The Thioredoxin-Thioredoxin Reductase System Can Function in Vivo as an Alternative System to Reduce Oxidized Glutathione in Saccharomyces cerevisiae

Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione E_h′. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular E_h′. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.     

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.     

Synthesis and role of glutathione in protection against oxidative stress in yeast

Summary

Glutathione (GSH) is an abundant and ubiquitous low-molecular-mass thiol with proposed roles in many cellular processes including amino acid transport, synthesis of proteins and nucleic acids, modulation of enzyme activity and metabolism of xenobiotics, carcinogens and reactive oxygen species. This review describes recent findings in the lower eukaryote Saccharomyces cerevisiae that are leading to a better understanding of the role of this peptide in eukaryotic cell metabolism. In particular, two gene products involved in maintaining the levels of reduced GSH have been studied; namely, GSH1 encoding γ-glutamylcysteine synthetase, the first step in the biosynthesis of GSH, and glutathione reductase, which recycles glutathione to its reduced form. These studies indicate that GSH is an essential metabolite in yeast, and that it is required for protection against oxidative stress produced by mitochondrial metabolism and exogenous reactive oxygen species. These findings are discussed in the light of analogous observations made in higher eukaryotes.     

Antioxidants and phase 2 enzymes in cardiomyocytes: Chemical inducibility and chemoprotection against oxidant and simulated ischemia-reperfusion injury

The increasing recognition of the role for oxidative stress in cardiac disorders has led to extensive investigation on the protection by exogenous antioxidants against oxidative cardiac injury. On the other hand, another strategy for protecting against oxidative cardiac injury may be through upregulation of the endogenous antioxidants and phase 2 enzymes in the myocardium by chemical inducers. However, our current understanding of the chemical inducibility of cardiac cellular antioxidants and phase 2 enzymes is very limited. In this study, using rat cardiac H9c2 cells we have characterized the concentration- and time-dependent induction of cellular antioxidants and phase 2 enzymes by 3H-1,2-dithiole-3-thione (D3T), and the resultant chemoprotective effects on oxidative cardiac cell injury. Incubation of H9c2 cells with D3T resulted in a marked concentration- and time-dependent induction of a number of cellular antioxidants and phase 2 enzymes, including catalase, reduced glutathione (GSH), GSH peroxidase, glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). D3T treatment of H9c2 cells also caused an increase in mRNA expression of catalase, gamma-glutamylcysteine ligase catalytic subunit, GR, GSTA1, M1 and P1, and NQO1. Moreover, both mRNA and protein expression of Nrf2 were induced in D3T-treated cells. D3T pretreatment led to a marked protection against H9c2 cell injury elicited by various oxidants and simulated ischemia-reperfusion. D3T pretreatment also resulted in decreased intracellular accumulation of reactive oxygen in H9c2 cells after exposure to the oxidants as well as simulated ischemia-reperfusion. This study demonstrates that a series of endogenous antioxidants and phase 2 enzymes in H9c2 cells can be induced by D3T in a concentration- and time-dependent fashion, and that the D3T-upregulated cellular defenses are accompanied by a markedly increased resistance to oxidative cardiac cell injury.     

Inhibition of glutathione disulfide reductase by glutathione

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.     

Determination of glutathione and glutathione disulfide in hepatocytes by liquid chromatography with an electrode modified with functionalized carbon nanotubes

Glutathione (GSH) and glutathione disulfide (GSSG) are important thiols, which provide defence against oxidative stress by scavenging free radicals or causing the reduction of hydrogen peroxide. The ratio GSH/GSSG is often used as a sensitive index of oxidative stress in vivo. In this paper, a direct electrochemical method using an electrode modified with functionalized carbon nanotubes as electrochemical detector (ED) for liquid chromatography (LC) was described. The electrochemical behaviors of GSH and GSSG on this modified electrode were investigated by cyclic voltammetry and it was found that the functionalized carbon nanotubes exhibited efficiently electrocatalysis on the current responses of GSH and GSSG. In LC-ED, both of the analytes showed good and stable current responses. The detection limit of GSH was 0.2 pmol on column and that of GSSG was 1.2 pmol on column, which were low enough for the analysis of real small samples. The method was sensitive enough to detect difference in concentration of GSH and GSSG in hepatocytes from animals with and without introduction of oxidation stress by glucose or hydrogenperoxide.     

Oxidative damage to proteins in yeast cells exposed to adaptive levels of H(2)O(2)

When yeast cells are exposed to sublethal concentrations of oxidants, they adapt to tolerate subsequent lethal treatments. Here, we show that this adaptation involves tolerance of oxidative damage, rather than protection of cellular constituents. o- and m-tyrosine levels are used as a sensitive measure of protein oxidative damage and we show that such damage accumulates in yeast cells exposed to H(2)O(2) at low adaptive levels. Glutathione represents one of the main cellular protections against free radical attack and has a role in adaptation to oxidative stress. Yeast mutants defective in glutathione metabolism are shown to accumulate significant levels of o- and m-tyrosine during normal aerobic growth conditions.     

Effects of sulfite on glutathione S-sulfonate and the glutathione status of lung cells

A mechanistic study was performed to elucidate the biochemical events connected with the cocarcinogenic effect of sulfur dioxide (SO2). Glutathione S-sulfonate (GSSO3H), a competitive inhibitor of the glutathione S-transferases, forms in lung cells exposed in culture to sulfite, the hydrated form of SO2. Changes in glutathione status (total GSH) were also observed during a 1-h exposure. Some cells were pretreated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione reductase. In human lung cells GSSO3H formed in a concentration-dependent manner, while glutathione (GSH) increased and glutathione disulfide (GSSG) decreased as the extracellular sulfite concentration was increased from 0 to 20 mM. The ratio of GSH/GSSG increased greater than 5-fold and the GSH/GSSO3H ratio decreased to 10 with increasing sulfite concentration. GSSO3H formed in rat lung cells exposed to sulfite, with no detectable effect on GSH and GSSG. GSSO3H also formed from cellular GSH mixed disulfides. GSSO3H formed rapidly, reaching its maximum value in 15 min. The viability of both cell types was unaffected except at 20 mM sulfite. GSSO3H incubated with human lung cells did not affect cellular viability. BCNU inhibited cellular GSSO3H reductase to the same extent as GSSG reductase. These results indicate that GSSO3H is formed in cells exposed to sulfite, and could be the active metabolite of sulfite responsible for the cocarcinogenic effect of SO2 by inhibiting conjugation of electrophiles by GSH.     

Mechanism of RPE cell death in α-crystallin deficient mice: a novel and critical role for MRP1-mediated GSH efflux

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.     

Dissecting the superoxide dismutase-ascorbate-glutathione-pathway in chloroplasts by metabolic modeling. Computer simulations as a step towards flux analysis

The present study introduces metabolic modeling as a new tool to analyze the network of redox reactions composing the superoxide dismutase-ascorbate (Asc)-glutathione (GSH) cycle. Based on previously determined concentrations of antioxidants and defense enzymes in chloroplasts, kinetic properties of antioxidative enzymes, and nonenzymatic rate constants of antioxidants with reactive oxygen, models were constructed to simulate oxidative stress and calculate changes in concentrations and fluxes of oxidants and antioxidants. Simulated oxidative stress in chloroplasts did not result in a significant accumulation of O2*- and H2O2 when the supply with reductant was sufficient. Model results suggest that the coupling between Asc- and GSH-related redox systems was weak because monodehydroascorbate radical reductase prevented dehydroascorbate (DHA) formation efficiently. DHA reductase activity was dispensable. Glutathione reductase was mainly required for the recycling of GSH oxidized in nonenzymatic reactions. In the absence of monodehydroascorbate radical reductase and DHA reductase, glutathione reductase and GSH were capable to maintain the Asc pool more than 99% reduced. This suggests that measured DHA/Asc ratios do not reflect a redox balance related to the Asc-GSH-cycle. Decreases in Asc peroxidase resulted in marked H2O2 accumulation without significant effects on the redox balance of Asc/DHA or GSH/GSSG. Simulated loss of SOD resulted in higher H2O2 production rates, thereby affecting all subsequent steps of the Asc-GSH-cycle. In conclusion, modeling approaches contribute to the theoretical understanding of the functioning of antioxidant systems by pointing out questions that need to be validated and provide additional information that is useful to develop breeding strategies for higher stress resistance in plants.     

Progressive iron accumulation induces a biphasic change in the glutathione content of neuroblastoma cells

Glutathione (GSH) constitutes the single most important antioxidant in neurons, whereas iron causes oxidative stress that leads to cell damage and death. Although GSH and iron produce opposite effects on redox cell status, no mechanistic relationships between iron and GSH metabolism are known. In this work, we evaluated in SH-SY5Y neuroblastoma cells the effects of iron accumulation on intracellular GSH metabolism. After 2 d exposure to increasing concentrations of iron, cells underwent concentration-dependent iron accumulation and a biphasic change in intracellular GSH levels. Increasing iron from 1 to 5 microM resulted in a marked increase in intracellular oxidative stress and increased GSH levels. Increased GSH levels were due to increased synthesis. Further increases in iron concentration led to significant reduction in both reduced (GSH) and total (GSH + (2 x GSSG)) glutathione. Cell exposure to high iron concentrations (20-80 microM) was associated with a marked decrease in the GSH/GSSG molar ratio and the GSH half-cell reduction potential. Moreover, increasing iron from 40 to 80 microM resulted in loss of cell viability. Iron loading did not change GSH reductase activity but induced significant increases in GSH peroxidase and GSH transferase activities. The changes in GSH homeostasis reported here recapitulate several of those observed in Parkinson's disease substantia nigra. These results support a model by which progressive iron accumulation leads to a progressive decrease in GSH content and cell reduction potential, which finally results in impaired cell integrity.     

Disruption of a glutathione reductase encoding gene in Acremonium chrysogenum leads to reduction of its growth, cephalosporin production and antioxidative ability which is recovered by exogenous methionine

Glutathione is a ubiquitous thiol in eukaryotic cells, and its high intracellular ratio of reduced form (GSH) to oxidized form (GSSG) is largely maintained by glutathione reductase (GR) using NADPH as electron donor. glrA, a glutathione reductase encoding gene, was found and cloned from Acremonium chrysogenum by searching its genomic sequence based on similarity. Its deduced protein exhibits high similarity to GRs of other eukaryotic organisms. Disruption of glrA resulted in lack of GR activity and accumulation of a high level of GSSG in A. chrysogenum. Overexpression of glrA dramatically enhanced GR activity and the ratio of GSH/GSSG in this fungus. The spore germination and hyphal growth of glrA disruption mutant was strongly reduced in chemical defined medium. Meanwhile, the mutant was more sensitive to hydrogen peroxide than the wild-type strain. We found that the glrA mutant recovered normal germination and growth by adding exogenous methionine (Met). Exogenous Met also enhanced the antioxidative ability of both the mutant and wild-type strain. GSH determination indicated that the total GSH and ratio of GSH/GSSG in the mutant or wild-type strain were significantly increased when addition of Met into the medium. The glrA mutant grew poorly and could not produce detectable cephalosporin in the fermentation medium without Met. However, its growth and cephalosporin production was restored with addition of exogenous Met. These results indicate that glrA is required for the normal growth and protection against oxidative damage in A. chrysogenum, and its absence can be complemented by exogenous Met.     

Glutathione and antioxidant enzymes in skeletal muscle: effects of fiber type and exercise intensity.

Glutathione status and antioxidant enzymes in various types of rat skeletal muscle were studied after an acute bout of exercise (Ex) at different intensities. Glutathione (GSH) and glutathione disulfide (GSSG) concentrations were the highest in soleus (SO) muscle, followed by those in deep (DVL) and then superficial (SVL) portions of vastus lateralis. In DVL, but not in SO or SVL, muscle GSH increased proportionally with Ex intensity and reached 1.8 +/- 0.08 mumol/g wet wt compared with 1.5 +/- 0.03 (P < 0.05) in resting controls (R). GSSG in DVL was increased from 0.10 +/- 0.01 mumol/g wet wt in R to 0.14 +/- 0.01 (P < 0.05) after Ex. Total glutathione (GSH + GSSG) contents in DVL were also significantly elevated with Ex, whereas GSH/GSSG ratio was unchanged. Activities of GSH peroxidase (GPX), GSSG reductase (GR), and catalase (CAT) were significantly higher in SO than in DVL and SVL, but there was no difference in superoxide dismutase activity between the three muscle types. Furthermore, Ex at moderate intensities elicited significant increases in GPX, GR, and CAT activities in DVL muscle. None of the antioxidant enzymes was affected by exercise in SO. It is concluded that rat DVL muscle is particularly vulnerable to exercise-induced free radical damage and that a disturbance of muscle GSH status is indicative of an oxidative stress.     

HIV-1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein-1 (Mrp1) in glial cells

Brain human immunodeficiency virus type-1 (HIV-1) infection is associated with oxidative stress, which may lead to HIV-1 encephalitis, a chronic neurodegenerative condition. In vitro , oxidative stress can be induced in glial cells by exposure to HIV-1 envelope protein glycoprotein (gp120). Multidrug resistance proteins (Mrps) are known to efflux endogenous substrates (i.e. GSH and GSSG) involved in cellular defense against oxidative stress. Altered GSH/GSSG export may contribute to oxidative damage during HIV-1 encephalitis. At present, it is unknown if gp120 exposure can alter the functional expression of Mrp isoforms. Heat-shock protein 70, inducible nitric oxide synthase, intracellular GSSG, 2',7'-dichlorofluorescein fluorescence, and extracellular nitrite were increased in primary cultures of rat astrocytes triggered with gp120, suggesting an oxidative stress response. RT-PCR and immunoblot analysis demonstrated increased Mrp1 mRNA (2.3-fold) and protein (2.2-fold), respectively, in gp120 treated astrocytes while Mrp4 mRNA or protein expression was not changed. Cellular retention of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, an established Mrp substrate, was reduced (twofold) in gp120-treated astrocytes, suggesting increased Mrp-mediated transport. In addition, GSH and GSSG export were enhanced in gp120-triggered cells. These data suggest that gp120 can up-regulate Mrp1, but not Mrp4, functional expression in cultured astrocytes. Our observation of increased GSH/GSSG efflux in response to gp120 treatment implies that Mrp isoforms may be involved in regulating the oxidative stress response in glial cells.     

Oxidation and release of glutathione from myocardium during early reperfusion

Glutathione (GSH) is an important intracellular defense against reactive oxygen metabolites. Reaction of GSH with peroxides generates oxidized glutathione (GSSG). We hypothesized that reperfusion would cause oxidation of GSH and release of GSSG as a potential marker of intracellular oxidative reactions. Ten dogs underwent 90 min left anterior descending (LAD) occlusion and 30 min reperfusion. Coronary sinus (CS) plasma was sampled from the great cardiac vein, which drains the LAD region, and from the aorta at pre-ischemia (I), 90 min ischemia, and during reperfusion (R). We found that both GSSG and GSH increased in coronary sinus plasma during early reperfusion. (Formula: see text) Measured GSSG did not arise from autoxidation of plasma GSH. GSH and GSSG release from myocardium not only may be evidence of intracellular oxidative injury, but loss of GSH also could impair metabolism of peroxides during early reperfusion and predispose to further injury.