Cross‐effects of dietary probiotic supplementation and rearing temperature on antioxidant responses in European seabass [Dicentrarchus labrax (Linnaeus, 1758)] juveniles

Temperature modulates the metabolism in both fish and bacteria and therefore the effect of probiotic bacteria on its host may vary accordingly. The current study aim was to evaluate the effect of probiotic supplementation (Bacillus sp., Lactobacillus sp., Enterococcus sp., Pediococcus sp.) in juvenile seabass, Dicentrarchus labrax, when reared under different temperatures (17, 20 and 23°C). A control diet was tested against a probiotic‐supplemented diet, with a concentration of 3 × 10⁹ CFU probiotic/kg diet. Antioxidant responses (TG, GSH, GSSG, GR, CAT and GSTs) and lipid peroxidation (LPO) were evaluated after 70 days of dietary probiotic supplementation. An effect of temperature was observed on LPO, which increased significantly in fish reared at 17°C (p < .05) compared to the 20 and 23°C groups. Total glutathione (TG) was significantly higher in the probiotic treatments in fish reared at 17 and 20°C (p < .05). In addition, a probiotic temperature interaction was observed for TG, reduced glutathione (GSH) levels, and for reduction of the oxidized glutathione ratio (GSH/GSSG; p < .05). In conclusion, the current study showed a strong temperature effect on oxidative stress responses, with an anti‐oxidant role of dietary probiotic supplementation at different rearing temperatures.     

Variation in glutathione status associated with induction and initiation of diapause in eggs of the bivoltine strain of the silkworm Bombyx mori

For the bivoltine (Dazao) strain of the silkworm Bombyx mori L., diapause expression in progeny is induced by exposure to conditions of 25 °C and continuous illumination (LL) during the maternal generation, whereas an environment of 15 °C and constant darkness (DD) results in nondiapause progeny. Initiation of diapause in progeny can be prevented by treatment of diapause‐programmed eggs with hydrochloric acid (HCl) at approximately 24 h post‐oviposition. To investigate whether glutathione is involved in the regulation of diapause induction and initiation in this species, measurements of total glutathione, reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG ratio, glutathione S‐transferase (GST) and peroxiredoxins (Prdx) are compared in eggs incubated under LL and DD conditions, and between diapause eggs and those treated with HCl. Compared with DD, eggs incubated under LL have higher total glutathione (GSH + 2GSSG), lower GSH, higher GSSG, a lower GSH/GSSG ratio, lower GST activity and higher Prdx activity at stages 20–25 of maternal embryogenesis. The lower ratio of GSH/GSSG is indicative of pro‐oxidative conditions during diapause induction, which may result from the stronger oxidation of GSH. Compared with HCl‐treated eggs, diapause eggs have lower total glutathione, no difference in GSH, lower GSSG, a higher GSH/GSSG ratio, no difference in GST activity and lower Prdx between 36 and 72 h post‐oviposition. The higher ratio GSH/GSSG is indicative of reducing conditions during diapause initiation, which may a result of the weaker oxidation of GSH. Moreover, variations of Prdx and GST suggest that Prdx rather than GST plays an important role in the oxidation of GSH during the induction and initiation of diapause.     

Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm,Bombyx moil

To explore whether glutathione regulates diapause determination and termina tion in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapanse and nondiapauseegg producers, as well as those in dia pause eggs incubated at different temperatures. The activity ofthioredoxin reductase （TrxR） was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione （GSH/GSSG） ratio was observed in the ovary of diapauseegg producers, due to weaker reduction of oxidized glutathione （GSSG） to the reduced glutathione （GSH） catalyzed by glutathione reductase （GR） and TrxR. This indicates an oxidative shift in the glutathione redox cy cle during diapause determination. Compared with the 25℃treated diapause eggs, the 5℃treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm.     

Oxidative folding of reduced and denatured huwentoxin-I

Huwentoxin-I, a neurotoxic peptide with 33 ammo acid residues and three disulfide bonds, was used to investigate the pathway of reduction/denaturation and of oxidative folding in small proteins with multiple disulfide bonds. Titration of thiol groups, reversed-phase HPLC, 1D NMR spectroscopy, and biological activity assays were used to monitor the extent of reduction/denaturation and renaturation of the toxin. The reduction and denaturation of huwentoxin-I resulted in a 100% loss of bioactivity as measured in a mouse phrenic nerve-diaphragm preparation. About 90% of full biological activity could be restored under optimized conditions of oxidative refolding of the reduced peptide. Several reaction conditions employing air oxidation, oxidized and reduced glutathione (GSSG and GSH), and cystine/cysteine were investigated in order to find optimal conditions for renaturation of huwentoxin-I. The best renaturation yield was achieved in 0.1 mM GSSG and 1 mM GSH at pH 8.5 and 4°C over 24 hr. High concentrations of glutathione and high temperatures reduced renaturation yields. Oxidative refolding of huwentoxin-I in air requires about 6 days for maximal yields and is inhibited by EDTA.     

Lipoxygenase-mediated glutathione oxidation and superoxide generation

Soybean lipoxygenase-mediated cooxidation of reduced glutathione (GSH) and concomitant superoxide generation was examined. The oxidation of GSH was dependent on the concentration of linoleic acid (LA), GSH, and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 0.42 mM LA, 2 mM GSH, and 50 pmole of enzyme/mL. The GSH oxidation was linear up to 10 minutes and exhibited a pH optimum of 9.0. The reaction displayed a K_m of 1.49 mM for GSH and V_max of 1.35 ± 0.02 μmoles/min/nmole of enzyme. Besides LA, arachidonic and γ-linolenic acids also supported the lipoxygenase-mediated GSH oxidation. Hydrogen peroxide and 13-hydroperoxylinoleic acid supported GSH cooxidation, but to a very limited extent. Oxidized glutathione (GSSG) was identified as the major product of the reaction based on the depletion of nicotinamide-adenine dinucleotide 3′-phosphate (NADPH) in the presence of glutathione reductase. The GSH oxidation was accompanied by the reduction of ferricytochrome c, which can be completely abolished by superoxide dismutase (SOD), suggesting the generation of superoxide anion radicals. Under optimal conditions, the rate of superoxide generation (measured as the SOD-inhibitable reduction of ferricytochrome c) was 10 ± 1.0 nmole/min/nmole of enzyme. These results clearly suggest that lipoxygenase is capable of oxidizing GSH to GSSG and simultaneously generating superoxide anion radicals, which may contribute to oxidative stress in cells under certain conditions.     

Human jejunal glutathione reductase: purification and evaluation of the NADPH- and glutathione-induced changes in redox state

Human proximal jejunal glutathione reductase (EC 1.6.4.2) was purified to homogeneity by affinity chromatography on 2', 5'-ADP-Sepharose 4B. In most of its molecular and kinetic properties, the enzyme resembled glutathione reductase from other sources: The subunit mass was 56 kDa; the isoelectric point and pH optimum were 6.75 and 7.25, respectively; Michaelis constants, determined at pH 7.4, 37 degrees C, fell within the range of previously reported values [Km(NADPH) = 20 microM, Km(GSSG) = 80 microM]. The response of the enzyme to reducing conditions, on the other hand, had unique features: Preincubation with 1 mM NADPH resulted in 90% loss of activity which could be partially reversed by 2 mM GSSG, but not GSH. (Treatment with GSSG regenerated 68% of the original activity.) Reduction by GSH also caused inactivation which potentially amounted to greater than 80%. This inactivation could not be reversed by GSSG. The protective effect of GSSG against inactivation by GSH was studied. Except where [GSSG] far exceeded [GSH], the presence of GSSG in the preincubation medium decreased the extent of inhibition without affecting the rate constant for approach to equilibrium activity. At [GSSG] greater than [GSH] a decrease in the rate constant for inactivation was also observed. The results were interpreted in terms of a three-step mechanism: (1) preequilibrium reduction of Eox to Ered; (2) rate-limiting change in conformation from Ered to E'red, and (3) irreversible conversion to catalytically inferior products.     

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.     

Dicoumarol Inhibits Multidrug Resistance Protein 1-Mediated Export Processes in Cultured Primary Rat Astrocytes

Dicoumarol is frequently used as inhibitor of the detoxifying enzyme NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1). In order to test whether dicoumarol may also affect the cellular glutathione (GSH) metabolism, we have exposed cultured primary astrocytes to dicoumarol and investigated potential effects of this compound on the cell viability as well as on the cellular and extracellular contents of GSH and its metabolites. Incubation of astrocytes with dicoumarol in concentrations of up to 100 µM did not acutely compromise cell viability nor was any GSH consumption or GSH oxidation to glutathione disulfide (GSSG) observed. However, unexpectedly dicoumarol inhibited the cellular multidrug resistance protein (Mrp) 1-dependent export of GSH in a time- and concentration-dependent manner with half-maximal effects observed at low micromolar concentrations of dicoumarol. Inhibition of GSH export by dicoumarol was not additive to that observed for the known Mrp1 inhibitor MK571. In addition, dicoumarol inhibited also the Mrp1-mediated export of GSSG during menadione-induced oxidative stress and the export of the GSH–bimane-conjugate (GS–B) that had been generated in the cells after exposure to monochlorobimane. Half-maximal inhibition of the export of Mrp1 substrates was observed at dicoumarol concentrations of around 4 µM (GSH and GSSG) and 30 µM (GS–B). These data demonstrate that dicoumarol strongly affects the GSH metabolism of viable cultured astrocytes by inhibiting Mrp1-mediated export processes and identifies for the first time Mrp1 as additional cellular target of dicoumarol.

     

Glutathione disulfide liposomes – A research tool for the study of glutathione disulfide associated functions and dysfunctions

Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4 h and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at ?80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions.     

2-Deoxyribose Deprives Cultured Astrocytes of their Glutathione

High concentrations of 2-deoxy-d-ribose (2dRib) have been reported to cause oxidative stress and to disturb the glutathione (GSH) metabolism of various cell types. Exposure of astrocyte-rich primary cultures to millimolar concentrations of 2dRib or its stereoisomer 2-deoxy-l-ribose, but not the incubation with ribose, 2-deoxyglucose, glucose, fructose or saccharose, lowered the cellular GSH content in a time and concentration dependent manner. After exposure for 4 h to 30 mM 2dRib the cells contained 2dRib in a concentration of about 24 mM. Under these conditions 2dRib did not compromise cell viability and the ability of the cells to synthesise GSH, nor were the cellular ratio of glutathione disulfide (GSSG) to GSH and the extracellular concentrations of GSH or GSSG increased. These data demonstrate that 2dRib deprives viable cultured astrocytes of GSH and suggest that a cellular reaction of GSH with 2dRib or its metabolites is involved in the deprivation of astrocytic GSH.     

Selenium-Induced Up-Regulation of the Antioxidant Defense and Methylglyoxal Detoxification System Reduces Salinity-Induced Damage in Rapeseed Seedlings

The present study investigates the regulatory role of exogenous selenium (Se) in the antioxidant defense and methylglyoxal (MG) detoxification systems in rapeseed seedlings exposed to salt stress. Twelve-day-old seedlings, grown in Petri dishes, were supplemented with selenium (25 μM Na₂SeO₄) and salt (100 and 200 mM NaCl) separately and in combination, and further grown for 48 h. The ascorbate (AsA) content of the seedlings decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) increased with an increase in the level of salt stress, while the GSH/GSSG ratio decreased. In addition, the ascorbate peroxidase (APX) and glutathione S-transferase (GST) activity increased significantly with increased salt concentration (both at 100 and 200 mM NaCl), while glutathione peroxidase (GPX) activity increased only at moderate salt stress (100 mM NaCl). Glutathione reductase (GR) activity remained unchanged at 100 mM NaCl, while it was decreased under severe (200 mM NaCl) salt stress. Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, whereas a sharp decrease of these activities was observed under severe salt stress (200 mM NaCl). Concomitant increases in the levels of H₂O₂ and lipid peroxidation (MDA) were also measured. Exogenous Se treatment alone had little effect on the non-enzymatic and enzymatic components. However, further investigation revealed that Se treatment had a synergistic effect: in salt-stressed seedlings, it increased the AsA and GSH contents; GSH/GSSG ratio; and the activities of APX, MDHAR, DHAR, GR, GST, GPX, CAT, Gly I, and Gly II. As a result, addition of Se in salt-stressed seedlings led to a reduction in the levels of H₂O₂ and MDA as compared to salt stress alone. These results suggest that the exogenous application of Se rendered the plants more tolerant to salt stress-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze–thawing process

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen is one of the main causes for the decline in motility and fertility of sperm during the freeze–thawing process. The aim of this study was thus to determine the effects of anti-oxidants on standard semen parameters, lipid peroxidation (LPO) and anti-oxidant activities after the freeze–thawing of ram semen. Ejaculates collected from four Akkaraman rams, were pooled and evaluated at 33 °C. Semen samples were diluted in a Tris-based extender containing the anti-oxidants glutathione (GSH) (5 mM), oxidized glutathione (GSSG) (5 mM) or cysteine (5 mM) and an extender containing no anti-oxidants (control), cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually for 20 s in a water bath (37 °C) for microscopic evaluation. The use of an extender supplemented with cysteine led to the highest (P < 0.01) post-thaw motility (61.0 ± 1.9%), compared to the other treatment groups. No significant differences were observed in viability, acrosome damage and total abnormalities, and following the hypo-osmotic swelling test (HOST), following supplementation with anti-oxidants after the thawing of the semen. Following the thawing process, the levels of malondialdehyde (MDA) did not change with the addition of anti-oxidants, compared to the control. The GSH level and glutathione peroxidase (GSH-PX) activity remained significantly higher upon the addition of GSH (3.33 ± 0.14 nmol/ml and 22.02 ± 1.27 IU/g protein) and GSSG (3.24 ± 0.08 nmol/ml and 20.17 ± 3.38 IU/g protein) compared to the other treatment (P < 0.001) groups. Only cysteine significantly elevated the activity of catalase (CAT, 842.40 ± 90.42 kU/l) following the freeze–thawing process. The Vitamin E (VitE) level was significantly higher, when compared to GSSG, cysteine and the control, when GSH (4.21 ± 0.20 mg/dl) was added to the freezing extender (P < 0.001). It could be concluded that future efforts aimed on improving the efficiency of cryopreservation of ram sperm should concentrate on the use of anti-oxidant additives. The results obtained provide a new approach to the cryopreservation of ram semen, and could positively contribute to intensive sheep production.     

Simultaneous determination of reduced glutathione,glutathione disulphide and glutathione sulphonamide in cells and physiological fluids by isotope dilution liquid chromatography–tandem mass spectrometry

A stable isotope dilution liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneously quantifying glutathione (GSH), glutathione disulphide (GSSG) and glutathione sulphonamide (GSA) from biological samples. GSA is a selective product of the reaction of GSH with hypochlorous acid and a potential biomarker of myeloperoxidase activity. GSH was detected as the N-ethylmaleimide alkylated adduct, as formation of this species prevented GSH oxidation occurring during sample processing. Synthesised stable isotope analogues were used as internal standards to accurately quantify each target species. The limit of quantification was determined as being 0.1 pmol for each species and excellent linearity was observed over relevant concentration ranges for biological samples. Relative standard deviations were <5% for within-day variation and <10% for between-day variation, except at the lower limit of quantification where they remained <20%. Accuracy was between 82% and 113%. We could detect GSA in neutrophils and endothelial cells treated with hypochlorous acid and in bronchoalveolar lavage fluid from children with cystic fibrosis. This is the first time GSA has been quantified in clinical material and suggests it is formed in vivo. The assay can now be used for investigating GSA as a biomarker of myeloperoxidase activity in inflammatory conditions, and is also applicable to measuring GSH:GSSG molar ratios as a general index of oxidative stress.     

Human sperm glutathione reductase activity in situ reveals limitation in the glutathione antioxidant defense system due to supply of NADPH

In order to characterize further the antilipoperoxidative enzyme system of human sperm, that part of the system designed to provide reducing equivalents for the reduction of highly reactive and potentially damaging lipid hydroperoxides to relatively inert hydroxylipids was examined. The substrate that provides the reducing equivalents directly to glutathione peroxidase (GPX) is reduced glutathione (GSH), which is in turn oxidized to glutathione disulfide (GSSG). The reducing equivalents needed for regeneration of GSH through the action of glutathione reductase (GRD) are provided by NADPH, produced by the action of glucose-6-phosphate dehydrogenase (G6P-DH) on substrates glucose-6-phosphate and NADP⁺. The kinetic properties of the enzymes GRD and G6P-DH were determined by standard enzyme activity assay at 24 and 37°C. At 37°C, the V_max for GRD was found to be 36 nmol/min · 10⁸ cells, with K_m values for GSSG and NAPH of 150 μM and 16 μM, respectively; the V_max for G6P-DH was 3.3 nmol/min · 10⁸ cells with K_m for NADP⁺ of 8 μM. This suggested that G6P-DH activity was limiting in this reductive pathway. The activity of GRD in situ in intact cells was estimated using the thiol-reactive fluorogenic probe ThioGlo-1, which is cell permeant and reacts rapidly with GSH to give a highly fluorescent adduct. Mixing a suspension of human sperm with the fluorogenic reagent at 37°C gave an initial rapid increase in fluorescence, followed by a slower one. The rapid phase is due to reaction with intracellular GSH already present; the slow phase is due to reaction with GSH generated by the GRD-catalyzed reduction of GSSG. Both rates showed first-order kinetics. Calculation of the maximal rate as NADPH oxidation, attributable to in situ GRD activity, gave the value of 1.0 nmol/min · 10⁸ cells, less than the maximum for NADPH production by the dehydrogenase. These results support the suggestion that NADPH production limits the capacity of the pathway leading to hydroperoxide reduction in human sperm. We propose that the antilipoperoxidative defense system of human sperm has just sufficient capacity to allow these cells to fulfill their function but is limited to allow their timely disposal from the female reproductive tract. Mol. Reprod. Dev. 49:400–407, 1998. © 1998 Wiley-Liss, Inc.     

Exposure of Cultured Astrocytes to Menadione Triggers Rapid Radical Formation,Glutathione Oxidation and Mrp1-Mediated Export of Glutathione Disulfide

Menadione (2-methyl-1,4-naphthoquinone) is a synthetic derivative of vitamin K that allows rapid redox cycling in cells and thereby generates reactive oxygen species (ROS). To test for the consequences of a treatment of brain astrocytes with menadione, we incubated primary astrocyte cultures with this compound. Incubation with menadione in concentrations of up to 30 µM did not affect cell viability. In contrast, exposure of astrocytes to 100 µM menadione caused a time-dependent impairment of cellular metabolism and cell functions as demonstrated by impaired glycolytic lactate production and strong increases in the activity of extracellular lactate dehydrogenase and in the number of propidium iodide-positive cells within 4 h of incubation. In addition, already 5 min after exposure of astrocytes to menadione a concentration-dependent increase in the number of ROS-positive cells as well as a concentration-dependent and transient accumulation of cellular glutathione disulfide (GSSG) were observed. The rapid intracellular GSSG accumulation was followed by an export of GSSG that was prevented in the presence of MK571, an inhibitor of the multidrug resistance protein 1 (Mrp1). Menadione-induced glutathione (GSH) oxidation and ROS formation were found accelerated after glucose-deprivation, while the presence of dicoumarol, an inhibitor of the menadione-reducing enzyme NQO1, did not affect the menadione-dependent GSSG accumulation. Our study demonstrates that menadione rapidly depletes cultured astrocytes of GSH via ROS-induced oxidation to GSSG that is subsequently exported via Mrp1.

     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.     

The interaction of reduced glutathione with active oxygen species generated by xanthine-oxidase-catalyzed metabolism of xanthine

The interaction of reduced glutathione (GSH) with active oxygen species generated during xanthine-oxidase-catalyzed metabolism of xanthine was investigated. The only GSH-derived product detected in this system was oxidized glutathione (GSSG). Catalase inhibited the oxidation of GSH to GSSG by more than 80%, whereas superoxide dismutase exerted a smaller but significant inhibition of GSSG formation. Hydroxyl radical (OH) scavengers or desferrioxamine (1 mM) had no effect on GSSG formation. Using EPR spectroscopy and the spin trap 5,5-dimethylpyrroline-N-oxide (DMPO), the production of superoxide was observed by the detection of a DMPO-OOH radical adduct. This spectrum was altered by the inclusion of GSH (5 - 20 mM) in the reaction mixture, indicating the generation of a different radical species consistent with DMPO-glutathionyl radical adduct generation.     

Hydrogen selenide ion adsorption to colloidal elemental selenium

Hydrogen selenide ion (HSe^?) has an important role in the metabolism of the essential trace element selenium. Several redox reactions of selenide were found to be dominated by the amount of colloidal elemental selenium (Se°) generated during the reaction. The following reaction of selenide with the disulfide, oxidized glutathione (GSSG), was used as an example: HSe^? + GSSG + H⁺ → Se° + 2 GSH. The resulting thiol is reduced glutathione (GSH; γ-glutamylcysteinylglycine). By following this reaction with polarography, it was seen that the ratio of colloidal selenium produced to selenide unreacted was a constant 2.1 ± 0.1, and was the only factor found to determine the extent of oxidation. This is best explained by the hypothesis that freshly generated colloidal selenium adsorbs selenide readily; no evidence for polyselenide formation was found. Adsorption of selenide should be considered in any reaction involving the oxidation of selenide to colloidal selenium.     

Ageing of tomato seeds involves glutathione oxidation

The effect of seed ageing on the oxidation of reduced glutathione (GSH) and the role of GSH oxidation in ageing-induced deterioration were studied in seeds of tomato ( Lycopersicon esculentum Mill. cv. Lerica, Moneymaker and Cromco). Both long-term storage at 15°C/30% relative humidity (RH) and artificial ageing at 20°C/75% RH, 30°C/45% RH and 60°C/45% RH resulted in a marked loss of GSH and a simultaneous, though not proportional, increase in its oxidized form GSSG. The glutathione thiol-disulfide status shifted towards a highly oxidized form, while the total glutathione pool decreased. The extent of GSH oxidation differed between ageing conditions and was not directly related to the extent of seed deterioration. Thiobarbituric acid-reactive substances did not increase in ageing tomato seeds, suggesting that lipid peroxidation did not take place. Hydration of seeds, either upon imbibition in water or by priming in an osmotic solution, resulted in a rapid decrease in GSSG, a shift of the glutathione redox couple to a mainly reduced status and an increase in the glutathione pool, in both control and aged seeds. The results indicate that, in tomato seeds, (1) seed ageing involves GSH oxidation into GSSG, which is indicative of oxidative stress, (2) ageing does not affect the GSSG reduction capacity upon subsequent imbibition, and (3) the lowered viability of aged seeds cannot directly be ascribed to the decreased GSH pool or To the highly oxidized glutathione redox status.     

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.