GSSG Content Research Articles

Conifer somatic embryogenesis: improvements by supplementation of medium with oxidation-reduction agents.

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue (ET) and continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment has been shown to decrease this recalcitrance. Glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid and dehydroascorbate analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiological concentrations. Major differences in stage-specific oxidation-reduction (redox) agents were observed. A simple bioassay was used to evaluate potential growth-promotion of natural and inorganic redox agents added to early-stage somatic embryo growth medium. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase initiation of loblolly pine. Low-cost reducing agents sodium dithionite and sodium thiosulfate increased ET initiation for loblolly pine and Douglas fir (Mirb) Franco. Germination medium supplementation with GSSG increased somatic embryo germination. Early-stage somatic embryos grown on medium with or without sodium thiosulfate did not differ in GSH or GSSG content, suggesting that sodium thiosulfate-mediated growth stimulation does not involve GSH or GSSG. We have developed information demonstrating that alteration of the redox environment in vitro can improve ET initiation, early-stage embryo development and somatic embryo germination in loblolly pine.

Gene knockout of glutathione reductase 3 results in increased sensitivity to salt stress in rice.

Glutathione reductase (GR) is one of important antioxidant enzymes in plants. This enzyme catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) with the accompanying oxidation of NADPH. Previously, we showed that salt-stress-responsive GR3 is a functional protein localized in chloroplasts and mitochondria in rice. To learn more about the role of GR3 in salt-stress tolerance, we investigated the response to 100mM NaCl treatment in wild-type rice (WT); GR3 knockout mutant of rice (gr3); and the functional gr3-complementation line (C1). Rice GR3 was primarily expressed in roots at the seedling stage and ubiquitously expressed in all tissues except the sheath at heading stage. GR3 promoter-GUS was expressed in the vascular cylinder and cortex of root tissues in rice seedlings, vascular tissue of nodes, embryo and aleurone layer of seeds, and young flowers. Under both normal and salt-stress conditions, total GR activity was decreased by 20% in gr3. Oxidative stress, indicated by malondialdehyde content, was greater in gr3 than the WT under salt stress. As compared with the WT, gr3 was sensitive to salt and methyl viologen; it showed inhibited growth, decreased maximal efficiency of photosystem II, decreased GSH and GSSG contents, and the ratio of GSH to GSSG. Conversely, the gr3-complementation line C1 rescued the tolerance to methyl viologen and salinity and recovered the growth and physiological damage caused by salinity. These results reveal that GR3 plays an important role in salt stress tolerance by regulating the GSH redox state in rice.

Exogenous glutathione confers high temperature stress tolerance in mung bean (Vigna radiata L.) by modulating antioxidant defense and methylglyoxal detoxification system

The present study investigates the role of exogenous glutathione (GSH) in conferring high temperature stress (HT, 42°C) tolerance in mung bean (Vigna radiata L. cv. Binamoog-1) seedlings by modulating the antioxidant defense and methylglyoxal (MG) detoxification systems. Six-day-old seedlings were exposed to HT stress with or without exogenous GSH (0.5mM for 24h as pretreatment) for 24 and 48h. Heat stress at any duration significantly increased lipid peroxidation (MDA), H2O2, MG, and Proline (Pro) content, generation rate of O2− and lipoxygenase (LOX) activity; decreased leaf chlorophyll (chl) and leaf relative water content (RWC), and the level of ascorbate (AsA); increased endogenous GSH and GSSG (glutathione disulfide); decreased the GSH/GSSG ratio. For both treatment durations, activities of ascorbate peroxidase (APX), glutathione reductase (GR), glutathione-S-transferase (GST) increased; the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), catalase (CAT) and glyoxalase I (Gly I) decreased; the activity of glyoxalase II (Gly II) increased at 48h. Mung bean seedlings pretreated with exogenous GSH under HT improved chl and leaf RWC; increased APX (only after 24h), MDHAR, DHAR, GR, GPX, GST (increased only after 24h), CAT, Gly I and Gly II activities; improved endogenous GSH content and the GSH/GSSG ratio; lowered GSSG content. Glutathione supplementation with drought stress significantly decreased MDA, H2O2 and MG content, O2− generation rate and LOX activity. Pretreatment with GSH resulted in better physiological performance, improved antioxidant and glyoxalase systems, and reduced MG and oxidative stress under 24h of HT stress, compared with that of 48h. The results suggest that exogenous GSH enhances mung bean seedling tolerance of short-term HT stress by modulating the antioxidant and glyoxalase systems and by improving physiological adaptation.

AMPK activation prevents prenatal stress-induced cognitive impairment: Modulation of mitochondrial content and oxidative stress

Prenatal stress induces cognitive functional impairment in offspring, an eventuality in which mitochondrial dysfunction and oxidative stress are believed to be closely involved. In this study, the involvement of the AMP-activated protein kinase (AMPK) pathway was investigated. A well-known activator, resveratrol (Res), was used to induce AMPK activation in SH-SY-5Y cells. Significant mitochondrial biogenesis and phase II enzyme activation, accompanied by decreased protein oxidation and GSSG content, were observed after Res treatment, and inhibition of AMPK with Compound c abolished the induction effects of Res. Further study utilizing a prenatal restraint stress (PRS) animal model indicated that maternal supplementation of Res may activate AMPK in the hippocampi of both male and female offspring, and that PRS-induced mitochondrial loss in the offspring hippocampus was inhibited by Res maternal supplementation. In addition, Res activated Nrf2-mediated phase II enzymes and reduced PRS-induced oxidative damage in both male and female offspring. Moreover, PRS markedly decreased mRNA levels of various neuron markers, as well as resultant offspring cognitive function, based on spontaneous alternation performance and Morris water maze tests, the results of which were significantly improved by maternal Res supplementation. Our results provide evidence indicating that AMPK may modulate mitochondrial content and phase II enzymes in neuronal cells, a process which may play an essential role in preventing PRS-induced cognitive impairment. Through the coupling of mitochondrial biogenesis and the Nrf2 pathway, AMPK may modulate oxidative stress and be a promising target against neurological disorders.

Reassessing cellular glutathione homoeostasis: novel insights revealed by genetically encoded redox probes.

Glutathione is the most abundant small molecule thiol in nearly all eukaryotes. Whole-cell levels of oxidized (GSSG) and reduced (GSH) glutathione are variable and responsive to genetic and chemical manipulations, which has led to their relative levels being widely used as a marker of the 'cellular redox state' and to indicate the level of 'oxidative stress' experienced by cells, tissues and organisms. However, the applicability of glutathione as a marker for a generalized 'cellular redox state' is questionable, especially in the light of recent observations in yeast cells. In yeast, whole-cell GSSG changes are almost completely dependent upon the activity of an ABC-C (ATP-binding cassette-C) transporter, Ycf1 (yeast cadmium factor 1), which mediates sequestration of GSSG to the vacuole. In the absence of Ycf1 whole-cell GSSG content is strongly decreased and extremely robust to perturbation. These observations are consistent with highly specific redox-sensitive GFP probe-based measurements of the cytosolic glutathione pool and indicate that cytosolic GSSG reductive systems are easily able to reduce nearly all GSSG formed, even following treatment with large concentrations of oxidant. In the present paper, I discuss the consequences of these new findings for our understanding of glutathione homoeostasis in the eukaryotic cell.

Exogenous Proline and Betaine-induced Upregulation of Glutathione Transferase and Glyoxalase I in Lentil (<i>Lens culinaris</i>) under Drought Stress</span>

This study was conducted to investigate the proline (Pro) and betaine (Bet) driven modulation of glutathione S-transferase (GST) and glyoxalase I (Gly I) in drought stressed lentil seedlings. Among the seven lentil varieties tested, BARI mosur 2 was found have higher activities of GST and Gly 1 which was selected for further time-course investigation. Drought stress resulted in a significant increase in glutathione (GSH), glutathione disulfide (GSSG) and hydrogen peroxide (H2O2) contents and GST and Gly I activities at different period  of the stress, while the GST activity decreased significantly at one and three days drought. However, exogenous application of 15 mM Bet or Pro with drought stress resulted in an increase in GSH content, maintenance of high activities of GST and Gly I as compared to the control and mostly also drought stressed plants, with a concomitant decrease in GSSG content and H2O2 level suggesting important role in protecting cell from the toxic effects of reactive oxygen species (ROS) and methyl glyoxal (MG) detoxification system. The application of Pro and Bet with the stress period revealed that they have important role in upregulating the homeostasis in lentil under stress condition. However, Pro exhibited better protection than Bet under drought stress. These findings suggest that both Pro and Bet provided a protective role in drought induced oxidative stress by reducing H2O2 levels and by increasing the antioxidant defense systems.

NADPH oxidase inhibitor, apocynin, improves renal glutathione status in Zucker diabetic fatty rats: A comparison with melatonin

Apocynin (4′-hydroxy-3′-methoxyacetophenone) is the most commonly used NADPH oxidase (Nox) inhibitor. However, its application raises serious controversies, as the compound has been reported to reveal some prooxidative effects. The aim of this study was to elucidate apocynin action on glutathione, the main intracellular antioxidant, metabolism in kidneys of Zucker diabetic fatty (ZDF) rat, a well established model of diabetes type 2. Additionally, apocynin effects were compared with those of melatonin.The experiments were performed on five groups of animals: (1) untreated lean (?/+) ZDF rats, (2) ZDF ?/+ rats treated with apocynin (2g/l) in drinking water, (3) untreated obese diabetic (fa/fa) ZDF rats, (4) ZDF fa/fa rats treated with apocynin (2g/l) in drinking water, and (5) ZDF fa/fa rats treated with melatonin (20mg/l) in drinking water. After 8weeks of the treatment, the following parameters were measured in kidneys: NADPH oxidase activity, the rate of hydroxyl free radicals (HFR) production, GSH and GSSG content and the activities of the enzymes of glutathione metabolism: γ-glutamylcysteine synthetase (GCS), glutathione reductase (GR) and glutathione peroxidase (GPx).Compared to ?/+ controls, ZDF fa/fa rats exhibited increased Nox activity, accelerated HFR generation and dramatically lowered GSH/GSSG ratio accompanied by increased GPx and diminished GCS activities. In case of diabetic animals, apocynin treatment resulted in attenuation of both Nox activity and HFR production, restoration of control GSH/GSSG ratio (due to both an increase in GSH and a decline in GSSG content), normalization of GPx activity and a slight increase in GCS activity. Similar observations were made upon melatonin application to ZDF fa/fa rats.Thus, it is concluded that, in the diabetic model studied, apocynin extends a beneficial effect on renal glutathione homeostasis. The mechanism of this phenomenon involves attenuation of glutathione peroxidase activity, which is overstimulated under conditions of oxidative stress accompanying diabetes.

Proteomic responses to lead-induced oxidative stress in Talinum triangulare Jacq. (Willd.) roots: identification of key biomarkers related to glutathione metabolisms

In this study, Talinum triangulare Jacq. (Willd.) treated with different lead (Pb) concentrations for 7 days has been investigated to understand the mechanisms of ascorbate-glutathione metabolisms in response to Pb-induced oxidative stress. Proteomic study was performed for control and 1.25 mM Pb-treated plants to examine the root protein dynamics in the presence of Pb. Results of our analysis showed that Pb treatment caused a decrease in non-protein thiols, reduced glutathione (GSH), total ascorbate, total glutathione, GSH/oxidized glutathione (GSSG) ratio, and activities of glutathione reductase and γ-glutamylcysteine synthetase. Conversely, cysteine and GSSG contents and glutathione-S-transferase activity was increased after Pb treatment. Fourier transform infrared spectroscopy confirmed our metabolic and proteomic studies and showed that amino, phenolic, and carboxylic acids as well as alcoholic, amide, and ester-containing biomolecules had key roles in detoxification of Pb/Pb-induced toxic metabolites. Proteomic analysis revealed an increase in relative abundance of 20 major proteins and 3 new proteins (appeared only in 1.25 mM Pb). Abundant proteins during 1.25 mM Pb stress conditions have given a very clear indication about their involvement in root architecture, energy metabolism, reactive oxygen species (ROS) detoxification, cell signaling, primary and secondary metabolisms, and molecular transport systems. Relative accumulation patterns of both common and newly identified proteins are highly correlated with our other morphological, physiological, and biochemical parameters.

Effect of D,L-buthionine-S,R-sulfoximine on the ratio of glutathione forms and the growth of Tatar buckwheat calli

We studied the intracellular content of reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase activity, glutathione-S-transferase, and ascorbate peroxidase in morphogenic and nonmorphogenic Tatar buckwheat calli during the culture cycle as well as under the treatment with D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthase, the first enzyme of glutathione biosynthesis. We found that, during passaging, cultures only slightly differed in total glutathione content; however, the content of GSH was higher in the morphogenic culture, whereas the content of GSSG was higher in the nonmorphogenic culture. In the morphogenic callus, the glutathione-S-transferase activity was 10-20 times higher and the glutathione reductase activity, was 2-2.5 times lower than in the nonmorphogenic callus. Under the treatment with BSO, the decrease in the GSH content in the morphogenic callus was temporary (on day 6-8 of passage), whereas that in the nonmorphogenic callus decreased within a day and remained lower than in the control throughout the entire passage. In the morphogenic callus, BSO did not affect the content of GSSG, whereas it caused GSSG accumulation in the nonmorphogenic callus. These differences are probably due to the fact that, in the BSO-containing medium, glutathione reductase is activated in the morphogenic callus and, conversely, inhibited in the nonmorphogenic callus. Although BSO caused a decrease in the total glutathione content only in the nonmorphogenic culture, the cytostatic effect of BSO was more pronounced in the morphogenic callus. In addition, BSO also had a negative effect on the differentiation ofproembryonic cell complexes in the morphogenic callus. The role of the glutathione redox status in maintaining the embryogenic activity of cultured plant cells is discussed.

Role of connexin 32 in acetaminophen toxicity in a knockout mice model

Gap junctional intercellular communication (GJIC), by which glutathione (GSH) and inorganic ions are transmitted to neighboring cells, is recognized as being largely involved in toxic processes of chemicals. We examined acetaminophen (APAP)-induced hepatotoxicity clinicopathologically using male wild-type mice and mice lacking the gene for connexin32, a major gap junction protein in the liver [knockout (Cx32KO) mice]. When APAP was intraperitoneally administered at doses of 100, 200, or 300mg/kg, hepatic centrilobular necrosis with elevated plasma aminotransferase activities was observed in wild-type mice receiving 300mg/kg, and in Cx32KO mice given 100mg/kg or more. At 200mg/kg or more, hepatic GSH and GSSG contents decreased significantly and the effect was more severe in wild-type mice than in Cx32KO mice. On the other hand, markedly decreased GSH staining was observed in the hepatic centrilobular zones of Cx32KO mice compared to that of wild-type mice. These results demonstrate that Cx32KO mice are more susceptible to APAP hepatotoxicity than wild-type mice, and indicate that the distribution of GSH of the centrilobular zones in the hepatic lobules, rather than GSH and GSSG contents in the liver, is important in APAP hepatotoxicity. In conclusion, Cx32 protects against APAP-induced hepatic centrilobular necrosis in mice, which may be through the GSH transmission to neighboring hepatocytes by GJIC.

English

Two cyanobacterial strains Synechocystis “AHZ-HB-MK” and “AHZ-HB-P2A” were isolated from tannery effluent form Lahore, Pakistan and their tolerance against K2CrO4was examined. Although growth was completely inhibited at 10 µg mL-1 in AHZ-HB-P2Astrain, cell growth of AHZ-HB-MK strain was observed even at a concentration of 100 µg mL-1. To show reasons for different tolerance between the two strains, we compared changes in nonprotein thiols, glutathione (GSH and GSSG), cysteine and cystine, after K2CrO4 treatment in both strains. After K2CrO4 treatment GSH completely disappeared in both strains, suggesting that GSH was involved in detoxification. GSSG content in former strain remarkably increased almost three and six times after 7-days treatment with 10 and 100 µg mL-1 K2CrO4, respectively while it decreased in the later strain. Cysteine content in the former strain increased almost three and eleven times after 1 day and 7 days treatment with 100 µg mL-1 K2CrO4, respectively, such increase in cysteine content was not observed in later strain. These data revealed that former strain could induce glutathione and cysteine but later strain could not. The difference in the ability to induce the nonprotein thiols between two strains must be responsible for differential tolerance in K2CrO4 observed in the growth inhibition.   Key words: Chromium, cysteine, cyanobacteria, glutathione, nonprotein thiol,Synechocystis.

Oxidative stress and apoptosis was induced by bio-insecticide spinosad in the liver of Oreochromis niloticus

This study was conducted to investigate acute toxic effects of spinosad on Glutathione-related oxidative stress markers, lipid peroxidation, heat shock proteins, apoptosis in the liver of Oreochromis niloticus selected as a model organism. The fish were exposed to sublethal spinosad concentrations (25, 50, 75mg/L) for 24–48–72h. tGSH, GSH, GSSG, and TBARS contents, GSH/GSSG ratio, and GPx, GR, GST and caspase enzyme activities were measured using spectrophotometrical methods, and Hsp70 content was measured by ELISA technique. The results demonstrated that spinosad exposure caused significant alterations in the GSH-related oxidative stress markers, and also caused increases in lipid peroxidation and stress proteins with inducing ROS generation in the liver. Apoptosis initiated with the induction of caspase-3 and Hsp70 could not protect the liver cells. Our results indicated that GSH-related antioxidant system tried to protect the liver cells from spinosad-induced hepatotoxicity however, the oxidative stress resulting from induction of ROS generation induced apoptosis in the liver of O. niloticus.

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.

Quercetin ameliorate insulin resistance and up-regulates cellular antioxidants during oleic acid induced hepatic steatosis in HepG2 cells

Hepatic lipid accumulation and oxidative stress contribute to non-alcoholic fatty liver disease (NAFLD). Thus, we hypothesized that the hypolipidemic and antioxidant activity of quercetin would attenuate events leading to NAFLD. Addition of 2.0mM oleic acid (OA) into the culture media induced fatty liver condition in HepG2 cells by 24h. It was marked by significant accumulation of lipid droplets as determined by Oil-Red-O (ORO) based colorimetric assay, increased triacylglycerol (TAG) and increased lipid peroxidation. The inflammatory cytokines TNF-α and IL-8 levels were significantly increased with decreased antioxidant molecules. OA induced insulin resistance which was evident by inhibition of glucose uptake and cell proliferation. Quercetin (10μM) increased cell proliferation by 3.05 folds with decreased TAG content (45%) and was effective in increasing insulin mediated glucose uptake by 2.65 folds. The intracellular glutathione content was increased by 2.0 folds without substantial increase in GSSG content. Quercetin (10μM) decreased TNF-α and IL-8 by 59.74% and 41.11% respectively and inhibited generation of lipid peroxides by 50.5%. In addition, RT-PCR results confirmed quercetin (10μM) inhibited TNF-alpha gene expression. Further, superoxide dismutase, catalase and glutathione peroxidase activities were increased by 1.68, 2.19 and 1.71 folds respectively. Albumin and urea content was increased while the alanine aminotransferase (ALAT) activity was significantly decreased by quercetin. Hence, quercetin effectively reversed NAFLD symptoms by decreased triacyl glycerol accumulation, insulin resistance, inflammatory cytokine secretion and increased cellular antioxidants in OA induced hepatic steatosis in HepG2 cells.

Neurotoxic effects of lambda-cyhalothrin modulated by piperonyl butoxide in the brain ofOreochromis niloticus

The objective of this research was to investigate the neurotoxic effects of pyrethroid pesticide lambda-cyhalothrin by the modulation of cytochrome P450 with piperonyl butoxide in the brain of juvenile Oreochromis niloticus. The fish were exposed to 0.48 μg L(-1) (1/6 of the 96-h LC50 ) lambda-cyhalothrin and 10 μg L(-1) piperonyl butoxide for 96 h and 15 days. tGSH, GSSG, TBARS contents, GPx, GR, GST, and AChE enzymes activities were determined by spectrophotometrical methods and Hsp70 content was analyzed by ELISA technique. Lambda-cyhalothrin had no significant effect on the components of GSH redox system, lipid peroxidation and Hsp70 levels but inhibited AChE activity. In the presence of piperonyl butoxide, lambda-cyhalothrin caused increases in tGSH, GSSG, TBARS and Hsp70 contents, GST activity, and decrease in AChE activity. Present results showed that in the presence of piperonyl butoxide, lambda-cyhalothrin caused neurotoxic effects by increasing oxidative stress. Adaptation to its oxidative stress effects may be supplied by GSH-related antioxidant system. Piperonyl butoxide revealed neurotoxic effect of lambda-cyhalothrin.

Cross Protection by Cold-shock to Salinity and Drought Stress-induced Oxidative Stress in Mustard (<i>Brassica campestris</i> L.) Seedlings

In the present study, cold-shock (6 ℃ , 5.5 h) induced salinity and drought tolerance and involvement of antioxidative and glyoxalase systems were investigated in mustard ( Brassica campestris L.) seedlings. Seven-day-old seedlings were subjected to salt (150 mmol/L NaCl, 48 h) and drought stress (induced by 20% PEG, 48 h) with or without cold pre-treatment. The results showed that both salt and drought stresses abruptly increased the hydrogen peroxide (H 2 O 2 ) and lipid peroxidation (malondialdehyde, MDA) levels. Ascorbate (AsA), reduced glutathione (GSH) and oxidized glutathione (GSSG) contents, GSH/GSSG ratio and the activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S -transferase (GST), glutathione peroxidase (GPX), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) showed both homogeneity and discrepancies in the responses of mustard seedlings to salinity and drought stresses. Drought stress treatment resulted in a significant increases in AsA content. The GSH and GSSG content increased in response to both salt and drought stresses, however, the GSH/GSSG ratio decreased significantly in response to drought stress. Salt stress treatment resulted in a significant increase of APX, MDHAR, GR, GST and Gly I activities, whereas, CAT and Gly II activities decreased. In contrast, drought stress treatment resulted in a significant increase in MDHAR, DHAR, GPX and Gly I activities; whereas, APX, CAT and Gly II activities decreased. Importantly, cold pre-treated salt and drought-stressed seedlings maintained higher level of AsA, GSH contents and GSH/GSSG ratio, higher activities of APX, DHAR, GR, GST, GPX, CAT, Gly I and Gly II, and lower the levels of GSSG, H 2 O 2 and MDA as compared to the control as well as in most cases seedlings subjected to salt and drought stress without cold pre-treatment. Our findings showed that a retention of the imprint of previous stress exposure (short-term cold-shock), induces salt and drought-induced oxidative stress tolerance by modulating antioxidative and glyoxalase systems.

Alternanthera sessilis and Alternanthera bettzickiana Improved Superoxide Dismutase and Catalase Activities in the Livers of Ovariectomized Mice

Background: Oxidative stress from reactive oxygen species can cause the damages to several cells or tissues. Ovariectomized (OVX) mice have been used as the oxidative stress model. Alternanthera sessilis (AS) and Alternanthera bettzickiana (AB), the Southeast Asia edible plants, have been traditionally used for promoting lactation, anti-inflammatory, wound healing, and antioxidation. Objective: Impacts of AS and AB crude extracts on the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) including the levels of malondialdehyde (MDA) formation and glutathione (GSH) content were investigated in the livers of ovariectomized induced oxidative stress in mice to clarify their anti-oxidative potentials, compared to 17b-estradiol (E2). Materials and Methods: Adult ICR mice were ovariectomized before treated with E2 (1 mg/kg/day, i.p.), or AS or AB (250, 500 mg/kg/day, p.o.), daily for eight weeks. The activity of CAT, SOD, and GPx and the levels of MDA and GSH in the mouse livers were determined. Results: Ovariectomy significantly reduced the activities of hepatic CAT, SOD, and GPx enzymes while the level of MDA was increased. Extensive decrease of total GSH content, reduced GSH, and the ratio of GSH/GSSG in the OVX mice demonstrated occurrence of hepatic oxidative stress. E2, AS, and AB restored the CAT and SOD activities while that of GPx activity was not modified. The MDA level was extensively suppressed by E2, AS, and AB. The significant increase of the GSH/GSSG ratio was achieved as a result from the marked abatement of GSSG content by E2, AS, and AB, though the level of total GSH content and the reduced GSH was not accrued. Conclusion: AS and AB possessed antioxidant benefit to improve the hepatic oxidant-antioxidant balance in the ovariectomized mice comparable to E2 did.

Moderate hypoxia/hyperoxia attenuates acute hypoxia-induced oxidative damage and improves antioxidant defense in lung mitochondria

A new mode of adaptive training was explored, which combines periods of hypoxia and hyperoxia (H/H) and is characterized by upregulation of adaptive ROS signals compared to classical intermittent hypoxic training. The purpose of this study was to determine the influence of repetitive moderate sessions of hypoxia and hyperoxia on pro-/antioxidant homeostasis in lung mitochondria of rats exposed to acute severe hypoxia. It was shown that H/H pretreatment [5 cycles of 5 min hypoxia (10% O2 in N2) alternated with 5 min hyperoxia (30% O2 in N2) daily for two weeks] reduced the acute hypoxia-induced basal and stimulated in vitro lipid peroxidation, increased the GSH/GSSG ratio, and decreased the GSSG content. The enhancement in the level of GSH and activities of MnSOD, GPx and GR in comparison with acute hypoxia as well as the maintenance of GST activity at control level confirm that mitochondrial protection during H/H may be mediated through the modulation of mitochondrial antioxidant levels. In lung H/H training caused the increase in MnSOD protein synthesis, at the same time, no changes in mRNA MnSOD expression was registered. This study supports the viewpoint that moderate periodic generation of free radical signal during changes in the oxygen level causes the induction of antioxidant enzyme protein synthesis that may be an important trigger for specific adaptations.

Investigation into the Effect of Molds in Grasses on Their Content of Low Molecular Mass Thiols

The aim of this study was to investigate the effect of molds on levels of low molecular mass thiols in grasses. For this purpose, the three grass species Lolium perenne, Festulolium pabulare and Festulolium braunii were cultivated and sampled during four months, from June to September. The same species were also grown under controlled conditions. High-performance liquid chromatography with electrochemical detection was used for quantification of cysteine, reduced (GSH) and oxidized (GSSG) glutathione, and phytochelatins (PC2, PC3, PC4 and PC5). Data were statistically processed and analyzed. Thiols were present in all examined grass species. The effect of fungicide treatments applied under field conditions on the content of the evaluated thiols was shown to be insignificant. Species influenced (p < 0.05) PC3 and GSSG content. F. pabulare, an intergeneric hybrid of drought- and fungi-resistant Festuca arundinacea, was comparable in PC3 content with L. perenne and F. braunii under field conditions. Under controlled conditions, however, F. pabulare had higher (p < 0.05) PC3 content than did L. perenne and F. braunii. Under field conditions, differences between the evaluated species were recorded only in GSSG content, but only sampling in June was significant. F. pabulare had higher (p < 0.05) GSSG content in June than did L. perenne and F. braunii.

Liv.52 up-regulates cellular antioxidants and increase glucose uptake to circumvent oleic acid induced hepatic steatosis in HepG2 cells

HepG2 cells were rendered steatotic by supplementing 2.0mM oleic acid (OA) in the culture media for 24h. OA induced hepatic steatosis in HepG2 cells was marked by significant accumulation of lipid droplets as determined by Oil-Red-O (ORO) based colorimetric assay, increased triacylglycerol (TAG) and increased lipid peroxidation. It was also marked by increased inflammatory cytokines TNF-α and IL-8 with decreased enzymic and non-enzymic antioxidant molecules and decreased cell proliferation associated with insulin resistance and DNA fragmentation. Addition of Liv.52 hydro-alcoholic extract (LHAE) 50μg/mL to the steatotic cells was effective in increasing the insulin mediated glucose uptake by 3.13 folds and increased cell proliferation by 3.81 folds with decreased TAG content (55%) and cytokines. The intracellular glutathione content was increased by 8.9 folds without substantial increase in GSSG content. LHAE decreased TNF-α and IL-8 by 51% and 6.5% folds respectively, lipid peroxidation by 65% and inhibited DNA fragmentation by 69%. The superoxide dismutase, catalase and glutathione peroxidase activities were increased by 88%, 128% and 64% respectively. Albumin and urea content was increased while the alanine aminotransferase (ALAT) activity was significantly decreased by LHAE. Hence, LHAE effectively attenuate molecular perturbations associated with non-alcoholic fatty liver disease (NAFLD) indications in HepG2 cells.