Ascorbate-dependent recycling of the vitamin E homologue Trolox by dihydrolipoate and glutathione in murine skin homogenates

In the redox antioxidant network, dihydrolipoate can synergistically enhance the ascorbate-dependent recycling of vitamin E. Since the major endogenous thiol antioxidant in biological systems is glutathione (GSH) it was of interest to compare the effects of dihydrolipoate with GSH on ascorbate-dependent recycling of the water-soluble homologue of vitamin E, Trolox, by electron spin resonance (ESR). Trolox phenoxyl radicals were generated by a horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) oxidation system. In the presence of dihydrolipoate, Trolox radicals were suppressed until both dihydrolipoate and endogenous levels of ascorbate in skin homogenates were consumed. Similar experiments made in the presence of GSH revealed that Trolox radicals reappeared immediately after ascorbate was depleted and that GSH was not able to drive the ascorbate-dependent Trolox recycling reaction. However, at higher concentrations GSH was able to increase ascorbate-mediated Trolox regeneration from the Trolox radical. ESR and spectrophotometric measurements demonstrated the ability of dihydrolipoate or GSH to react with dehydroascorbate, the two-electron oxidation product of ascorbate in this system. Dihydrolipoate regenerated greater amounts of ascorbate at a much faster rate than equivalent concentrations of GSH. Thus the marked difference between the rate and efficiency of ascorbate generation by dihydrolipoate as compared with GSH appears to account for the different kinetics by which these thiol antioxidants influence ascorbate-dependent Trolox recycling.     

Cooperative interaction between ascorbate and glutathione during mitochondrial impairment in mesencephalic cultures

A decrease in total glutathione, and aberrant mitochondrial bioenergetics have been implicated in the pathogenesis of Parkinson's disease. Our previous work exemplified the importance of glutathione (GSH) in the protection of mesencephalic neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. Additionally, reactive oxygen species (ROS) generation was an early, contributing event in malonate toxicity. Protection by ascorbate was found to correlate with a stimulated increase in protein-glutathione mixed disulfide (Pr-SSG) levels. The present study further examined ascorbate-glutathione interactions during mitochondrial impairment. Depletion of GSH in mesencephalic cells with buthionine sulfoximine potentiated both the malonate-induced toxicity and generation of ROS as monitored by dichlorofluorescein diacetate (DCF) fluorescence. Ascorbate completely ameliorated the increase in DCF fluorescence and toxicity in normal and GSH-depleted cultures, suggesting that protection by ascorbate was due in part to upstream removal of free radicals. Ascorbate stimulated Pr-SSG formation during mitochondrial impairment in normal and GSH-depleted cultures to a similar extent when expressed as a proportion of total GSH incorporated into mixed disulfides. Malonate increased the efflux of GSH and GSSG over time in cultures treated for 4, 6 or 8 h. The addition of ascorbate to malonate-treated cells prevented the efflux of GSH, attenuated the efflux of GSSG and regulated the intracellular GSSG/GSH ratio. Maintenance of GSSG/GSH with ascorbate plus malonate was accompanied by a stimulation of Pr-SSG formation. These findings indicate that ascorbate contributes to the maintenance of GSSG/GSH status during oxidative stress through scavenging of radical species, attenuation of GSH efflux and redistribution of GSSG to the formation of mixed disulfides. It is speculated that these events are linked by glutaredoxin, an enzyme shown to contain both dehydroascorbate reductase as well as glutathione thioltransferase activities.     

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.     

Interference of plasmatic reduced glutathione and hemolysis on glutathione disulfide levels in human blood

The blood reduced glutathione (GSH)/GSH disulfide (GSSG) ratio is an index of the oxidant/antioxidant balance of the whole body. Nevertheless, data indicating GSH and GSSG physiological levels are still widely divergent, especially those on GSSG, probably due to its low concentration. Standardization in methodological protocols and sample manipulation could help to minimize these discrepancies. Therefore, we have investigated how plasma reduced GSH, which is rapidly oxidized after blood withdrawal, could alter the blood GSSG measurement if the sample is not suitably processed. We have observed that an increase in plasma GSH concentration, due to red blood cell hemolysis, is responsible for a significant overestimation of blood GSSG level. Our results show that, before performing blood GSSG determination, thiols have to be rapidly blocked, to avoid possible pitfalls in GSSG measurement, in particular when hemolysis is present.     

Macrophage uptake and recycling of ascorbic acid: response to activation by lipopolysaccharide

To test whether ascorbic acid might be involved in the antioxidant defenses of inflammatory cells, we studied ascorbate uptake and recycling by quiescent and lipopolysaccharide-activated RAW264.7 murine macrophages. These cells concentrated ascorbate 100-fold in overnight culture, achieving steady-state concentrations of more than 10 mM at extracellular concentrations of 20-100 muM. This steep gradient was generated by high-affinity sodium-dependent ascorbate transport. The latter likely reflects function of the SVCT2 (SLC23A2), since this protein was detected on immunoblots. Dehydroascorbate, the two-electron oxidized form of ascorbate, was also taken up and reduced to ascorbate by the cells. Dehydroascorbate reduction required rapid recycling of GSH from GSSG by glutathione reductase. Activation of ascorbate-containing macrophages with lipopolysaccharide transiently depleted intracellular ascorbate without affecting GSH. Recovery of intracellular ascorbate required function of the SVCT2 transporter, the activity of which was modestly enhanced by lipopolysaccharide. Lipopolysaccharide treatment nearly doubled intracellular GSH concentrations over 2 h. Despite lipopolysaccharide-induced oxidant stress, this GSH increase was associated with a comparable increase in reduction of dehydroascorbate to ascorbate. These results show that macrophages maintain millimolar concentrations of ascorbate through function of the SVCT2 and that activated cells have an enhanced ability to transport and recycle ascorbate, possibly reflecting its role as an intracellular antioxidant.     

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane,by ascorbate/dihydrolipoate in living HL-60 cells

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.     

The biosynthesis of ascorbate protects isolated rat hepatocytes from cumene hydroperoxide-mediated oxidative stress

Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.     

Lipoic acid and vitamin C potentiate nitric oxide synthesis in human aortic endothelial cells independently of cellular glutathione status

Vitamin C and thiol agents improve vasomotor function. To determine whether these compounds directly affect endothelial function, nitric oxide (NO) synthesis was measured in human aortic endothelial cells treated with ascorbic acid or the thiol modulating agents lipoic acid or L-2-oxothiazolidine-4-carboxylic acid (OTC). A dose-dependent increase in A23187-stimulated NO synthesis and elevated cGMP levels were observed in all cases except for OTC. Cellular GSH levels were not significantly increased, and the GSH/GSSG ratio was not significantly affected by treatment of the cells with lipoic acid, OTC, or ascorbic acid. Thus, vitamin C and lipoic acid potentiate endothelial NO synthesis and bioactivity by mechanisms that appear to be independent of cellular GSH levels and redox environment.     

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.     

Insulin neuroprotection against oxidative stress in cortical neurons--involvement of uric acid and glutathione antioxidant defenses

In this study we investigated the effect of insulin on neuronal viability and antioxidant defense mechanisms upon ascorbate/Fe2+-induced oxidative stress, using cultured cortical neurons. Insulin (0.1 and 10 microM) prevented the decrease in neuronal viability mediated by oxidative stress, decreasing both necrotic and apoptotic cell death. Moreover, insulin inhibited ascorbate/Fe2+-mediated lipid and protein oxidation, thus decreasing neuronal oxidative stress. Increased 4-hydroxynonenal (4-HNE) adducts on GLUT3 glucose transporters upon exposure to ascorbate/Fe2+ were also prevented by insulin, suggesting that this peptide can interfere with glucose metabolism. We further analyzed the influence of insulin on antioxidant defense mechanisms in the cortical neurons. Oxidative stress-induced decreases in intracellular uric acid and GSH/GSSG levels were largely prevented upon treatment with insulin. Inhibition of phosphatidylinositol-3-kinase (PI-3K) or mitogen-induced extracellular kinase (MEK) reversed the effect of insulin on uric acid and GSH/GSSG, suggesting the activation of insulin-mediated signaling pathways. Moreover, insulin stimulated glutathione reductase (GRed) and inhibited glutathione peroxidase (GPx) activities under oxidative stress conditions, further supporting that insulin neuroprotection was related to the modulation of the glutathione redox cycle. Thus, insulin may be useful in preventing oxidative stress-mediated injury that occurs in several neurodegenerative disorders.     

Changes in the antioxidant systems as part of the signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobacco Bright-Yellow 2 cells

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for the activation of the hypersensitive reaction, a defense response induced in the noncompatible plant-pathogen interaction. However, its involvement in activating programmed cell death (PCD) in plant cells has been questioned. In this paper, the involvement of the cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition to the culture medium of NO and/or ROS generators. The individual increase in NO or ROS had different effects on the studied parameters than the simultaneous increase in the two reactive species. NO generation did not cause an increase in phenylalanine ammonia-lyase (PAL) activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death when it was generated at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed. The involvement of ASC and GSH as part of the transduction pathway leading to PCD is discussed.     

Antioxidant capacity of Lemna gibba L. exposed to wastewater treatment

In this study, the amounts of antioxidant vitamins (A, E and C), selenium (Se), reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio and malondialdehyde (MDA) that is the indicator of lipid peroxidation were determined in Lemna gibba L. plants placed in the secondary clarifier and grown in natural water. The amounts of antioxidant vitamins (A, E and C), GSH, GSSG and MDA were determined with HPLC (high performance liquid chromatography) and the amounts of Se were determined fluorimetrically. While significant decrease in amounts of antioxidant vitamins (A, E and C) and Se takes place between the first and the second or third day depending on species and insignificant decrease takes place after that day, the amount of GSH/GSSG ratio decreases until the second day (p < 0.05) and insignificantly increases after that day. Amounts of vitamins (A, E and C), Se and GSH/GSSG ratio for the plants placed in the secondary clarifier are much less than that for control group while an opposite trend was observed in MDA level. The MDA for the plants placed in secondary clarifier has maximum value at the second day, which can be considered as the maximum stress occurring at that day. Consequently, the first two days of treatment time can be taken as acclimation time and after the acclimation time the plant lives in the hostile environmental condition with the certain amount of oxidative stress. As a result, it is determined that wastewater decreases the lifetime of plant by causing metabolic stress on it and affects antioxidant capacity. The lifetime of the plants in the secondary clarifier was determined to be five days since fading toward yellow color (necrosis) in the plants was observed at the fifth day.     

Riboflavin spraying impairs the antioxidant defense system but induces waterlogging tolerance in tobacco

Riboflavin, which causes plants to produce reactive oxygen species (ROS) when exposed to light, is an excellent photosensitizer for biocidal reactions. This study explores the possible protective role of riboflavin against waterlogging stress in tobacco plants. Tobacco seedlings (4 weeks old) were divided into four groups and pretreated with 0, 0.2, 0.5 or 1.0 mM riboflavin for 1 week, after which all groups were exposed to waterlogging stress for 7 days. We observed delayed leaf senescence and extended survival time, suggesting that riboflavin can confer increased waterlogging tolerance to plants as compared with the control (0 mM riboflavin). Enhanced stomatal closure was observed in the riboflavin-pretreated tobacco. We evaluated the levels of oxidative damage (H₂O₂ and lipid peroxidation), antioxidant enzyme (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) activity and antioxidant metabolites (including ascorbate and glutathione) in tobacco leaves that were pretreated with riboflavin. However, the results show that riboflavin pretreatment caused a decrease in chlorophyll content, antioxidant enzyme activity and redox values (AsA/DHA and GSH/GSSG), while causing a significant increase in lipid peroxidation, H₂O₂ accumulation and total ascorbate or glutathione content. In addition, the survival time and stomatal aperture of riboflavin-treated plants were significantly modified by exogenous application of GSH, well-known ROS scavenger. To explain the stomatal closure observed in tobacco plants, we propose a “damage avoidance” hypothesis based on riboflavin-mediated ROS toxicity. The protective function of the photosensitizer riboflavin may be highly significant for farming in frequently waterlogged areas.     

Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study

Since the higher redox potential of quinone molecules has been correlated with enhanced cellular deleterious effects, we studied the ability of the association of ascorbate with several quinones derivatives (having different redox potentials) to cause cell death in K562 human leukaemia cell line. The rationale is that the reduction of quinone by ascorbate should be dependent of the quinone half-redox potential thus determining if reactive oxygen species (ROS) are formed or not, leading ultimately to cell death or cell survival. Among different ROS that may be formed during redox cycling between ascorbate and the quinone, the use of different antioxidant compounds (mannitol, desferal, N-acetylcysteine, catalase and superoxide dismutase) led to support H2O2 as the main oxidizing agent. We observed that standard redox potentials, oxygen uptake, free ascorbyl radical formation and cell survival were linked. The oxidative stress induced by the mixture of ascorbate and the different quinones decreases cellular contents of ATP and GSH while caspase-3-like activity remains unchanged. Again, we observed that quinones having higher values of half-redox potential provoke a severe depletion of ATP and GSH when they were associated with ascorbate. Such a drop in ATP content may explain the lack of activation of caspase-3. In conclusion, our results indicate that the cytotoxicity of the association quinone/ascorbate on K562 cancer cells may be predicted on the basis of half-redox potentials of quinones.     

Comparison between the effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state in ex vivo culture of CD34(+) cells

Hypoxia maintained biological characteristics of CD34(+) cells through keeping lower intracellular reactive oxygen specials (ROS) levels. The effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state were compared in this study. Hypoxia decreased the mRNA expression of both catalase (CAT) and glutathione peroxidase (GPX), but not affected mRNAs expression of superoxide dismutase (SOD). While the cellular GPX activities under hypoxia were apparently less than those under normoxia, neither SOD activities nor CAT activities were affected by hypoxia. The analysis of glutathione redox status and ROS products showed the lower oxidized glutathione (GSSG) levels, the higher reduced glutathione (GSH) levels, the higher GSH/GSSG ratios, and the less O(2)- and H(2)O(2) generation under hypoxia (versus normoxia). Meanwhile more primary CD34(+)CD38(-) cells were obtained when cultivation was performed under hypoxia or with N-acetyl cysteine (the precursor of GSH) under normoxia. These results demonstrated the different responses of anti-oxidative mechanism between normoxia and hypoxia. Additionally, the present study suggested that the GSH-GPX antioxidant system played an important role in HSPCs preservation by reducing peroxidation.     

A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L. seeds

The ascorbate and glutathione systems have been studied during the first stages of germination in orthodox seeds of the gymnosperm Pinus pinea L. (pine). The results indicate that remarkable changes in the content and redox balance of these metabolites occur in both the embryo and endosperm; even if with different patterns for the two redox pairs. Dry seeds are devoid of the ascorbate reduced form (ASC) and contain only dehydroascorbic acid (DHA). By contrast, glutathione is present both in the reduced (GSH) and in the oxidized (GSSG) forms. During imbibition the increase in ASC seems to be mainly caused by the reactivation of its biosynthesis. On the other hand, the GSH rise occurring during the first 24 h seems to be largely due to GSSG reduction, even if GSH biosynthesis is still active in the seeds. The enzymes of the ascorbate--glutathione cycle also change during germination, but in different ways. ASC peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities progressively rise both in the embryo and in endosperm. These changes are probably required for counteracting production of reactive oxygen species caused by recovery of oxidative metabolism. The two enzymes involved in the ascorbate recycling, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and DHA reductase (EC 1.8.5.1), show different behaviour: the DHA reductase activity decreases, while that of AFR reductase remains unchanged. The relationship between ascorbate and glutathione metabolism and their relevance in the germination of orthodox seeds are also discussed.     

Regeneration of vitamin E in human platelets

Human platelets possess active lipoxygenase and cyclooxygenase which convert arachidonic acid to (12S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) plus (12S)-12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and thromboxane B2 plus 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), respectively. When platelet homogenates were incubated with arachidonate, there was a rapid consumption of platelet tocopherol. Time course analysis revealed that within 0.5 min, over half of arachidonate and tocopherol were metabolized. Mass formation of 12-HPETE and 12-HETE or thromboxane B2 and HHT exceeded that of the mass of tocopherol oxidized. Preincubation with the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) completely abolished this arachidonate-induced tocopherol oxidation whereas cyclooxygenase inhibitors (indomethacin and aspirin) further potentiated tocopherol oxidation, indicating that this oxidation is closely linked with platelet 12-lipoxygenase activity. Incubation with lipoxygenase metabolites of arachidonic acid showed that only 12-HPETE caused a rapid tocopherol oxidation which was followed by a gradual tocopherol regeneration. By using nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor which is also a strong reductant, over 60% of the arachidonate-induced oxidized tocopherol was regenerated. Tocopherol regeneration declined with increasing oxidation time induced by arachidonate, and after 30-60 min virtually no regeneration could be observed, suggesting that the precursor molecule was unstable. We postulate that the precursor molecule is the tocopheroxyl radical. In the presence of ETYA, a lipoxygenase inhibitor without antioxidant properties, either ascorbate or GSH provided significant tocopherol regeneration. Kinetic studies showed that tocopherol regeneration after the addition of ascorbate was essentially completed by 1 min. By contrast, GSH addition caused a steady increase in tocopherol which peaked after 10 min of its addition. To determine whether this rapid regeneration is chemical or enzymic, regeneration was studied in the presence of chloroform and methanol. Comparison of various reductants in this denaturing condition for enzymes showed that ascorbate and NDGA afforded significant regeneration whereas GSH was ineffective, indicating that there are distinct enzymic and non-enzymic mechanisms for tocopherol regeneration. This study provides direct evidence from mass analysis that tocopherol can be regenerated in human cell homogenates. This finding implies that maintenance of membrane tocopherol status may be an essential function of ascorbate and GSH which operate in concert to ensure maximum membrane protection against oxidative damage.     

Roles of sedentary aging and lifelong physical activity in exchange of glutathione across exercising human skeletal muscle

Reactive oxygen species (ROS) are important signaling molecules with regulatory functions, and in young and adult organisms, the formation of ROS is increased during skeletal muscle contractions. However, ROS can be deleterious to cells when not sufficiently counterbalanced by the antioxidant system. Aging is associated with accumulation of oxidative damage to lipids, DNA, and proteins. Given the pro-oxidant effect of skeletal muscle contractions, this effect of age could be a result of excessive ROS formation. We evaluated the effect of acute exercise on changes in blood redox state across the leg of young (23±1 years) and older (66±2 years) sedentary humans by measuring the whole blood concentration of the reduced (GSH) and oxidized (GSSG) forms of the antioxidant glutathione. To assess the role of physical activity, lifelong physically active older subjects (62±2 years) were included. Exercise increased the venous concentration of GSSG in an intensity-dependent manner in young sedentary subjects, suggesting an exercise-induced increase in ROS formation. In contrast, venous GSSG levels remained unaltered during exercise in the older sedentary and active groups despite a higher skeletal muscle expression of the superoxide-generating enzyme NADPH oxidase. Arterial concentration of GSH and expression of antioxidant enzymes in skeletal muscle of older active subjects were increased. The potential impairment in exercise-induced ROS formation may be an important mechanism underlying skeletal muscle and vascular dysfunction with sedentary aging. Lifelong physical activity upregulates antioxidant systems, which may be one of the mechanisms underlying the lack of exercise-induced increase in GSSG.     

Effect of antioxidant-enriched diets on glutathione redox status in tissue homogenates and mitochondria of the senescence-accelerated mouse

The main purpose of this study was to investigate whether consumption of diets enriched in antioxidants attenuates the level of oxidative stress in the senescence-accelerated mouse (SAM). In separate and independent studies, two different dietary mixtures, one enriched with vitamin E, vitamin C, L-carnitine, and lipoic acid (Diet I) and another diet including vitamins E and C and 13 additional ingredients containing micronutrients with bioflavonoids, polyphenols, and carotenoids (Diet II), were fed for 8 and 10 months, respectively. The amounts of glutathione (GSH) and glutathione disulfides (GSSG) and GSH:GSSG ratios were determined in plasma, tissue homogenates, and mitochondria isolated from five different tissues of SAM (P8) mice. Both diets had a reductive effect in plasma; however Diet I had relatively little effect on the glutathione redox status in tissue homogenates or mitochondria. Remarkably, Diet II caused a large increase in the amount of glutathione and a marked reductive shift in glutathione redox state in mitochondria. Overall, the effects of Diet II were tissue and gender specific. Results indicated that the glutathione redox state in mitochondria and tissues can be altered by supplemental intake of a relatively complex mixture of dietary antioxidants that contains substances known to induce phase 2 enzymes, glutathione, and antioxidant defenses. Whether corresponding attenuations occur in age-associated deleterious changes in physiological functions or life span remains unknown.     

Spermidine application enhances tomato seedling tolerance to salinity-alkalinity stress by modifying chloroplast antioxidant systems

The purpose of this study was to elucidate whether exogenous spermidine (Spd) protection of tomato (Solanum lycopersicum L.) seedlings under salinity-alkalinity stress is associated with antioxidant enzymes in the chloroplast. The effects of exogenous Spd on antioxidant enzyme activity and antioxidant content in the chloroplast were evaluated in seedlings of salt-sensitive ecotype (Zhongza 9) grown in a 75 mM salinity-alkalinity solution, with or without 0.25 mM Spd foliar spraying. Results showed that salinity-alkalinity stress increased MDA content, superoxide anion O₂^?- generation rate, superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) activities and ratio of AsA/DHA and reduced contents of ascorbate (AsA), dehydroascorbate (DHA), AsA+DHA, glutathione (GSH), oxidized glutathione (GSSG), GSH+GSSG, dehydroascorbate reductase (DHAR) activity and ratio of GSH/GSSG in chloroplasts. The exogenous Spd application combined with salinity-alkalinity stress decreased the O₂^?- generation rate and MDA content compared to salinity-alkalinity stress alone. The exogenous Spd also increased AsA-GSH cycle components and increased all antioxidant enzyme activities in most cases. Therefore, exogenous Spd alleviates salinity-alkalinity stress damage using antioxidant enzymes and non-enzymatic systems in chloroplasts.