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

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.     

Glutathione utilization by Candida albicans requires a functional glutathione degradation (DUG) pathway and OPT7, an unusual member of the oligopeptide transporter family

Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1-OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a K(m) of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence.     

A sensitive fluorimetric microassay for the determination of glutathione peroxidase activity. Application to human blood platelets

The method proposed for measuring glutathione peroxydase (GSH-Px) activity is based on the determination of oxidized glutathione (GSSG) using o-phtalaldehyde (OPT) as a fluorescent reagent. This method makes it possible to study the kinetics of both substrates (peroxide and reduced glutathione, GSH), and allosteric kinetics were found for GSH, with human platelets as the source of GSH-Px. Different methods for platelet disruption were compared. The reference values obtained for GSH-Px activity in human blood platelets by this fluorimetric procedure and the conventional enzymatic method were very similar and significantly higher than those previously reported; the reasons for this difference are discussed.     

The role of oxidative stress in acrolein-induced DNA damage in HepG2 cells

This study evaluated the role of oxidative stress in acrolein-induced DNA damage, using HepG2 cells. Using the standard single cell gel electrophoresis (SCGE) assay, a significant dose-dependent increment in DNA migration was detected at lower concentrations of acrolein; but at the higher tested concentrations, a reduction in the migration was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of acrolein. These results indicated that acrolein caused DNA strand breaks and DNA-protein crosslinks (DPC). To elucidate the oxidatively generated DNA damage mechanism, the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) were used to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH), respectively. The present study showed that acrolein induced the increased levels of ROS and depletion of GSH in HepG2 cells. Moreover, acrolein significantly caused 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) formation in HepG2 cells. These results demonstrate that the DNA damage induced by acrolein in HepG2 cells is related to the oxidative stress.     

The role of oxidative stress in acrolein-induced DNA damage in HepG2 cells

This study evaluated the role of oxidative stress in acrolein-induced DNA damage, using HepG2 cells. Using the standard single cell gel electrophoresis (SCGE) assay, a significant dose-dependent increment in DNA migration was detected at lower concentrations of acrolein; but at the higher tested concentrations, a reduction in the migration was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of acrolein. These results indicated that acrolein caused DNA strand breaks and DNA-protein crosslinks (DPC). To elucidate the oxidatively generated DNA damage mechanism, the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) were used to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH), respectively. The present study showed that acrolein induced the increased levels of ROS and depletion of GSH in HepG2 cells. Moreover, acrolein significantly caused 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) formation in HepG2 cells. These results demonstrate that the DNA damage induced by acrolein in HepG2 cells is related to the oxidative stress.     

Rethinking cystic fibrosis pathology: the critical role of abnormal reduced glutathione (GSH) transport caused by CFTR mutation

Though the cause of cystic fibrosis (CF) pathology is understood to be the mutation of the CFTR protein, it has been difficult to trace the exact mechanisms by which the pathology arises and progresses from the mutation. Recent research findings have noted that the CFTR channel is not only permeant to chloride anions, but other, larger organic anions, including reduced glutathione (GSH). This explains the longstanding finding of extracellular GSH deficit and dramatically reduced extracellular GSH:GSSG (glutathione disulfide) ratio found to be chronic and progressive in CF patients. Given the vital role of GSH as an antioxidant, a mucolytic, and a regulator of inflammation, immune response, and cell viability via its redox status in the human body, it is reasonable to hypothesize that this condition plays some role in the pathogenesis of CF. This hypothesis is advanced by comparing the literature on pathological phenomena associated with GSH deficiency to the literature documenting CF pathology, with striking similarities noted. Several puzzling hallmarks of CF pathology, including reduced exhaled NO, exaggerated inflammation with decreased immunocompetence, increased mucus viscoelasticity, and lack of appropriate apoptosis by infected epithelial cells, are better understood when abnormal GSH transport from epithelia (those without anion channels redundant to the CFTR at the apical surface) is added as an additional explanatory factor. Such epithelia should have normal levels of total glutathione (though perhaps with diminished GSH:GSSG ratio in the cytosol), but impaired GSH transport due to CFTR mutation should lead to progressive extracellular deficit of both total glutathione and GSH, and, hypothetically, GSH:GSSG ratio alteration or even total glutathione deficit in cells with redundant anion channels, such as leukocytes, lymphocytes, erythrocytes, and hepatocytes. Therapeutic implications, including alternative methods of GSH augmentation, are discussed.     

Flow cytometric analysis of protein thiol groups in relation to the cell cycle and the intracellular content of glutathione in rat hepatocytes

The relationship between protein thiols (PSH) and cell proliferation was examined in ethanol-fixed rat hepatocytes. A new protocol was developed for simultaneous measurement of protein thiol vs. DNA content by flow cytometry. The fluorescent dye o-phthalaldehyde (OPT) was used for flow cytometric measurements of protein thiol groups. The influence of nonprotein thiols was examined by monitoring the cell cycle of cells in which the glutathione content (GSH) was modified by treatment with buthionine sulphoximine (BSO). Three rat liver cell lines (IAR 20, IAR 6.1, IAR 6.1RT7) were used: these cell lines possess different growth characteristics and degrees of tumorigenicity, which made it possible to analyse changes in PSH during normal and deranged cell proliferation. The effects on the cell cycle of the changes in PSH due to the depletion of GSH were measured by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and flow cytometry. The data obtained can be summarised as follows: a) OPT fluorescence increases with increasing DNA content in all rat liver cell lines examined; b) the greatest variation in PSH content occurs in G1. There is a smaller variation in G2 + M, and PSH levels are relatively invariant throughout S-phase; c) a higher content of PSH is found in the tumorigenic cell lines; d) the amount and distribution of PSH is not affected by BSO treatment; e) kinetic studies indicate that BSO treatment has no effect on the ability of the IAR rat liver cell lines to progress through the cycle.     

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells

AimsMenadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).Main methodsBAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.Key findingsIntracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.SignificanceWe conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.     

The nitric oxide-iron interplay in mammalian cells: transport and storage of dinitrosyl iron complexes.

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl-dithiolato-Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl-diglutathionyl-Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.     

Molecular and structural basis of glutathione import in Gram‐positive bacteria via GshT and the cystine ABC importer TcyBC of Streptococcus mutans

Glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions across all branches of life. Despite recent advances in our understanding of the transport mechanisms responsible for maintaining the spatiotemporal homeostasis of GSH and its conjugates in eukaryotes and Gram‐negative bacteria, the molecular and structural basis of GSH import into Gram‐positive bacteria has remained largely uncharacterized. Here, we employ genetic, biochemical and structural studies to investigate a possible glutathione import axis in Streptococcus mutans, an organism that has hitherto served as a model system. We show that GshT, a type 3 solute binding protein, displays physiologically relevant affinity for GSH and glutathione disulfide (GSSG). The crystal structure of GshT in complex with GSSG reveals a collapsed structure whereby the GS‐I‐leg of GSSG is accommodated tightly via extensive interactions contributed by the N‐ and C‐terminal lobes of GshT, while the GS‐II leg extends to the solvent. This can explain the ligand promiscuity of GshT in terms of binding glutathione analogues with substitutions at the cysteine‐sulfur or the glycine‐carboxylate. Finally, we show that GshT primes glutathione import via the l ‐cystine ABC transporter TcyBC, a membrane permease, which had previously exclusively been associated with the transport of l ‐cystine.     

Glutathione metabolism in cultured Sertoli cells and spermatogenic cells from hamsters

Isolated spermatocytes and spermatids from hamsters contained a large amount of glutathione (GSH) (approximately 40 and 30 nmol GSH/mg protein, respectively), but showed a spontaneous decrease of GSH content during prolonged incubation (t1/2 approximately 35 h). Incubation of the germ cells in the presence of the glutathione biosynthesis inhibitor buthionine sulphoximine (BSO) provided evidence that the cells can perform glutathione synthesis. This synthesis, however, was not sufficient to maintain the GSH content of the isolated cells, or to restore the cellular GSH pool after depletion caused by exposure of the cells to the glutathione S-transferase substrate, diethyl maleate (DEM). Cultured Sertoli cells, containing approximately 10 nmol GSH/mg protein, had a more active BSO-sensitive GSH synthesis system. The Sertoli cells, but also tubule fragments containing Sertoli cells and germ cells, were able to restore their GSH pool after DEM-induced depletion. DEM treatment of the tubule fragments resulted in a 90% decrease of the GSH content of the spermatocytes and spermatids present within the fragments. The GSH levels of the tubule fragments and the enclosed germ cells were restored during a subsequent incubation in the absence of DEM. As indicated above, such a recovery was not observed for isolated spermatocytes and spermatids. The results illustrate the importance of Sertoli cell-germ cell interaction, and point to a role of Sertoli cells in glutathione synthesis by the germ cells.     

Neuroprotection against neuroblastoma cell death induced by depletion of mitochondrial glutathione

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.     

Turnover and functions of glutathione studied with isolated hepatic and renal cells

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.     

A novel cis-acting cysteine-responsive regulatory element of the gene for the high-affinity glutathione transporter of Saccharomyces cerevisiae

We cloned a DNA fragment from Saccharomyces cerevisiae that complemented the deficiency in high-affinity glutathione transport activity conferred by a gsh11 mutation, and found that the ORF responsible was YJL212c, which had already been designated as OPT1 and HGT1 by others. Northern analysis clearly demonstrated that this ORF, now referred to as OPT1/ HGT1/ GSH11, was induced by sulfur starvation and repressed by adding cysteine to the growth medium. Reporter gene assays showed that a segment spanning the region between positions -371 and -355 was essential for the regulation of this gene. A sequence of 9 nt, CCGCCACAC (from -364 to -356), in this region was shown to be required for protein binding, using an electrophoretic mobility shift assay. Based on these results, we propose that CCGCCACAC comprises the core of a cis-acting element involved in cysteine-responsive gene regulation in S. cerevisiae.     

Lack of association between glutathione content and development of thermal tolerance in human fibroblasts

Thermal tolerance is a transient state of heat resistance occurring in cells and tissues after exposure to sublethal heat or certain chemicals. Although the mechanism of such resistance is unknown, it has been recently shown that preceding its development, cellular glutathione (GSH) levels rise. We have used a glutathione synthetase-deficient [GSH(-)] human fibroblast line to study the relationship between glutathione content and thermal tolerance. The GSH(-) cells had approximately 6% as much GSH as normal fibroblasts. Normal and GSH(-) fibroblasts showed similar survival after exposure to 45 degrees C. Exposure of normal fibroblasts to heat (45 degrees C for 15 min) led to a prompt rise in cellular GSH content as well as development of transient thermal tolerance. Similar treatment of GSH(-) fibroblasts produced no change in the very low GSH levels but was associated with a degree of thermal tolerance similar to that of normal cells. Thermal tolerance decayed more rapidly in GSH(-) cells than in normal fibroblasts. We conclude that the development of thermal tolerance in human fibroblasts is independent of GSH content.     

The nitric oxide–iron interplay in mammalian cells: Transport and storage of dinitrosyl iron complexes

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl–dithiolato–Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl–diglutathionyl–Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.     

Complete glutathione system in probiotic Lactobacillus fermentum ME-3

There is much information about glutathione (GSH) in eukaryotic cells, but relatively little is known about GSH in prokaryotes. Without GSH and glutathione redox cycle lactic acid bacteria (LAB) cannot protect themselves against reactive oxygen species. Previously we have shown the presence of GSH in Lactobacillus fermentum ME-3 (DSM14241). Results of this study show that probiotic L. fermentum ME-3 contains both glutathione peroxidase and glutathione reductase. We also present that L. fermentum ME-3 can transport GSH from environment and synthesize GSH. This means that it is characterized by a complete glutathione system: synthesis, uptake and redox turnover ability that makes L. fermentum ME-3 a perfect protector against oxidative stress. To our best knowledge studies on existence of the complete glutathione system in probiotic LAB strains are still absent and glutathione synthesis in them has not been demonstrated.     

Dual role of glutathione in selenite-induced oxidative stress and apoptosis in human hepatoma cells

It is well known that glutathione, the major intracellular antioxidant, is closely involved in the metabolism and bioactivity of selenium. In the present study, glutathione was demonstrated to play a dual role on selenite (Se)-induced oxidative stress and apoptosis in human hepatoma HepG(2) cells. The experiment was carried out in two different modes to modulate intracellular reduced glutathione (GSH) content. In Mode A (pretreatment), cells were pretreated with N-acetylcysteine (NAC), buthionine sulfoximine (BSO), or GSH prior to Se exposure. In Mode B (simultaneous treatment), cells were treated with Se and NAC, BSO, or GSH simultaneously. It was found that Se-induced oxidative stress and apoptosis are closely related to the intracellular level of GSH. Both the increase and depletion of GSH content significantly enhanced Se-induced oxidative stress and apoptosis in HepG(2) cells. Results from this study clearly demonstrated that GSH has a dual role in the effects of Se on cancer cells: (i) GSH acts as a pro-oxidant, facilitating Se-induced oxidative stress, and (ii) GSH acts as an antioxidant, protecting against Se-induced oxidative stress and apoptosis. Understanding such a unique association between GSH and Se may help to explain the controversy in the literature over the complex relationship between selenium and glutathione, and ultimately the capability of selenium to prevent cancer.