Showing papers on "Glutathione published in 1985"

Determination of glutathione and glutathione disulfide in biological samples.

Abstract: Publisher Summary There are a number of procedures, for example, chemical, enzymatic, and chromatographic for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in biological samples. Enzymatic and chromatographic methods for the determination of glutathione in biological samples are described in this chapter. Because GSH readily oxidizes nonenzymatically and because it is a good substrate of γ-glutamyl transpeptidase, the biological samples are acidified quickly to reduce oxidation of GSH to GSSG and to mixed disulfides, and to inactivate γ-glutamyl transpeptidase. Glutathione oxidizes rapidly at pH values greater than 7. Acid treatment inactivates γ-glutamyl transpeptidase, which catalyzes the reactions that decrease the levels of both GSH and GSSG. The optimum method for treating biological samples depends upon the tissue and the experimental system. The discussed 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)-GSSG reductase recycling assay for total glutathione is a specific, sensitive, rapid, and reliable procedure. However, because the method depends on an accurate standard curve, appropriate standards containing the protein precipitating agent are essential.

[59] Glutathione reductase

Abstract: Publisher Summary Glutathione reductase is a flavoprotein catalyzing the NADPH-dependent reduction of glutathione disulfide (GSSG) to glutathione (GSH). The reaction is essential for the maintenance of glutathione levels. Glutathione has a major role as a reductant in oxidation–reduction processes, and serves in detoxication and several other cellular functions of great importance. A purification method of this enzyme from calf liver and rat liver is described in this chapter. Similar methods are used for the purification of the enzyme from yeast, porcine, and human erythrocytes. All the steps are carried out at about 5°. The purification method from calf liver consists of various steps including preparation of cytosol fraction, chromatography on DEAE-sephadex, precipitation with ammonium sulfate, and chromatography on hydroxyapatite. The purification of glutathione reductase from rat liver is usually combined with the preparation of glutathione transferases, thioltransferase, and glyoxalase I.

The Regulation of Hepatic Glutathione

Abstract: Glutathione (GSH) is a peptide composed of glutamate, cysteine, and glycine that exists in thiol·reduced (GSH) and disulfide-oxidized (GSSG) forms. GSH has been the subject of intense interest in the past decade and numerous symposia and reviews have been written about its function and regulation since 1 980 ( 1 -7). We propose to review some of the recent exciting developments in this field, with a particular focus on the regulation of hepatic GSH. We will not exhaustively review the literature and therefore apologize for inadvertent or intentional omissions. We hope to bring a personal and different perspective to this subject. A few words about the functions of GSH will focus on the importance of elucidating the physiology and biochemistry of the regulation of this vital substance . GSH is a fairly ubiquitous substance in aerobic life forms and tissues and generally exists in millimolar concentrations . The liver is among the organs with the highest content of GSH. The heterogeneity of GSH content in tissues has been observed. Thus , periportal hepatocytes may contain approximately twice the centrilobular concentration, enterocytes at the villus tip have a higher content than the crypts, and proximal tubular cells of kidney have more GSH than other parts of the nephron (8-1 0) . GSH plays a critical role in detoxification reactions. It is a specific substrate for GSH peroxidase ( 1 1 ) and GSH S-transferases ( 12), and it participates in microsomal peroxidase and radical scavenging reactions ( 1 3, 14). In this regard, probably the key function of GSH is reducing hydrogen peroxide (H202), a reaction catalyzed by GSH peroxidase. H202 production is a by-

Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic.

Abstract: Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte-derived free radicals. This is the first demonstration that short-lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.

Origin and turnover of mitochondrial glutathione.

Abstract: Mitochondrial glutathione in liver does not arise by intramitochondrial synthesis, but rather from the cytoplasm, by a process characterized by slow net transport and more rapid exchange transport.

Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis.

Abstract: A polypeptide of Mr 26,000 and pI 6.7 that was markedly increased in rat livers bearing hyperplastic nodules (HNs) induced by chemical carcinogens was identified immunochemically as the subunit of neutral glutathione (GSH) transferase (GSHTase; RX:glutathione R-transferase, EC 2.5.1.18; also called GSH S-transferase) purified from placenta (GSHTase-P) and was demonstrated immunohistochemically to be localized in preneoplastic foci and HNs. In the present study, GSHTase-P has been purified from the HN-bearing liver, and the distribution and inducibility have been examined quantitatively using anti-GSHTase-P antibody. Elevation of GSHTase-P in the HN-bearing livers was also confirmed by in vitro translation of mRNAs isolated from the HN-bearing livers. The purified GSHTase-P was homogeneous in size but had two charge isomers on two-dimensional gel electrophoresis. In normal tissues, including liver, placenta, and fetal liver, the protein content of GSHTase-P was generally low but was significantly high in kidney and pancreas. In contrast, the amount of GSHTase-P in HN-bearing livers (primary hepatomas) and transplantable Morris hepatoma 5123D were several 10-fold higher than that in normal liver but were undetectably low in transplantable Yoshida ascites hepatoma AH 130. Different from ordinary drug-metabolizing enzymes, GSHTase-P was uninducible by administration of drugs and carcinogens prior to appearance of the preneoplastic foci and HNs. In addition, species specificity of GSHTase-P was low as it was crossreactive among rat, hamster, and human.

Oxidative stress: damage to intact cells and organs

Abstract: Oxidative cell damage can be monitored by detection of (a) photoemission of singlet molecular oxygen formed from radical interactions (so-called low-chemical chemiluminescence), (b) end products of lipid peroxidation, such as ethane, and (c) glutathione disulphide release. These methods, preferably used in a complementary fashion, provide insight into the pro-oxidant-antioxidant balance in the intact cell or organ. Recent work from this laboratory on the metabolism of hydroperoxides and aldehydes as well as on redox cycling of the quinone menadione is presented. The comparison of GSSG transport systems in liver and heart reveals a limitation of capacity in the latter, thus making GSSG export potentially critical in the heart. As part of an inter-organ feedback system between extrahepatic tissues and liver, the newly described hormone stimulation of GSH release from liver is also presented.

Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity.

Abstract: N-Acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, rapidly reacts at physiological pH with glutathione (GSH) forming an acetaminophen-glutathione conjugate and stoichiometric amounts of acetaminophen and glutathione disulfide (GSSG). The same reaction products are formed in isolated hepatocytes incubated with NAPQI. In hepatocytes which have been treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) in order to inhibit glutathione reductase, the initial rise in GSSG concentration in the presence of NAPQI is maintained, whereas GSSG is rapidly reduced back to GSH in untreated hepatocytes. Oxidation by NAPQI of GSH to GSSG and the reduction of GSSG back to GSH by the NADPH-dependent glutathione reductase appear to be responsible for the rapid oxidation of NADPH that occurs in hepatocytes incubated with NAPQI in that the effect is blocked by pretreatment of cells with BCNU. When added to hepatocytes, NAPQI not only reacts with GSH but also causes a loss in protein thiol groups. The loss in protein thiols occurs more rapidly in cells pretreated with BCNU or diethylmaleate. Whereas both of these treatments enhance cytotoxicity caused by NAPQI, BCNU pretreatment has no effect on the covalent binding of [14C-ring]NAPQI to cellular proteins. Furthermore, dithiothreitol added to isolated hepatocytes after maximal covalent binding of [14C-ring]NAPQI but preceding cell death protects cells from cytotoxicity and regenerates protein thiols. Thus, the toxicity of NAPQI to isolated hepatocytes may result primarily from its oxidative effects on cellular proteins.

gamma-Glutamyl transpeptidase from kidney.

Abstract: Publisher Summary This chapter focuses on mammalian γ-glutamyl transpeptidase, emphasizing the rat kidney enzyme, the most extensively studied transpeptidase. γ-glutamyl transpeptidase plays a key role in the γ-glutamyl cycle, a pathway for the synthesis and degradation of glutathione. Glutathione is synthesized from its constituent amino acids within the cells by the successive actions of γ-glutamylcysteine and glutathione synthetases. The reactions allow the storage of cysteine as glutathione and γ-glutamyl transpeptidase catalyzes the first step in the pathway that leads to release of the cysteine moiety from the tripeptide. Homogenates of animal tissues exhibit a wide range of transpeptidase activity. Low transpeptidase activity in a tissue homogenate is often misleading because specific localization studies indicate that there are specific regions of intense enzyme activity in many tissues. In most mammals, the kidney exhibits by far the highest activity.

Influence of Acidosis on Lipid Peroxidation in Brain Tissues In Vitro

Abstract: To study the influence of acidosis on free radical formation and lipid peroxidation in brain tissues, homogenates fortified with ferrous ions and, in some experiments, with ascorbic acid were equilibrated with 5–15% O2 at pH values of 7.0, 6.5, 6.0, and 5.0, with subsequent measurements of thiobarbituric acid-reactive (TBAR) material, as well as of water- and lipid-soluble antioxidants (glutathione, ascorbate, and α-tocopherol) and phospholipid-bound fatty acids (FAs). Moderate to marked acidosis (pH 6.5–6.0) was found to grossly exaggerate the formation of TBAR material and the decrease in α-tocopherol content and to enhance degradation of phospholipid-bound, polyenoic FAs. These effects were reversed at pH 5.0, suggesting a pH optimum at pH 6.0–6.5. It is concluded that acidosis of a degree encountered in ischemic brain tissues has the potential of triggering increased free radical formation. This effect may involve increased formation of the protonated form of superoxide radicals, which is strongly prooxidant and lipid soluble, and/or the decompartmentalization of iron bound to cellular macromolecules like ferritin. (Less)

Glutathione transferases from human liver.

Abstract: Publisher Summary This chapter investigates glutathione transferases derived from human liver. The glutathione transferases are a group of related enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds bearing an electrophilic center. The proteins also act as intracellular binding proteins for a large number of lipophilic substances, including bilirubin. Human glutathione transferases have been purified from liver, erythrocytes, placenta, and lung. A simple and rapid procedure for the purification of basic (α-ɛ) and neutral (μ) glutathione transferases from human liver cytosol is described in the chapter. The enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The steps of the purification procedure include (1) preparation of cytosol fraction, (2) chromatography on Sephadex G-25, (3) chromatography on DEAE-cellulose, and (4) chromatography on Sephadex G-25.

Stimulation of Glutathione Synthesis in Photorespiring Plants by Catalase Inhibitors

Abstract: The effect of various herbicides on glutathione levels in barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), soybean (Glycine max [L.] Merr.), and corn (Zea mays L.) was examined. Illumination of excised barley, tobacco, and soybean plants for 8 hours in solution containing 2 millimolar aminotriazole (a catalase inhibitor) resulted in an increase in leaf glutathione from 250 to 400 nanomoles per gram fresh weight to 600 to 1800 nanomoles per gram fresh weight, depending on the species tested. All of this increase could be accounted for as oxidized glutathione. Between 25 and 50% of this oxidized glutathione was reduced when plants were darkened for 16 hours, but there was no significant decline in total glutathione. Another catalase inhibitor, thiosemicarbazide, was as effective as aminotriazole in elevating glutathione in soybean but was less effective in barley and tobacco. Glyphosate, an inhibitor of aromatic amino acid biosynthesis, had no significant effect on glutathione levels in any of the plants examined. Whereas methyl viologen (paraquat), which is a sink for photosystem I electrons, caused oxidation of leaf glutathione in all of the plants but did not increase the total amount of glutathione present.

Glutathione biosynthesis; gamma-glutamylcysteine synthetase from rat kidney.

Abstract: Publisher Summary Glutathione is synthesized by the consecutive actions of γ-glutamylcysteine synthetase and glutathione synthetase. The most highly purified preparations of the γ-glutamylcysteine synthetase enzyme have been obtained from rat kidney and rat and sheep erythrocytes. This chapter presents the isolation procedure of this enzyme. The assay method (ADP formation) is discussed. The enzyme activity is measured in reaction mixtures containing L-glutamate, L-α-aminobutyrate, and ATP by a coupled enzyme procedure in which the rate of formation of ADP, in the presence of pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, and NADH, is obtained from the decrease in the absorbance of NADH at 340 nm. The physical properties of the enzyme are reviewed in the chapter. The molecular weight of the rat kidney enzyme is about 104,000. The enzyme dissociates under denaturing conditions to yield two subunits of molecular weights about 73,000 and 27,700. Treatment of the enzyme with dithiothreitol followed by gel electrophoresis under nondenaturing conditions leads to partial dissociation into subunits.

Differential Potentiation of Alkylating and Platinating Agent Cytotoxicity in Human Ovarian Carcinoma Cells by Glutathione Depletion

Abstract: We have determined the effect of glutathione (GSH) depletion on the cytotoxicity of three nitrogen mustards, six platinum complexes, and mitomycin C in a human ovarian carcinoma cell line. GSH levels in COLO 316 cells were depleted by exposure of cell monolayers to 0.5 mm d,l-buthionine- S,R -sulfoximine. GSH depletion significantly potentiated the cytotoxicity of l-phenylalanine mustard, chlorambucil, and mechlorethamine as determined by clonogenic assay on plastic plates. The dose modification factors were 2.6, 2.6, and 1.9, respectively. The same level of GSH depletion had a minimal effect on the cytotoxicity of cis -diamminedichloroplatinum(II) (cis-DDP), carboplatin, dichloro(ethylenediamine)platinum(II), 1,2-diaminocyclohexylplatinum(II) malonate, and iproplatin. The dose modification factors of GSH depletion for these drugs were 1.4 or less. trans -Diamminedichloroplatinum(II) was, however, markedly potentiated by GSH depletion with a dose modification factor of 2.7. Mitomycin C was minimally potentiated by GSH depletion. We have also generated cis-DDP-resistant cells from COLO 316 and 2008 human ovarian carcinoma cells by in vitro selection with cis-DDP. These cis-DDP-resistant cells had identical levels of GSH as the parental cells. GSH depletion sensitized these cells only to the same degree as the parental cells and did not reverse the resistant phenotype. Our results indicate that intracellular GSH levels are not an important determinant of the cytotoxicity of cis -platinum(II) or cis -platinum(IV) complexes in COLO 316 and 2008 cells. In addition, altered GSH metabolism does not appear to be a component of the cis-DDP-resistant phenotype in these cells.

Increased loss and decreased synthesis of hepatic glutathione after acute ethanol administration. Turnover studies.

Abstract: The effect of acute ethanol administration on rates of synthesis and utilization of hepatic glutathione (GSH) was studied in rats after a pulse of [35S]cysteine. A 35% decrease in hepatic GSH content 5h after administration of 4 g of ethanol/kg body wt. was accompanied by a 33% increase in the rate of GSH utilization. The decrease occurred without increases in hepatic oxidized glutathione (GSSG) or in the GSH/GSSG ratio. The rate of non-enzymic condensation of GSH with acetaldehyde could account for only 6% of the rate of hepatic GSH disappearance. The increased loss of [35S]GSH induced by ethanol was not accompanied by an increased turnover; rather, a 30% inhibition of GSH synthesis balanced the increased rate of loss, leaving the turnover rate unchanged. The rate of acetaldehyde condensation with cysteine in vitro occurred at about one-third of the rate of GSH loss in ethanol-treated animals. However, ethanol induced only a minor decrease in liver cysteine content, which did not precede, but followed, the decrease in GSH. The characteristics of 2-methylthiazolidine-4-carboxylic acid, the condensation product between acetaldehyde and cysteine, were studied and methodologies were developed to determine its presence in tissues. It was not found in the liver of ethanol-treated animals. Ethanol administration led to a marked increase (47%) in plasma GSH in the post-hepatic inferior vena cava, but not in its pre-hepatic segment. Data suggest that an increased loss of GSH from the liver constitutes an important mechanism for the decrease in GSH induced by ethanol. In addition, an inhibition of GSH synthesis is observed.

A common biochemical pattern in preneoplastic hepatocyte nodules generated in four different models in the rat.

Abstract: Hepatocyte nodules, structures consistently seen in every model of liver carcinogenesis well before the first appearance of cancer, were examined with respect to some Phase I and Phase II components considered to be important in the metabolism of carcinogens and other xenobiotics. Phase I components are those related to the metabolism of xenobiotics and include microsomal cytochromes P-450 and mixed-function oxygenase activities. Phase II components are those related to the conjugation and detoxification reactions of xenobiotics and their metabolites and include glutathione S-transferases and glutathione. Nodules were induced by the resistant hepatocyte, choline-deficient, methionine-low diet, phenobarbital and orotic acid models of liver carcinogenesis. Also, nodules generated by the resistant hepatocyte model were examined after transplantation to the spleen of syngeneic animals. The hepatocyte nodules show a common biochemical pattern, consisting of decreased microsomal cytochromes P-450, cytochrome b5, and aminopyrine N-demethylase activity and increased glutathione and γ-glutamyltransferase in whole homogenates and glutathione S-transferase activity in the cytosol. This similarity, appropriate to a resistance phenotype, adds additional support for the hypothesis that hepatocyte nodules may be a common step in liver carcinogenesis in several different models.