diet, increased the time taken for oxidation of lower density lipoproteins (lag time), and inhibited the formation of atherosclerosis better than low selenium/glutathione yeast supplementation. It was concluded that the yeast prepared to provide high selenium and high glutathione was the best for effecting beneficial changes in glutathione, cholesterol, atherosclerosis, and for demonstrating an antioxidant effect. The high selenium and low glutathione yeast was the best for improving selenium and glucose levels. PMID:17880156

  20. Farnesol-induced apoptosis in Candida albicans is mediated by Cdr1-p extrusion and depletion of intracellular glutathione.

    PubMed

    Zhu, Jingsong; Krom, Bastiaan P; Sanglard, Dominique; Intapa, Chaidan; Dawson, Clinton C; Peters, Brian M; Shirtliff, Mark E; Jabra-Rizk, Mary Ann

    2011-01-01

    Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent. PMID:22205973

  1. Radiographic changes and lung function in relation to activity of the glutathione transferases theta and mu among asbestos cement workers.

    PubMed

    Jakobsson, K; Rannug, A; Alexandrie, A K; Warholm, M; Rylander, L; Hagmar, L

    1995-05-01

    Experimental data indicate that active oxygen species may be casually involved in the development of asbestos-related disease. Thus, it was hypothesized that individual differences in glutathione transferase activity, which may affect the ability to inactivate molecules formed in relation to oxidative stress, could influence the biological response to asbestos exposure. We could, however, not demonstrate an increased risk for radiographic changes or reduced lung function among asbestos cement workers deficient for glutathione transferase theta (GSTT1), glutathione transferase mu (GSTM1), or having a combined deficiency of enzyme activity. PMID:7618163

  2. Effect of tocotrienol on the activities of cytosolic glutathione-dependent enzymes in rats treated with 2-acetylaminofluorene.

    PubMed

    Shamaan, N A; Wan Ngah, W Z; Ibrahim, R; Jarien, Z; Top, A G; Abdul Kadir, K

    1993-04-01

    The effect of tocotrienol on the activities of glutathione S-transferases (GSTs), glutathione reductase (GR) and glutathione peroxidase (GPx) in rats given 2-acetylaminofluorene (AAF) was investigated over a 20 week period. Liver and kidney GST and liver GR activities were significantly increased after AAF administration. Kidney GPx activities were significantly affected; activity assayed with cumene hydroperoxide (cu-OOH) was increased but activity assayed with H2O2 was reduced. Supplementation of the diet with tocotrienol in the AAF-treated rats reduced the increase in enzyme activities. Tocotrienol on its own had no effect on the enzyme activities. PMID:8471073

  3. Ratios of biliary glutathione disulfide (GSSG) to glutathione (GSH): a potential index to screen drug-induced hepatic oxidative stress in rats and mice.

    PubMed

    Cao, Lei; Waldon, Daniel; Teffera, Yohannes; Roberts, John; Wells, Mary; Langley, Meghan; Zhao, Zhiyang

    2013-03-01

    Hepatotoxicity of drug candidates is one of the major concerns in drug screening in early drug discovery. Detection of hepatic oxidative stress can be an early indicator of hepatotoxicity and benefits drug selection. The glutathione (GSH) and glutathione disulfide (GSSG) pair, as one of the major intracellular redox regulating couples, plays an important role in protecting cells from oxidative stress that is caused by imbalance between prooxidants and antioxidants. The quantitative determination of the GSSG/GSH ratios and the concentrations of GSH and GSSG have been used to indicate oxidative stress in cells and tissues. In this study, we tested the possibility of using the biliary GSSG/GSH ratios as a biomarker to reflect hepatic oxidative stress and drug toxicity. Four compounds that are known to alter GSH and GSSG levels were tested in this study. Diquat (diquat dibromide monohydrate) and acetaminophen were administered to rats. Paraquat and tert-butyl hydroperoxide were administered to mice to induce changes of biliary GSH and GSSG. The biliary GSH and GSSG were quantified using calibration curves prepared with artificial bile to account for any bile matrix effect in the LC-MS analysis and to avoid the interference of endogenous GSH and GSSG. With four examples (in rats and mice) of drug-induced changes in the kinetics of the biliary GSSG/GSH ratios, this study showed the potential for developing an exposure response index based on biliary GSSG/GSH ratios for predicting hepatic oxidative stress. PMID:23377112

  4. Evaluation of aflatoxin B/sub 1/ mutagenesis: addition of glutathione and glutathione-S-transferase to the Salmonella mutagenicity assay

    SciTech Connect

    Jorgensen, K.V.; Clayton, J.W.; Price, R.L.

    1987-01-01

    The effects of glutathione (GSH) and the combination of GSH and glutathione-S-transferase (GST) on aflatoxin B/sub 1/ (AFB/sub 1/) mutagenesis in the Salmonella mutagenicity assay using Salmonella typhimurium strains TA98 and TA100 were tested. Ten concentrations of AFB/sub 1/ (0-1.0 ..mu..g/plate) were added to a liver microsomal homogenate (S9 mix) or to S9 mix containing GSH or S9 mix containing the combination of GSH + GST. One third of the samples were plated directly. Two-thirds were incubated for 30 min at 37/sup 0/C prior to plating, and of those, half included bacteria. The results show that the addition of GSH and GSH + GST affected AFB/sub 1/ mutagenesis by forming the AFB/sub 1/-GSH conjugate and decreasing the availability of AFB/sub 1/-8,9-epoxide. The effect of GST on GSH activity varied with the strain because of the different amounts of S9 mix used. The formation of the AFB/sub 1/-GSH conjugate was verified by using reverse-phase high-performance liquid chromatography for quantitation of AFB/sub 1/ and detection of AFB/sub 1/-GSH.

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

    PubMed Central

    Riechers, D E; Irzyk, G P; Jones, S S; Fuerst, E P

    1997-01-01

    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. PMID:9276955

  6. Glutathione-functionalized graphene quantum dots as selective fluorescent probes for phosphate-containing metabolites

    NASA Astrophysics Data System (ADS)

    Liu, Jing-Jing; Zhang, Xiao-Long; Cong, Zhong-Xiao; Chen, Zhi-Tao; Yang, Huang-Hao; Chen, Guo-Nan

    2013-02-01

    Bright blue fluorescent glutathione-functionalized graphene quantum dots (GQDs@GSH) were prepared by a one-step pyrolysis method with a fluorescence quantum yield as high as 33.6%. Futhermore, the obtained GQDs@GSH can be used as a probe to estimate the ATP level in cell lysates and human blood serum.Bright blue fluorescent glutathione-functionalized graphene quantum dots (GQDs@GSH) were prepared by a one-step pyrolysis method with a fluorescence quantum yield as high as 33.6%. Futhermore, the obtained GQDs@GSH can be used as a probe to estimate the ATP level in cell lysates and human blood serum. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr33794d

  7. Water-soluble Pd nanoparticles capped with glutathione: synthesis, characterization, and magnetic properties.

    PubMed

    Sharma, Sachil; Kim, Bit; Lee, Dongil

    2012-11-13

    The synthesis, characterization, and magnetic properties of water-soluble Pd nanoparticles capped with glutathione are described. The glutathione-capped Pd nanoparticles were synthesized under argon and air atmospheres at room temperature. Whereas the former exhibits a bulklike lattice parameter, the lattice parameter of the latter is found to be considerably greater, indicating anomalous lattice expansion. Comparative structural and compositional studies of these nanoparticles suggest the presence of oxygen in the core lattice when Pd nanoparticles are prepared under an air atmosphere. Both Pd nanoparticles prepared under argon and air show ferromagnetism at 5 K, but the latter exhibits significantly greater coercivity (88 Oe) and magnetization (0.09 emu/g at 50 kOe). The enhanced ferromagnetic properties are explained by the electronic effect of the incorporated oxygen that increases the 4d density of holes at the Pd site and localizes magnetic moments. PMID:23092154

  8. Fluorescent glutathione probe based on MnO2-phenol formaldehyde resin nanocomposite.

    PubMed

    Wang, Xudong; Wang, Dan; Guo, Yali; Yang, Chengduan; Liu, Xiaoyu; Iqbal, Anam; Liu, Weisheng; Qin, Wenwu; Yan, Dan; Guo, Huichen

    2016-03-15

    MnO2-phenol formaldehyde resin (MnO2-PFR) nanocomposite is successfully prepared by a simple chemical reduction process. The resultant MnO2-PFR nanocomposite is well characterized. The absorption band of non-fluorescent MnO2 nanosheets overlaps well with the fluorescence emission of PFR nanoparticles. The green fluorescence of PFR in this nanocomposite can be effectively quenched by fluorescence resonance energy transfer from PFR to MnO2. In the presence of glutathione (GSH), the fluorescence of PFR could be recovered due to MnO2 was reduced to Mn(2+) by GSH. The nanocomposite can be use for detecting glutathione in blood serum. PMID:26426853

  9. Pharmacogenetics of azathioprine in inflammatory bowel disease: a role for glutathione-S-transferase?

    PubMed

    Stocco, Gabriele; Pelin, Marco; Franca, Raffaella; De Iudicibus, Sara; Cuzzoni, Eva; Favretto, Diego; Martelossi, Stefano; Ventura, Alessandro; Decorti, Giuliana

    2014-04-01

    Azathioprine is a purine antimetabolite drug commonly used to treat inflammatory bowel disease (IBD). In vivo it is active after reaction with reduced glutathione (GSH) and conversion to mercaptopurine. Although this reaction may occur spontaneously, the presence of isoforms M and A of the enzyme glutathione-S-transferase (GST) may increase its speed. Indeed, in pediatric patients with IBD, deletion of GST-M1, which determines reduced enzymatic activity, was recently associated with reduced sensitivity to azathioprine and reduced production of azathioprine active metabolites. In addition to increase the activation of azathioprine to mercaptopurine, GSTs may contribute to azathioprine effects even by modulating GSH consumption, oxidative stress and apoptosis. Therefore, genetic polymorphisms in genes for GSTs may be useful to predict response to azathioprine even if more in vitro and clinical validation studies are needed. PMID:24707136

  10. Biochemical properties of an omega-class glutathione S-transferase of the silkmoth, Bombyx mori.

    PubMed

    Yamamoto, Kohji; Nagaoka, Sumiharu; Banno, Yutaka; Aso, Yoichi

    2009-05-01

    A cDNA encoding an omega-class glutathione S-transferase of the silkmoth, Bombyx mori (bmGSTO), was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and deduced for amino acid sequence, which revealed 40, 40, and 39% identities to omega-class GSTs from human, pig, and mouse, respectively. A recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rbmGSTO was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. This enzyme was shown to have high affinity for organophosphorus insecticide and was present abundantly in silkmoth strain exhibiting fenitrothion resistance. These results indicate that bmGSTO could be involved in the increase in level of insecticide resistance for lepidopteran insects. PMID:19022397

  11. Glutathione peroxidase in yeast. Presence of the enzyme and induction by oxidative conditions.

    PubMed

    Galiazzo, F; Schiesser, A; Rotilio, G

    1987-09-30

    The presence of glutathione peroxidase activity is reported for the first time for a wild type strain of Saccharomyces cerevisiae. Both forms of enzyme, i.e. that specifically active toward H2O2 alone and that decomposing also organic peroxides, were found to be present. The H2O2 specific form disappeared when cells were grown in the absence of oxygen, while the other form was much increased under the same conditions. Addition of copper to the culture greatly increased both forms. The results show that glutathione peroxidase is to be included, as an important component that is also highly responsive to oxidative environments, in the enzyme defense system of yeast against oxidative damage. PMID:3311044

  12. Isolation and identification of a pyrrolic glutathione conjugate metabolite of the pyrrolizidine alkaloid monocrotaline.

    PubMed

    Lamé, M W; Morin, D; Jones, A D; Segall, H J; Wilson, D W

    1990-05-01

    This report describes the isolation and identification of a monocrotaline-derived, glutathione-conjugated pyrrole obtained from the bile of male Sprague-Dawley rats. Bile obtained from rats given an intravenous bolus of 14C-monocrotaline was fractionated using a series of chromatographic separations. Initial purification with cholestyramine resin removed bile acid and pigment contaminants. Subsequent anion exchange and reversed-phase HPLC separations yielded several fractions that contained the 14C label and tested positive for pyrroles using Ehrlich's reagent. These fractions were analyzed using fast-atom-bombardment tandem mass spectrometry (FAB MS/MS). In addition to glutathione-conjugated dehydroretronecine, at least one other pyrrole present had similar ionic properties. The latter was not present in amounts sufficient for positive identification. PMID:2111054

  13. Development of a glutathione production process from proteinaceous biomass resources using protease-displaying Saccharomyces cerevisiae.

    PubMed

    Hara, Kiyotaka Y; Kim, Songhee; Yoshida, Hideyo; Kiriyama, Kentaro; Kondo, Takashi; Okai, Naoko; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2012-02-01

    Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources. PMID:22075633

  14. Blocking Lactate Export by Inhibiting the Myc Target MCT1 Disables Glycolysis and Glutathione Synthesis

    PubMed Central

    Doherty, Joanne R.; Yang, Chunying; Scott, Kristen E. N.; Cameron, Michael D.; Fallahi, Mohammad; Li, Weimin; Hall, Mark A.; Amelio, Antonio L.; Mishra, Jitendra K.; Li, Fangzheng; Tortosa, Mariola; Genau, Heide Marika; Rounbehler, Robert J.; Lu, Yunqi; Dang, Chi. V.; Kumar, K. Ganesh; Butler, Andrew A.; Bannister, Thomas D.; Hooper, Andrea T.; Unsal-Kacmaz, Keziban; Roush, William R.; Cleveland, John L.

    2014-01-01

    Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1, and that elevated MCT1 levels are manifest in premalignant and neoplastic Eμ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, and reductions in glucose transport, and in levels of ATP, NADPH and glutathione. Reductions in glutathione then lead to increases in hydrogen peroxide, mitochondrial damage and, ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies. PMID:24285728

  15. Enhanced success of Mexican bean beetle (coleoptera: Coccinellidae) on glutathione-enriched soybean leaves

    SciTech Connect

    Hughes, P.R.; Chiment, J.J. )

    1988-01-01

    Artificial augmentation of soybean leaves with reduced glutathione (GSH) elicited all of the same responses from Mexican bean beetle (MBB), Epilachna varivestis Mulsant, as did fumigation with the air pollutant sulfur dioxide. Larval growth, rate of development, and survivorship as well as adult fecundity and longevity were all significantly greater on excised leaves that had been allowed to imbibe a solution of the tripeptide. In addition, adults showed a strong preference for feeding on the treated leaves over nontreated leaves. Increased fecundity after feeding on treated leaves was a consequence of the earlier and longer period of egg laying rather than a change in the rate of egg production. The effects of GSH treatment were even more distinct than those produced by exposure of plants to the pollutant. These results establish the very close correlation between changes in foliar glutathione and alteration of MBB success on this plant in response to air pollution.

  16. Sonochemical synthesis of highly fluorescent glutathione-stabilized Ag nanoclusters and S2- sensing

    NASA Astrophysics Data System (ADS)

    Zhou, Tingyao; Rong, Mingcong; Cai, Zhimin; Yang, Chaoyong James; Chen, Xi

    2012-06-01

    A facile one-pot sonochemical approach is presented to prepare highly blue-emitting Ag nanoclusters (AgNCs) using glutathione as a stabilizing agent in aqueous solution. The as-prepared AgNCs can be applied in the selective detection of S2- with a limit of detection of 2 nM based on fluorescence quenching.A facile one-pot sonochemical approach is presented to prepare highly blue-emitting Ag nanoclusters (AgNCs) using glutathione as a stabilizing agent in aqueous solution. The as-prepared AgNCs can be applied in the selective detection of S2- with a limit of detection of 2 nM based on fluorescence quenching. Electronic supplementary information (ESI) available: Experimental procedures of fluorescent AgNCs synthesis and Fig. S1-S6. See DOI: 10.1039/c2nr30718a

  17. A fluorescence enhancement probe based on BODIPY for the discrimination of cysteine from homocysteine and glutathione.

    PubMed

    Gong, Deyan; Tian, Yuejun; Yang, Chengduan; Iqbal, Anam; Wang, Zhiping; Liu, Weisheng; Qin, Wenwu; Zhu, Xiangtao; Guo, Huichen

    2016-11-15

    Herein, a fluorescent probe BODIPY-based glyoxal hydrazone (BODIPY-GH) (1) for cysteine based on inhibiting of intramolecular charge transfer (ICT) quenching process upon reaction with the unsaturated aldehyde has been synthesized, which exhibits longer excitation wavelength, selective and sensitive colorimetric and fluorimetric response toward cysteine in natural media. The probe shows highly selectivity towards cysteine over homocysteine and glutathione as well as other amino acids with a significant fluorescence enhancement response within 15min In the presence of 50 equiv. of homocysteine, the emission increased slightly within 15min and completed in 2.5h to reach its maximum intensity. Therefore, the discrimination of cysteine from homocysteine and glutathione can be achieved through detection of probe 1. It shows low cytotoxicity and excellent membrane permeability toward living cells, which was successfully applied to detect and image intracellular cysteine effectively by confocal fluorescence imaging. PMID:27176916

  18. Effect of glutathione on peroxyoxalate chemiluminescence of hypericin as the fluorophore

    NASA Astrophysics Data System (ADS)

    Kazemi, Sayed Yahya; Abedirad, Seyed Mohammad

    2014-01-01

    Herein, the effect of amino acid Glutathione (GSH) on Peroxyoxalate Chemiluminescence was studied for the first time. Hypericin (HYP) was employed as the efficient fluorophore. The investigated parameters included rise and fall rate constant for the chemiluminescence burst, theoretical and experimental maximum intensity, the time-needed to reach maximum intensity and the total light yield emission which theoretically was evaluated using the pooled intermediate model by a computerized non-linear least-squares curve fitting program (KINFIT). Furthermore, based on observed quenching effect of GSH, the Stern-Volmer plot in quencher concentration range of 2.8 × 10-6 to 3.4 × 10-5 M with KQ value of 1.59 × 104 was calculated. The bimolecular quenching rate constant (Kq) was also estimated about 2.8 × 1012 and M-1 S-1. Moreover the system was applied successfully to determine glutathione in biological samples.

  19. Mercury(II) Complex Formation With Glutathione in Alkaline Aqueous Solution

    SciTech Connect

    Mah, V.; Jalilehvand, F.

    2009-05-19

    The structure and speciation of the complexes formed between mercury(II) ions and glutathione (GSH = L-glutamyl-L-cysteinyl-glycine) have been studied for a series of alkaline aqueous solutions (C{sub Hg{sup 2+}} {approx} 18 mmol dm{sup -3} and C{sub GSH} = 40-200 mmol dm{sup -3} at pH {approx} 10.5) by means of extended X-ray absorption fine structure (EXAFS) and {sup 199}Hg NMR spectroscopy at ambient temperature. The dominant complexes are [Hg(GS){sub 2}]{sup 4-} and [Hg(GS){sub 3}]{sup 7-}, with mean Hg-S bond distances of 2.32(1) and 2.42(2) {angstrom} observed in digonal and trigonal Hg-S coordination, respectively. The proportions of the Hg{sup 2+}-glutathione complexes were evaluated by fitting linear combinations of model EXAFS oscillations representing each species to the experimental EXAFS spectra. The [Hg(GS){sub 4}]{sup 10-} complex, with four sulfur atoms coordinated at a mean Hg-S bond distance of 2.52(2) {angstrom}, is present in minor amounts (<30%) in solutions containing a large excess of glutathione (C{sub GSH} {ge} 160 mmol dm{sup -3}). Comparable alkaline mercury(II) cysteine (H{sub 2}Cys) solutions were also investigated and a reduced tendency to form higher complexes was observed, because the deprotonated amino group of Cys{sup 2-} allows the stable [Hg(S,N-Cys){sub 2}]{sup 2-} chelate to form. The effect of temperature on the distribution of the Hg{sup 2+}-glutathione complexes was studied by comparing the EXAFS spectra at ambient temperature and at 25 K of a series of glycerol/water (33/67, v/v) frozen glasses with and C{sub Hg{sup 2+}} {approx} 7 mmol dm{sup -3} and C{sub GSH} = 16-81 mmol dm{sup -3}. Complexes with high Hg-S coordination numbers, [Hg(GS){sub 3}]{sup 7-} and [Hg(GS){sub 4}]{sup 10-}, became strongly favored when just a moderate excess of glutathione (C{sub GSH} {ge} 28 mmol dm{sup -3}) was used in the glassy samples, as expected for a stepwise exothermic bond formation. Addition of glycerol had no effect on the Hg(II)-glutathione

  20. Glutathione-coated luminescent gold nanoparticles: a surface ligand for minimizing serum protein adsorption.

    PubMed

    Vinluan, Rodrigo D; Liu, Jinbin; Zhou, Chen; Yu, Mengxiao; Yang, Shengyang; Kumar, Amit; Sun, Shasha; Dean, Andrew; Sun, Xiankai; Zheng, Jie

    2014-08-13

    Ultrasmall glutathione-coated luminescent gold nanoparticles (GS-AuNPs) are known for their high resistance to serum protein adsorption. Our studies show that these NPs can serve as surface ligands to significantly enhance the physiological stability and lower the serum protein adsorption of superparamagnetic iron oxide nanoparticles (SPIONs), in addition to rendering the NPs the luminescence property. After the incorporation of GS-AuNPs onto the surface of SPIONs to form the hybrid nanoparticles (HBNPs), these SPIONs' protein adsorption was about 10-fold lower than those of the pure glutathione-coated SPIONs suggesting that GS-AuNPs are capable of providing a stealth effect against serum proteins. PMID:25029478

  1. Glutathione-Coated Luminescent Gold Nanoparticles: A Surface Ligand for Minimizing Serum Protein Adsorption

    PubMed Central

    2015-01-01

    Ultrasmall glutathione-coated luminescent gold nanoparticles (GS-AuNPs) are known for their high resistance to serum protein adsorption. Our studies show that these NPs can serve as surface ligands to significantly enhance the physiological stability and lower the serum protein adsorption of superparamagnetic iron oxide nanoparticles (SPIONs), in addition to rendering the NPs the luminescence property. After the incorporation of GS-AuNPs onto the surface of SPIONs to form the hybrid nanoparticles (HBNPs), these SPIONs’ protein adsorption was about 10-fold lower than those of the pure glutathione-coated SPIONs suggesting that GS-AuNPs are capable of providing a stealth effect against serum proteins. PMID:25029478

  2. Reactivity of the glutathione species towards the reduction of ormaplatin (or tetraplatin).

    PubMed

    Dong, Jingran; Huo, Shuying; Shen, Shigang; Xu, Jianzhong; Shi, Tiesheng; Elding, Lars I

    2016-09-01

    The reduction of ormaplatin (tetraplatin), a prototype for Pt(IV) anticancer prodrugs, by glutathione (GSH) was kinetically characterized over a wide pH range at 25.0°C and 1.0M ionic strength. The reduction follows overall second-order kinetics, giving rise to the oxidized glutathione as the oxidation product, which was identified by high-resolution mass spectrometry. The reaction mechanism put forward involves parallel attacks by all the GSH species on the Pt(IV) prodrug as rate-determining steps. All rate constants for the rate-determining steps have been derived for the first time, enabling the construction of the reactivity of GSH species versus their pH distribution diagram. The diagram clearly displays that only one out of the five GSH species is the mainly responsible for the reduction of ormaplatin at the physiological pH of 7.4. PMID:27481559

  3. Apocynin increases glutathione synthesis and activates AP-1 in alveolar epithelial cells.

    PubMed

    Lapperre, T S; Jimenez, L A; Antonicelli, F; Drost, E M; Hiemstra, P S; Stolk, J; MacNee, W; Rahman, I

    1999-01-25

    Apocynin (4-hydroxy-3-methoxy-acetophenone) is a potent intracellular inhibitor of superoxide anion production in neutrophils. In this study, we studied the effect of apocynin on the regulation of the antioxidant glutathione (GSH) and activation of the transcription factor AP-I in human alveolar epithelial cells (A549). Apocynin enhanced intracellular GSH by increasing gamma-glutamylcysteine synthetase activity in A549 cells. Apocynin also increased the expression of gamma-GCS heavy subunit mRNA. This was associated with increased AP-1 DNA binding as measured by the electrophoretic mobility shift assay. These data indicate that apocynin displays antioxidant properties, in part, by increasing glutathione synthesis through activation of AP-1. PMID:9989612

  4. Glutathione transferase activity and formation of macromolecular adducts in two cases of acute methyl bromide poisoning.

    PubMed Central

    Garnier, R; Rambourg-Schepens, M O; Müller, A; Hallier, E

    1996-01-01

    OBJECTIVES: To determine the activity of glutathione transferase and to measure the S-methylcysteine adducts in blood proteins, after acute inhalation exposure to methyl bromide. To examine the influence of the polymorphism of glutathione-S-transferase theta (GSTT1) on the neurotoxicity of methyl bromide. METHODS: Two workers acutely exposed to methyl bromide with inadequate respiratory protective devices were poisoned. Seven weeks after the accident, blood samples were drawn from both patients, for measurement of glutathione transferase activity in erythrocytes (conjugator status--that is, GSTT1 phenotype) and measurement of binding products of methyl bromide with blood proteins. Conjugator status was determined by a standard procedure. The binding product of methyl bromide, S-methylcysteine, was measured in globin and albumin. RESULTS: Duration and intensity of exposure were identical for both patients as they worked together with the same protective devices and with similar physical effort. However, one patient had very severe poisoning, whereas the other only developed mild neurotoxic symptoms. The first patient was a "conjugator" with normal glutathone transferase activity, whereas this activity was undetectable in the erythrocytes of the second patient, who consequently had higher concentrations of S-methylcysteine adduct in albumin (149 v 91 nmol/g protein) and in globin (77 v 30 nmol/g protein). CONCLUSIONS: Methyl bromide is genotoxic and neurotoxic. Its genotoxicity seems to be the consequence of the alkylating activity of the parent compound, and conjugation to glutathione has a protective effect. The data presented here suggest a different mechanism for methyl bromide neurotoxicity which could be related to the transformation of methylglutathione into toxic metabolites such as methanethiol and formaldehyde. If such metabolites are the ultimate toxic species, N-acetylcysteine treatment could have a toxifying rather than a detoxifying effect. PMID:8704864

  5. A comparison of erythrocyte glutathione S-transferase activity from human foetuses and adults.

    PubMed Central

    Strange, R C; Johnston, J D; Coghill, D R; Hume, R

    1980-01-01

    Glutathione S-transferase activity was measured in partially purified haemolysates of erythrocytes from human foetuses and adults. Enzyme activity was present in erythrocytes obtained between 12 and 40 weeks of gestation. The catalytic properties of the enzyme from foetal cells were similar to those of the enzyme from adult erythrocytes, indicating that probably only one form of the erythrocytes enzyme exists throughout foetal and adult life. PMID:7396875

  6. Green tea supplementation increases glutathione and plasma antioxidant capacity in adults with the metabolic syndrome.

    PubMed

    Basu, Arpita; Betts, Nancy M; Mulugeta, Afework; Tong, Capella; Newman, Emily; Lyons, Timothy J

    2013-03-01

    Green tea, a popular polyphenol-containing beverage, has been shown to alleviate clinical features of the metabolic syndrome. However, its effects in endogenous antioxidant biomarkers are not clearly understood. Thus, we tested the hypothesis that green tea supplementation will upregulate antioxidant parameters (enzymatic and nonenzymatic) in adults with the metabolic syndrome. Thirty-five obese participants with the metabolic syndrome were randomly assigned to receive one of the following for 8 weeks: green tea (4 cups per day), control (4 cups water per day), or green tea extract (2 capsules and 4 cups water per day). Blood samples and dietary information were collected at baseline (0 week) and 8 weeks of the study. Circulating carotenoids (α-carotene, β-carotene, lycopene) and tocopherols (α-tocopherol, γ-tocopherol) and trace elements were measured using high-performance liquid chromatography and inductively coupled plasma mass spectroscopy, respectively. Serum antioxidant enzymes (glutathione peroxidase, glutathione, catalase) and plasma antioxidant capacity were measured spectrophotometrically. Green tea beverage and green tea extract significantly increased plasma antioxidant capacity (1.5 to 2.3 μmol/L and 1.2 to 2.5 μmol/L, respectively; P < .05) and whole blood glutathione (1783 to 2395 μg/g hemoglobin and 1905 to 2751 μg/g hemoglobin, respectively; P < .05) vs controls at 8 weeks. No effects were noted in serum levels of carotenoids and tocopherols and glutathione peroxidase and catalase activities. Green tea extract significantly reduced plasma iron vs baseline (128 to 92 μg/dL, P < .02), whereas copper, zinc, and selenium were not affected. These results support the hypothesis that green tea may provide antioxidant protection in the metabolic syndrome. PMID:23507223

  7. Glutathione-supported arsenate reduction coupled to arsenolysis catalyzed by ornithine carbamoyl transferase

    SciTech Connect

    Nemeti, Balazs; Gregus, Zoltan

    2009-09-01

    Three cytosolic phosphorolytic/arsenolytic enzymes, (purine nucleoside phosphorylase [PNP], glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase) have been shown to mediate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in a thiol-dependent manner. With unknown mechanism, hepatic mitochondria also reduce AsV. Mitochondria possess ornithine carbamoyl transferase (OCT), which catalyzes phosphorolytic or arsenolytic citrulline cleavage; therefore, we examined if mitochondrial OCT facilitated AsV reduction in presence of glutathione. Isolated rat liver mitochondria were incubated with AsV, and AsIII formed was quantified. Glutathione-supplemented permeabilized or solubilized mitochondria reduced AsV. Citrulline (substrate for OCT-catalyzed arsenolysis) increased AsV reduction. The citrulline-stimulated AsV reduction was abolished by ornithine (OCT substrate inhibiting citrulline cleavage), phosphate (OCT substrate competing with AsV), and the OCT inhibitor norvaline or PALO, indicating that AsV reduction is coupled to OCT-catalyzed arsenolysis of citrulline. Corroborating this conclusion, purified bacterial OCT mediated AsV reduction in presence of citrulline and glutathione with similar responsiveness to these agents. In contrast, AsIII formation by intact mitochondria was unaffected by PALO and slightly stimulated by citrulline, ornithine, and norvaline, suggesting minimal role for OCT in AsV reduction in intact mitochondria. In addition to OCT, mitochondrial PNP can also mediate AsIII formation; however, its role in AsV reduction appears severely limited by purine nucleoside supply. Collectively, mitochondrial and bacterial OCT promote glutathione-dependent AsV reduction with coupled arsenolysis of citrulline, supporting the hypothesis that AsV reduction is mediated by phosphorolytic/arsenolytic enzymes. Nevertheless, because citrulline cleavage is disfavored physiologically, OCT may have little role in AsV reduction in vivo.

  8. Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases.

    PubMed Central

    Alfenito, M R; Souer, E; Goodman, C D; Buell, R; Mol, J; Koes, R; Walbot, V

    1998-01-01

    Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1. PMID:9668133

  9. Peroxynitrite is a critical mediator of acetaminophen hepatotoxicity in murine livers: protection by glutathione.

    PubMed

    Knight, Tamara R; Ho, Ye-Shih; Farhood, Anwar; Jaeschke, Hartmut

    2002-11-01

    Acetaminophen (AAP) overdose causes formation of nitrotyrosine, a footprint of peroxynitrite, in centrilobular hepatocytes. The importance of peroxynitrite for the pathophysiology, however, is unclear. C3Heb/FeJ mice were treated with 300 mg/kg AAP. To accelerate the restoration of hepatic glutathione (GSH) levels as potential endogenous scavengers of peroxynitrite, some groups of animals received 200 mg of GSH/kg i.v. at different time points after AAP. AAP induced severe liver cell damage at 6 h. Total liver and mitochondrial glutathione levels decreased by >90% at 1 h but recovered to 75 and 45%, respectively, of untreated values at 6 h after AAP. In addition, the hepatic and mitochondrial glutathione disulfide (GSSG) content was significantly increased over baseline, suggesting a mitochondrial oxidant stress. Moreover, centrilobular hepatocytes stained for nitrotyrosine. Treatment with GSH at t = 0 restored hepatic GSH levels and completely prevented the mitochondrial oxidant stress, peroxynitrite formation, and liver cell injury. In contrast, treatment at 1.5 and 2.25 h restored hepatic and mitochondrial GSH levels but did not prevent the increase in GSSG formation. Nitrotyrosine adduct formation and liver injury, however, was substantially reduced. GSH treatment at 3 h after AAP was ineffective. Similar results were obtained when these experiments were repeated with glutathione peroxidase-deficient animals. Our data suggest that early GSH treatment (t = 0) prevented cell injury by improving the detoxification of the reactive metabolite of AAP. Delayed GSH treatment enhanced hepatic GSH levels, which scavenged peroxynitrite in a spontaneous reaction. Thus, peroxynitrite is an important mediator of AAP-induced liver cell necrosis. PMID:12388625

  10. No Evidence of Exogenous Origin for the Abnormal Glutathione Redox State in Schizophrenia

    PubMed Central

    Ballesteros, Alejandro; Jiang, Pan; Summerfelt, Ann; Du, Xiaoming; Chiappelli, Joshua; O’Donnell, Patricio; Kochunov, Peter; Hong, L. Elliot

    2013-01-01

    Schizophrenia has been associated with low glutathione (GSH), one of the most important substrates for natural defense against oxidative stress. This abnormality is often attributed to genetic or other pathological causes. However, low GSH in schizophrenia could also be due to insufficient antioxidant consumption or other exogenous factors. We evaluated GSH in relation to diet, smoking, and medication status in schizophrenia patients. We recruited 54 participants (29 schizophrenia patients and 25 normal controls). The Antioxidant Dietary Source Questions was used to estimate the total antioxidant capacity (TAC) from participants’ diet. GSH and the oxidized form of glutathione (GSSG) were assayed. We found that GSH was significantly lower (p < 0.001) while %GSSG was 2 to 5 fold higher (p = 0.023) in patients compared with controls. No evidence for lower TAC dietary intake was found in schizophrenia patients compared with controls; rather nominally higher TAC level was found in the patients diet (p=0.02). Analysis of consumption of individual food categories also failed to find evidence of reduced dietary antioxidant intake in schizophrenia patients. Smoking and medications did not significantly predict the GSH deficit either. However, there was a significant smoking by diagnosis interaction on GSH (p=0.026) such that smoking was associated with higher GSH level in controls while smoking in patients was not associated with this effect. Schizophrenia patients may have an impaired upregulation of GSH synthesis that normally occurs due to smoking-induced antioxidative response. Lower GSH was independently present in patients on clozapine (p = 0.005) and patients on other antipsychotics (p < 0.001) compared with controls. In conclusion, none of the exogenous sources played a major role in explaining abnormalities in the glutathione pathway in patients. The state of abnormal glutathione redox may therefore be a part of schizophrenia pathophysiology. PMID:23466187

  11. Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells

    PubMed Central

    Downs, Charles A.; Kreiner, Lisa; Zhao, Xing-Ming; Trac, Phi; Johnson, Nicholle M.; Hansen, Jason M.; Brown, Lou Ann

    2015-01-01

    Amiloride-sensitive epithelial Na+ channels (ENaC) regulate fluid balance in the alveoli and are regulated by oxidative stress. Since glutathione (GSH) is the predominant antioxidant in the lungs, we proposed that changes in glutathione redox potential (Eh) would alter cell signaling and have an effect on ENaC open probability (Po). In the present study, we used single channel patch-clamp recordings to examine the effect of oxidative stress, via direct application of glutathione disulfide (GSSG), on ENaC activity. We found a linear decrease in ENaC activity as the GSH/GSSG Eh became less negative (n = 21; P < 0.05). Treatment of 400 μM GSSG to the cell bath significantly decreased ENaC Po from 0.39 ± 0.06 to 0.13 ± 0.05 (n = 8; P < 0.05). Likewise, back-filling recording electrodes with 400 μM GSSG reduced ENaC Po from 0.32 ± 0.08 to 0.17 ± 0.05 (n = 10; P < 0.05), thus implicating GSSG as an important regulatory factor. Biochemical assays indicated that oxidizing potentials promote S-glutathionylation of ENaC and irreversible oxidation of cysteine residues with N-ethylmaleimide blocked the effects of GSSG on ENaC Po. Additionally, real-time imaging studies showed that GSSG impairs alveolar fluid clearance in vivo as opposed to GSH, which did not impair clearance. Taken together, these data show that glutathione Eh is an important determinant of alveolar fluid clearance in vivo. PMID:25713321

  12. Escherichia coli Resistance to Chlorine and Glutathione Synthesis in Response to Oxygenation and Starvation

    PubMed Central

    Saby, Sébastien; Leroy, Pierre; Block, Jean-Claude

    1999-01-01

    Reduced glutathione (GSH) levels and resistance to chlorine were measured for two isogenic Escherichia coli strains stressed by oxygenation and/or starvation. The E. coli mutant deficient in GSH was not more sensitive to the oxidant than its parent strain when the bacteria were cultured with a low oxygenation rate. Starvation or oxygenation increased the resistance of the parent strain to chlorine, while the resistance of the deficient strain remained unchanged. PMID:10584025

  13. Deficiency of glutathione transferase zeta causes oxidative stress and activation of antioxidant response pathways.

    PubMed

    Blackburn, Anneke C; Matthaei, Klaus I; Lim, Cindy; Taylor, Matthew C; Cappello, Jean Y; Hayes, John D; Anders, M W; Board, Philip G

    2006-02-01

    Glutathione S-transferase (GST) zeta (GSTZ1-1) plays a significant role in the catabolism of phenylalanine and tyrosine, and a deficiency of GSTZ1-1 results in the accumulation of maleylacetoacetate and its derivatives maleylacetone (MA) and succinylacetone. Induction of GST subunits was detected in the liver of Gstz1(-/-) mice by Western blotting with specific antisera and high-performance liquid chromatography analysis of glutathione affinity column-purified proteins. The greatest induction was observed in members of the mu class. Induction of NAD(P)H:quinone oxidoreductase 1 and the catalytic and modifier subunits of glutamate-cysteine ligase was also observed. Many of the enzymes that are induced in Gstz1(-/-) mice are regulated by antioxidant response elements that respond to oxidative stress via the Keap1/Nrf2 pathway. It is significant that diminished glutathione concentrations were also observed in the liver of Gstz1(-/-) mice, which supports the conclusion that under normal dietary conditions, the accumulation of electrophilic intermediates such as maleylacetoacetate and MA results in a high level of oxidative stress. Elevated GST activities in the livers of Gstz1(-/-) mice suggest that GSTZ1-1 deficiency may alter the metabolism of some drugs and xenobiotics. Gstz1(-/-) mice given acetaminophen demonstrated increased hepatotoxicity compared with wild-type mice. This toxicity may be attributed to the increased GST activity or the decreased hepatic concentrations of glutathione, or both. Patients with acquired deficiency of GSTZ1-1 caused by therapeutic exposure to dichloroacetic acid for the clinical treatment of lactic acidosis may be at increased risk of drug- and chemical-induced toxicity. PMID:16278372

  14. MIF protein are theta-class glutathione S-transferase homologs.

    PubMed Central

    Blocki, F. A.; Ellis, L. B.; Wackett, L. P.

    1993-01-01

    MIF proteins are mammalian polypeptides of approximately 13,000 molecular weight. This class includes human macrophage migration inhibitory factor (MIF), a rat liver protein that has glutathione S-transferase (GST) activity (TRANSMIF), and the mouse delayed early response gene 6 (DER6) protein. MIF proteins were previously linked to GSTs by demonstrating transferase activity and observing N-terminal sequence homology with a mu-class GST (Blocki, F.A., Schlievert, P.M., & Wackett, L.P., 1992, Nature 360, 269-270). In this study, MIF proteins are shown to be structurally related to the theta class of GSTs. This is established in three ways. First, unique primary sequence patterns are developed for each of the GST gene classes. The patterns identify the three MIF proteins as theta-like transferase homologs. Second, pattern analysis indicates that GST members of the theta class contain a serine residue in place of the N-terminal tyrosine that is implicated in glutathione deprotonation and activation in GSTs of known structure (Liu, S., et al., 1992, J. Biol. Chem. 267, 4296-4299). The MIF proteins contain a threonine at this position. Third, polyclonal antibodies raised against recombinant human MIF cross-react on Western blots with rat theta GST but not with alpha and mu GSTs. That MIF proteins have glutathione-binding ability may provide a common structural key toward understanding the varied functions of this widely distributed emerging gene family. Because theta is thought to be the most ancient evolutionary GST class, MIF proteins may have diverged early in evolution but retained a glutathione-binding domain. PMID:8298459

  15. Proteomic Analysis of Ketogulonicigenium vulgare under Glutathione Reveals High Demand for Thiamin Transport and Antioxidant Protection

    PubMed Central

    Ma, Qian; Zhang, Weiwen; Zhang, Lu; Qiao, Bin; Pan, Chensong; Yi, Hong; Wang, Lili; Yuan, Ying-jin

    2012-01-01

    Ketogulonicigenium vulgare, though grows poorly when mono-cultured, has been widely used in the industrial production of the precursor of vitamin C with the coculture of Bacillus megaterium. Various efforts have been made to clarify the synergic pattern of this artificial microbial community and to improve the growth and production ability of K. vulgare, but there is still no sound explanation. In previous research, we found that the addition of reduced glutathione into K. vulgare monoculture could significantly improve its growth and productivity. By performing SEM and TEM, we observed that after adding GSH into K. vulgare monoculture, cells became about 4–6 folds elongated, and formed intracytoplasmic membranes (ICM). To explore the molecular mechanism and provide insights into the investigation of the synergic pattern of the co-culture system, we conducted a comparative iTRAQ-2-D-LC-MS/MS-based proteomic analysis of K. vulgare grown under reduced glutathione. Principal component analysis of proteomic data showed that after the addition of glutathione, proteins for thiamin/thiamin pyrophosphate (TPP) transport, glutathione transport and the maintenance of membrane integrity, together with several membrane-bound dehydrogenases had significant up-regulation. Besides, several proteins participating in the pentose phosphate pathway and tricarboxylic acid cycle were also up-regulated. Additionally, proteins combating intracellular reactive oxygen species were also up-regulated, which similarly occurred in K. vulgare when the co-cultured B. megaterium cells lysed from our former research results. This study reveals the demand for transmembrane transport of substrates, especially thiamin, and the demand for antioxidant protection of K. vulgare. PMID:22384164

  16. Chemoselective Protection of Glutathione in the Preparation of Bioconjugates: The Case of Trypanothione Disulfide.

    PubMed

    Antoniou, Antonia I; Pepe, Dionissia A; Aiello, Donatella; Siciliano, Carlo; Athanassopoulos, Constantinos M

    2016-05-20

    A novel synthetic route to the chemoselectively protected N,S-ditritylglutathione monomethyl ester is described involving the chemical modification of the commercially available glutathione (GSH). The synthetic value of this building block in the facile preparation of GSH bioconjugates in a satisfying overall yield was exemplified by the case of trypanothione disulfide (TS2), a GSH-spermidine bioconjugate, involved in the antioxidative stress protection system of parasitic protozoa, such as trypanosoma and leishmania parasites. PMID:27137354

  17. Glutathione precursor, N-acetyl-cysteine, improves mismatch negativity in schizophrenia patients.

    PubMed

    Lavoie, Suzie; Murray, Micah M; Deppen, Patricia; Knyazeva, Maria G; Berk, Michael; Boulat, Olivier; Bovet, Pierre; Bush, Ashley I; Conus, Philippe; Copolov, David; Fornari, Eleonora; Meuli, Reto; Solida, Alessandra; Vianin, Pascal; Cuénod, Michel; Buclin, Thierry; Do, Kim Q

    2008-08-01

    In schizophrenia patients, glutathione dysregulation at the gene, protein and functional levels, leads to N-methyl-D-aspartate (NMDA) receptor hypofunction. These patients also exhibit deficits in auditory sensory processing that manifests as impaired mismatch negativity (MMN), which is an auditory evoked potential (AEP) component related to NMDA receptor function. N-acetyl-cysteine (NAC), a glutathione precursor, was administered to patients to determine whether increased levels of brain glutathione would improve MMN and by extension NMDA function. A randomized, double-blind, cross-over protocol was conducted, entailing the administration of NAC (2 g/day) for 60 days and then placebo for another 60 days (or vice versa). 128-channel AEPs were recorded during a frequency oddball discrimination task at protocol onset, at the point of cross-over, and at the end of the study. At the onset of the protocol, the MMN of patients was significantly impaired compared to sex- and age- matched healthy controls (p=0.003), without any evidence of concomitant P300 component deficits. Treatment with NAC significantly improved MMN generation compared with placebo (p=0.025) without any measurable effects on the P300 component. MMN improvement was observed in the absence of robust changes in assessments of clinical severity, though the latter was observed in a larger and more prolonged clinical study. This pattern suggests that MMN enhancement may precede changes to indices of clinical severity, highlighting the possible utility AEPs as a biomarker of treatment efficacy. The improvement of this functional marker may indicate an important pathway towards new therapeutic strategies that target glutathione dysregulation in schizophrenia. PMID:18004285

  18. Serum Malondialdehyde Concentration and Glutathione Peroxidase Activity in a Longitudinal Study of Gestational Diabetes

    PubMed Central

    Miranda, María; Muriach, María; Romero, Francisco J.; Villar, Vincent M.

    2016-01-01

    Aims The main goal of this study was to evaluate the presence of oxidative damage and to quantify its level in gestational diabetes. Methods Thirty-six healthy women and thirty-six women with gestational diabetes were studied in the three trimesters of pregnancy regarding their levels of oxidative stress markers. These women were diagnosed with diabetes in the second trimester of pregnancy. Blood glucose levels after 100g glucose tolerance test were higher than 190, 165 or 145 mg/dl, 1, 2 or 3 hours after glucose intake. Results The group of women with gestational diabetes had higher serum malondialdehyde levels, with significant differences between groups in the first and second trimester. The mean values of serum glutathione peroxidase activity in the diabetic women were significantly lower in the first trimester. In the group of women with gestational diabetes there was a negative linear correlation between serum malondialdehyde concentration and glutathione peroxidase activity in the second and third trimester. Conclusions In this observational and longitudinal study in pregnant women, the alterations attributable to oxidative stress were present before the biochemical detection of the HbA1c increase. Usual recommendations once GD is detected (adequate metabolic control, as well as any other normally proposed to these patients) lowered the concentration of malondialdehyde at the end of pregnancy to the same levels of the healthy controls. Serum glutathione peroxidase activity in women with gestational diabetes increased during the gestational period. PMID:27228087

  19. Potent and selective inhibitors of glutathione S-transferase omega 1 that impair cancer drug resistance.

    PubMed

    Tsuboi, Katsunori; Bachovchin, Daniel A; Speers, Anna E; Spicer, Timothy P; Fernandez-Vega, Virneliz; Hodder, Peter; Rosen, Hugh; Cravatt, Benjamin F

    2011-10-19

    Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC(50) = 21 nM) and in situ (IC(50) = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in chemotherapy resistance. PMID:21899313

  20. Potent and Selective Inhibitors of Glutathione S-transferase Omega 1 that Impair Cancer Drug Resistance

    PubMed Central

    Tsuboi, Katsunori; Bachovchin, Daniel A.; Speers, Anna E.; Spicer, Timothy P.; Fernandez-Vega, Virneliz; Hodder, Peter; Rosen, Hugh; Cravatt, Benjamin F.

    2011-01-01

    Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC50 = 21 nM) and in situ (IC50 = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in the detoxification of chemo-therapeutic agents PMID:21899313

  1. [Activity of salivary glutathione-dependent enzymes in patients with periodontitis].

    PubMed

    Gavriliuk, L A; Shevchenko, N V; Spineĭ, A F; Vartichan, A I; Godorozha, P D; Lysyĭ, L T

    2008-07-01

    Forty-five patients aged 20-47 years who had mild, moderate, or severe periodontitis and 32 healthy individuals (a control group) were studied during 10-15-day treatment with traditional therapy and combined therapy including the traditional approach and the antihomotoxic agent Traumeel S ointment as a supplement. Increased free radical generation and lipid peroxidation were considered to play an important role in the pathogenesis of periodontitis. Salivary indices are a reflection of a patient's metabolic state and have clinical diagnostic values in patients with oral tissue inflammation. The activities of antioxidative enzymes (glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase) and the content of reduced glutathione (GSH) were determined in the saliva of patients with periodontitis during traditional and complex (traditional + Traumeel S) therapies. Inflammation led to metabolic disturbances and antioxidative defense system imbalance in patients with periodontitis. The findings suggest that the complex therapy with Traumeel S restored antioxidative defense balance and it was more effective than the traditional therapy in patients with periodontitis. An analysis showed a direct correlation between the activity of antioxidative enzymes and clinical characteristics of the disease. These results reflect the activity of a pathological process and the imbalance of antioxidative defense in patients with periodontitis. PMID:18756728

  2. Application of glutathione to roots selectively inhibits cadmium transport from roots to shoots in oilseed rape

    PubMed Central

    Nakamura, Shin-ichi

    2013-01-01

    Glutathione is a tripeptide involved in various aspects of plant metabolism. This study investigated the effects of the reduced form of glutathione (GSH) applied to specific organs (source leaves, sink leaves, and roots) on cadmium (Cd) distribution and behaviour in the roots of oilseed rape plants (Brassica napus) cultured hydroponically. The translocation ratio of Cd from roots to shoots was significantly lower in plants that had root treatment of GSH than in control plants. GSH applied to roots reduced the Cd concentration in the symplast sap of root cells and inhibited root-to-shoot Cd translocation via xylem vessels significantly. GSH applied to roots also activated Cd efflux from root cells to the hydroponic solution. Inhibition of root-to-shoot translocation of Cd was visualized, and the activation of Cd efflux from root cells was also shown by using a positron-emitting tracer imaging system (PETIS). This study investigated a similar inhibitory effect on root-to-shoot translocation of Cd by the oxidized form of glutathione, GSSG. Inhibition of Cd accumulation by GSH was abolished by a low-temperature treatment. Root cells of plants exposed to GSH in the root zone had less Cd available for xylem loading by actively excluding Cd from the roots. Consequently, root-to-shoot translocation of Cd was suppressed and Cd accumulation in the shoot decreased. PMID:23364937

  3. A small molecule that induces reactive oxygen species via cellular glutathione depletion.

    PubMed

    Kawamura, Tatsuro; Kondoh, Yasumitsu; Muroi, Makoto; Kawatani, Makoto; Osada, Hiroyuki

    2014-10-01

    Induction of excessive levels of reactive oxygen species (ROS) by small-molecule compounds has been considered a potentially effective therapeutic strategy against cancer cells, which are often subjected to chronic oxidative stress. However, to elucidate the mechanisms of action of bioactive compounds is generally a time-consuming process. We have recently identified NPD926, a small molecule that induces rapid cell death in cancer cells. Using a combination of two comprehensive and complementary approaches, proteomic profiling and affinity purification, together with the subsequent biochemical assays, we have elucidated the mechanism of action underlying NPD926-induced cell death: conjugation with glutathione mediated by GST, depletion of cellular glutathione and subsequent ROS generation. NPD926 preferentially induced effects in KRAS-transformed fibroblast cells, compared with their untransformed counterparts. Furthermore, NPD926 sensitized cells to inhibitors of system x(c)⁻, a cystine-glutamate antiporter considered to be a potential therapeutic target in cancers including cancer stem cells. These data show the effectiveness of a newly identified ROS inducer, which targets glutathione metabolism, in cancer treatment. PMID:25011393

  4. Glutathione-Mediated Detoxification of Halobenzoquinone Drinking Water Disinfection Byproducts in T24 Cells

    PubMed Central

    Li, Jinhua; Wang, Wei; Zhang, Hongquan; Le, X. Chris; Li, Xing-Fang

    2014-01-01

    Halobenzoquinones (HBQs) are a new class of drinking water disinfection byproducts (DBPs) and are capable of producing reactive oxygen species and causing oxidative damage to proteins and DNA in T24 human bladder carcinoma cells. However, the exact mechanism of the cytotoxicity of HBQs is unknown. Here, we investigated the role of glutathione (GSH) and GSH-related enzymes including glutathione S-transferase (GST) and glutathione peroxidase (GPx) in defense against HBQ-induced cytotoxicity in T24 cells. The HBQs are 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), 2,3,6-trichloro-1,4-benzoquinone (TriCBQ), and 2,6-dibromobenzoquinone (DBBQ). We found that depletion of cellular GSH could sensitize cells to HBQs and extracellular GSH supplementation could attenuate HBQ-induced cytotoxicity. HBQs caused significant cellular GSH depletion and increased cellular GST activities in a concentration-dependent manner. Our mass spectrometry study confirms that HBQs can conjugate with GSH, explaining in part the mechanism of GSH depletion by HBQs. The effects of HBQs on GPx activity are compound dependent; DCMBQ and DBBQ decrease cellular GPx activities, whereas DCBQ and TriCBQ have no significant effects. Pearson correlation analysis shows that the cellular GSH level is inversely correlated with ROS production and cellular GST activity in HBQ-treated cells. These results support a GSH and GSH-related enzyme-mediated detoxification mechanism of HBQs in T24 cells. PMID:24812012

  5. Catalase and glutathione peroxidase are equally active in detoxification of hydrogen peroxide in human erythrocytes

    SciTech Connect

    Gaetani, G.F.; Galiano, S.; Canepa, L.; Ferraris, A.M.; Kirkman, H.N.

    1989-01-01

    Genetic deficiencies of glucose-6-phosphate dehydrogenase (G6PD) and NADPH predispose affected erythrocytes to destruction from peroxides. Conversely, genetic deficiencies of catalase do not predispose affected erythrocytes to peroxide-induced destruction. These observations have served to strengthen the assumption that the NADPH/glutathione/glutathione peroxidase pathway is the principal means for disposal of H/sub 2/O/sub 2/ in human erythrocytes. Recently, however, mammalian catalase was found to have tightly bound NADPH and to require NADPH for the prevention and reversal of inactivation by its toxic substrate (H/sub 2/O/sub 2/). Since both catalase and the glutathione pathway are dependent on NADPH for function, this finding raises the possibility that both mechanisms destroy H/sub 2/O/sub 2/ in human erythrocytes. A comparison of normal and acatalasemic erythrocytes in the present study indicated that catalase accounts for more than half of the destruction of H/sub 2/O/sub 2/ when H/sub 2/O/sub 2/ is generated at a rate comparable to that which leads to hemolysis in G6PD- deficient erythrocytes.

  6. Expression of glutathione transferases in corneal cell lines, corneal tissues and a human cornea construct.

    PubMed

    Kölln, Christian; Reichl, Stephan

    2016-06-15

    Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas. PMID:27113863

  7. Glutathione reductase 2 maintains the function of photosystem II in Arabidopsis under excess light.

    PubMed

    Ding, Shunhua; Jiang, Rui; Lu, Qingtao; Wen, Xiaogang; Lu, Congming

    2016-06-01

    Glutathione reductase plays a crucial role in the elimination of H(2)O(2) molecules via the ascorbate-glutathione cycle. In this study, we used transgenic Arabidopsis plants with decreased glutathione reductase 2 (GR2) levels to investigate whether this GR2 activity protects the photosynthetic machinery under excess light. The transgenic plants were highly sensitive to excess light and accumulated high levels of H(2)O(2). Photosystem II (PSII) activity was significantly decreased in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements demonstrated inhibition of electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants. Immunoblot and blue native gel analysis showed that the levels of PSII proteins and PSII complexes were decreased in transgenic plants. Analyses of the repair of photodamaged PSII and in vivo pulse labeling of thylakoid proteins showed that the repair of photodamaged PSII is inhibited due to the inhibition of the synthesis of the D1 protein de novo in transgenic plants. Taken together, our results suggest that under excess light conditions, GR2 plays an important role in maintaining both the function of the acceptor side of PSII and the repair of photodamaged PSII by preventing the accumulation of H(2)O(2). In addition, our results provide details of the role of H(2)O(2) in vivo accumulation in photoinhibition in plants. PMID:26906429

  8. Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

    SciTech Connect

    Low, Wai Yee; Feil, Susanne C.; Ng, Hooi Ling; Gorman, Michael A.; Morton, Craig J.; Pyke, James; McConville, Malcolm J.; Bieri, Michael; Mok, Yee-Foong; Robin, Charles; Gooley, Paul R.; Parker, Michael W.; Batterham, Philip

    2010-06-14

    GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 {angstrom} resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional {sup 1}H,{sup 15}N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.

  9. Glutathione and iron at the crossroad of redox metabolism in rats infected by Trypanosoma evansi.

    PubMed

    Anschau, Valesca; Dafré, Alcir Luiz; Perin, Ana Paula; Iagher, Fabíola; Tizatto, Mayara Vieira; Miletti, Luiz Claudio

    2013-06-01

    The aim of this study was to evaluate the changes in hematological and biochemical parameters of blood during acute Trypanosoma evansi infection in Wistar rats. The end points studied were hematologic parameters, red blood cell fragility, iron content, and glutathione and lipid peroxidation levels. Forty-eight animals were infected with trypomastigotes and distributed into five groups according to the level of parasitemia. Twelve non-inoculated animals were used as control. Parasitemia increased progressively, reaching highest scores at 15 days post-inoculation. At this point, several deleterious effects were observed such as an increase in iron content, in osmotic fragility, and in lipid peroxidation index, while glutathione decreased drastically. These changes were highly correlated to parasitemia (p < 0.0001) and among each other (p ≤ 0.001). Hematological indices (Hb, packed cell volume (PCV), red blood cells (RBC), and mean corpuscular hemoglobin concentration) were also correlated to parasitemia (p ≤ 0.0003) but failed to correlate to the other variables. Along with increase in iron, RBC fragility produced a decrease in RBC, PCV, and Hb, but not in mean corpuscular volume. Decrease in glutathione was negatively correlated to the end products of lipid peroxidation, clearly indicating the establishment of a pro-oxidant condition. The results show that the infection causes hematological impairments, increases iron and osmotic fragility, along with marked oxidative stress in red blood cells of rats inoculated with T. evansi. PMID:23529337

  10. Fluorometric microplate assay to measure glutathione S-transferase activity in insects and mites using monochlorobimane.

    PubMed

    Nauen, Ralf; Stumpf, Natascha

    2002-04-15

    Elevated levels of glutathione S-transferases (GSTs) play a major role as a mechanism of resistance to insecticides and acaricides in resistant pest insects and mites, respectively. Such compounds are either detoxicated directly via phase I metabolism or detoxicated by phase II metabolism of metabolites as formed by microsomal monooxygenases. Here we used monochlorobimane (MCB) as an artificial substrate and glutathione to determine total GST activity in equivalents of single pest insects and spider mites in a sensitive 96-well plate-based assay system by measuring the enzymatic conversion of MCB to its fluorescent bimane-glutathione adduct. The differentiation by their GST activity between several strains of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), with different degrees of resistance to numerous acaricides was more sensitive with MCB compared to the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Compared to an acaricide-susceptible reference strain, one field population of T. urticae showed a more than 10-fold higher GST activity measured with MCB, in contrast to a less than 2-fold higher activity when CDNB was used. Furthermore, we showed that GST activity can be sensitively assessed with MCB in homogenates of pest insects such as Heliothis virescens, Spodoptera frugiperda (Lepidoptera: Noctuidae), Plutella xylostella (Lepidoptera: Yponomeutidae), and Myzus persicae (Hemiptera: Aphididae). PMID:11950219

  11. Modulation of Brain Glutathione Reductase and Peroxiredoxin 2 by α-Tocopheryl Phosphate.

    PubMed

    Uchoa, Mariana Figueiroa; de Souza, Luiz Felipe; Dos Santos, Danubia Bonfanti; Peres, Tanara Vieira; Mello, Danielle Ferraz; Leal, Rodrigo Bainy; Farina, Marcelo; Dafre, Alcir Luiz

    2016-08-01

    α-Tocopheryl phosphate (αTP) is a phosphorylated form of α-tocopherol. Since it is phosphorylated in the hydroxyl group that is essential for the antioxidant property of α-tocopherol, we hypothesized that αTP would modulate the antioxidant system, rather than being an antioxidant agent per se. α-TP demonstrated antioxidant activity in vitro against iron-induced oxidative stress in a mitochondria-enriched fraction preparation treated with 30 or 100 µM α-TP. However, this effect was not observed ex vivo with mitochondrial-enriched fraction from mice treated with an intracerebroventricular injection of 0.1 or 1 nmol/site of αTP. Two days after treatment (1 nmol/site αTP), peroxiredoxin 2 (Prx2) and glutathione reductase (GR) expression and GR activity were decreased in cerebral cortex and hippocampus. Glutathione content, glutathione peroxidase, and thioredoxin reductase activities were not affected by αTP. In conclusion, the persistent decrease in GR and Prx2 protein content is the first report of an in vivo effect of αTP on protein expression in the mouse brain, potentially associated to a novel and biologically relevant function of this naturally occurring compound. PMID:26749581

  12. Purification of a glutathione S-transferase that mediates fosfomycin resistance in bacteria.

    PubMed Central

    Arca, P; Hardisson, C; Suárez, J E

    1990-01-01

    The enzyme that modifies fosfomycin by formation of an adduct with glutathione was purified 12-fold with a 56% activity yield by passage through DEAE Sephacel and high-performance liquid chromatography molecular exclusion columns. Its functional form was a homodimer of two 16,000-dalton polypeptides, which possibly showed an antiparallel alpha tertiary structure and which lacked marked hydrophobic regions. Visualization of the reaction was achieved by precolumn derivatization of glutathione and the adduct, separation by high-performance liquid chromatography, and fluorescence detection of both compounds. Temperature and pH optima were 20 to 30 degrees C and 8.25, respectively; Mn2+, Fe2+, and Co2+ enhanced the rate of modification; and Km values were 9.4 and 11 mM for fosfomycin and glutathione, respectively. Phosphoenolpyruvate did not interfere with fosfomycin modification. The enzyme was stable at 4 degrees C for at least 6 months but progressively lost its activity upon being heated for 60 min at temperatures over 30 degrees C. Images PMID:2193621

  13. Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

    SciTech Connect

    Biterova, Ekaterina I.; Barycki, Joseph J.

    2009-12-01

    Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl{sub 2} (2.1 {angstrom}; R = 18.2%, R{sub free} = 21.9%), and in complex with glutamate, MgCl{sub 2}, and ADP (2.7 {angstrom}; R = 19.0%, R{sub free} = 24.2%). Inspection of these structures reveals an unusual binding pocket for the {alpha}-carboxylate of the glutamate substrate and an ATP-independent Mg{sup 2+} coordination site, clarifying the Mg{sup 2+} dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

  14. Optical biosensor consisting of glutathione-S-transferase for detection of captan.

    PubMed

    Choi, Jeong-Woo; Kim, Young-Kee; Song, Sun-Young; Lee, In-ho; Lee, Won-Hong

    2003-10-15

    The optical biosensor consisting of a glutathione-S-transferase (GST)-immobilized gel film was developed to detect captan in contaminated water. The sensing scheme was based on the decrease of yellow product, s-(2,4-dinitrobenzene) glutathione, produced from substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), due to the inhibition of GST reaction by captan. Absorbance of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The enzyme reactor of the sensor system was fabricated by the gel entrapment technique for the immobilized GST film. The immobilized GST had the maximum activity at pH 6.5. The optimal concentrations of substrates were determined with 1 mM for both of CDNB and GSH. The optimum concentration of enzyme was also determined with 100 microg/ml. The activity of immobilized enzyme was fairly sustained during 30 days. The proposed biosensor could successfully detect the captan up to 2 ppm and the response time to steady signal was about 15 min. PMID:12941561

  15. Increased glutathione contributes to stress tolerance and global translational changes in Arabidopsis.

    PubMed

    Cheng, Mei-Chun; Ko, Ko; Chang, Wan-Ling; Kuo, Wen-Chieh; Chen, Guan-Hong; Lin, Tsan-Piao

    2015-09-01

    Although glutathione is well known for its reactive oxygen species (ROS) scavenging function and plays a protective role in biotic stress, its regulatory function in abiotic stress still remains to be elucidated. Our previous study showed that exogenously applied reduced glutathione (GSH) could improve abiotic stress tolerance in Arabidopsis. Here, we report that endogenously increased GSH also conferred tolerance to drought and salt stress in Arabidopsis. Moreover, both exogenous and endogenous GSH delayed senescence and flowering time. Polysomal profiling results showed that global translation was enhanced after GSH treatment and by the induced increase of GSH level by salt stress. By performing transcriptomic analyses of steady-state and polysome-bound mRNAs in GSH-treated plants, we reveal that GSH has a substantial impact on translation. Translational changes induced by GSH treatment target numerous hormones and stress signaling molecules, which might contribute to the enhanced stress tolerance in GSH-treated plants. Our translatome analysis also revealed that abscisic acid (ABA), auxin and jasmonic acid (JA) biosynthesis, as well as signaling genes, were activated during GSH treatment, which has not been reported in previously published transcriptomic data. Together, our data suggest that the increased glutathione level results in stress tolerance and global translational changes. PMID:26213235

  16. Sulforaphane reduces the alterations induced by quinolinic acid: modulation of glutathione levels.

    PubMed

    Santana-Martínez, R A; Galván-Arzáte, S; Hernández-Pando, R; Chánez-Cárdenas, M E; Avila-Chávez, E; López-Acosta, G; Pedraza-Chaverrí, J; Santamaría, A; Maldonado, P D

    2014-07-11

    Glutamate-induced excitotoxicity involves a state of acute oxidative stress, which is a crucial event during neuronal degeneration and is part of the physiopathology of neurodegenerative diseases. In this work, we evaluated the ability of sulforaphane (SULF), a natural dietary isothiocyanate, to induce the activation of transcription factor Nrf2 (a master regulator of redox state in the cell) in a model of striatal degeneration in rats infused with quinolinic acid (QUIN). Male Wistar rats received SULF (5mg/kg, i.p.) 24h and 5min before the intrastriatal infusion of QUIN. SULF increased the reduced glutathione (GSH) levels 4h after QUIN infusion, which was associated with its ability to increase the activity of glutathione reductase (GR), an antioxidant enzyme capable to regenerate GSH levels at 24h. Moreover, SULF treatment increased glutathione peroxidase (GPx) activity, while no changes were observed in γ-glutamyl cysteine ligase (GCL) activity. SULF treatment also prevented QUIN-induced oxidative stress (measured by oxidized proteins levels), the histological damage and the circling behavior. These results suggest that the protective effect of SULF could be related to its ability to preserve GSH levels and increase GPx and GR activities. PMID:24814729

  17. Role of glutathione in the antiulcer effect of hot water extract of black tea (Camellia sinensis).

    PubMed

    Maity, S; Vedasiromoni, J R; Ganguly, D K

    1998-11-01

    The role of a hot water extract of black tea (Camellia sinensis (L). O. Kuntze Theaceae) in the gastric cytoprotective mechanisms was studied using gastric mucosal lesions produced by various ulcerogens in rats as an experimental model. Prior oral administration of black tea extract (BTE) at 20 ml/kg, i.g. once a day for 7 days significantly reduced the incidence of gastric erosions and severity induced by ethanol, diethyldithiocarbamate (DDC) and diethylmaleate (DEM). This treatment also favorably altered the changes in acid and peptic activity of gastric juice in these ulcerogen-treated animals. Singular administration of succimer (60 mg/kg, i.g.), the standard sulfhydryl containing antiulcer drug used as a reference drug, was also effective. The levels of glutathione and glutathione peroxidase were significantly decreased after treatment with ethanol, DDC and DEM, and this decrease was prevented by BTE pretreatment in the aforesaid manner. Other major features of BTE-induced reversal of ulcerogenic agents include a significant decrease in the protein content and a marked increase in hexosamine and sialic acid content. These results suggest a major role for glutathione, an endogenous antioxidant, in the cytoprotection against ulceration afforded by BTE. PMID:9869262

  18. Glutathione--hydroxyl radical interaction: a theoretical study on radical recognition process.

    PubMed

    Fiser, Béla; Jójárt, Balázs; Csizmadia, Imre G; Viskolcz, Béla

    2013-01-01

    Non-reactive, comparative (2 × 1.2 μs) molecular dynamics simulations were carried out to characterize the interactions between glutathione (GSH, host molecule) and hydroxyl radical (OH(•), guest molecule). From this analysis, two distinct steps were identified in the recognition process of hydroxyl radical by glutathione: catching and steering, based on the interactions between the host-guest molecules. Over 78% of all interactions are related to the catching mechanism via complex formation between anionic carboxyl groups and the OH radical, hence both terminal residues of GSH serve as recognition sites. The glycine residue has an additional role in the recognition of OH radical, namely the steering. The flexibility of the Gly residue enables the formation of further interactions of other parts of glutathione (e.g. thiol, α- and β-carbons) with the lone electron pair of the hydroxyl radical. Moreover, quantum chemical calculations were carried out on selected GSH/OH(•) complexes and on appropriate GSH conformers to describe the energy profile of the recognition process. The relative enthalpy and the free energy changes of the radical recognition of the strongest complexes varied from -42.4 to -27.8 kJ/mol and from -21.3 to 9.8 kJ/mol, respectively. These complexes, containing two or more intermolecular interactions, would be the starting configurations for the hydrogen atom migration to quench the hydroxyl radical via different reaction channels. PMID:24040010

  19. Purification, crystallization and preliminary X-ray analysis of glutathione peroxidase Gpx3 from Saccharomyces cerevisiae

    SciTech Connect

    Yang, Zhu; Zhou, Cong-Zhao

    2006-06-01

    The glutathione peroxidase Gpx3 from the yeast S. cerevisiae has been overexpressed, purified, crystallized and diffracted to 2.6 Å resolution. Gpx3 is a monomer in solution which is different from its counterparts in mammals. The glutathione peroxidase Gpx3 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. Both gel-filtration and dynamic light-scattering (DLS) results indicate that Gpx3 is a monomer in solution at a concentration of about 2 mg ml{sup −1}, whereas glutathione peroxidases are normally tetrameric or dimeric. X-ray diffraction data from a single crystal of Gpx3 have been collected to 2.6 Å resolution. The crystals are triclinic and belong to space group P1, with unit-cell parameters a = 38.187, b = 43.372, c = 56.870 Å, α = 71.405, β = 73.376, γ = 89.633°. There are two Gpx3 monomers in a crystallographic asymmetric unit. Preliminary analyses show that the yeast Gpx3 is quite different from those of mammals.

  20. Biochemical manipulation of intracellular glutathione levels influences cytotoxicity to isolated human lymphocytes by sulfur mustard

    SciTech Connect

    Gross, C.L.; Innace, J.K.; Hovatter, R.C.; Meier, H.L.; Smith, W.J.

    1993-12-31

    Glutathione (GSH) is the major nonprotein thiol that can protect cells from damage due to electrophilic alkylating agents by forming conjugates with the agent. Sulfur mustard (HD) is an electrophilic alkylating agent that has potent mutagenic, carcinogenic, cytotoxic, and vesicant properties. Compounds that elevate or reduce intracellular levels of GSH may produce changes in cytotoxicity induced by sulfur mustard. Pretreatment of human peripheral blood lymphocytes (PBL) for 72 hr with 1 mM buthionine sulfoximine (BSO), which reduces intracellular GSH content to approximately 26% of control, appears to sensitize these in vitro cells to the cytotoxic effects of 10 AM HD but not to higher HD concentrations. Pretreatment of PBL for 48 hr with 10 mM N-acetyl cysteine (NA C), which elevates intracellular glutathione levels to 122% of control, appears to partially protect these in vitro cells from the cytotoxic effects of 10 LAIHD but not to higher HD concentrations. Augmentation of intracellular levels of glutathione may provide partial protection against cytotoxicity of sulfur mustard.

  1. Role of Glutathione in the Morphogenesis of the Bacterial Spore Coat

    PubMed Central

    Cheng, H. M.; Aronson, A. I.; Holt, S. C.

    1973-01-01

    There is a marked increase in the half-cystine content of bacterial spores, especially the coat layers at the time of formation of the outer coat. When a cysteine auxotroph of Bacillus cereus T is grown on limiting cysteine, the spores contain the normal content of half-cystine, suggesting an alternate source. Glutathione appears to be such a supply of cysteine since it is hydrolyzed during sporulation and there are increased activities of the hydrolyzing enzymes at the same time. In addition, a cysteine auxotroph with a second alteration, a temperature-sensitive glutathione disulfide reductase, produces lysozyme-sensitive spores at 40 C. These spores appear to be defective in the formation of outer spore coat. During sporulation at 40 C, the double mutant accumulates oxidized glutathione which is a poor substrate for the hydrolytic enzymes. As a result, sporulating cells are deficient in half-cystines which are essential for outer spore coat morphogenesis. This alteration can be overcome by a shift to 30 C or by addition of cystinyl-pencillamine or cysteinyl-glycine to cultures sporulating at 40 C. PMID:4632393

  2. Long-term tocotrienol supplementation and glutathione-dependent enzymes during hepatocarcinogenesis in the rat.

    PubMed

    Rahmat, A; Wan Ngah, W Z; Gapor, A; Khalid, B A

    1993-09-01

    The effects of long-term administration of tocotrienol on hepatocarcinogenesis in rats induced by diethyl nitrosamine (DEN) and 2-acetylaminofluorene (AAF) were investigated by the determination of plasma and liver gamma-glutamyl transpeptidase (GGT), cytosolic glutathione reductase (GSSG-Rx), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST). Twenty-eight male Rattus norwegicus rats (120-160g) were divided according to treatments into four groups: control group, tocotrienol - supplemented diet group (30mg/kg food), DEN/AAF-treated group and DEN/AAF treated plus tocotrienol-supplemented-diet group (30mg/kg food). The rats were sacrificed after nine months. The results obtained indicated no difference in the morphology and histology of the livers of control and tocotrienol-treated rats. Greyish-white neoplastic nodules (two per liver) were found in all the DEN/ AAF treated rats (n-10) whereas only one nodule was found in one of the carcinogen treated rats receiving tocotrienol supplementation (n-6). Histological examination showed obvious cellular damage for both the DEN/AAF-treated rats and the tocotrienol-supplemented rats but were less severe in the latter. Treatment with DEN/AAF caused increases in GGT, GSH-Px, GST and GSSG-Rx activities when compared to controls. These increases were also observed when tocotrienol was supplemented with DEN/AAF but the increases were less when compared to the rats receiving DEN/AAF only. PMID:24352144

  3. 3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.

    PubMed

    Pettigrew, N E; Brush, E J; Colman, R F

    2001-06-26

    The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects. PMID:11412109

  4. GLUTATHIONE (GSH) CONCENTRATIONS VARY WITH THE CELL CYCLE IN MATURING HAMSTER OOCYTES, ZYGOTES AND PRE-IMPLANATION EMBRYOS

    EPA Science Inventory

    Abstract
    Glutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. Previous observations that GSH concentrations are higher in mature than immature o...

  5. CHANGES IN GLUTATHIONE SYSTEM AND LIPID PEROXIDATION IN RAT BLOOD DURING THE FIRST HOUR AFTER CHLORPYRIFOS EXPOSURE.

    PubMed

    Rosalovsky, V P; Grabovska, S V; Salyha, Yu T

    2015-01-01

    Chlorpyrifos (CPF) is a highly toxic organophosphate compound, widely used as an active substance of many insecticides. Along with the anticholinesterase action, CPF may affect other biochemical mechanisms, particularly through disrupting pro- and antioxidant balance and inducing free-radical oxidative stress. Origins and occurrence of these phenomena are still not fully understood. The aim of our work was to investigate the effects of chlorpyrifos on key parameters of glutathione system and on lipid peroxidation in rat blood in the time dynamics during one hour after exposure. We found that a single exposure to 50 mg/kg chlorpyrifos caused a linear decrease in butyryl cholinesterase activity, increased activity of glutathione peroxidase and glutathione reductase, alterations in the levels of glutathione, TBA-active products and lipid hydroperoxides during 1 hour after poisoning. The most significant changes in studied parameters were detected at the 15-30th minutes after chlorpyrifos exposure. PMID:26717603

  6. Inhibition of liver glutathione S-transferase activity in rats by hypolipidemic drugs related or unrelated to clofibrate.

    PubMed

    Foliot, A; Touchard, D; Mallet, L

    1986-05-15

    The effects of in vivo administration of six hypolipidemic drugs on rat liver glutathione S-transferase activity were compared. This activity was measured with sulfobromophthalein (BSP), 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Except for the nicotinic acid derivative ethanolamine oxiniacate, all the compounds tested significantly reduced it, whether or not they were related to clofibrate. The hepatic glutathione concentration either remained unchanged or only increased slightly after treatment with the various drugs. When measured, the maximal excretion rate of bile BSP dropped significantly, but not that of phenol-3,6-dibromophthalein (DBSP). Hepatic dye uptake and storage were not impaired. These results show that hypolipidemic drugs of the peroxisome proliferator type inhibit rat liver glutathione S-transferase activity and may reduce transport of anions conjugated with glutathione before excretion. PMID:3707598

  7. One-dimensional and two-dimensional nuclear magnetic resonance studies of the reaction of phenyldichloroarsine with glutathione

    SciTech Connect

    Dill, K.; Adams, E.R.; O'Connor, R.J.; Chong, S.; McGown, E.L.

    1987-09-01

    /sup 14/C-labeled phenyldichloroarsine (PDA) enters the red blood cell and forms a 1:2 adduct with intracellular glutathione. Upon gel filtration of the hemolysate, (/sup 14/C)PDA was recovered with the glutathione-containing fractions. One-dimensional and two-dimensional nuclear magnetic resonance spectroscopy were used to confirm the structure of the adduct and elucidate its stereochemistry, stability, and reactivity.

  8. Complementary DNA cloning, messenger RNA expression, and induction of alpha-class glutathione S-transferases in mouse tissues.

    PubMed

    Buetler, T M; Eaton, D L

    1992-01-15

    Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S-transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed. PMID:1728405

  9. Hepatic glutathione metabolism and lipid peroxidation in response to excess dietary selenomethionine and selenite in mallard ducklings

    USGS Publications Warehouse

    Hoffman, D.J.; Heinz, G.H.; Krynitsky, A.J.

    1989-01-01

    Selenium from selenomethionine accumulated in a dose-dependent manner in the liver, resulting in a decrease in hepatic-reduced glutathione with a corresponding decrease in total hepatic thiols. There was a dose-dependent increase in the oxidized to reduced glutathione ratio, and an increase in lipid peroxidation. These findings indicate that Se in the diet at 10 ppm and higher causes significant sublethal alterations in mallard ducklings, and 20-40 ppm causes significant hepatotoxicity.

  10. Glutathione modulation during sensitization as well as challenge phase regulates airway reactivity and inflammation in mouse model of allergic asthma.

    PubMed

    Nadeem, Ahmed; Siddiqui, Nahid; Alharbi, Naif O; Alharbi, Mohammad M; Imam, Faisal; Sayed-Ahmed, Mohamed M

    2014-08-01

    Glutathione, being a major intracellular redox regulator has been shown to be implicated in regulation of airway reactivity and inflammation. However, no study so far has investigated the effect of glutathione depletion/repletion during sensitization and challenge phases separately, which could provide an important insight into the pathophysiology of allergic asthma. The aim of the present study was to evaluate the role of glutathione depletion/repletion during sensitization and challenge phases separately in a mouse model of allergic asthma. Buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase or N-acetyl cysteine (NAC), a thiol donor were used for depletion or repletion of glutathione levels respectively during both sensitization and challenge phases separately followed by assessment of airway reactivity, inflammation and oxidant-antioxidant balance in allergic mice. Depletion of glutathione with BSO during sensitization as well as challenge phase worsened allergen induced airway reactivity/inflammation and caused greater oxidant-antioxidant imbalance as reflected by increased NADPH oxidase expression/reactive oxygen species (ROS) generation/lipid peroxides formation and decreased total antioxidant capacity. On the other hand, repletion of glutathione pool by NAC during sensitization and challenge phases counteracted allergen induced airway reactivity/inflammation and restored oxidant-antioxidant balance through a decrease in NADPH oxidase expression/ROS generation/lipid peroxides formation and increase in total antioxidant capacity. Taken together, these findings suggest that depletion or repletion of glutathione exacerbates or ameliorates allergic asthma respectively by regulation of airway oxidant-antioxidant balance. This might have implications towards increased predisposition to allergy by glutathione depleting environmental pollutants. PMID:24742380

  11. The role of mitochondrial phospholipid hydroperoxide glutathione peroxidase in cancer therapy

    NASA Astrophysics Data System (ADS)

    Wang, Hong

    Phospholipid hydroperoxide glutathione peroxidase (PhGPx) is a unique selenoenzyme that directly detoxifies lipid hydroperoxides in situ . It therefore plays an important role in the protection of cellular membranes. PhGPx is expressed in most mammalian tissues. It is present as a mitochondrial form (L-PhGPx) and a cytosolic form (S-PhGPx). Overexpression of PhGPx has been shown to significantly protect cells from oxidative damage. The hypothesis of this thesis is that mitochondrial PhGPx (L-PhGPx) may play an important role in the resistance of cells to certain oxidative stress- mediated cancer therapies. A human breast carcinoma MCF-7 cell line was used as a cell model system in this research. It was stably transfected with human L-PhGPx sense cDNA. Four clones (P-1, P-2, P-3, and P-4) with 3- to 7-fold increases in PhGPx activity were selected for study. Overexpression of L-PhGPx did not significantly influence other cellular antioxidants, including superoxide dismutases, cytosolic glutathione peroxidase, catalase, glutathione reductase, and glutathione. However, L-PhGPx did decrease the rate of cell growth. Cell plating efficiency was inversely correlated with effective PhGPx activity, which is defined as the product of cellular PhGPx activity and total glutathione. The biological functions of L-PhGPx have been investigated in cancer treatment, including photodynamic therapy (PDT) and hyperthermia (HT). Both PDT and HT can induce oxidative stress. Overexpression of L-PhGPx in MCF-7 cells significantly increased the resistance of cells to PDT- and HT-mediated cytotoxicity. The effective PhGPx activity had a remarkable inverse linear correlation (r = -0.80) to the rate of removal of lipid hydroperoxides in living cells, and correlated positively with cell survival after photooxidation (r = 0.91). L-PhGPx protected mitochondrial function by preserving the mitochondrial membrane potential. These data demonstrate that L-PhGPx provides significant protection against

  12. Determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection.

    PubMed

    Janes, Lucija; Lisjak, Klemen; Vanzo, Andreja

    2010-08-01

    A modified preparation of sample was developed for the determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection, using on-line pre-column derivatization. Ice-cold deoxygenated methanol was used to deactivate the oxidation enzymes in juices or wines and keep the glutathione stable. The optimum recovery of glutathione content in grape juice and wine was obtained when either the sample of grape juice or wine was mixed in ice-cold deoxygenated methanol in the ratio 10:90 (v:v) and further diluted in sodium acetate buffer in the ratio 1:1 (v:v). The optimized method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation data the method is appropriate for the determination of glutathione content in grape juice and wine. Glutathione contents in grape juices made from White Muscat grapes and Sauvignon Blanc wines were analysed. The average glutathione content in 28 young Sauvignon Blanc wines was 12.5 mg L(-1). PMID:20678636

  13. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis.

    PubMed Central

    Godwin, A K; Meister, A; O'Dwyer, P J; Huang, C S; Hamilton, T C; Anderson, M E

    1992-01-01

    Exposure of human ovarian tumor cell lines to cisplatin led to development of cell lines that exhibited increasing degrees of drug resistance, which were closely correlated with increase of the levels of cellular glutathione. Cell lines were obtained that showed 30- to 1000-fold increases in resistance; these cells also had strikingly increased (13- to 50-fold) levels of glutathione as compared with the drug-sensitive cells of origin. These levels of resistance to cisplatin and the cellular glutathione levels are substantially greater than previously reported. Very high cisplatin resistance was associated with enhanced expression of mRNAs for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase; immunoblots showed increase of gamma-glutamylcysteine synthetase but not of glutathione synthetase. Glutathione S-transferase activity was unaffected, as determined with chlorodinitrobenzene as a substrate. These studies suggest the potential value of examining regulation of glutathione synthesis as an indicator of clinical prognosis. The highly resistant cell lines are proving useful for studying the multiple mechanisms by which tumor cells acquire drug- and radiation-resistance. Images PMID:1348364

  14. Carbon dot cluster as an efficient "off-on" fluorescent probe to detect Au(III) and glutathione.

    PubMed

    Gu, Jiangjiang; Hu, Donghua; Wang, Weina; Zhang, Qiuhong; Meng, Zhen; Jia, Xudong; Xi, Kai

    2015-06-15

    In this paper, we reported for the first time that Au(III) decorated carbon dot cluster (Au(III)/CDC) was synthesized to detect glutathione through fluorescence "off-on" approach. The "off" process was realized by the introduction of Au(III) on luminescent carbon dots (CDs), which formed the complex of Au(III)/CDC and quenched the fluorescence of CDs efficiently. This "off" process was used to detect Au(III) with the selectivity among 21 metal ions and the limitation was 0.48 μM (S/N=3). Au(III) could be removed from the complex by biothiol in the solution, which restored the fluorescence of CDC to achieve the "on" process. This process was selective for biothiols (especially for glutathione) among saccharides, dopamine and amino acids and the limit of detection was 2.02 μM (S/N=3). Due to the dependence of the fluorescence restoration on the concentration of glutathione, Au(III)/CDC was applied as the fluorescence sensor for detection of glutathione in the solution and cellular cytosol. By referring to the fluorescence change in the solution, the intracellular glutathione with/without oxygen stress was evaluated. As compared with the commercial assay, our Au(III)/CDC based assay was simple, facile and low cost, which would be useful to measure intracellular glutathione at different cellular states. PMID:25558871

  15. Effects of cyanobacterial lipopolysaccharides from microcystis on glutathione-based detoxification pathways in the zebrafish (Danio rerio) embryo.

    PubMed

    Jaja-Chimedza, Asha; Gantar, Miroslav; Mayer, Gregory D; Gibbs, Patrick D L; Berry, John P

    2012-06-01

    Cyanobacteria ("blue-green algae") are recognized producers of a diverse array of toxic secondary metabolites. Of these, the lipopolysaccharides (LPS), produced by all cyanobacteria, remain to be well investigated. In the current study, we specifically employed the zebrafish (Danio rerio) embryo to investigate the effects of LPS from geographically diverse strains of the widespread cyanobacterial genus, Microcystis, on several detoxifying enzymes/pathways, including glutathione-S-transferase (GST), glutathione peroxidase (GPx)/glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), and compared observed effects to those of heterotrophic bacterial (i.e., E. coli) LPS. In agreement with previous studies, cyanobacterial LPS significantly reduced GST in embryos exposed to LPS in all treatments. In contrast, GPx moderately increased in embryos exposed to LPS, with no effect on reciprocal GR activity. Interestingly, total glutathione levels were elevated in embryos exposed to Microcystis LPS, but the relative levels of reduced and oxidized glutathione (i.e., GSH/GSSG) were, likewise, elevated suggesting that oxidative stress is not involved in the observed effects as typical of heterotrophic bacterial LPS in mammalian systems. In further support of this, no effect was observed with respect to CAT or SOD activity. These findings demonstrate that Microcystis LPS affects glutathione-based detoxification pathways in the zebrafish embryo, and more generally, that this model is well suited for investigating the apparent toxicophore of cyanobacterial LPS, including possible differences in structure-activity relationships between heterotrophic and cyanobacterial LPS, and teleost fish versus mammalian systems. PMID:22822454

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

    PubMed Central

    Van der Aar, E M; Bouwman, T; Commandeur, J N; Vermeulen, N P

    1996-01-01

    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. PMID:8973562

  17. Deficient synthesis of glutathione underlies oxidative stress in aging and can be corrected by dietary cysteine and glycine supplementation1234

    PubMed Central

    Patel, Sanjeet G; Guthikonda, Anuradha P; Reid, Marvin; Balasubramanyam, Ashok; Taffet, George E; Jahoor, Farook

    2011-01-01

    Background: Aging is associated with oxidative stress, but underlying mechanisms remain poorly understood. Objective: We tested whether glutathione deficiency occurs because of diminished synthesis and contributes to oxidative stress in aging and whether stimulating glutathione synthesis with its precursors cysteine and glycine could alleviate oxidative stress. Design: Eight elderly and 8 younger subjects received stable-isotope infusions of [2H2]glycine, after which red blood cell (RBC) glutathione synthesis and concentrations, plasma oxidative stress, and markers of oxidant damage (eg, F2-isoprostanes) were measured. Elderly subjects were restudied after 2 wk of glutathione precursor supplementation. Results: Compared with younger control subjects, elderly subjects had markedly lower RBC concentrations of glycine (486.7 ± 28.3 compared with 218.0 ± 23.7 μmol/L; P < 0.01), cysteine (26.2 ± 1.4 compared with 19.8 ± 1.3 μmol/L; P < 0.05), and glutathione (2.08 ± 0.12 compared with 1.12 ± 0.18 mmol/L RBCs; P < 0.05); lower glutathione fractional (83.14 ± 6.43% compared with 45.80 ± 5.69%/d; P < 0.01) and absolute (1.73 ± 0.16 compared with 0.55 ± 0.12 mmol/L RBCs per day; P < 0.01) synthesis rates; and higher plasma oxidative stress (304 ± 16 compared with 346 ± 20 Carratelli units; P < 0.05) and plasma F2-isoprostanes (97.7 ± 8.3 compared with 136.3 ± 11.3 pg/mL; P < 0.05). Precursor supplementation in elderly subjects led to a 94.6% higher glutathione concentration, a 78.8% higher fractional synthesis rate, a 230.9% higher absolute synthesis rate, and significantly lower plasma oxidative stress and F2-isoprostanes. No differences in these measures were observed between younger subjects and supplemented elderly subjects. Conclusions: Glutathione deficiency in elderly humans occurs because of a marked reduction in synthesis. Dietary supplementation with the glutathione precursors cysteine and glycine fully restores glutathione synthesis and

  18. Defining the cytosolic pathway of glutathione degradation in Arabidopsis thaliana: role of the ChaC/GCG family of γ-glutamyl cyclotransferases as glutathione-degrading enzymes and AtLAP1 as the Cys-Gly peptidase.

    PubMed

    Kumar, Shailesh; Kaur, Amandeep; Chattopadhyay, Banani; Bachhawat, Anand K

    2015-05-15

    Glutathione homoeostasis is critical to plant life and its adaptation to stress. The γ-glutamyl cycle of glutathione biosynthesis and degradation plays a pre-eminent role in glutathione homoeostasis. The genes encoding two enzymatic steps of glutathione degradation, the γ-glutamyl cyclotransferase (GGCT; acting on γ-glutamyl amino acids) and the Cys-Gly dipeptidase, have, however, lacked identification. We have investigated the family of GGCTs in Arabidopsis thaliana. We show through in vivo functional assays in yeast that all three members of the ChaC/GCG subfamily show significant activity towards glutathione but no detectable activity towards γ-glutamyl methionine. Biochemical characterization of the purified recombinant enzymes GGCT2;2 and GGCT2;3 further confirmed that they act specifically to degrade glutathione to yield 5-oxoproline and Cys-Gly peptide and show no significant activity towards γ-glutamyl cysteine. The Km for glutathione was 1.7 and 4.96 mM for GGCT2;2 and GGCT2;3 respectively and was physiologically relevant. Evaluation of representative members of other subfamilies indicates the absence of GGCTs from plants showing significant activity towards γ-glutamyl-amino acids as envisaged in the classical γ-glutamyl cycle. To identify the Cys-Gly peptidase, we evaluated leucine aminopeptidases (LAPs) as candidate enzymes. The cytosolic AtLAP1 (A. thaliana leucine aminopeptidase 1) and the putative chloroplastic AtLAP3 displayed activity towards Cys-Gly peptide through in vivo functional assays in yeast. Biochemical characterization of the in vitro purified hexameric AtLAP1 enzyme revealed a Km for Cys-Gly of 1.3 mM that was physiologically relevant and indicated that AtLAP1 represents a cytosolic Cys-Gly peptidase activity of A. thaliana. The studies provide new insights into the functioning of the γ-glutamyl cycle in plants. PMID:25716890

  19. Co-variation of glutathione transferase expression and cytostatic drug resistance in HeLa cells: establishment of class Mu glutathione transferase M3-3 as the dominating isoenzyme.

    PubMed Central

    Hao, X Y; Widersten, M; Ridderström, M; Hellman, U; Mannervik, B

    1994-01-01

    Qualitative and quantitative analyses of glutathione, glutathione transferases (GSTs) and other glutathione-linked enzymes in HeLa cells have been made in order to study their significance in cellular resistance to electrophilic cytotoxic agents. The cytosolic concentrations of three GSTs, GST M1-1 (53 +/- 9 ng/mg of cytosolic protein), GST P1-1 (11 +/- 3 ng/mg) and GST A1-1 (1.1 +/- 0.4 ng/mg) were quantified by isoenzyme-specific enzyme-linked immunoassays. Electrophoretic analysis and immunoblotting demonstrated another component, GST M3-3, which was identified by amino acid sequence analysis. GST M3-3 was quantified (1550 +/- 250 ng/mg) by slot-blot immunoanalysis and was the most abundant GST in HeLa cells. An additional cytosolic 13 kDa protein with high affinity for immobilized glutathione or S-hexyglutathione was found to be identical with a macrophage migration-inhibitory factor, previously identified as a lymphokine. Cells grown in roller bottles (HR) rather than in ordinary culture flasks contain a significantly lower concentration of all the GSTs and were found to be more sensitive to the cytostatic agents doxorubicin (2.3-fold), cisplatin (1.7-fold) and melphalan (1.4-fold). The cytosolic concentrations of glutathione reductase and glyoxalase I were also lower in HR cells, whereas the total glutathione concentration was unchanged and the glutathione peroxidase activity was increased. The results indicate that GSTs contribute to the cellular resistance phenotype. Images Figure 1 Figure 2 Figure 4 PMID:8280111

  20. Glutathione conjugates recognize the Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Puder, M; Soberman, R J

    1997-04-18

    Leukotriene (LT) C4 and other glutathione conjugates are synthesized intracellularly and then move to the plasma membrane for export. The intracellular proteins that bind these molecules and the significance of these interactions are poorly understood. To identify the binding sites of membrane-associated proteins that recognize these molecules, we utilized photoaffinity probes to label the inner leaflet of erythrocytes. The predominant molecule labeled with S-(p-nitrobenzyl)glutathione-[125I]4-azidosalicylic acid (PNBG-[125I]ASA) or LTC4-[125I]4-azidosalicylic acid (LTC4-[125I]ASA) was 38 kDa. The protein was labeled with PNBG-[125I]ASA, electroblotted to polyvinylidene difluoride membranes, digested in situ with lysyl endopeptidase, and two radiolabeled peptides isolated by reverse phase-high performance liquid chromatography. These contained an identity of 7/11 with amino acids 119-129, and 11/11 with amino acids 67-77 of human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Photoaffinity labeling with PNBG-[125I]ASA was blocked completely by 100 microM ATP and greater than 50% with 100 microM NAD+. LTC4-[125I]ASA binding to the NAD+ site was confirmed by V8 protease digestion of purified GAPDH labeled with LTC4-[125I]ASA or PNBG-[125I]ASA, with both labels localized to the 6.8-kDa N-terminal fragment. Photoaffinity labeling of HL-60 cells with LTC4-125I-ASA identified GAPDH as the predominant cytoplasmic binding protein in these cells. These data indicate that GAPDH is a membrane-associated and cytoplasmic protein which binds glutathione conjugates including LTC4. PMID:9099752

  1. A biophysically based mathematical model for the catalytic mechanism of glutathione reductase.

    PubMed

    Pannala, Venkat R; Bazil, Jason N; Camara, Amadou K S; Dash, Ranjan K

    2013-12-01

    Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as the reducing cofactor, and thereby maintains a constant GSH level in the system. GSH scavenges superoxide (O2(*-)) and hydroxyl radicals (OH) nonenzymatically or by serving as an electron donor to several enzymes involved in reactive oxygen species (ROS) detoxification. In either case, GSH oxidizes to GSSG and is subsequently regenerated by the catalytic action of GR. Although the GR kinetic mechanism has been extensively studied under various experimental conditions with variable substrates and products, the catalytic mechanism has not been studied in terms of a mechanistic model that accounts for the effects of the substrates and products on the reaction kinetics. The aim of this study is therefore to develop a comprehensive mathematical model for the catalytic mechanism of GR. We use available experimental data on GR kinetics from various species/sources to develop the mathematical model and estimate the associated model parameters. The model simulations are consistent with the experimental observation that GR operates via both ping-pong and sequential branching mechanisms based on relevant concentrations of its reaction substrate GSSG. Furthermore, we show the observed pH-dependent substrate inhibition of GR activity by GSSG and bimodal behavior of GR activity with pH. The model presents a unique opportunity to understand the effects of products on the kinetics of GR. The model simulations show that under physiological conditions, where both substrates and products are present, the flux distribution depends on the concentrations of both GSSG and NADP(+), with ping-pong flux operating at low levels and sequential flux dominating at higher levels. The kinetic model of GR may serve as a key module for the development of integrated models for ROS-scavenging systems to understand protection of cells under normal and oxidative stress

  2. Rapid method for glutathione quantitation using high-performance liquid chromatography with coulometric electrochemical detection.

    PubMed

    Bayram, Banu; Rimbach, Gerald; Frank, Jan; Esatbeyoglu, Tuba

    2014-01-15

    A rapid, sensitive, and direct method (without derivatization) was developed for the detection of reduced glutathione (GSH) in cultured hepatocytes (HepG2 cells) using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The method was validated according to the guidelines of the U.S. Food and Drug Administration in terms of linearity, lower limit of quantitation (LOQ), lower limit of detection (LOD), precision, accuracy, recovery, and stabilities of GSH standards and quality control samples. The total analysis time was 5 min, and the retention time of GSH was 1.78 min. Separation was carried out isocratically using 50 mM sodium phosphate (pH 3.0) as a mobile phase with a fused-core column. The detector response was linear between 0.01 and 80 μmol/L, and the regression coefficient (R(2)) was >0.99. The LOD for GSH was 15 fmol, and the intra- and interday recoveries ranged between 100.7 and 104.6%. This method also enabled the rapid detection (in 4 min) of other compounds involved in GSH metabolism such as uric acid, ascorbic acid, and glutathione disulfite. The optimized and validated HPLC-ECD method was successfully applied for the determination of GSH levels in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor, and α-lipoic acid (α-LA), an inducer of GSH synthesis. As expected, the amount of GSH concentration-dependently decreased with BSO and increased with α-LA treatments in HepG2 cells. This method could also be useful for the quantitation of GSH, uric acid, ascorbic acid, and glutathione disulfide in other biological matrices such as tissue homogenates and blood. PMID:24328299

  3. Comparison of human liver and small intestinal glutathione S-transferase-catalyzed busulfan conjugation in vitro.

    PubMed

    Gibbs, J P; Yang, J S; Slattery, J T

    1998-01-01

    The apparent oral clearance of busulfan has been observed to vary as much as 10-fold in the population of children and adults receiving high-dose busulfan. The only identified elimination pathway for busulfan involves glutathione conjugation. The reaction is predominantly catalyzed by glutathione S-transferase (GST) A1-1, which is present in both liver and intestine. The purpose of this study was to compare busulfan Vmax/Km in cytosol prepared from adult human liver and small intestine. Tetrahydrothiophenium ion formation rate per milligram of cytosolic protein was constant along the length (assessed in 30-cm segments) of three individual small intestines. A 30-cm-long intestinal segment 90-180 cm from the pylorus was chosen to be representative of intestinal cytosolic busulfan conjugating activity. Busulfan Vmax/Km (mean +/- SD) in cytosol prepared from 23 livers and 12 small intestines was 0.166 +/- 0.066 and 0.176 +/- 0.085 microl/min/mg cytosolic protein, respectively, in incubations with 5 microM busulfan, 1 mM glutathione, and 2 mg of cytosolic protein. The relative content of GSTalpha (A1-1, A1-2, and A2-2) was compared for human liver and intestinal cytosol using Western blot. The levels of GSTalpha in liver and intestinal cytosol were 1.12 +/- 0.56 and 1.36 +/- 0.32 integrated optimal density units/5 microg cytosolic protein, respectively. Busulfan conjugation in vitro was comparable per milligram of cytosolic protein in liver and intestinal cytosol. PMID:9443852

  4. Glutathione-Responsive Multilayer Coated Gold Nanoparticles for Targeted Gene Delivery.

    PubMed

    Yu, Feifei; Huang, Jingbin; Yu, Yuan; Lu, Ying; Chen, Yan; Zhang, He; Zhou, Guichen; Sun, Zhiguo; Liu, Junjie; Sun, Duxin; Zhang, Guoqing; Zou, Hao; Zhong, Yanqiang

    2016-03-01

    Efficient gene release after intracellular uptake is very important for non-viral gene delivery systems. To construct a glutathione-responsive gene delivery system, we developed gold-cysteamine (AuCM)/plasmid DNA (pDNA)/poly TAT (pTAT)/hyaluronic acid (HA) nanocomplexes (AuCM/pDNA/pTAT/HA) in this study. The AuCM/pDNA/pTAT/HA nanocomplexes possessed a small size less than 200 nm and negative zeta potential of -17 ± 4 mV. The multilayer structure was verified by UV-Vis spectra, surface charges, dynamic light scattering. Morphology was observed by transmission electron microscope. The AuCM/pDNA/pTAT/HA nanocomplexes could completely protect pDNA against enzymatic degradation. These nanocomplexes showed effective cellular uptake in CD44 receptors over-expressed HepG 2 cells in a HA/CD44 interaction dependent manner. Moreover, transfection efficacy was significantly enhanced in AuCM/pDNA/pTAT/HA treated HepG 2 cells compared with AuCM/pDNA/pTAT, and was further enhanced in the presence of GSH, indicating that AuCM/pDNA/pTAT/HA was glutathione-responsive. Biodistribution revealed that AuCM/pDNA/pTAT/HA nanocomplexes mainly accumulated in liver. In conclusion, AuCM/pDNA/pTAT/HA nanocomplexes may serve as glutathione-responsive gene carriers for actively targeting gene delivery to CD44 receptors over-expressed liver cancers. PMID:27280248

  5. Glutathione enzyme and selenoprotein polymorphisms associate with mercury biomarker levels in Michigan dental professionals.

    PubMed

    Goodrich, Jaclyn M; Wang, Yi; Gillespie, Brenda; Werner, Robert; Franzblau, Alfred; Basu, Niladri

    2011-12-01

    Mercury is a potent toxicant of concern to both the general public and occupationally exposed workers (e.g., dentists). Recent studies suggest that several genes mediating the toxicokinetics of mercury are polymorphic in humans and may influence inter-individual variability in mercury accumulation. This work hypothesizes that polymorphisms in key glutathione synthesizing enzyme, glutathione S-transferase, and selenoprotein genes underlie inter-individual differences in mercury body burden as assessed by analytical mercury measurement in urine and hair, biomarkers of elemental mercury and methylmercury, respectively. Urine and hair samples were collected from a population of dental professionals (n=515), and total mercury content was measured. Average urine (1.06±1.24 microg/L) and hair mercury levels (0.49±0.63 microg/g) were similar to national U.S. population averages. Taqman assays were used to genotype DNA from buccal swab samples at 15 polymorphic sites in genes implicated in mercury metabolism. Linear regression modeling assessed the ability of polymorphisms to modify the relationship between mercury biomarker levels and exposure sources (e.g., amalgams, fish consumption). Five polymorphisms were significantly associated with urine mercury levels (GSTT1 deletion), hair mercury levels (GSTP1-105, GSTP1-114, GSS 5'), or both (SEPP1 3'UTR). Overall, this study suggests that polymorphisms in selenoproteins and glutathione-related genes may influence elimination of mercury in the urine and hair or mercury retention following exposures to elemental mercury (via dental amalgams) and methylmercury (via fish consumption). PMID:21967774

  6. Multiple isoforms of mitochondrial glutathione S-transferases and their differential induction under oxidative stress.

    PubMed Central

    Raza, Haider; Robin, Marie-Anne; Fang, Ji-Kang; Avadhani, Narayan G

    2002-01-01

    The mitochondrial respiratory chain, which consumes approx. 85-90% of the oxygen utilized by cells, is a major source of reactive oxygen species (ROS). Mitochondrial genetic and biosynthetic systems are highly susceptible to ROS toxicity. Intramitochondrial glutathione (GSH) is a major defence against ROS. In the present study, we have investigated the nature of the glutathione S-transferase (GST) pool in mouse liver mitochondria, and have purified three distinct forms of GST: GSTA1-1 and GSTA4-4 of the Alpha family, and GSTM1-1 belonging to the Mu family. The mitochondrial localization of these multiple GSTs was confirmed using a combination of immunoblot analysis, protease protection assay, enzyme activity, N-terminal amino acid sequencing, peptide mapping and confocal immunofluorescence analysis. Additionally, exogenously added 4-hydroxynonenal (HNE), a reactive byproduct of lipid peroxidation, to COS cells differentially affected the cytosolic and mitochondrial GSH pools in a dose- and time-dependent manner. Our results show that HNE-mediated mitochondrial oxidative stress caused a decrease in the GSH pool, increased membrane lipid peroxidation, and increased levels of GSTs, glutathione peroxidase and Hsp70 (heat-shock protein 70). The HNE-induced oxidative stress persisted for longer in the mitochondrial compartment, where the recovery of GSH pool was slower than in the cytosolic compartment. Our study, for the first time, demonstrates the presence in mitochondria of multiple forms of GSTs that show molecular properties similar to those of their cytosolic counterparts. Our results suggest that mitochondrial GSTs may play an important role in defence against chemical and oxidative stress. PMID:12020353

  7. The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione.

    PubMed

    Seres, T; Knickelbein, R G; Warshaw, J B; Johnston, R B

    2000-09-15

    During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage. PMID:10975851

  8. Induction of mutagenic DNA damage by chromium(VI) and glutathione

    SciTech Connect

    Liu, Shaojun; Dixon, K.

    1996-12-31

    Certain chromium (Cr) compounds are known to be carcinogenic in humans and mutagenic in cell culture. However, the mechanism of Cr mutagenesis is not well understood. It appears that intracellular reduction of Cr by agents such as glutathione plays a role in the induction of DNA damage. We have used a simian virus 40-based shuttle vector to investigate the relationship between chromium-induced DNA damage and Cr mutagenicity. The treatment of the plasmid pZ189 with Cr(VI) plus glutathione (GSH) induced DNA strand breaks and reduced the plasmid biological activity, whereas Cr(III) treatment with or without GSH did not give rise to such DNA damage. When Cr(VI)/GSH-or Cr(III)/GSH-treated pZ189 was replicated in mammalian cells, a dose-dependent increase in mutant frequency was observed with Cr(VI)/GSH-treated pZ189, but not with Cr(III)/GSH-treated plasmid. About 43% of the mutants from Cr(VI)/GSH-treated pZ189 were deletion mutants. The remainder were base substitution mutants, mostly GC {r_arrow} AT transitions and GC {r_arrow} TA transversion. This pattern of mutagenesis is similar to that observed with other agents that cause oxidative DNA damage such as ionizing radiation and H{sub 2}O{sub 2}. These results support the hypothesis that Cr mutagenesis can be induced by the generation of reactive oxygen intermediates during the reduction of Cr(VI) by glutathione. 45 refs., 4 figs., 4 tabs.

  9. The role of the glutathione system in seizures induced by diphenyl diselenide in rat pups.

    PubMed

    Prigol, Marina; Brüning, César Augusto; Nogueira, Cristina W; Zeni, Gilson

    2011-08-15

    The present study investigated the role of the glutathione system in seizures induced by diphenyl diselenide (PhSe)(2) (50 mg/kg) in rat pups (post natal day, 12-14). Reduced glutathione (GSH) (300 nmol/site; i.c.v.), administered 20 min before (PhSe)(2), abolished the appearance of seizures, protected against the inhibition of catalase and δ-aminolevulinic dehydratase (δ-ALA-D) activities and increased glutathione peroxidase (GPx) activity induced by (PhSe)(2). Administration of l-buthionine sulfoximine (BSO, a GSH-depleting compound) (3.2 μmol/site; i.c.v.) 24h before (PhSe)(2) increased the percentage (42-100%) of rat pups which had seizure episodes, reduced the onset for the first convulsive episode. In addition, BSO increased thiobarbituric acid reactive species (TBARS) levels and decreased GSH content, catalase, δ-ALA-D and Na(+), K(+)-ATPase activities. Treatment with sub effective doses of GSH (10 nmol/site) and d-2-amino-7-phosphonoheptanoic acid (AP-7, an antagonist of the glutamate site at the NMDA receptor; 5mg/kg, i.p.) abolished the appearance of seizures induced by (PhSe)(2) in rat pups. Sub effective doses of GSH and kynurenic acid (an antagonist of strychnine-insensitive glycine site at the NMDA receptor; 40 mg/kg, i.p.) were also able in abolishing the appearance of seizures induced by (PhSe)(2). In conclusion, administration of GSH protected against seizure episodes induced by (PhSe)(2) in rat pups by reducing oxidative stress and, at least in part, by acting as an antagonist of glutamate and glycine modulatory sites in the NMDA receptor. PMID:21620807

  10. The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase

    PubMed Central

    Jiang, Li-Juan; Maret, Wolfgang; Vallee, Bert L.

    1998-01-01

    The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC 1.1.1.14) in vitro has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in this process. Reduced glutathione (GSH) and glutathione disulfide (GSSG) are critical modulators of both the rate of zinc transfer and the ultimate number of zinc atoms transferred. GSSG increases the rate of zinc transfer 3-fold, and its concentration is the major determinant for efficient zinc transfer. GSH has a dual function. In the absence of GSSG, it inhibits zinc transfer from MT, indicating that MT is in a latent state under the relatively high cellular concentrations of GSH. In addition, it primes MT for the reaction with GSSG by enhancing the rate of zinc transfer 10-fold and by increasing the number of zinc atoms transferred to four. 65Zn-labeling experiments confirm the release of one zinc from MT in the absence of glutathione and the more effective release of zinc in the presence of GSH and GSSG. In vivo, MT may keep the cellular concentrations of free zinc very low and, acting as a temporary cellular reservoir, release zinc in a process that is dynamically controlled by its interactions with both GSH and GSSG. These results suggest that a change of the redox state of the cell could serve as a driving force and signal for zinc distribution from MT. PMID:9520392

  11. The DinB Superfamily Includes Novel Mycothiol, Bacillithiol and Glutathione S-transferases

    PubMed Central

    Newton, Gerald L.; Leung, Stephan S.; Wakabayashi, Judy I.; Rawat, Mamta; Fahey, Robert C.

    2011-01-01

    The superfamily of glutathione S-transferases has been the subject of extensive study but Actinobacteria produce mycothiol (MSH) in place of glutathione and no mycothiol S-transferase (MST) has been identified. Using mycothiol and monochlorobimane as substrates a MST activity was detected in extracts of Mycobacterium smegmatis and purified sufficiently to allow identification of MSMEG_0887, a member the DUF664 family of the DinB superfamily, as the MST. The identity of the M. smegmatis and homologous Mycobacterium tuberculosis (Rv0443) enzymes was confirmed by cloning and the expressed proteins were found to be active with MSH but not bacillithiol (BSH) or glutathione (GSH). Bacillus subtilis YfiT is another member of the DinB superfamily but this bacterium produces BSH. The YfiT protein was shown to have S-transferase activity with monochlorobimane when assayed with BSH but not with MSH or GSH. Enterococcus faecalis EF_3021 shares some homology with MSMEG_0887 but this organism produces GSH but not MSH or BSH. Cloned and expressed EF_0321 was active with monochlorobimane and GSH but not with MSH or BSH. MDMPI_2 is another member of the DinB superfamily and has been previously shown to have mycothiol-dependent maleylpyruvate isomerase activity. Three of the eight families of the DinB superfamily include proteins shown to catalyze thiol-dependent metabolic or detoxification activities. Since more than two-thirds of the sequences assigned to the DinB superfamily are members of these families it seems likely that such activity is dominant in the DinB superfamily. PMID:22059487

  12. Effect of glutathione S-transferase M1 polymorphisms on biomarkers of exposure and effects.

    PubMed Central

    Srám, R J

    1998-01-01

    Genotypes responsible for interindividual differences in ability to activate or detoxify genotoxic agents are recognized as biomarkers of susceptibility. Among the most studied genotypes are human glutathione transferases. The relationship of genetic susceptibility to biomarkers of exposure and effects was studied especially in relation to the genetic polymorphism of glutathione S-transferase M1 (GSTM1). For this review papers reporting the effect of GSTM1 genotype on DNA adducts, protein adducts, urine mutagenicity, Comet assay parameters, chromosomal aberrations, sister chromatid exchanges (SCE), micronuclei, and hypoxanthine-guanine phosphoribosyl transferase mutations were assessed. Subjects in groups occupationally exposed to polycyclic aromatic hydrocarbons, benzidine, pesticides, and 1,3-butadiene were included. As environmentally exposed populations, autopsy donors, coal tar-treated patients, smokers, nonsmokers, mothers, postal workers, and firefighters were followed. From all biomarkers the effect of GSTM1 and N-acetyl transferase 2 was seen in coke oven workers on mutagenicity of urine and of glutathione S-transferase T1 on the chromosomal aberrations in subjects from 1,3-butadiene monomer production units. Effects of genotypes on DNA adducts were found from lung tissue of autopsy donors and from placentas of mothers living in an air-polluted region. The GSTM1 genotype affected mutagenicity of urine in smokers and subjects from polluted regions, protein adducts in smokers, SCE in smokers and nonsmokers, and Comet assay parameters in postal workers. A review of all studies on GSTM1 polymorphisms suggests that research probably has not reached the stage where results can be interpreted to formulate preventive measures. The relationship between genotypes and biomarkers of exposure and effects may provide an important guide to the risk assessment of human exposure to mutagens and carcinogens. PMID:9539016

  13. Cyclophosphamide-induced apoptosis in COV434 human granulosa cells involves oxidative stress and glutathione depletion.

    PubMed

    Tsai-Turton, Miyun; Luong, Brian T; Tan, Youming; Luderer, Ulrike

    2007-07-01

    The anticancer drug cyclophosphamide induces granulosa cell apoptosis and is detoxified by glutathione (GSH) conjugation. We previously showed that both cyclophosphamide treatment and GSH depletion induced granulosa cell apoptosis in rats, but the role of GSH in apoptosis in human ovarian cells has not been studied. Using the COV434 human granulosa cell line, we tested the hypotheses that (1) GSH depletion or treatment with 4-hydroperoxycyclophosphamide (4HC), a preactivated form of cyclophosphamide, induces apoptosis, (2) GSH depletion potentiates 4HC-induced apoptosis, and (3) 4HC-induced apoptosis is mediated by GSH depletion and oxidative stress. Cells were treated with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, with or without follicle stimulating hormone (FSH) or serum. A significant increase in the number of apoptotic cells, assessed by terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate nick-end labeling (TUNEL) and Hoechst 33342 staining, occurred with BSO treatment. Treatment with 4HC dose-dependently induced apoptosis by TUNEL, Hoechst staining, and caspase 3 activation. Treatment with 4HC caused an increase in reactive oxygen species generation, measured by dichlorofluorescein fluorescence, oxidative DNA damage, measured by 8-hydroxyguanosine immunostaining, and an oxidation of the redox potential for the oxidized glutathione/reduced glutathione couple. Total intracellular GSH declined after 4HC treatment, preceding the onset of cell death. Treatment with antioxidants inhibited 4HC-induced apoptosis. Combined treatment with BSO and 4HC caused greater induction of apoptosis than either treatment alone. These findings are consistent with roles for oxidative stress and GSH depletion in mediating the induction of apoptosis in COV434 cells by cyclophosphamide. PMID:17434952

  14. High temperature nucleation and growth of glutathione protected ~Ag75 clusters.

    PubMed

    Chakraborty, Indranath; Udayabhaskararao, Thumu; Pradeep, Thalappil

    2012-07-11

    We report the first high temperature solution state synthesis of glutathione (-SG) protected atomically precise silver clusters. Noble metal cluster synthesis from metal ions generally requires ice cold temperatures as they are extremely sensitive and high temperature routes are used only for core reduction methods, starting from nanoparticles. The clusters formed by the new route have distinct features in their absorption profile and they exhibit red luminescence. They are characterised by other spectroscopic and microscopic techniques and a tentative formula of Ag(75)(SG)(40) has been assigned. PMID:22648389

  15. Induction of glutathione-S-transferase activity by antioxidants in hepatocyte culture.

    PubMed

    Chen, L H; Shiau, C C

    1989-01-01

    Twelve male Sprague-Dawley rats were used for the study. Six rats were injected with benzo(a)pyrene (BP); the other six rats served as the control. Twenty-four hours after injection, hepatocytes were isolated and cultured. The cultured plates were divided into 5 groups and treated with absolute ethanol (control), butylated hydroxytoluene, vitamin E, ascorbic acid or vitamin Elascorbic acid. After 48 hours, the hepatocytes were harvested for enzyme activation determination. With both control and BP-injected rats, each antioxidant treatment significantly increased glutathione-S-transferase activity. The results suggest that antioxidants may have a detoxifying effect against BP-induced carcinogenesis. PMID:2817788

  16. Polyamines regulate cell growth and cellular methylglyoxal in high-glucose medium independently of intracellular glutathione.

    PubMed

    Kwak, Min-Kyu; Lee, Mun-Hyoung; Park, Seong-Jun; Shin, Sang-Min; Liu, Rui; Kang, Sa-Ouk

    2016-03-01

    Polyamines can presumably inhibit protein glycation, when associated with the methylglyoxal inevitably produced during glycolysis. Herein, we hypothesized a nonenzymatic interaction between putrescine and methylglyoxal in putrescine-deficient or -overexpressing Dictyostelium cells in high-glucose medium, which can control methylglyoxal production. Putrescine was essentially required for growth rescue accompanying methylglyoxal detoxification when cells underwent growth defect and cell cycle G1-arrest when supplemented with high glucose. Furthermore, methylglyoxal regulation by putrescine seemed to be a parallel pathway independent of the changes in cellular glutathione content in high-glucose medium. Consequently, we suggest that Dictyostelium cells need polyamines for normal growth and cellular methylglyoxal regulation. PMID:26898161

  17. Photoactivation of hypericin down-regulates glutathione S-transferase activity in nasopharyngeal cancer cells.

    PubMed

    Du, H Y; Olivo, M; Tan, B K H; Bay, B H

    2004-04-30

    Photodynamic therapy (PDT) is a new modality of treatment for cancer. Hypericin is a photosensitizer, which is known to generate reactive oxygen species upon activation with light. We observed that photoactivated hypericin induces the generation of reactive oxygen intermediates in nasopharyngeal cancer (NPC) cells in vitro. There was also significant reduction of Glutathione S-transferase (GST) activity in HK1 and CNE-2 NPC cells and in tumor tissues from the NPC/HK1 murine tumor model by hypericin-mediated PDT. As antioxidants protect cells against phototoxicity, down-regulation of GST activity would potentiate the efficacy of hypericin-PDT treatment. PMID:15072826

  18. Biological evaluation of some uracil derivatives as potent glutathione reductase inhibitors

    NASA Astrophysics Data System (ADS)

    Güney, Murat; Ekinci, Deniz; Ćavdar, Huseyin; Şentürk, Murat; Zilbeyaz, Kani

    2016-04-01

    Discovery of glutathione reductase (GR) inhibitors has become very popular recently due to antimalarial and anticancer activities. In this study, GR inhibitory capacities of some uracil derivatives (UDCs) (1-4) were reported. Some commercially available molecules (5-6) were also tested for comparison reasons. The novel UDCs were obtained in high yields using simple chemical procedures and exhibited much potent inhibitory activities against GR at low nanomolar concentrations with IC50 values ranging from 2.68 to 166.6 nM as compared with well-known agents.

  19. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth

    PubMed Central

    Jin, Lingtao; Li, Dan; Alesi, Gina N.; Fan, Jun; Kang, Hee-Bum; Lu, Zhou; Boggon, Titus J.; Jin, Peng; Yi, Hong; Wright, Elizabeth R.; Duong, Duc; Seyfried, Nicholas T.; Egnatchik, Robert; DeBerardinis, Ralph J.; Magliocca, Kelly R.; He, Chuan; Arellano, Martha L.; Khoury, Hanna J.; Shin, Dong M.; Khuri, Fadlo R.; Kang, Sumin

    2015-01-01

    SUMMARY How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) is commonly upregulated in human cancers. GDH1 is important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate (α-KG) and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a ROS scavenging enzyme glutathione peroxidase 1 (GPx1). Targeting GDH1 by shRNA or a small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth. PMID:25670081

  20. LPS alters pattern of sickness behavior but does not affect glutathione level in aged male rats.

    PubMed

    Wrotek, Sylwia; Jędrzejewski, Tomasz; Nowakowska, Anna; Kozak, Wiesław

    2016-08-01

    Behavioral symptoms of sickness, such as fever and motor activity are a coordinated set of changes that develop during infection. The aim of study was to compare the sickness behaviour (SB) in healthy old and young rats treated with pyrogenic dose of endotoxin and to check their glutathione level. Before experimentation male Wistar rats were selected according to standard body mass, motor activity, and white blood cells count. Intraperitoneal injection of lipopolysaccharide (LPS) from E. coli was used to provoke SB. The level of liver glutathione, interleukin (IL) -6, deep body temperature (Tb) and motor activity were measured. Glutathione level in old and young rats did not differ significantly. In both young and old rats LPS administration provoked fever (the mean value of Tb was 38.06 ± 0.01 °C in old rats, and 38.19 ± 0.06 °C in young rats). LPS injection affected night-time activity in both groups (12 h averages were 1.56 ± 0.40 counts in old LPS-treated rats vs 2.74 ± 0.53 counts in not-treated old rats and 3.44 ± 0.60 counts for young LPS-treated vs 4.28 ± 0.57 counts for young not-treated rats). The injection of LPS provoked an elevation of plasma IL-6 concentration (from values below the lowest detectable standard in not-treated groups of animals to 6322.82 ± 537.00 pg/mL in old LPS-treated rats and 7415.62 ± 451.88 pg/mL in young LPS-treated rats). Based on these data, we conclude that good health of aged rats prevents decrease in the glutathione level. Old rats are still able to develop SB in response to pyrogenic dose of LPS, although its components have changed pattern compared to young animals. PMID:26829940

  1. Screening of potential cancer preventing chemicals for induction of glutathione in rat liver cells.

    PubMed

    White, E L; Ross, L J; Schmid, S M; Kelloff, G J; Steele, V E; Hill, D L

    1998-01-01

    With BRL 3A hepatocytes, a series of selected, potentially chemopreventive chemicals was evaluated for their capacity to elevate glutathione (GSH) levels. Since sodium selenite consistently increased GSH levels by approximately 70%, it was selected as a positive control. Of 62 test chemicals, eighteen stimulated GSH levels by >30%, but eleven of these had only a modest effect or displayed considerable toxicity. At non-toxic concentrations, seven compounds had substantial activity: black tea extract (decaffeinated), trans-chalcone, N-ethyl-9-cis-retinamide, indole-3-carbinol, dehydroepiandrosterone (DHEA) curcumin and N-(4-carboxyphenyl)retinamide. These should be considered for further development as cancer preventive agents. PMID:9468590

  2. Glutathion S-transferase activity and DDT-susceptibility of Malaysian mosquitos.

    PubMed

    Lee, H L; Chong, W L

    1995-03-01

    Comparative DDT-susceptibility status and glutathion s-transferase (GST) activity of Malaysian Anopheles maculatus, Culex quinquefasciatus and Aedes aegypti was investigated to ascertain the role of this enzyme in DDT resistance. The standardised WHO dose-mortality bioassay tests were used to determine DDT susceptibility in these mosquitos, whilst GST microassay (Brogdon and Barber, 1990) was conducted to measure the activity of this enzyme in mosquito homogenate. It appeared that DDT susceptibility status of Malaysian mosquitos was not correlated with GST activity. PMID:8525405

  3. Preparation, characterization and electrochemical application of Ag-ZnO nanoplates for voltammetric determination of glutathione and tryptophan using modified carbon paste electrode.

    PubMed

    Beitollahi, Hadi; Gholami, Abbas; Ganjali, Mohammad Reza

    2015-12-01

    Ag-ZnO nanoplates and 2-chlorobenzoyl ferrocene were synthesized and used to construct a modified carbon paste electrode. The electrooxidation of glutathione at the surface of the modified electrode was studied. Under the optimized conditions, the square wave voltammetric (SWV) peak current of glutathione increased linearly with glutathione concentrations in the range of 5.0 × 10(-8) to 2.0 × 10(-4)M with sensitivity of 0.659 μA μM(-1) and method detection limit of 20.0 nM was obtained for glutathione. The prepared modified electrode exhibits a very good resolution between the voltammetric peaks of glutathione and tryptophan which makes it suitable for the detection of glutathione in the presence of tryptophan in real samples. PMID:26354245

  4. Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1

    PubMed Central

    2011-01-01

    Background Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast Hansenula polymorpha is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of H. polymorpha DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (GSH2) and its precursor cysteine (MET4) was studied. Results Glutathione producing capacity of H. polymorpha DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSGin, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSGex, per liter of the culture medium. Conclusions H. polymorpha is an competitive glutathione producer as compared to other known yeast and bacteria strains (Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione. PMID:21255454

  5. Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its d,l-polylactide microparticle formulation

    PubMed Central

    Bartolini, D.; Piroddi, M.; Tidei, C.; Giovagnoli, S.; Pietrella, D.; Manevich, Y.; Tew, K.D.; Giustarini, D.; Rossi, R.; Townsend, D.M.; Santi, C.; Galli, F.

    2015-01-01

    Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this “depowered” GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage

  6. 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.

    PubMed Central

    Robinson, Anna; Huttley, Gavin A; Booth, Hilary S; Board, Philip G

    2004-01-01

    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

  7. Effects of protein deficiency and food restriction on lung ascorbic acid and glutathione in rats exposed to ozone

    SciTech Connect

    Dubick, M.A.; Heng, H.; Rucker, R.B.

    1985-08-01

    Weanling (52 +/- 4 g) or adult (259 +/- 16 g) male Sprague-Dawley rats were fed ad libitum casein-based diets containing 4 or 16% protein. A third group (food restricted) was fed daily the 16% protein diet, but at the food intake level of the 4% protein group. After 3 wk (weanling) or 5 wk (adults), half of the rats in each group were continuously exposed to 0.64 ppm ozone for 7 d. Ascorbic acid and reduced glutathione levels were then measured. In the heart and liver from weanling rats, ascorbic acid concentrations were lower in the protein-deficient group than in either control group. In the liver from weanling rats glutathione concentrations were also reduced in response to protein deficiency. Exposure to ozone produced no additional response. For adult rats the response for liver glutathione was similar to that of the weanlings. The liver ascorbate concentration, however, was consistently lower in adult rats compared to weanlings exposed to ozone. In lungs from adult rats, the ascorbic acid concentration was lower in the protein-deficient group than in either control group. On a whole-organ basis, both ascorbic acid and glutathione were usually higher in lungs from rats exposed to ozone than from those exposed to air. Interestingly, protein deficiency did not appear to compromise the lung's ability to maintain, in relative terms, the ascorbic acid or glutathione concentration in response to ozone.

  8. Glutathione depletion impairs transcriptional activation of heat shock genes in primary cultures of guinea pig gastric mucosal cells.

    PubMed

    Rokutan, K; Hirakawa, T; Teshima, S; Honda, S; Kishi, K

    1996-05-15

    When primary cultures of guinea pig gastric mucosal cells were exposed to heat (43 degree C), ethanol, hydrogen peroxide (H2O2), or diamide, heat shock proteins (HSP90, HSP70, HSP60, and HSC73) were rapidly synthesized. The extent of each HSP induction varied with the type of stress. Ethanol, H2O2, and diamide increased the syntheses of several other undefined proteins besides the HSPs. However, none of these proteins were induced by exposure to heat or the reagents, when intracellular glutathione was depleted to <10% of the control level by pretreatment with DL-buthionine-[S,R]-sulfoximine. Gel mobility shift assay using a synthetic oligonucleotide coding HSP70 heat shock element showed that glutathione depletion inhibited the heat- and the reagent-initiated activation of the heat shock factor 1 (HSF1) and did not promote the expression of HSP70 mRNA. Immunoblot analysis with antiserum against HSF1 demonstrated that the steady-state level of HSF1 was not changed in glutathione-depleted cells, but glutathione depletion inhibited the nuclear translocation of HSF1 after exposure to heat stress. These results suggest that intracellular glutathione may support early and important biochemical events in the acquisition by gastric mucosal cells of an adaptive response to irritants. PMID:8636403

  9. Sensory-motor performance after acute glutathione depletion by L-buthionine sulfoximine injection into substantia nigra pars compacta.

    PubMed

    Díaz-Hung, Mei-Li; Blanco, Lisette; Pavón, Nancy; León, Rilda; Estupiñan, Bárbara; Orta, Eduardo; Martínez, Klaudia; Fernández, Isabel

    2014-09-01

    Glutathione is the major antioxidant in the living cells. Its deficit has been linked to neurodegenerative disorders as Parkinson's disease but its role in the etiology of nigral degeneration and sensory-motor performance has been poorly explored. To evaluate the effect of glutathione depletion on nigro-striatal oxidative metabolism and sensory-motor performance in rats, l-buthionine sulfoximine (15 mM) or saline solution was injected into substantia nigra pars compacta (SNpc). Then, oxidative metabolism was studied 24h and 7 days later in SNpc and corpus striatum (CS). Tyrosine hydroxylase and GFAP immunohistochemistry assays were carried out at 7 days. In addition, animals were evaluated in open field, adhesive removal, staircase and traverse beam tests. Glutathione depletion induced compensatory response in catalase activity and glial response in the in SNpc and no oxidative damage was observed. However, a loss in dopaminergic cells was found. At the same time, animals with glutathione depletion have shown poor performance in behavioral tests except for staircase test. These results suggest that glutathione depletion can be related to sensory-motor dysfunction. PMID:24912031

  10. 1,4-Naphthoquinones and Others NADPH-Dependent Glutathione Reductase-Catalyzed Redox Cyclers as Antimalarial Agents

    PubMed Central

    Belorgey, Didier; Lanfranchi, Don Antoine; Davioud-Charvet, Elisabeth

    2013-01-01

    The homodimeric flavoenzyme glutathione reductase catalyzes NADPH-dependent glutathione disulfide reduction. This reaction is important for keeping the redox homeostasis in human cells and in the human pathogen Plasmodium falciparum. Different types of NADPH-dependent disulfide reductase inhibitors were designed in various chemical series to evaluate the impact of each inhibition mode on the propagation of the parasites. Against malaria parasites in cultures the most potent and specific effects were observed for redox-active agents acting as subversive substrates for both glutathione reductases of the Plasmodium-infected red blood cells. In their oxidized form, these redox-active compounds are reduced by NADPH-dependent flavoenzyme-catalyzed reactions in the cytosol of infected erythrocytes. In their reduced forms, these compounds can reduce molecular oxygen to reactive oxygen species, or reduce oxidants like methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Furthermore, studies on a fluorinated suicide-substrate of the human glutathione reductase indicate that the glutathione reductase-catalyzed bioactivation of 3-benzylnaphthoquinones to the corresponding reduced 3-benzoyl metabolites is essential for the observed antimalarial activity. In conclusion, the antimalarial lead naphthoquinones are suggested to perturb the major redox equilibria of the targeted cells. These effects result in development arrest of the parasite and contribute to the removal of the parasitized erythrocytes by macrophages. PMID:23116403

  11. Glutathione transferase from Plasmodium falciparum--interaction with malagashanine and selected plant natural products.

    PubMed

    Mangoyi, Rumbidzai; Hayeshi, Rose; Ngadjui, Bonventure; Ngandeu, Francois; Bezabih, Merhatibebe; Abegaz, Berhanu; Razafimahefa, Solofoniaina; Rasoanaivo, Philippe; Mukanganyama, Stanley

    2010-12-01

    A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC(50) values of 8.5, 12, 50 and 69 μM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The K(i) for GSH and CDNB were -0.015 μM and 0.011 μM, respectively. Malagashanine (100 µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t(1/2) of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5 mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds. PMID:20521884

  12. A simple microfluidic integrated with an optical sensor for micro flow injection colorimetric determination of glutathione.

    PubMed

    Supharoek, Sam-ang; Youngvises, Napaporn; Jakmunee, Jaroon

    2012-01-01

    A simple and inexpensive method for fabricating a microfluidic platform was developed. A printed circuit board (PCB) was used to make a master mold for replicating a polydimethylsiloxane (PDMS) microchannel. The master mold was fabricated by a simple photolithographic method, employing a photoresist dry film. The process did not use hazardous chemicals, a clean room or any expensive instrument. The PDMS microchannel was clamped with polymethylmethacrylate (PMMA) plates, where a light emitting diode (LED) as a light source and a light dependent resistor (LDR) as a light sensor were attached to form a simple optical sensor. The system was successfully employed as a micro flow injection analysis for the determination of glutathione in dietary supplement samples. A linear calibration graph in the range of 5.0 - 60.0 mg L(-1) glutathione was obtained with a detection limit of 0.01 mg L(-1). The system provided a sample throughput of 48 h(-1), with microliter consumption of the reagent. PMID:22790365

  13. The gut microbiota modulates host amino acid and glutathione metabolism in mice.

    PubMed

    Mardinoglu, Adil; Shoaie, Saeed; Bergentall, Mattias; Ghaffari, Pouyan; Zhang, Cheng; Larsson, Erik; Bäckhed, Fredrik; Nielsen, Jens

    2015-10-01

    The gut microbiota has been proposed as an environmental factor that promotes the progression of metabolic diseases. Here, we investigated how the gut microbiota modulates the global metabolic differences in duodenum, jejunum, ileum, colon, liver, and two white adipose tissue depots obtained from conventionally raised (CONV-R) and germ-free (GF) mice using gene expression data and tissue-specific genome-scale metabolic models (GEMs). We created a generic mouse metabolic reaction (MMR) GEM, reconstructed 28 tissue-specific GEMs based on proteomics data, and manually curated GEMs for small intestine, colon, liver, and adipose tissues. We used these functional models to determine the global metabolic differences between CONV-R and GF mice. Based on gene expression data, we found that the gut microbiota affects the host amino acid (AA) metabolism, which leads to modifications in glutathione metabolism. To validate our predictions, we measured the level of AAs and N-acetylated AAs in the hepatic portal vein of CONV-R and GF mice. Finally, we simulated the metabolic differences between the small intestine of the CONV-R and GF mice accounting for the content of the diet and relative gene expression differences. Our analyses revealed that the gut microbiota influences host amino acid and glutathione metabolism in mice. PMID:26475342

  14. A bacterial glutathione transporter (Escherichia coli CydDC) exports reductant to the periplasm.

    PubMed

    Pittman, Marc S; Robinson, Hilary C; Poole, Robert K

    2005-09-16

    Glutathione (GSH), a major biological antioxidant, maintains redox balance in prokaryotes and eukaryotic cells and forms exportable conjugates with compounds of pharmacological and agronomic importance. However, no GSH transporter has been characterized in a prokaryote. We show here that a heterodimeric ATP-binding cassette-type transporter, CydDC, mediates GSH transport across the Escherichia coli cytoplasmic membrane. In everted membrane vesicles, GSH is imported via an ATP-driven, protonophore-insensitive, orthovanadate-sensitive mechanism, equating with export to the periplasm in intact cells. GSH transport and cytochrome bd quinol oxidase assembly are abolished in the cydD1 mutant. Glutathione disulfide (GSSG) was not transported in either Cyd(+) or Cyd(-) strains. Exogenous GSH restores defective swarming motility and benzylpenicillin sensitivity in a cydD mutant and also benzylpenicillin sensitivity in a gshA mutant defective in GSH synthesis. Overexpression of the cydDC operon in dsbD mutants defective in disulfide bond formation restores dithiothreitol tolerance and periplasmic cytochrome b assembly, revealing redundant pathways for reductant export to the periplasm. These results identify the first prokaryotic GSH transporter and indicate a key role for GSH in periplasmic redox homeostasis. PMID:16040611

  15. pH-dependent immobilization of urease on glutathione-capped gold nanoparticles.

    PubMed

    Garg, Seema; De, Arnab; Mozumdar, Subho

    2015-05-01

    Urease is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. Although the enzyme serves a significant role in several detoxification and analytical processes, its usability is restricted due to high cost, availability in small amounts, instability, and a limited possibility of economic recovery from a reaction mixture. Hence, there is a need to develop an efficient, simple, and reliable immobilization strategy for the enzyme. In this study, the carboxyl terminated surface of glutathione-capped gold nanoparticles have been utilized as a solid support for the covalent attachment of urease. The immobilization has been carried out at different pH conditions so as to elucidate its effect on the immobilization efficiency and enzyme bioactivity. The binding of the enzyme has been quantitatively and qualitatively analyzed through techniques like ultraviolet-visible spectroscopy, intrinsic steady state fluorescence, and circular dichorism. The bioactivity of the immobilized enzyme was investigated with respect to the native enzyme under different thermal conditions. Recyclability and shelf life studies of the immobilized enzyme have also been carried out. Results reveal that the immobilization is most effective at pH of 7.4 followed by that in an acidic medium and is least in alkaline environment. The immobilized enzyme also exhibits enhance activity in comparison to the native form at physiological temperature. The immobilized urease (on gold glutathione nanoconjugates surface) can be effectively employed for biosensor fabrication, immunoassays and as an in vivo diagnostic tool in the future. PMID:25227875