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.     

Effect of cadmium on antioxidative enzymes,glutathione content,and glutathionylation in tall fescue

The aim of this work was to assess the effect of different Cd2+concentrations on some antioxidant enzymes in Festuca arundinacea. Increased activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione S-transferase, and glutathione reductase were ascertained in response to low Cd²⁺ concentrations (0–20 μM), whereas the enzyme activities were less increased or decreased at a higher Cd²⁺ dosage (50 μM) and a longer exposure. The content of reduced glutathione (GSH) decreased significantly with increasing Cd²⁺ concentrations, whereas the content of oxidized glutathione (GSSG) increased proportionally to the amount of Cd²⁺ applied. Further experiments, performed by incubating the enzyme extracts with oxidized glutathione, evidenced that the addition of GSSG to the incubation mixtures caused significant decreases of some enzymatic activities. Finally, the effect of glutathione S-transferase, FaGST I, extracted from fescue seedlings and purified till homogeneity, on these enzyme activities was investigated. It was found that FaGST I enhanced the decreased enzymatic activities caused by GSSG.     

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.     

Influence of Complete and Pronounced Incomplete Cerebral Ischemia and Subsequent Recirculation on Cortical Concentrations of Oxidized and Reduced Glutathione in the Rat

Abstract: The influence of complete and pronounced incomplete cerebral ischemia on cortical concentrations of reduced (GSH) and oxidized (GSSG) glutathione was studied in lightly anaesthetized (70% N₂ O) rats. GSH was extracted with HCl-methanol-perchloric acid and GSSG with trichloroacetic acid in the presence of N-ethylmaleimide and measured fluorometrically, giving normal concentrations in cortical tissue of about 2 and 0.01 μmol.g^?1 respectively. Reversible complete ischemia was induced by increasing the intracranial pressure to above the systolic blood pressure by infusing mock CSF into the cisterna magna. Reversible pronounced incomplete ischemia was induced by bilateral carotid artery clamping combined with hypovolemic hypotension. Whether complete or incomplete, a 30-min ischemic period caused a similar decrease in cortical GSH concentration (to about 90% of control) without any concomitant accumulation of GSSG in the tissue (or in CSF). Prolongation of the ischemic period (complete ischemia) to maximally 120 min caused an almost linear decrease of the tissue glutathione concentration to 45% of the preischemic value. During subsequent recirculation following a 30 min period of either complete or pronounced incomplete ischemia, there was a further decrease in cortical GSH concentrations without a reciprocal increase in GSSG concentrations. Lipid peroxidation (verified by determination of malondialdehyde production) induced in brain cortical tissue in vitro caused oxidation of tissue GSH with accumulation of GSSG. As the observed decrease in GSH during brain ischemia in vivo was not accompanied by any reciprocal increase in GSSG the results fail to support the hypothesis that peroxidative damage occurs during or following brain ischemia. The finding of an unchanged GSSG concentration does, however, not exclude the possibility of an increased turnover rate in the glutathione reductase reaction. It is concluded that the observed decrease in tissue GSH concentration mainly reflects a decrease in the glutathione pool size, due to an imbalance between breakdown and synthesis secondary to tissue energy failure.     

Detection of oxidized and reduced glutathione with a recycling postcolumn reaction

A rapid, sensitive, and selective method for the quantitation of both oxidized (GSSG) and reduced (GSH) glutathione in biological materials is described. Oxidized and reduced glutathione are resolved by anion-exchange high-performance liquid chromatography and detected with an in-line, recycling postcolumn reaction. The recycling reaction specifically amplifies the response to oxidized and reduced glutathione 20-100 times over that obtained with a stoichiometric reaction, permitting the detection of 2 pmol glutathione. Oxidized and reduced glutathione levels were measured in rat liver and in dog heart mitochondria. Special precautions are necessary to avoid artifacts which lead to either underestimation or overestimation of GSSG levels. GSH/GSSG ratios of approximately 100-300 were observed in samples prepared from rapidly frozen rat liver. Somewhat higher GSH/GSSG ratios were observed in isolated dog heart mitochondria.     

Glutathione disulfide reduction in tumor mitochondria after t-butyl hydroperoxide treatment.

Treatment of isolated mitochondria from rat hepatoma tumor cells (AS-30D) with the oxidant, t-butyl hydroperoxide (tBuOOH, 1 or 5 mumol/ml) resulted in the oxidation of glutathione (GSH to GSSG) and the formation of protein-glutathione mixed disulfides (ProSSG). The GSSG was retained inside of the hepatoma mitochondria. In the presence of ADP+succinate (5 or 10 mM), or ketoglutarate (10 mM) or malate (5 mM), the GSSG was reduced to GSH, but the amount of ProSSG stayed constant. With saline or ADP+glutamate (10 mM)/malate (0.1 mm) no reduction of GSSG to GSH occurred. The presence of antimycin (5 micrograms/ml) with ADP+succinate inhibited reduction. At a concentration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 0.5 mM) which inhibited a major portion of the glutathione reductase activity, the reduction of GSSG to replenish GSH was also inhibited. NADPH may play a critical role as well, for the addition of 2.4 mM NADPH to permeabilized hepatoma mitochondria fostered the reduction of GSSG after tBuOOH treatment. Therefore, hepatoma mitochondria possess a glutathione reductase-dependent system to reduce GSSG to GSH. The reaction only occurs with actively respiring mitochondria.     

Glutathione production in copper-deficient isolated rat hepatocytes.

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.     

Determination of reduced, oxidized, and protein-bound glutathione in human plasma with precolumn derivatization with monobromobimane and liquid chromatography

This assay measures reduced (GSH), oxidized (GSSG, GSSR), and protein-bound (glutathione-protein mixed disulfides, ProSSG) glutathione in human plasma. Oxidized glutathione and ProSSG are converted to GSH in the presence of NaBH4, and, after precolumn derivatization with monobromobimane, GSH is quantitated by reversed-phase liquid chromatography and fluorescence detection. The NaBH4 concentration is optimized so that total recovery of oxidized glutathione is obtained and no interference with the formation/stability of the GSH-bimane adduct occurs. The presence of 50 microM dithioerythritol prevents reduced recovery at low concentrations of GSH, and the standard curve for GSH is linear over a wide concentration range and is super-imposed upon that obtained with GSSG. Selective determination of oxidized glutathione exploits the fact that N-ethylmaleimide (NEM) blocks free sulfhydryl groups and excess NEM is inactivated by the subsequent addition of NaBH4. To measure total glutathione including the protein-bound forms, the protein is solubilized with dimethyl sulfoxide, which is compatible with the other reagents and slightly increases the yield of the fluorescent GSH derivative. The assay is characterized by a sensitivity (less than 2 pmol) sufficiently high to detect the various forms of glutathione in plasma, by an analytical recovery of GSH and GSSG close to 100%, and by a within-day precision corresponding to a coefficient of variation of 7%. The assay was used to determine the dynamic relationships among various glutathione species in human plasma.     

Measurement of oxidized glutathione by enzymatic recycling coupled to bioluminescent detection

A new method is described for the quantification of oxidized glutathione (GSSG) in tissues by enzymatic recycling coupled to NADPH bioluminescent detection. Tissue samples are treated with metaphosphoric acid. In a first step, after derivatization of GSH with 4-chloro-7-trifluoromethyl-1-methylquinolinium (CFQ), GSSG is recycled in the presence of dithionitrobenzoic acid (DTNB) and NADPH by glutathione reductase. In a second step, the GSSG-dependent NADPH consumption is measured by luminescence with NADPH:FMN oxidoreductase-bacterial luciferase. The coefficient of variation for GSSG measurements on repeated assays (n = 5) is 2 and 3% for standards and tissue samples, respectively. The sensitivity of this method is at the picomole level and is convenient for determination of GSSG physiological concentrations in tissues: GSSG levels measured in rat liver and kidney ranged from 76 to 215 and 52 to 170 nmol/g wet weight, respectively.     

Reduced circulating oxidized LDL is associated with hypocholesterolemia and enhanced thiol status in Gilbert syndrome

A protective association between bilirubin and atherosclerosis/ischemic heart disease clearly exists in vivo. However, the relationship between bilirubin and in vivo oxidative stress parameters in a clinical population remains poorly described. The aim of this study was to assess whether persons expressing Gilbert syndrome (GS; i.e., unconjugated hyperbilirubinemia) are protected from thiol oxidation and to determine if this, in addition to their improved lipoprotein profile, could explain reduced oxidized low-density lipoprotein (oxLDL) status in them. Forty-four matched GS and control subjects were recruited and blood was prepared for the analysis of lipid profile and multiple plasma antioxidants and measures of oxidative stress. GS subjects possessed elevated plasma reduced thiol (8.03±1.09 versus 6.75±1.39 nmol/mg protein; P<0.01) and glutathione concentrations (12.7±2.39 versus 9.44±2.45 μM; P<0.001). Oxidative stress status (reduced:oxidized glutathione; GSH:GSSG) was significantly improved in GS (0.49±0.16 versus 0.32±0.12; P<0.001). Protein carbonyl concentrations were negatively associated with bilirubin concentrations and were significantly lower in persons with >40 μM bilirubin versus controls (<17.1 μmol/L; P<0.05). Furthermore, absolute oxLDL concentrations were significantly lower in GS subjects (P<0.05). Forward stepwise regression analysis revealed that bilirubin was associated with increased GSH:GSSG ratio and reduced thiol concentrations, which, in addition to reduced circulating LDL, probably decreased oxLDL concentrations within the cohort. In addition, a marked reduction in total cholesterol concentrations in hyperbilirubinemic Gunn rats is presented (Gunn 0.57±0.09 versus control 1.69±0.40 mmol/L; P<0.001), arguing for a novel role for bilirubin in modulating lipid status in vivo. These findings implicate the physiological importance of bilirubin in protecting from atherosclerosis by reducing thiol and subsequent lipoprotein oxidation, in addition to reducing circulating LDL concentrations.     

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.     

Determination of reduced and oxidized glutathione in erythrocytes by high-performance liquid chromatography with ultraviolet absorbance detection

A method is described for simultaneous quantitation of reduced (GSH) and oxidized (GSSG) glutathione in erythrocytes by HPLC. They were determined by standard addition method. Blood samples were collected in tubes containing 1,10-phenanthroline. The separated erythrocytes were hemolyzed with water containing standard. After deproteinization, GSH and GSSG were converted to N-(2,4-dinitrophenyl) derivatives and analyzed by HPLC with UV detection. The coefficients of variation of GSH and GSSG on replicate assays were 6% and 8%, respectively. The stabilities of GSH and GSSG and of the derivatives were also examined. The present method appears to be satisfactory for determination of these physiological concentrations in erythrocytes.     

Effects of age and caloric restriction on glutathione redox state in mice

The main purpose of this study was to determine whether the aging process in the mouse is associated with a pro-oxidizing shift in the redox state of glutathione and whether restriction of caloric intake, which results in the extension of life span, retards such a shift. Amounts of reduced and oxidized forms of glutathione (GSH and GSSG, respectively) and protein-glutathione mixed disulfides (protein-SSG) were measured in homogenates and mitochondria of liver, kidney, heart, brain, eye, and testis of 4, 10, 22, and 26 month old ad libitum-fed (AL) mice and 22 month old mice fed a diet containing 40% fewer calories than the AL group from the age of 4 months. The concentrations of GSH, GSSG, and protein-SSG vary greatly (approximately 10-, 30-, and 9-fold, respectively) from one tissue to another. During aging, the ratios of GSH:GSSG in mitochondria and tissue homogenates decreased, primarily due to elevations in GSSG content, while the protein-SSG content increased significantly. Glutathione redox potential in mitochondria became less negative, i.e., more pro-oxidizing, as the animal aged. Caloric restriction (CR) lowered the GSSG and protein-SSG content. Results suggest that the aging process in the mouse is associated with a gradual pro-oxidizing shift in the glutathione redox state and that CR attenuates this shift.     

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.     

Glutathione and glutathione disulfide affect adventitious root formation and growth in tomato seedling cuttings

To study the relationship between glutathione and rooting, tomato seedling cuttings, grown on basal- or on auxin-supplemented media, were treated with the reduced (GSH) or oxidized (GSSG) form of this antioxidant. In turn, the consequences of the depletion of GSH pool on rooting were tested using l-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis. Effects of the aforementioned treatments on rooting response were assessed. GSH treatment promoted root formation on cuttings grown on both basal- and auxin-supplemented media. Whereas GSSG did not affect the number of roots formed by cuttings grown on basal medium, it strongly enhanced the rooting stimulatory effect of auxin treatment. GSH depletion resulting from BSO application did not change the number of roots formed. All the tested compounds, namely GSH, GSSG, BSO and auxin, had a strong inhibitory effect on the elongation of regenerated roots. Supplementing the rooting medium with glutathione efficiently increased the GSH level in the rooting zones, while addition of BSO led to a strong decrease in endogenous GSH level. Neither of the treatments affected the level of GSSG. Exogenous auxin affect neither GSH nor GSSG levels in rooting zones; however, in the regenerated roots, GSH level was significantly higher when the organs were formed on auxin-supplemented medium. Patterns of GSH distribution in the roots regenerated on basal- and auxin-enriched media were studied using the GSH-specific dye monochlorobimane and confocal laser scanning microscopy. GSH was found in the root apical meristem and in the elongation zone. Auxin did not change the GSH distribution; however, the number of fluorescent cells was higher when roots were regenerated on auxin-supplemented medium.     

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.     

Glutathione reductase activity in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum

Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min^-1·mg protein^-1, and the apparent K_m for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn⁺² ions, however, preincubation with oxidized glutathione rendered its resistance to Zn⁺² inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min^-1·mg protein^-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min^-1·mg protein^-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N₂-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB 5-5-dithiobis-(2-nitrobenzoic acid) - EDTA ethylenediaminetetra-acetic acid - GR glutathione reductase (EC1.6.4.2) - GSH reduced glutathione - GSSG oxidized glutathione - OPT O-phtaldialdehyde - SOD superoxide dismutase (EC 1.15.1.1)     

Circulating glutathione concentrations in marine,semiaquatic, and terrestrial mammals

An important low molecular weight antioxidant in biological systems is glutathione; its efficiency depends on the equilibrium between its reduced (GSH) and oxidized (GSSG) forms. The oxidized:total glutathione (GSSG:GSH‐Eq) ratio can be used as an indicator of oxidative stress. Previous studies suggest that marine mammals, unlike terrestrial mammals, do not show adverse effects in tissues exposed to ischemia/reperfusion during the peripheral vasoconstriction associated with breath‐hold diving. This is due, in part, to higher antioxidant enzyme activities in marine mammals compared with terrestrial mammals. The objective of this study was to compare circulating glutathione levels among mammals with different diving capacities. Circulating GSH‐Eq, GSH, and GSSG concentrations in erythrocyte samples from northern elephant seals (Mirounga angustirostris), bottlenose dolphins (Tursiops truncatus), neotropical otters (Lontra longicaudis annectens), domestic pigs (Sus scrofa), and humans were quantified using spectrophotometry. Higher GSH‐Eq and GSH concentrations and a lower GSSG:GSH‐Eq index were found in erythrocytes from northern elephant seals and bottlenose dolphins as compared to otters, domestic pigs, and humans. Results suggest that marine mammals, independent of their diving capacity, possess a highly developed antioxidant system, including GSH; continuous availability of GSH could allow these species to avoid oxidative damage and tolerate ischemia/reperfusion and hypoxia/reoxygenation events associated with diving.     

Glutathione metabolism in soybean callus-cultures as affected by salinity

Induction and growth of soybean callus cultures were influenced by NaCl, especially at the highest concentration tested (150 mM). Protein content was raised as NaCl was increased in the Murashige and Skoog medium. Total sulfhydryl group (-SH) and glutathione (GSH) concentrations were also increased in NaCl treated cultures. The affinity (Km) of glutathione reductase (GR) for oxidized glutathione (GSSG) was gradually increased as NaCl level was raised in the medium. The GSH/GSSG ratio was raised significantly as the result of GR activity. The increase in GR activity may constitute an adaptive response of soybean callus to NaCl. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glutathione levels during the mosquito life span with emphasis on senescence

Glutathione levels were determined in mosquitoes of all ages of the life span. Specific analyses for reduced (GSH) and oxidized (GSSG) glutathione were used and validated to ensure minimal autoxidation of GSH and conversion of these forms. Indeed GSH accounted for greater than 97% of the total glutathione (GSH + GSSG) content in all samples. Marked changes occurred during the life span, and the highest levels of GSH and total glutathione were found during larval growth and metamorphosis (P less than 0.001). Thereafter the levels decreased in the early adult, plateaued in the mature, and decreased 46% in the old and very old mosquito (P less than 0.001). This aging-specific decrease was a general phenomenon, for it occurred in all body regions of both sexes. Starvation up to 3 days did not affect the GSH levels. The importance of these changes in glutathione is its relationship to the reducing and biosynthetic capacities of different life span stages. Of special interest is the senescence decrease which can lead to lower biosynthetic activity and also impaired detoxification capacity.