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PubMed2000

Glutathione depletion leads to delayed growth stasis in Saccharomyces cerevisiae: evidence of a partially overlapping role for thioredoxin.

Disruption of the first enzyme of glutathione biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a glutathione auxotrophy phenotype on plates. However, growth experiments in liquid medium revealed that the cessation of growth resulting from glutathione depletion in these yeasts is very delayed in S. cerevisiae compared to S. pombe. Glutathione metabolism was investigated to understand this delayed growth stasis in S. cerevisiae. The assimilation of reduced and oxidized glutathione, the intracellular storage pools of glutathione and the turnover of this compound were investigated and found to be similar in both yeasts. A possible overlapping role of intracellular thioredoxin in causing delayed stasis was studied. Yeast thioredoxin was overexpressed in S. cerevisiae and was found to partially relieve the dependence of S. cerevisiae glutathione auxotrophs on extracellular glutathione in glucose-grown cultures, as well as in glycerol-grown cultures where conditions of increased glutathione requirements exists in the cell. By partially, but not completely, compensating for glutathione deficiency in this yeast, thioredoxin thus appeared to be the major factor that was causing the delayed growth stasis following glutathione depletion in this yeast.

Culture Media↗

PubMed1993

Deficient glutathione peroxidase activity in preeclampsia is associated with increased placental production of thromboxane and lipid peroxides.

OBJECTIVE: Thromboxane and lipid peroxide levels are abnormally increased in preeclamptic placentas. The cause of this increase is not known. Peroxides stimulate prostaglandin H2 synthase to increase thromboxane and oxygen radicals, which increase lipid peroxides. Glutathione peroxidase inactivates peroxides, thereby decreasing peroxide stimulation of prostaglandin H synthase. If glutathione peroxidase activity were deficient, then peroxides could increase, leading to increased stimulation of prostaglandin H synthase, resulting in increased production of thromboxane and lipid peroxides. The following study tested this hypothesis. STUDY DESIGN: Placental tissues from 11 normal and 11 preeclamptic women were immediately frozen in liquid nitrogen after delivery. One gram of tissue from each placenta was homogenized for analysis. Placental tissues were also obtained from six normal pregnancies for incubation with a glutathione peroxidase inhibitor, N-ethylmaleimide. Samples were analyzed for glutathione peroxidase activity, lipid peroxides by hydrogen peroxide equivalents, thromboxane by thromboxane B2, and prostacyclin by 6-keto-prostaglandin F1 alpha. RESULTS: Glutathione peroxidase activity was significantly lower in preeclamptic than in normal placentas (9.41 +/- 0.05 vs 13.41 +/- 0.63 units/gm, p < 0.001, mean +/- SE). Lipid peroxides and thromboxane were significantly higher in preeclamptic than in normal placentas (hydrogen peroxide equivalents 4.23 +/- 0.32 vs 2.84 +/- 0.27 mumol/gm, p < 0.01; thromboxane B2 215 +/- 31 vs 138 +/- 15 ng/gm, p < 0.05), whereas prostacyclin was significantly lower (6-keto-prostaglandin F1 alpha 23.3 +/- 2.2 vs 41.6 +/- 6.0 ng/gm, p < 0.01). Inhibition of glutathione peroxidase activity in normal placentas resulted in a dose-response increase in placental production of both lipid peroxides and thromboxane without affecting prostacyclin, so the ratio of thromboxane to prostacyclin increased progressively with inhibition of glutathione peroxidase. CONCLUSIONS: Glutathione peroxidase activity is significantly lower, and lipid peroxides and thromboxane are significantly higher, in preeclamptic placentas compared with normal placentas. Inhibition of glutathione peroxidase activity in normal placentas results in significantly increased production of lipid peroxides and thromboxane and an increase in the thromboxane to prostacyclin ratio. We speculate that in normal placentas, glutathione peroxidase limits prostaglandin H synthase activity by reducing the amount of peroxide present, thus reducing peroxide stimulation of prostaglandin H synthase. In preeclampsia deficient glutathione peroxidase activity results in in increased peroxide levels leading to increased stimulation of prostaglandin H synthase, which results in increased production of lipid peroxides and thromboxane.

Case-Control Studies↗

PubMed1978

Glutathione peroxidase activities from rat liver.

There are two enzymes in rat liver with glutathione peroxidase activity when cumene hydroperoxide is used as substrate. One is the selenium-requiring glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) and the other appears to be independent of dietary selenium. Activities of the two enzymes vary greatly among tissues and among animals. The molecular weight of the enzyme with selenium-independent glutathione peroxidase activity was estimated by gel filtration to be 35 000, and the subunit molecular weight was estimated by dodecyl sulfate-polyacrylamide gel electrophoresis to be 17 000. Double reciprocal plots of enzyme activity as a function of substrate concentration produced intersecting lines which are suggestive of a sequential reaction mechanism. The Km for glutathione was 0.20 mM and the Km for cumene hydroperoxide was 0.57 mM. The enzyme was inhibited by N-ethylmaleimide, but not by iodoacetic acid. Inhibition by cyanide was competitive with respect to glutathione and the Ki for cyanide was 0.95 mM. This selenium-independent glutathione peroxidase also catalyzes the conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. Along with other similarities to glutathione S-transferase, this suggests that the selenium-independent glutathione peroxidase and glutathione S-transferase activities in rat liver are of the same enzyme.

Animals↗

PubMed1985

Alteration of hepatic glutathione S-transferases and release into serum after treatment with bromobenzene, carbon tetrachloride, or N-nitrosodimethylamine.

The effects of bromobenzene, carbon tetrachloride, and N-nitrosodimethylamine (DMN) on hepatic glutathione S-transferase activity were studied in untreated and in phenobarbital- or ethanol-treated rats. In phenobarbital-treated rats, the isozymic composition of the hepatic cytosolic glutathione S-transferases was changed after giving hepatotoxic chemicals; glutathione S-transferases 2-2(AA), 3-3(A), 1-2(B), 3-4(C), and 4-4 + 5-5(D + E) were present in cytosol from control rats, but only glutathione S-transferases cochromatographing with transferases 4-4 + 5-5(D + E) were detected in rats given carbon tetrachloride or bromobenzene. A marked decrease in hepatic and an increase in serum glutathione S-transferase activity were also observed after carbon tetrachloride or bromobenzene treatment, but little change was seen after giving DMN. On the contrary, in untreated or ethanol-treated rats, DMN administration decreased hepatic glutathione S-transferase activity and caused an elevation in serum glutathione S-transferase activity. The isozymic composition of the hepatic cytosolic glutathione S-transferases after giving DMN to untreated rats was also altered, but the alteration was much less than that observed after giving carbon tetrachloride or bromobenzene to phenobarbital-treated rats. The elevation in serum glutathione S-transferase activity was accompanied by an increase in both serum glutamate-pyruvate transaminase activity and serum bilirubin concentrations. Thus, hepatic glutathione S-transferase activity was altered and released into serum after giving hepatotoxic chemicals, and the alteration in glutathione S-transferase activity was dependent on treatment with phenobarbital or ethanol.

Animals↗

PubMed1995

1-naphthylisothiocyanate-induced elevation of biliary glutathione.

1-Naphthylisothiocyanate (ANIT) has been used for many years to study cholangiolitic hepatotoxicity in laboratory animals. Hallmarks of ANIT hepatotoxicity include portal edema and inflammation with bile duct epithelial and hepatic parenchymal cell necrosis. In rats, ANIT hepatotoxicity is dependent upon hepatic glutathione. Studies in vitro have demonstrated that ANIT combines reversibly with glutathione and suggest that intracellular formation and secretion of this glutathione-ANIT conjugate from hepatic parenchymal cells may be responsible for the efflux of glutathione observed upon exposure to ANIT. In vivo, glutathione conjugates produced within hepatic parenchymal cells are typically transported into bile for elimination. Therefore, large concentrations of ANIT in bile may result from hepatic parenchymal cell secretion of a reversible glutathione-ANIT conjugate. To investigate this hypothesis, bile and plasma concentrations of ANIT were determined in rats 1, 4, 8, 12 and 24 hr after administration (100 mg/kg, p.o.). Liver and bile glutathione concentrations were also evaluated. Plasma ANIT concentrations ranged between 2 and 5 microM at 1, 4, 8 and 12 hr and were 0.9 microM at 24 hr after administration. ANIT concentrations in bile at 1, 4, 8 and 12 hr were 60, 28, 21 and 22 microM, respectively. Thus, ANIT was concentrated in bile. Hepatic glutathione was not affected by ANIT during the first 12 hr after administration; however, a moderate elevation occurred by 24 hr. In contrast, a marked elevation in bile glutathione concentration (two times control) occurred 1, 4 and 8 hr after ANIT administration. Thus, the early accumulation of ANIT in bile was coincident with an elevation in bile glutathione. These findings support the hypothesis that glutathione functions to concentrate ANIT in bile. The large concentration of this toxicant in bile may be injurious to bile epithelium, a primary cellular target in ANIT hepatotoxicity.

1-Naphthylisothiocyanate↗

PubMed1984

Comparative effects of antioxidants on enzymes involved in glutathione metabolism.

The present study was designed to examine changes in glutathione metabolism in the liver of mice as influenced by supplementation of their diet with 1 of 4 antioxidants: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), vitamin E and selenium. In addition to determination of the acid-soluble thiol levels, 5 different enzymes involved with glutathione utilization and synthesis were measured: glutathione transferase, gamma-glutamyl transpeptidase, selenium-dependent glutathione peroxidase, gamma-glutamylcysteine synthetase and glutathione reductase. All 4 antioxidants produced significant increases in glutathione transferase activity, with BHA and BHT being much more effective than the other two. With the exception of vitamin E, BHA, BHT and selenium all resulted in a slight enhancement in the activity of glutathione reductase as well as in the acid-soluble thiol level. On the other hand, the induction of gamma-glutamyl transpeptidase and gamma-glutamylcysteine synthetase was responsive to only vitamin E and selenium supplementation, respectively. Although the influence of each of these antioxidants in glutathione metabolism appears to be specific and somewhat compartmentalized, the overall impression is that of an increased capacity for glutathione-conjugate formation and recovery of reduced glutathione. These biochemical changes in glutathione metabolism may be relevant to the anticarcinogenic effects observed with BHA, BHT and selenium.

Animals↗

PubMed1987

Glutathione S-transferases in human prostate.

A number of human prostatic tissue biopsies have been analyzed for glutathione S-transferase activity, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Samples from nine patients (age range 61-90) with benign prostatic hypertrophy who had received no prior chemotherapy had a mean glutathione S-transferase activity of 137 +/- 44 nmol/min per mg with a range of 97-237. A qualitative comparison of the glutathione S-transferase of normal prostate and benign prostatic hypertrophy samples was carried out. Approximately 260-fold purification was achieved using glutathione-Sepharose affinity chromatography, with glutathione S-transferase accounting for approximately 0.19-0.33% of the total protein. Substrate specificity determinations suggested similar, but not identical, glutathione S-transferase subunits in normal prostate and benign prostatic hypertrophy. One- and two-dimensional electrophoresis (isoelectric focusing and 12.5% SDS-polyacrylamide gel electrophoresis) identified at least seven stained polypeptides in the purified glutathione S-transferase preparations. These ranged in Mr from approximately 24,000 to 28,500 and in pI from near neutral to basic. Western blot analysis using polyclonal antibodies raised against rat liver glutathione S-transferase suggested crossreactivity with five of the human isoenzymes in both normal prostate and benign prostatic hypertrophy. One of the glutathione S-transferases, present in both normal prostate and benign prostatic hypertrophy, had an Mr of approx. 24,000 and a near-neutral pI and crossreacted immunologically with a polyclonal antibody raised against human placental glutathione S-transferase (Yf, subunit 7 or pi). These data suggest that four glutathione S-transferases are expressed in human prostate, with subunits from each of the major classes alpha, mu and pi. These are characterized as Ya, Yb, Yb' and Yf (analogous alternative nomenclature subunits 1, 3, 4 and 7).

Aged↗

PubMed1991

The effect of glutathione monoethyl ester on the potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion by diethyl maleate in the mouse.

Depletion of hepatic glutathione in the mouse by pretreatment with diethyl maleate (DEM) is known to potentiate the acute toxicities of many dimethyl-substituted organothiophosphate insecticides. However, certain studies have raised doubts regarding the participation of glutathione in the detoxification of methyl parathion in the mouse, and hence the putative mechanism of action of DEM-induced potentiation of this insecticide. The present study evaluates the hypothesis that DEM potentiates the acute toxicities of methyl parathion, methyl paraoxon, and fenitrothion by a mechanism other than glutathione depletion. One hour following pretreatment of mice with DEM (0.75 ml/kg i.p.), glutathione was markedly depleted and the acute toxicities of methyl parathion, methyl paraoxon and fenitrothion were potentiated. Administration of glutathione monoethyl ester (20 mmol/kg p.o.) to DEM-pretreated mice attenuated DEM-depletion of hepatic glutathione, or maintained glutathione at or above control levels. However, glutathione monoethyl ester did not alter the DEM-induced potentiation of the lethality of these insecticides. Furthermore, administration of glutathione monoethyl ester to naive mice increased hepatic glutathione levels, but did not affect the percentage of animals succumbing to a challenge dose of methyl parathion, methyl paraoxon, or fenitrothion. These data indicate that DEM potentiates the toxicity of methyl parathion, methyl paraoxon or fenitrothion by a mechanism unrelated to hepatic glutathione content.

Animals↗

PubMed2006

Nitric oxide modulates glutathione synthesis during endotoxemia.

Nitric oxide is known to modulate intracellular glutathione levels, but the relationship between nitric oxide synthesis and glutathione metabolism during endotoxemia is unknown. The present study was designed to examine the effects of increased nitric oxide formation on hepatic glutathione synthesis and antioxidant defense in endotoxemic mice. Our results demonstrate that hepatic glutathione synthesis is decreased for 24 h following injection of lipopolysaccharide (LPS). Administration of the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTZ), failed to normalize hepatic glutathione concentration, and suggests that decreased gamma-glutamylcysteine ligase activity is primarily responsible for the decrease in hepatic glutathione levels during endotoxemia. Inhibition of nitric oxide synthesis prevented the endotoxin-induced changes in hepatic and plasma glutathione status and up-regulated liver glutathione and cysteine synthesis pathways at the level of gene expression. Furthermore, whereas the activity of glutathione peroxidase and glutathione S-transferase decreased during endotoxemia, both of these changes were prevented by inhibition of nitric oxide synthesis. In conclusion, increased nitric oxide synthesis during endotoxemia causes marked changes in glutathione flux and defenses against oxidative stress in the liver.

Animals↗

PubMed2002

Hypoxia/reoxygenation-induced damage to mitochondrial activity is determined by glutathione threshold in astroglia-rich cell cultures.

It has been shown that astrocytes play an important role during ischemia/reperfusion and in neurodegenerative diseases by supporting neuronal functions, but the effect of these pathophysiological conditions on this particular cell type is still unclear. Here, we investigated the ischemia/reperfusion-induced damage to astroglia-rich cells. For that purpose, we studied the effects of substrate deprivation and hypoxia/reoxygenation on total cellular glutathione contents, and mitochondrial function. Substrate deprivation as well as increasing time of cultivation in vitro (from 2 to 4 weeks) induced a decrease in the total glutathione content. Three qualitative distinct concentration ranges of the glutathione pool with respect to the effect of hypoxia/reoxygenation on the glutathione content were found: (i) high glutathione levels above 40 nmol per mg protein remained unchanged during hypoxia/reoxygenation. (ii) Hypoxia/reoxygenation was accompanied by higher glutathione levels in comparison to controls at intermediate initial glutathione concentrations of about 20 up to 40 nmol per mg protein. (iii) Below an initial glutathione threshold concentration of about 20 nmol per mg protein, hypoxia/reoxygenation led to a stronger decrease of glutathione levels in comparison to controls. Decrease of mitochondrial respiratory chain activity during hypoxia/reoxygenation only occurred at low initial glutathione concentrations below 20 nmol per mg protein. Our data emphasize the important role of glutathione with respect to the defense of mitochondria against oxidative stress in astroglia cells during hypoxia/reoxygenation.

Animals↗

PubMed2000

Metabolism and functions of glutathione in brain.

The tripeptide glutathione is the thiol compound present in the highest concentration in cells of all organs. Glutathione has many physiological functions including its involvement in the defense against reactive oxygen species. The cells of the human brain consume about 20% of the oxygen utilized by the body but constitute only 2% of the body weight. Consequently, reactive oxygen species which are continuously generated during oxidative metabolism will be generated in high rates within the brain. Therefore, the detoxification of reactive oxygen species is an essential task within the brain and the involvement of the antioxidant glutathione in such processes is very important. The main focus of this review article will be recent results on glutathione metabolism of different brain cell types in culture. The glutathione content of brain cells depends strongly on the availability of precursors for glutathione. Different types of brain cells prefer different extracellular glutathione precursors. Glutathione is involved in the disposal of peroxides by brain cells and in the protection against reactive oxygen species. In coculture astroglial cells protect other neural cell types against the toxicity of various compounds. One mechanism for this interaction is the supply by astroglial cells of glutathione precursors to neighboring cells. Recent results confirm the prominent role of astrocytes in glutathione metabolism and the defense against reactive oxygen species in brain. These results also suggest an involvement of a compromised astroglial glutathione system in the oxidative stress reported for neurological disorders.

Animals↗

PubMed1997

Neutrophil antioxidant capacity during the respiratory burst: loss of glutathione induced by chloramines.

Low-molecular weight antioxidants in rat peritoneal neutrophils undergo rapid redox recycling, so measurements were made of their initial content and subsequent changes during the respiratory burst, when superoxide formation is maximized. Endogenous vitamin E, ascorbate and total glutathione (reduced + oxidized) were not significantly changed during 30 min of respiratory burst, which was stimulated by phorbol 12-myristate 13-acetate (PMA). When de novo synthesis of glutathione was inhibited by buthionine-[S,R] sulfoximine (BSO), the glutathione content rapidly decreased in activated neutrophils but not in resting cells. The lost total glutathione was recovered neither from the incubation medium nor as a protein-bound form, which suggests that irreversible oxidation of glutathione occurs. Furthermore, the glutathione loss continues even 30 min after PMA stimulation, when the respiratory burst has almost ceased. The decrease of glutathione was prevented by added catalase, or by addition of NaN3 or KCN which inhibits myeloperoxidase (MPO). Superoxide dismutase had no protective effects. These findings suggest the involvement of an MPO-H2O2-halide system in the accelerated consumption of glutathione during the respiratory burst. Additional studies showed that neutrophil-derived chloramines found in the extracellular medium could lead to intracellular glutathione loss. Incubation of resting cells with chemically produced membrane permeable monochloramine in the presence of BSO resulted in a decrease of glutathione, whereas membrane-impermeable taurine-chloramine was less effective. We conclude that chloramines are responsible for accelerated glutathione turnover in neutrophils during the respiratory burst. Permeable extracellular chloramines derived from the respiratory burst activity, such as monochloramine, can reenter cells and react with thiols.

Animals↗

PubMed1996

Effects of intracerebroventricular administration of 5-(glutathion-S-yl)-alpha-methyldopamine on brain dopamine, serotonin, and norepinephrine concentrations in male Sprague-Dawley rats.

alpha-Methyldopamine (alpha-MeDA) is a metabolite of the serotonergic neurotoxicants 3,4-(+/-)-(methylenedioxy)amphetamine (MDA) and 3,4-(+/-)-(methylenedioxy)methamphetamine (MDMA). alpha-MeDA readily oxidizes, and in the presence of glutathione (GSH) it forms 5-(glutathion-S-yl)-alpha-methyldopamine [5-(glutathion-S-yl)-alpha-MeDA]. Since GSH conjugates of many polyphenols are biologically (re)active, we investigated the role of 5-(glutathion-S-yl)-alpha-MeDA in the acute and long-term neurochemical changes observed after administration of MDA. Intracerebroventricular (icv) administration of 5-(glutathion-S-yl)-alpha-MeDA (720 nmol) to male Sprague-Dawley rats produced behavioral changes similar to those reported after subcutaneous administration of MDA. Thus, animals became hyperactive and aggressive and displayed forepaw treading and Straub tails, behaviors usually seen after administration of serotonin (5-HT) releasers, and consistent with a role for 5-(glutathion-S-yl)-alpha-MeDA in some of the behavioral alterations seen after administration of MDA and MDMA. In addition to the behavioral changes, 5-(glutathion-S-yl)-alpha-MeDA also caused short-term alterations in the dopaminergic, serotonergic, and noradrenergic systems. An increase in dopamine synthesis appears to be a prerequisite for the long-term depletion of brain 5-HT following MDMA administration. However, although 5-(glutathion-S-yl)-alpha-MeDA reproduced some of the effects of MDA on the dopaminergic system and was capable of causing acute increases in 5-HT turnover, a single icv injection of 5-(glutathion-S-yl)-alpha-MeDA did not result in long-term serotonergic toxicity. Thus, although acute stimulation of dopamine turnover may be necessary for long-term serotonergic toxicity, such changes are not sufficient to produce these effects. The effects of a multiple dosing schedule of 5-(glutathion-S-yl)-alpha-MeDA will therefore require investigation before we can define a role for this metabolite in MDA and MDMA mediated neurotoxicity. MDA also produces a pressor response that is related to its ability to release neuronal norepinephrine stores, and 5-(glutathion-S-yl)-alpha-MeDA caused comparable depletions of brain norepinephrine concentrations, indicating that both compounds produce similar effects on the noradrenergic system.

Animals↗

PubMed2002

Regulation of intracellular glutathione levels in erythrocytes infected with chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum.

Malaria is one of the most devastating tropical diseases despite the availability of numerous drugs acting against the protozoan parasite Plasmodium in its human host. However, the development of drug resistance renders most of the existing drugs useless. In the malaria parasite the tripeptide glutathione is not only involved in maintaining an adequate intracellular redox environment and protecting the cell against oxidative stress, but it has also been shown that it degrades non-polymerized ferriprotoporphyrin IX (FP IX) and is thus implicated in the development of chloroquine resistance. Glutathione levels in Plasmodium -infected red blood cells are regulated by glutathione synthesis, glutathione reduction and glutathione efflux. Therefore the effects of drugs that interfere with these metabolic processes were studied to establish possible differences in the regulation of the glutathione metabolism of a chloroquine-sensitive and a chloroquine-resistant strain of Plasmodium falciparum. Growth inhibition of P. falciparum 3D7 by D,L-buthionine-( S, R )sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), and by Methylene Blue (MB), an inhibitor of gluta thione reductase (GR), was significantly more pronounced than inhibition of P. falciparum Dd2 growth by these drugs. These results correlate with the higher levels of total glutathione in P. falciparum Dd2. Short-term incubations of Percoll-enriched trophozoite-infected red blood cells in the presence of BSO, MB and N, N (1)-bis(2-chloroethyl)- N -nitrosourea and subsequent determinations of gamma-GCS activities, GR activities and glutathione disulphide efflux revealed that maintenance of intracellular glutathione in P. falciparum Dd2 is mainly dependent on glutathione synthesis whereas in P. falciparum 3D7 it is regulated via GR. Generally, P. falciparum Dd2 appears to be able to sustain its intracellular glutathione more efficiently than P. falciparum 3D7. In agreement with these findings is the differential susceptibility to oxidative stress of both parasite strains elicited by the glucose/glucose oxidase system.

Animals↗

PubMedDate not supplied

Role of glutathione redox dysfunction in diabetic wounds.

We propose that diabetic foot ulcers and diabetic mouse wounds have insufficient glutathione to maintain correct cellular redox potential. Therefore, tissue samples from the wound edge of diabetic foot ulcers, diabetic mice wounds and nondiabetic mice wounds were obtained. Levels of glutathione, cysteine, and mixed protein disulfide were determined and topical application of esterified glutathione in carboxymethylcellulose or carboxymethylcellulose alone was applied to the mice wounds. Diabetic foot ulcer mean glutathione levels were 150.6 pmol/mg in the controls and 53.4 pmol/mg at the wound edge (p < 0.05), while mean cysteine levels were 22.3 pmol/mg in the control and 10.5 pmol/mg at the wound edge (p < 0.05). The mixed protein disulfide levels were elevated in the wounds (14.6 pmol/mg), but not in the control (6.9 pmol/mg) (p < 0.05). The glutathione levels were lower in the diabetic mouse wounds (155 pmol/mg) than the nondiabetic mouse wounds (205 pmol/mg) (p=0.04). The diabetic mouse treated with carboxymethylcellulose alone healed slower (19.5 +/- 2.2 days) than the nondiabetic mouse DM (11.5 +/- 0.5 days) (p < 0.001). The diabetic mouse that received topical glutathione healed significantly faster (12.5 +/- 0.8 days) than the carboxymethylcellulose-treated mice (19.5 +/- 2.2 days) (p < 0.001). Glutathione levels in the diabetic mouse (26.0 pmol/mg) were lower than in the nondiabetic mouse (311.7 pmol/mg) (p < 0.05) after glutathione treatment. In the glutathione-treated diabetic mouse, the oxidized glutathione was higher (26.7%) than in the nondiabetic mouse (9.9%) (p=0.05). These data suggest that cellular redox dysfunction and lower glutathione levels are present in diabetic foot ulcers and diabetic mouse wounds.

Adult↗

PubMed2006

Virus-induced changes in the subcellular distribution of glutathione precursors in Cucurbita pepo (L.).

Changes in glutathione contents occur in plants during environmental stress situations, such as pathogen attack, as the formation of reactive oxygen species leads to the activation of the antioxidative defence system. As glutathione is synthesized out of its constituents cysteine, glycine, and glutamate the availability of these components will limit glutathione synthesis in plants especially during stress situations and therefore the ability of the plant to fight oxidative stress. To gain a deeper insight into possible limitations of glutathione synthesis during pathogen attack the present investigations were aimed to study how the subcellular distribution of glutathione precursors correlates with the subcellular distribution of glutathione during virus attack in plants. Selective antibodies against cysteine, glutamate, and glycine were used to study the impact of Zucchini yellow mosaic virus (ZYMV) infection on glutathione precursor contents within different cell compartments of cells from Cucurbita pepo (L.) plants with the transmission electron microscope (TEM). Generally, levels of cysteine and glutamate were found to be strongly decreased in most cell compartments of younger and older leaves including glutathione-producing cell compartments such as plastids and the cytosol. The strongest decrease of cysteine was found in plastids (- 54 %) and mitochondria (- 51 %) of younger leaves and in vacuoles (- 37 %) and plastids (- 29 %) of older leaves. The strongest decrease of glutamate in younger leaves occurred in peroxisomes (- 67 %) and nuclei (- 58 %) and in peroxisomes (- 64 %) and plastids (- 52 %) of the older ones. Glycine levels were found to be strongly decreased (- 63 % in mitochondria and - 53 % in plastids) in most cell compartments of older leaves and strongly increased (about 50 % in plastids and peroxisomes) in all cell compartments of the younger ones. These results indicate that low glycine contents in the older leaves were responsible for low levels of glutathione in these organs during ZYMV infection rather than limited amounts of cysteine or glutamate. Glutathione precursors were virtually absent in cell walls and intercellular spaces and play therefore no important role during ZYMV attack in the apoplast. While glutamate was absent in vacuoles, elevated levels of glycine (up to 30 %) and decreased cysteine contents (up to - 37 %) were observed in vacuoles during ZYMV infection. The impact of the present results on the current knowledge about glutathione synthesis and degradation on the cellular level during ZYMV infection are discussed.

Cucurbita↗

PubMed1990

High-affinity transport of glutathione is part of a multicomponent system essential for mitochondrial function.

Glutathione, an essential cellular antioxidant required for mitochondrial function, is not synthesized by mitochondria but is imported from the cytosol. Rat liver mitochondria have a multicomponent system that underlies the remarkable ability of mitochondria to take up and retain glutathione. At external glutathione levels of less than 1 mM, glutathione is transported into the mitochondrial matrix by a high-affinity component (Km, approximately 60 microM; V max, approximately 0.5 nmol/min per mg of protein), which is saturated at levels of 1-2 mM and stimulated by ATP. Another component has lower affinity (Km, approximately 5.4 mM; Vmax, approximately 5.9 nmol/min per mg of protein) and is stimulated by ATP and ADP. Both components are inhibited by carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), glutamate, and ophthalmic acid. Increase of extramitochondrial glutathione promotes uptake and exchange; the intermembranous space seems to function as a recovery zone that promotes efficient recycling of matrix glutathione. The findings are in accord with in vivo data showing that (i) rapid exchange occurs between mitochondrial and cytosolic glutathione, (ii) lowering of cytosolic glutathione levels (produced by administration of buthionine sulfoximine) decreases export of glutathione from mitochondria to cytosol, and (iii) administration of glutathione esters increases glutathione levels in mitochondria more than those in the cytosol.

Animals↗

PubMed1995

Multiple canalicular transport mechanisms for glutathione S-conjugates. Transport on both ATP- and voltage-dependent carriers.

A large number of structurally distinct electrophiles are conjugated to glutathione within hepatocytes, and the resulting glutathione S-conjugates are selectively transported across the canalicular membrane into bile. To test the hypothesis that a single multi-specific, ATP-dependent carrier mediates biliary secretion of glutathione S-conjugates, the present study compared the driving forces and substrate specificity for canalicular transport of S-ethylglutathione (ethyl-SG), a low molecular weight and relatively hydrophilic thioether, and S-(2,4-dinitrophenyl)-glutathione (DNP-SG), a larger and more hydrophobic anion, using isolated rat liver canalicular membrane vesicles. In agreement with previous findings, DNP-SG transport was stimulated by ATP, although there was considerable transport in the absence of ATP. ATP-independent DNP-SG transport was unaffected by a Na+ gradient, was enhanced by a valinomycin-induced K+ diffusion potential, and was saturable, with both high affinity (Km = 8 +/- 2 microM) and low affinity (Km = 0.5 +/- 0.1 mM) components. High affinity ATP-independent DNP-SG uptake was cis-inhibited by GSH, GSH monoethyl ester, glutathione S-conjugates, other gamma-glutamyl compounds, sulfobromophthalein, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In contrast, ATP-dependent DNP-SG uptake was unaffected by GSH, GSH ester, S-methyl glutathione, or S-carbamidomethyl glutathione, but was strongly inhibited by sulfobromophthalein, DIDS, and by high molecular weight and relatively hydrophobic glutathione S-conjugates. Transport of the low molecular weight ethyl-SG conjugate was only minimally stimulated by ATP (10-20%). ATP-independent ethyl-SG uptake was electrogenic, saturable (Km = 10 +/- 1 microM) and was inhibited by GSH and all glutathione S-conjugates tested. These findings indicate the presence of multiple canalicular transport mechanisms for glutathione S-conjugates and demonstrate that the physicochemical properties of the S moiety are major determinants of transport. Relatively high molecular weight hydrophobic conjugates are substrates for both ATP-dependent and -independent mechanisms, whereas low molecular weight glutathione S-conjugates are transported largely by electrogenic carriers.

Adenosine Triphosphate↗