Redox state of glutathione in human plasma

Thiol and disulfide forms of glutathione (GSH) and cysteine (Cys) were measured in plasma from 24 healthy individuals aged 25-35 and redox potential values (E(h)) for thiol/disulfide couples were calculated using the Nernst equation. Although the concentration of GSH (2.8 +/- 0.9 microM) was much greater than that of GSSG (0.14 +/- 0.04 microM), the redox potential of the GSSG/2GSH pool (-137 +/- 9 mV) was considerably more oxidized than values for tissues and cultured cells (-185 to -258 mV). This indicates that a rapid oxidation of GSH occurs upon release into plasma. The difference in values between individuals was remarkably small, suggesting that the rates of reduction and oxidation in the plasma are closely balanced to maintain this redox potential. The redox potential for the Cys and cystine (CySS) pool (-80 +/- 9 mV) was 57 mV more oxidized, showing that the GSSG/2GSH and the CySS/2Cys pools are not in redox equilibrium in the plasma. Potentials for thiol/disulfide couples involving CysGly were intermediate between the values for these couples. Regression analyses showed that the redox potentials for the different thiol/disulfide couples within individuals were correlated, with the E(h) for CySS-mono-Gly/(Cys. CysGly) providing the best correlation with other low molecular weight pools as well as protein disulfides of GSH, CysGly and Cys. These results suggest that E(h) values for GSSG/2GSH and CySS-mono-Gly/(Cys. CysGly) may provide useful means to quantitatively express the oxidant/antioxidant balance in clinical and epidemiologic studies.     

Glutathione and thioredoxin redox during differentiation in human colon epithelial (Caco-2) cells

Cellular redox, maintained by the glutathione (GSH)- and thioredoxin (Trx)-dependent systems, has been implicated in the regulation of a variety of biological processes. The redox state of the GSH system becomes oxidized when cells are induced to differentiate by chemical agents. The aim of this study was to determine the redox state of cellular GSH/glutathione disulfide (GSH/GSSG) and Trx as a consequence of progression from proliferation to contact inhibition and spontaneous differentiation in colon carcinoma (Caco-2) cells. Results showed a significant decrease in GSH concentration, accompanied by a 40-mV oxidation of the cellular GSH/GSSG redox state and a 28-mV oxidation of the extracellular cysteine/cystine redox state in association with confluency and increase in differentiation markers. The redox state of Trx did not change. Thus the two central cellular antioxidant and redox-regulating systems (GSH and Trx) were independently controlled. According to the Nernst equation, a 30-mV oxidation is associated with a 10-fold change in the reduced/oxidized ratio of a redox-sensitive dithiol motif. Therefore, the measured 40-mV oxidation of the cellular GSH/GSSG couple or the 28-mV oxidation of the extracellular cysteine/cystine couple should be sufficient to function in signaling or regulation of differentiation in Caco-2 cells.     

Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis

Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis.     

Stimulation of colonic mucosal growth associated with oxidized redox status in rats

Limited data in animal models suggest that colonic mucosa undergoes adaptive growth following massive small bowel resection (SBR). In vitro data suggest that intestinal cell growth is regulated by reactive oxygen species and redox couples [e.g., glutathione (GSH)/glutathione disulfide (GSSG) and cysteine (Cys)/cystine (CySS) redox]. We investigated the effects of SBR and alterations in redox on colonic growth indexes in rats after either small bowel transection (TX) or 80% midjejunoileal resection (RX). Rats were pair fed +/- blockade of endogenous GSH synthesis with buthionine sulfoximine (BSO). Indexes of colonic growth, proliferation, and apoptosis and GSH/GSSG and Cys/CySS redox potentials (E(h)) were determined. RX significantly increased colonic crypt depth, number of cells per crypt, and epithelial cell proliferation [crypt cell bromodeoxyuridine (BrdU) incorporation]. Administration of BSO markedly decreased colonic mucosal GSH, GSSG, and Cys concentrations in both TX and RX groups, with a resultant oxidation of GSH/GSSG and Cys/CySS E(h). BSO did not alter colonic crypt cell apoptosis but significantly increased all colonic mucosal growth indexes (crypt depth, cells/crypt, and BrdU incorporation) in both TX and RX groups in a time- and dose-dependent manner. BSO significantly decreased plasma GSH and GSSG, oxidized GSH/GSSG E(h), and increased plasma Cys and CySS concentrations. Collectively, these data provide in vivo evidence indicating that oxidized colonic mucosal redox status stimulates colonic mucosal growth in rats. The data also suggest that GSH is required to maintain normal colonic and plasma Cys/CySS homeostasis in these animal models.     

Redox biology of the intestine

The intestinal tract, known for its capability for self-renew, represents the first barrier of defence between the organism and its luminal environment. The thiol/disulfide redox systems comprising the glutathione/glutathione disulfide (GSH/GSSG), cysteine/cystine (Cys/CySS) and reduced and oxidized thioredoxin (Trx/TrxSS) redox couples play important roles in preserving tissue redox homeostasis, metabolic functions, and cellular integrity. Control of the thiol-disulfide status at the luminal surface is essential for maintaining mucus fluidity and absorption of nutrients, and protection against chemical-induced oxidant injury. Within intestinal cells, these redox couples preserve an environment that supports physiological processes and orchestrates networks of enzymatic reactions against oxidative stress. In this review, we focus on the intestinal redox and antioxidant systems, their subcellular compartmentation, redox signalling and epithelial turnover, and contribution of luminal microbiota, key aspects that are relevant to understanding redox-dependent processes in gut biology with implications for degenerative digestive disorders, such as inflammation and cancer.     

Extracellular thiol/disulfide redox state affects proliferation rate in a human colon carcinoma (Caco2) cell line

Redox mechanisms function in regulation of cell growth, and variation in redox state of plasma thiol/disulfide couples occurs in various physiologic conditions, including diabetes, chemotherapy, and aging. The present study was designed to determine whether a systematic variation in extracellular thiol/disulfide redox state (E(h)) over a range (0 mV to -150 mV) that occurs in human plasma altered proliferation of cultured cells. Experiments were performed with a human colon carcinoma cell line (Caco2), which grows slowly in the absence of serum and responds to peptide growth factors with increased rate of cell division. The extracellular redox states were established by varying concentrations of cysteine and cystine, maintaining constant pool size in terms of cysteine equivalents. Incorporation of 5-bromo-2-deoxyuridine (BrdU) was used to measure DNA synthesis and was lowest at the most oxidized extracellular E(h) (0 mV). Incorporation increased as a function of redox state, attaining a 100% higher value at the most reduced condition (-150 mV). Addition of insulin-like growth factor-1 (IGF-1) or epidermal growth factor (EGF) increased the rate of BrdU incorporation at more oxidizing redox conditions (0 to -80 mV) but had no effect at -150 mV. Cellular GSH was not significantly affected by variation in extracellular E(h). In the absence of growth factors, extracellular E(h) values were largely maintained for 24 h. However, IGF-1 or EGF stimulated a change in extracellular redox to values similar to that for cysteine/cystine redox in plasma of young, healthy individuals. The results show that extracellular thiol/disulfide redox state modulates cell proliferation rate and that this control interacts with growth factor signaling apparently independently of cellular glutathione.     

Redox biology of the intestine

Abstract

The intestinal tract, known for its capability for self-renew, represents the first barrier of defence between the organism and its luminal environment. The thiol/disulfide redox systems comprising the glutathione/glutathione disulfide (GSH/GSSG), cysteine/cystine (Cys/CySS) and reduced and oxidized thioredoxin (Trx/TrxSS) redox couples play important roles in preserving tissue redox homeostasis, metabolic functions, and cellular integrity. Control of the thiol-disulfide status at the luminal surface is essential for maintaining mucus fluidity and absorption of nutrients, and protection against chemical-induced oxidant injury. Within intestinal cells, these redox couples preserve an environment that supports physiological processes and orchestrates networks of enzymatic reactions against oxidative stress. In this review, we focus on the intestinal redox and antioxidant systems, their subcellular compartmentation, redox signalling and epithelial turnover, and contribution of luminal microbiota, key aspects that are relevant to understanding redox-dependent processes in gut biology with implications for degenerative digestive disorders, such as inflammation and cancer.     

Impact of sex,age and diet on the cysteine/cystine and glutathione/glutathione disulfide plasma redox couples in mice

Age, sex and diet are well-established risk factors for several diseases. In humans, each of these variables has been linked to differences in plasma redox potentials (E_h) of the glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox couples. Mice have been very useful for modeling human disease processes, but it is unknown if age, sex and diet affect redox couples in mice as they do in humans. The purpose of the present study was to examine the effects of these factors on plasma redox potentials in C57BL/6J mice. We found that age had no effect on either redox couple in either sex. Plasma E_h Cys/CySS and E_h GSH/GSSG were both more oxidized (more positive) in females than in males. A 24-hour fast negated the sex differences in both redox potentials by oxidizing both redox couples in male mice, while having no effect on E_h Cys/CySS and a smaller effect on E_h GSH/GSSG in female mice. A diet with excess sulfur amino acids reduced the plasma E_h Cys/CySS in females to a level comparable to that seen in male mice. Thus, sex-specific differences in plasma E_h Cys/CySS could be normalized by two different dietary interventions. Some of these findings are consistent with reported human studies, while others are not. Most strikingly, mice do not exhibit age-dependent oxidation of plasma redox potentials. Care must be taken when designing and interpreting mouse studies to investigate redox regulation in humans.     

Glutathione redox potential in response to differentiation and enzyme inducers

The reduced glutathione (GSH)/oxidized glutathione (GSSG) redox state is thought to function in signaling of detoxification gene expression, but also appears to be tightly regulated in cells under normal conditions. Thus it is not clear that the magnitude of change in response to physiologic stimuli is sufficient for a role in redox signaling under nontoxicologic conditions. The purpose of this study was to determine the change in 2GSH/GSSG redox during signaling of differentiation and increased detoxification enzyme activity in HT29 cells. We measured GSH, GSSG, cell volume, and cell pH, and we used the Nernst equation to determine the changes in redox potential Eh of the 2GSH/GSSG pool in response to the differentiating agent, sodium butyrate, and the detoxification enzyme inducer, benzyl isothiocyanate. Sodium butyrate caused a 60-mV oxidation (from -260 to -200 mV), an oxidation sufficient for a 100-fold change in protein dithiols:disulfide ratio. Benzyl isothiocyanate caused a 16-mV oxidation in control cells but a 40-mV oxidation (to -160 mV) in differentiated cells. Changes in GSH and mRNA for glutamate:cysteine ligase did not correlate with Eh; however, correlations were seen between Eh and glutathione S-transferase (GST) and nicotinamide adenine dinucleotide phosphate (NADPH):quinone reductase activities (N:QR). These results show that 2GSH/GSSG redox changes in response to physiologic stimuli such as differentiation and enzyme inducers are of a sufficient magnitude to control the activity of redox-sensitive proteins. This suggests that physiologic modulation of the 2GSH/GSSG redox poise could provide a fundamental parameter for the control of cell phenotype.     

Ameliorative potential of S-allyl cysteine on oxidative stress in STZ induced diabetic rats

Increased oxidative stress and impaired antioxidant defense mechanism are important factors in the pathogenesis and progression of diabetes mellitus and other oxidant-related diseases. The present study was undertaken to evaluate the possible protective effects of S-allyl cysteine (SAC) against oxidative stress in streptozotocin (STZ) induced diabetic rats. SAC was administered orally for 45 days to control and STZ induced diabetic rats. The effects of SAC on glucose, plasma insulin, thiobarbituric acid reactive substances (TBARS), hydroperoxide, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH/GSSG ratio were studied. The levels of glucose, TBARS, hydroperoxide, and GSSG were increased significantly whereas the levels of plasma insulin, reduced glutathione, GSH/GSSG ratio, superoxide dismutase, catalase and GPx were decreased in STZ induced diabetic rats. Administration of SAC to diabetic rats showed a decrease in plasma glucose, TBARS, hydroperoxide and GSSG. In addition, the levels of plasma insulin, superoxide dismutase, catalase, GPx and reduced glutathione (GSH) were increased in SAC treated diabetic rats. The above findings were supported by histological observations of the liver and kidney. The antioxidant effect of SAC was compared with glyclazide, a well-known antioxidant and antihyperglycemic drug. The present study indicates that the SAC possesses a significant favorable effect on antioxidant defense system in addition to its antidiabetic effect.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Differential oxidation of thioredoxin-1, thioredoxin-2, and glutathione by metal ions

Metal toxicity often includes the generation of reactive oxygen species (ROS) and subsequent oxidative stress, but whether metals have different effects on the major thiol antioxidant systems is unknown. Here, we examine the effects of arsenic, cadmium, cesium, copper, iron, mercury, nickel, and zinc on glutathione (GSH), cytoplasmic thioredoxin-1 (Trx1), and mitochondrial thioredoxin-2 (Trx2) redox states. GSH/GSSG redox states were determined by HPLC, and Trx1 and Trx2 redox states were determined by Redox Western blot methods. Copper, iron, and nickel showed significant oxidation of GSH but relatively little oxidation of either Trx1 or Trx2. Arsenic, cadmium, and mercury showed little oxidation of GSH but significantly oxidized both Trx1 and Trx2. The magnitude of effects of arsenic, cadmium, and mercury was greater for the mitochondrial Trx2 (>60 mV) compared to the cytoplasmic Trx1 (20 to 40 mV). Apoptosis signal-regulating kinase 1 (ASK1) may be activated by two different pathways, one dependent upon GSH and glutaredoxin and the other independent of GSH and dependent upon thioredoxin. ASK1 activation and cell death were observed with metals that oxidized thioredoxins but not with metals that oxidized GSH. These findings show that metals have differential oxidative effects on the major thiol antioxidant systems and that activation of apoptosis may be associated with metal ions that oxidize thioredoxin and activate ASK1. The differential oxidation of the major thiol antioxidant systems by metal ions suggest that the distinct thiol/disulfide redox couples represented by GSH/GSSG and the thioredoxins may convey different levels of control in apoptotic and toxic signaling pathways.     

The Extracellular Redox State Modulates Mitochondrial Function,Gluconeogenesis, and Glycogen Synthesis in Murine Hepatocytes

Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.     

Role of disulfide exchange in alpha 1-protease inhibitor.

The major endogenous inhibitor of neutrophil elastase in the plasma, alpha 1-protease inhibitor (alpha 1-PI), has a single cysteine residue which has been shown to form mixed disulfides with a number of thiols in vitro. Under normal physiological conditions, the plasma concentrations of reduced and oxidized thiols are such that a major fraction of alpha 1-PI in the circulation in vivo is in the form of mixed disulfides [Laurell, C.-B. (1979) in The Chemistry and Physiology of Human Plasma Proteins (Bing, D. H., Ed.) pp 329-341, Pergamon, New York]. We show here that the mixed disulfide between glutathione or cysteine and alpha 1-PI (alpha 1-PI-SSG or alpha 1-PI-SScys) has an intrinsic fluorescence which distinguishes it from the reduced form of alpha 1-PI. By employing the fluorescence difference, we have measured the ratio of alpha 1-PI-SH to mixed disulfide alpha 1-PI in redox buffers of different ratios of reduced to oxidized glutathione (GSH to GSSG) or reduced to oxidized cysteine (cys to cysSScys) and have calculated an equilibrium constant and redox potential of 0.74 +/- 0.08 and 8 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SSG couple and of 0.32 +/- 0.02 and 29 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SScys couple. We are unable to detect any change in Trp fluorescence in the complex of alpha 1-PI and elastase when the preformed complex is added to the same GSH/GSSG or cys/cysSScys redox buffers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E(GSSG/2 GSH)) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E(CySS/2 Cys)). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a 'thiol-disulphide redox environment' (E(thiol-disulphide)), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E(CySS/2 Cys) to E(thiol-disulphide) in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Age-associated analysis of oxidative stress parameters in human plasma and erythrocytes

Oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Rarely, the oxidative stress parameters in healthy humans related to the individual age have been reported. The purpose of this study was to examine the redox status in plasma and erythrocytes of healthy individuals and determine correlations between these parameters and the aging process. The following parameters were used: malondialdehyde (MDA), protein carbonyls (PCO), 4-hydroxy-2,3-trans-nonenal (HNE), reduced glutathione (GSH), glutathione disulfide (GSSG) and uric acid (UA) in blood and plasma samples of 194 healthy women and men of ages ranging from 18 to 84 years. The results indicate that the balance of oxidant and antioxidant systems in plasma shifts in favor of accelerated oxidation during ageing. That is demonstrated by increases of MDA, HNE, GSSG and by the slight decrease of erythrocytic GSH with age. As the content of UA is more determined by metabolic and nutritional influences than by the balance between prooxidants and antioxidants there was no significant age-related change observed. For plasma concentrations of HNE the first time age-dependent reference values for healthy humans are presented.     

Oxidation of glutathione and cysteine in human plasma associated with smoking

Cigarette smoking contributes to the development or progression of numerous chronic and age-related disease processes, but detailed mechanisms remain elusive. In the present study, we examined the redox states of the GSH/GSSG and Cys/CySS couples in plasma of smokers and nonsmokers between the ages of 44 and 85 years (n = 78 nonsmokers, n = 43 smokers). The Cys/CySS redox in smokers (−64 ± 16 mV) was more oxidized than nonsmokers (− 76 ± 11 mV; p < .001), with decreased Cys in smokers (9 ± 5 μM) compared to nonsmokers (13 ± 6 μM; p < .001). The GSH/GSSG redox was also more oxidized in smokers (−128 ± 18 mV) than in nonsmokers (−137 ± 17 mV; p = .01) and GSH was lower in smokers (1.8 ± 1.3 μM) than in nonsmokers (2.4 ± 1.0; p < .005). Although the oxidation of GSH/GSSG can be explained by the role of GSH in detoxification of reactive species in smoke, the more extensive oxidation of the Cys pool shows that smoking has additional effects on sulfur amino acid metabolism. Cys availability and Cys/CySS redox are known to affect cell proliferation, immune function, and expression of death receptor systems for apoptosis, suggesting that oxidation of Cys/CySS redox or other perturbations of cysteine metabolism may have a key role in chronic diseases associated with cigarette smoking.     

Age-related changes in the glutathione redox system

The effect of aging on the glutathione redox system was evaluated in this study. For this purpose, we determined reduced glutathione (GSH) and oxidized glutathione (GSSG) in whole blood, glutathione peroxidase (GPx) and glutathione reductase (GSSGR) in erythrocytes and selenium (Se) in plasma in 176 healthy individuals. We also calculated GSH/GSSG molar ratios. These subjects were divided into five groups: group 1 (n=25; 0.2-1 years old); group 2 (n=28; 2-11 years old); group 3 (n=23; 12-24 years old); group 4 (n=40; 25-40 years old); group 5 (n=60; 41-69 years old). GSH levels in groups 1 and 5 were significantly lower than the other groups (p<0.001). Conversely, GSSG levels were significantly high in these periods (p<0.001). The GSH/GSSG molar ratio was found to be low both in the first year of life and in the oldest group (p<0.001, respectively). GPx activity in group 5 was increased as compared to the other groups (p<0.001). GSSGR activity was significantly lower in the oldest groups than in the other groups (p<0.001). Se levels were found to be low in the oldest group (p<0.001). Selenium levels of women in group 5 were significantly high as compared to the men (p<0.01). We found negative correlations between age and GSH levels (r=0.402; p<0.001), selenium levels (r=0.454; p<0.001), GSH/GSSG molar ratio (r=0.557; p<0.001) and GSSGR activity (r=0.556; p<0.001). There were positive correlations between age and GPx (r=0.538; p<0.001) and GSSG level (r=0.551; p<0.001). In conclusion, our findings show that the glutathione redox system is affected by age. Oxidative stress increases during the aging process. There is no effect of aging on the glutathione redox system according to sex except for the Se level.     

The effect of aging on glutathione and cysteine levels in different regions of the mouse brain

A general glutathione (GSH) deficiency occurs in many tissues of the aging mouse. However, there is no information on GSH in the aging brain even though it has been involved in a number of neurobiologic reactions. To this end, C57BL/6 mice, 3-31 months old, representing the growth, maturation, and aging periods of the life-span were studied. Brain cortex, hippocampus, and stem samples were dissected, processed, and analyzed specifically for reduced and oxidized glutathione (GSH, GSSG) and cyst(e)ine using high performance liquid chromatography with dual electrochemical detection. The GSH content of each brain region varied in the order brain cortex greater than brain hippocampus greater than brainstem. However, the GSH profiles of all regions were the same through the life-span, namely, high values during growth dropping to a maturation plateau and then decreasing 30% during aging. In contrast to GSH, the order of cysteine levels was brain cortex less than brain hippocampus less than brainstem and no life-span changes occurred in any region. In addition, the brain GSSG and cystine contents of all regions were very low and did not change during the life-span. Thus, the GSH loss was not accountable by oxidation to GSSG or degradation to cyst(e)ine. Altogether these results demonstrated a GSH deficiency in brain tissues of aging mice like that found previously in other tissues. These findings suggest an increased susceptibility of the aging brain to oxidative damage.     

Pseudomonas aeruginosa pyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells

Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H(2)O(2) formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O(2)(-). Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.