On-line derivatization for continuous and automatic monitoring of brain extracellular glutamate levels in anesthetized rats: a microdialysis study

Glutamate is an important excitatory amino acid in central nervous system. We developed a method for in vivo, continuous and automatic monitoring of brain extracellular glutamate, as well as other amino acids in anesthetized rat. This method involves the use of microdialysis perfusion technique and a high-performance liquid chromatography system equipped with a fluorescence detector. The microdialysate (perfused at a flow-rate of 1 μl/min) was on-line derivatized with o-phthaldehyde (perfused at 2 μl/min) through a mixing tee prior to the injection onto the HPLC column. The efficiency of this on-line derivatization was equivalent to that performed with an off-line manner. The effect of cerebral ischemia (2 h) and reperfusion (2 h) in brain cortex of anesthetized rats was monitored using this method. In addition to glutamate, extracellular concentrations of other amino acids, such as aspartate, glutamine, glycine, taurine and γ-aminobutyric acid, were also simultaneously monitored with this on-line method. Since monitoring of extracellular amino acids by microdialysis perfusion is intensively used in neuroscience investigations, this simple and convenient method would be useful in the future applications.     

In vivo, continuous and automatic monitoring of extracellular ascorbic acid by microdialysis and on-line liquid chromatography

A system for in vivo, automatic, continuous monitoring of organ extracellular ascorbic acid in anesthetized rat is described. This system involves microdialysis perfusion and a LC system equipped with an electrochemical detector. Microdialysate, eluted from a microdialysis probe implanted in the brain cortex or in the left ventricular myocardium of anesthetized rats was collected in the sample loop of an on-line injector for direct injection onto the LC system. This automated method provides a shortened sample processing time. This system was utilized to investigate the effect of cerebral ischemia on cortex extracellular ascorbic acid and the effect of myocardial ischemia on left ventricular myocardium extracellular ascorbic acid in anesthetized rats. Basal ascorbic acid concentrations in the cortex and left ventricular myocardium ranged from 9.7 to 15.4 μM (mean±S.D. 12.7±2.5 μM from the results of eight rats) and from 9.3 to 36.0 μM (mean±S.D., 24.3±8.9 μM from the results of twelve rats), respectively. Cerebral ischemia significantly elevated ascorbic acid levels in the cortex extracellular space, while myocardial ischemia did not significantly alter ascorbic acid levels in the left ventricular myocardium extracellular space.     

Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures

Arsenate is a toxic compound that has been connected with neuropathies and impaired cognitive functions. To test whether arsenate affects the viability and the GSH metabolism of brain astrocytes, we have used primary astrocyte cultures as model system. Incubation of astrocytes for 2 h with arsenate in concentrations of up to 10 mM caused an almost linear increase in the cellular arsenic content, but did not acutely compromise cell viability. The presence of moderate concentrations of arsenate caused a time- and concentration-dependent loss of GSH from viable astrocytes which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for arsenate in a concentration of about 0.3 mM. The arsenate-induced stimulated GSH export from astrocytes was prevented by MK571, an inhibitor of the multidrug resistance protein 1. Exposure of astrocytes to arsenite increased the specific cellular arsenic content and stimulated GSH export to values that were similar to those observed for arsenate-treated cells, while dimethylarsinic acid was less efficiently accumulated by the cells and did not modulate cellular and extracellular GSH levels. The observed strong stimulation of GSH export from astrocytes by arsenate suggests that disturbances of the astrocytic GSH metabolism may contribute to the observed arsenic-induced neurotoxicity.     

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2′,5′-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2′,5′-dihydroxychalcone (2′,5′-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 μm), indicating competitive inhibition. In contrast, 2′,5′-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.     

Use of Monochlorobimane to Determine the In Vivo Effect of Cadmium,Copper and Lead on Glutathione Status in Mercenaria mercenaria Brown Cells

This study was undertaken to demonstrate the synthesis of glutathione (GSH) in Mercenaria mercenaria brown cells to test the hypothesis that failure to achieve 100% mortality with metal treatment is the result of high concentrations of GSH heterogeneously distributed in the brown cell population and to determine the effect of Cd²⁺, Cu²⁺, and Pb²⁺ on the GSH status in brown cells. The monochlorobimane (MCB) assay appeared to be selective for GSH in brown cells and a close relationship between the levels of GSH measured by MCB and a standard enzymatic method was found. The fluorescent GSH-bimane adduct, once formed within the cell, was not released from the cell. The technique was used to establish that GSH was synthesized in brown cells and was heterogeneously distributed in the brown cell population. Metal concentrations as high as 40 mM cadmium, 6.0 μM copper, or 20 mM lead did not deplete but caused decreases in brown cell GSH concentration that differed significantly (P < 0.05) from controls. The decrease caused by cadmium, copper, and lead was not in a dose dependent manner, whereas, the decrease caused by N-ethylmaleimide (NEM) was. It appears that the partial kill phenomenon associated with metal toxicity may be due to the heterogeneous distribution of GSH in the brown cell population.     

Response to copper stress in aposymbiotically grown lichen mycobiont Cladonia cristatella: uptake,viability, ergosterol and production of non-protein thiols

The mycobiont of lichens usually determines the morphology of the symbiotic organism and is also dominates in terms of biomass. However, its role for sensitivity or tolerance of lichens to heavy metals is almost unknown. In the present study, the influence of copper (Cu) on the aposymbiotically-grown mycobiont of Cladonia cristatella was assessed. Intracellular Cu uptake was correlated with increasing Cu concentrations over a 24-h exposure time. Viability, measured as the degree of reduction of triphenyltetrazolium chloride to triphenyl formazan, as well as to ergosterol levels, decreased with growing Cu concentrations tested. Reduced glutathione (GSH) was found to be the most abundant low-molecular-weight thiol in the hyphae of C. cristatella and its intracellular content increased at concentrations of 10 μm Cu. Higher Cu concentrations caused a significant decrease in GSH, possibly due to heavy metal-induced oxidation of GSH to glutathione disulphide (GSSG). Free cysteine levels were relatively constant. As expected, we did not observe the production of phytochelatins in the mycobiont, contrary to what is found in intact lichens and axenic cultures of their photobionts.     

A Txnrd1-dependent metabolic switch alters hepatic lipogenesis,glycogen storage,and detoxification

Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug metabolism. We show that hepatocyte-specific disruption of Txnrd1, encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however, their constitutive metabolic state supported more robust GSH biosynthesis, glutathionylation, and glucuronidation systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage.     

Adaptation of subcellular glutathione detoxification system to stress conditions in choline-deficient diet induced rat fatty liver

The response of fatty liver to stress conditions (t-butyl hydroperoxide [t-BH] or 36 h of fasting) was investigated by assessing intracellular glutathione (GSH) compartmentation and redox status, GSH peroxidase (GSH-Px) and reductase (GSSG-Rx) activities, lipid peroxidation (TBARs) and serum ALT levels in rats on a choline-deficient diet. Baseline cytosolic GSH was similar between fatty and normal livers, while the mitochondrial GSH content was significantly lower in fatty livers. With the except of cytosolic GSH-Px activity, steatosis was associated with significantly higher GSH-related enzymes activities. Liver TBARs and serum ALT levels were also higher. Administration of t-BH significantly decreased the concentration of cytosolic GSH, increased GSSG levels in all the compartments, and increased TBARs levels in cytosol and mitochondria and serum ALT; all these alterations were more marked in rats with fatty liver. Fasting decreased the concentration of GSH in all the compartments both in normal and fatty livers, increased GSSG, TBARs and ALT levels, and decreased by 50% the activities of GSH-related enzymes. Administration of diethylmaleimide (DEM) resulted in cytosolic and microsomal GSH pool depletion. Administration of t-BH to DEM-treated rats further affected cytosolic GSH and enhanced ALT levels, whereas the application of fasting to GSH depleted rats mainly altered the mitochondrial GSH system, especially in fatty livers. This study shows that fatty livers have a weak compensation of hepatic GSH regulation, which fails under stress conditions, thus increasing the fatty liver's susceptibility to oxidative damage. Differences emerge among subcellular compartments which point to differential adaptation of these organelles to fatty degeneration.     

Control of wild carrot somatic embryo development by antioxidants : a probable mode of action of 2,4-dichlorophenoxyacetic Acid

As we previously reported for glutathione (GSH), both ascorbic acid (AA) and vitamin E were observed to suppress wild carrot (Daucus carota L.) somatic embryogenesis with little concomitant effect on biomass. Endogenous concentrations of AA were lower during embryo development than during cell proliferation, exhibiting a temporal pattern nearly identical to that of GSH. GSSG (oxidized GSH) reductase was found to be considerably more active in proliferating than in developing cultures, whereas no difference was evident in the case of dehydroascorbate (DHA) reductase. Both GSH and AA concentrations in these cells are governed by 2,4-D. These results show that redox status is a strong determinant of proliferative versus developmental growth and indicate that the mode of action of 2,4-D in this system may be explained at least in part by its influence on endogenous antioxidant levels.     

OXIDIZED AND REDUCED GLUTATHIONE IN THE RAT BRAIN UNDER NORMOXIC AND HYPOXIC CONDITIONS

In order to test the proposition that hypoxia leads to a change in the concentration ratio of reduced (GSH) and oxidized (GSSG) glutathione in the brain, enzymatic, fluorometric assays were worked out for measuring GSH and GSSG. In lightly anaesthetized and immobilized rats. GSH concentrations in the cerebral cortex and the cerebellum were close to 2 μmol.g^-1 while a slightly lower concentration (approx 1.4μmol.g^-1) was found in the brain stem. In order to avoid artefactual oxidation of GSH during sample preparation for GSSG determination the tissue was extracted with trichloroacetic acid, following alkylation of SH groups with N-ethylmaleimide. With these precautions GSSG concentrations were approx 0.7% of the corresponding GSH concentrations. However. the results indicated that the true GSSG concentrations may be even lower. During hypoxia there was neither a decrease in GSH nor an increase in GSSG concentrations in cortical tissue or cisternal CSF.     

Glutathione and ischemia-reperfusion injury in the perfused rat liver.

Using the isolated perfused rat liver, we investigated the relationship of glutathione (GSH) with reactive oxygen species (ROS) generation and liver cell damage during ischemia/reperfusion in normal and GSH-depleted conditions. Lucigenin-enhanced chemiluminescence was used as a sensitive index of tissue ROS generation. After 30 minutes of equilibration, livers were subjected to global ischemia for various times (60 or 90 minutes) and then reperfused for another 120 minutes. Intracellular ROS levels increased sharply at the onset of reperfusion and then declined slowly. After 30 to 60 minutes of reperfusion, ROS levels started to increase progressively in a linear fashion. However, sinusoidal glutathione disulfide release did not increase during reperfusion in the same livers, suggesting that intracellular ROS generation is too low to cause a significant increase in GSH oxidation. Pretreatment with phorone (300 mg/kg intrapentoneally [ip]), which reduced hepatic GSH by 90%, did not cause any difference in intracellular ROS generation compared with the control livers. There were also no significant differences in lactate dehydrogenase and thiobarbituric acid reactive substances (TBARS) release between the control and phorone-treated livers during reperfusion after various times of ischemia. These data indicate that ROS generation in the normal isolated perfused liver during ischemia/reperfusion is extremely low and intracellular GSH does not serve as a major intracellular defense system against such a low oxidative stress.     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

Extracellular Neuroactive Amino Acids in the Rat Striatum During Ischaemia: Comparison Between Penumbral Conditions and Ischaemia with Sustained Anoxic Depolarisation

Abstract: Changes in the extracellular levels of excitatory and inhibitory amino acid transmitters were studied in the rat striatum during penumbral ischaemia using intracerebral microdialysis. Effects of penumbral forebrain ischaemia were compared with those of ischaemia with sustained anoxic depolarisation and K⁺ (100 m M ). Comparisons were also made between different groups of animals at 2 and 24 h after dialysis probe implantation. The K⁺ stimulus did not provoke any release of excitatory amino acids in the 24-h group, probably reflecting a decrease of functional synapses adjacent to the probe. During 30 min of penumbral ischaemia, excitatory amino acids did not reach critical concentrations in the extracellular fluid, and increases in levels of inhibitory/modulatory amino acids were similar. On the other hand, severe transient ischaemia resulted in massive synchronous release of many neuroactive excitatory and inhibitory compounds, in both the 2- and 24-h groups. These and other data suggest that changes during severe ischaemia may arise from both neurotransmitter and metabolic pools. It is concluded that is- chaemic damage in the penumbra may not be related to extracellular neuroactive amino acid changes generated within this region.     

In Situ Microdialysis for Monitoring of Extracellular Glutathione Levels in Normal,Ischemic and Post-Ischemic Skeletal Muscle

Microdialysis probes were inserted into the tibialis anterior muscle and into the femoral vein of anaesthetised Sprague-Dawley rats for monitoring of reduced (GSH) and oxidized (GSSG) extracellular glutathione. The dialysates were analysed using HPLC. The levels of GSH and GSSG were high immediately after implantation in the skeletal muscle and declined to steady state levels after 90 minutes into the same range as that found in the venous dialysate. Total ischemia was induced two hours after implantation of the dialysis probe after steady state levels had been reached. The extracellular levels of GSH increased during total ischemia and had doubled at the end of the ischemic period compared to preischemic values. During the following initial 30 minutes of reperfusion the levels increased further to four-fold the preischemic levels. The levels of GSSG also increased (100%) during the initial 30 minutes of reperfusion. The extracellular GSH levels remained elevated for 1 hour of reperfusion, but the GSSG levels returned to preischemic levels. The results indicate that intermittent hypoxia or anoxia in muscle tissue through hypoperfusion or ischemia decreases intracellular GSH stores by leakage, reducing the intracellular antioxidative capacity and increasing the risk for oxidative reperfusion injury upon final normalization of tissue blood supply.     

In Situ Microdialysis for Monitoring of Extracellular Glutathione Levels in Normal, Ischemic and Post-Ischemic Skeletal Muscle

Microdialysis probes were inserted into the tibialis anterior muscle and into the femoral vein of anaesthetised Sprague-Dawley rats for monitoring of reduced (GSH) and oxidized (GSSG) extracellular glutathione. The dialysates were analysed using HPLC. The levels of GSH and GSSG were high immediately after implantation in the skeletal muscle and declined to steady state levels after 90 minutes into the same range as that found in the venous dialysate. Total ischemia was induced two hours after implantation of the dialysis probe after steady state levels had been reached. The extracellular levels of GSH increased during total ischemia and had doubled at the end of the ischemic period compared to preischemic values. During the following initial 30 minutes of reperfusion the levels increased further to four-fold the preischemic levels. The levels of GSSG also increased (100%) during the initial 30 minutes of reperfusion. The extracellular GSH levels remained elevated for 1 hour of reperfusion, but the GSSG levels returned to preischemic levels. The results indicate that intermittent hypoxia or anoxia in muscle tissue through hypoperfusion or ischemia decreases intracellular GSH stores by leakage, reducing the intracellular antioxidative capacity and increasing the risk for oxidative reperfusion injury upon final normalization of tissue blood supply.     

On-line monitoring of PHB production by mixed microbial cultures using respirometry,titrimetry and chemometric modelling

This work describes a method for on-line monitoring of biomass production, acetate consumption and intracellular polyhydroxybutyrate (PHB) storage by mixed microbial cultures (MMC). The method is based on reliable and easily available on-line measurements, namely pH, dissolved oxygen, dissolved carbon dioxide, on-line respirometry and on-line titrimetric analysis. Biomass production refers to active biomass growth and also to the synthesis of extracellular polymeric substances (EPS). The composition and kinetics of EPS synthesis has high variability depending on the culture enrichment protocol. Since the metabolism for EPS production is rather difficult to define, it was not possible to develop a reliable estimation model based on metabolic principles only. Instead, projection of latent structures (PLS) linear regression constrained by steady state carbon balance was employed. PHB concentration and biomass production rate were directly estimated by the PLS model, whereas acetate concentration was indirectly estimated through the carbon balance. The method was validated experimentally with data of four experiments carried out in a SBR. Accurate on-line estimations were obtained with regression coefficients (r²) of 0.986 and 0.980 for biomass concentration, 0.976 and 0.999 for PHB and 0.992 and 0.999 for acetate concentration in calibration and validation, respectively. These results confirm the ability of the proposed methodology for on-line monitoring of the state variables in PHB production process by MMC.     

Method of intracranial pressure monitoring and cerebrospinal fluid sampling in swine

Cerebral oedema and encephalopathy have been noted to occur frequently in patients severely ill or dying after trauma, ischaemia, infections or even metabolic disorders. The objective of the present study was to establish continuous monitoring of the intracranial pressure (ICP) and sampling of cerebrospinal fluid (CSF) for further investigations in swine. ICP monitoring was established in eight pigs by using a ventricular drainage system, implemented after paramedian trepanation of the os frontale. CSF and serum samples were taken for measurement of the levels of glucose and protein. Operating time was 21+/-8 min for the trepanation until ICP monitoring was performed. No complications occurred during surgery. Continuous monitoring of ICP and CSF sampling was easy to perform, and without any side-effects in any animal. At autopsy, no iatrogenic lesions were found and monitoring catheters were still in place. For several types of research requiring ICP monitoring and sampling of CSF, this method can be used successfully.     

Glutamate induces release of glutathione from cultured rat astrocytes – a possible neuroprotective mechanism?

Glutamate is the major excitatory amino acid of the mammalian brain but can be toxic to neurones if its extracellular levels are not tightly controlled. Astrocytes have a key role in the protection of neurones from glutamate toxicity, through regulation of extracellular glutamate levels via glutamate transporters and metabolic and antioxidant support. In this study, we report that cultures of rat astrocytes incubated with high extracellular glutamate (5 mM) exhibit a twofold increase in the extracellular concentration of the tripeptide antioxidant glutathione (GSH) over 4 h. Incubation with glutamate did not result in an increased release of lactate dehydrogenase, indicating that the rise in GSH was not because of membrane damage and leakage of intracellular pools. Glutamate-induced increase in extracellular GSH was also independent of de novo GSH synthesis, activation of NMDA and non-NMDA glutamate receptors or inhibition of extracellular GSH breakdown. Dose–response curves indicate that GSH release from rat astrocytes is significantly stimulated even at 0.1 mM glutamate. The ability of astrocytes to increase GSH release in the presence of extracellular glutamate could be an important neuroprotective mechanism enabling neurones to maintain levels of the key antioxidant, GSH, under conditions of glutamate toxicity.     

Chronic Inflammation Alters Production and Release of Glutathione and Related Thiols in Human U373 Astroglial Cells

Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer’s disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1β and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1β and TNF-α (0.01–1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1β and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1β and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1β and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD.     

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.