Gamma-glutamylcysteine Synthetase Activity Research Articles

Emodin Alleviates Sodium Taurocholate-Induced Pancreatic Ductal Cell Damage by Inhibiting the S100A9/VNN1 Signaling Pathway.

Because the pathogenesis of the disease is unclear, the treatment of patients with acute pancreatitis, especially severe acute pancreatitis, is still a major challenge for clinicians. Emodin is an anthraquinone compound extracted from rhubarb that can alleviate the damage to pancreatic ductal epithelial cells induced by adenosine triphosphate, but whether it has a similar protective effect on sodium taurocholate (STC)-stimulated pancreatic ductal cells and the underlying mechanism has not yet been reported. A model of STC-induced HPDE6-C7 human pancreatic ductal epithelial cell injury was established, and then apoptosis and the levels of reactive oxygen species (ROS), glutathione, gamma-glutamylcysteine synthetase, and inflammatory cytokines were assessed in the presence or absence of emodin pretreatment. S100 calcium binding protein A9 (S100A9) and Vanin1 (VNN1) protein expression was also measured. Emodin significantly increased HPDE6-C7 cell viability, inhibited apoptosis and ROS release, and elevated glutathione levels and gamma-glutamylcysteine synthetase activity. Furthermore, emodin downregulated S100A9 and VNN1 protein expression and inhibited the production of inflammatory factors, such as interleukin (IL)-1β, IL-6, IL-8, and IL-18. Emodin attenuates STC-induced pancreatic ductal cell injury possibly by inhibiting S100A9/VNN1-mediated ROS release. This finding provides evidence for the future development of emodin as a therapeutic agent.

Exploiting Rhizobium for Cadmium Sulphide Nanoparticle Synthesis: Heterologous Expression of an Escherichia coli DH10B Enzyme, YbdK [EC: 6.3.2.2] in Sinorhizobium fredii NGR234

Escherichia coli DH10B has 1.1 kb ybdK gene which is responsible for encoding YbdK enzyme that possess a Gamma glutamyl cysteine synthetase activity. ybdK gene was ligated downstream of a constitutive derepressed lac promoter of a low copy number plasmid vector pBBR1MCS-2, giving rise to a recombinant plasmid pPAT. Sinorhizobium fredii NGR234 transformed with pPAT showed an augmented production of glutathione which in turn increased the production of cadmium sulphide nanoparticles to some extent. Also, a heterologous expression of YbdK in Sinorhizobium fredii NGR234 improved the oxidation status of bacterial cells which is confirmed by fluorescence microscopy images and fluorometry. Genetically modified (GM) cells stained by DCFDA showed a significant decrease in fluorescence compared to wild type (WT) cells. Physical and chemical properties of the nanoparticles produced by the pPAT transformed Sinorhizobium fredii NGR234 differed significantly compared to wild type (WT) Sinorhizobium fredii NGR234. Comparative analysis of the nanoparticles by FTIR and SEM analysis revealed the functional groups attached to nanoparticles and average nanoparticle size respectively. Nanoparticles synthesized by genetically modified (GM) bacteria were about 3 times smaller in size compared to those produced by wild type (WT) rhizobium. FTIR analysis revealed an augmented presence of peptide with the nanoparticles produced by GM bacteria compared to those produced by the WT bacteria. XRD data revealed that biosynthesized CdS nanoparticles are face centered crystalline particles which was confirmed by comparing the peaks to standard JCPDS data (JCPDS card no. 10-454).

Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at clinically relevant concentrations.

Mitoxantrone (MTX) is used to treat several types of cancers and to improve neurological disability in multiple sclerosis. Unfortunately, cardiotoxicity is a severe and common adverse effect in MTX-treated patients. Herein, we aimed to study early and late mechanisms of MTX-induced cardiotoxicity using murine HL-1 cardiomyocytes. Cells were exposed to MTX (0.1, 1 or 10µM) during short (2, 4, 6, or 12h) or longer incubation periods (24 or 48h). At earlier time points, (6 and 12h) cytotoxicity was already observed for 1 and 10µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12h to MTX (pH gradients 4-7 and 6-11). Of note, the expression of the regulatory protein 14-3-3 protein epsilon was increased by a factor of two and three, after exposure to 1 and 10µM MTX, respectively. At earlier time-points, 10µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48h), MTX-induced cytotoxicity was concentration and time-dependent, according to the MTT reduction and lactate dehydrogenase leakage assays, while caspase-9, -8 and -3 activities increased at 24h. Regarding cellular redox status, total glutathione increased in 1µM MTX (24h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10µM MTX, oxidized glutathione was significantly higher than control at 48h. Moreover, MTX was able to significantly decrease proteasomal chymotrypsin-like activity in a concentration and time-independent manner. In summary, MTX significantly altered proteomic, energetic and oxidative stress homeostasis in cardiomyocytes at clinically relevant concentrations and our data clearly demonstrate that MTX causes early cardiotoxicity that needs further study.

Effects of sodium selenite on the antioxidant capacity and the fruit yield and quality of strawberry under cadmium stress

This study investigated the effects of sodium selenite (Na2SeO3) on the antioxidant capacity and the fruit yield and quality of cadmium-stressed strawberry. The results showed that cadmium (Cd) stress notably decreased the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione synthetic enzyme gamma-glutamylcysteine synthetase (γ-ECS), plant height and plant biomass, and fruit yield and quality, improved the activities of ascorbate oxidase (AO), glutathione reductase (GR) and ascorbate synthetic enzyme L-galactono-1,4-lactone dehydrogenase (GalLDH), the ratios of AsA/DHA and GSH/GSSG and the contents of malondialdehyde (MDA), hydrogen peroxide (H2O2) and Cd, compared with control. Compared with Cd stress alone, 5 and 10 μM Na2SeO3 notably improved the activities of above enzymes except AO, the ratios of AsA/DHA and GSH/GSSG, plant height and biomass, Se contents in leaves and fruits, average fruit weight and the contents of Vc, soluble sugar and total soluble solid in fruits. 15 μM Na2SeO3 notably improved the activities of above enzymes except AO, the ratio of GSH/GSSG, plant height, Se contents in leaves and fruits, average fruit weight and Vc content in fruits. 20 μM Na2SeO3 only notably improved Se contents in leaves and fruits, average fruit weight and Vc content in fruits. Meanwhile, 5, 10 and 15 μM Na2SeO3 significantly reduced Cd stress-induced the contents of MDA, H2O2 and Cd, 20 μM Na2SeO3 only significantly reduced Cd stress-induced the contents of Cd in leaves and fruits, compared with Cd stress alone.Our findings indicated that only low concentrations of Se were effective in conferring Cd tolerance in strawberry plants by up-regulating their antioxidant capacity and reducing Cd accumulation, which further improved the fruit yield and quality of strawberry.

Hydrogen Peroxide is Involved in the Regulation of Ascorbate and Glutathione Metabolism in Wheat Leaves under Water Stress

This study investigated the regulation of ascorbic acid and glutathione metabolism by hydrogen peroxide in wheat leaves under water stress. The results showed that H2O2 level, the activities of ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, L-galactono-1,4-lactone dehydrogenase and gamma-glutamylcysteine synthetase, and the contents of reduced ascorbate, reduced glutathione, total ascorbate and total glutathione were increased by water stress. And above increases were suppressed by pre-treatment with NADPH oxidase inhibitor diphenyleneiodonium chloride. Application of DPI also reduced H2O2 level, the activities of γ-ECS, GalLDH, APX, GR, DHAR and MDHAR, and the contents of AsA, GSH, total ascorbate and total glutathione, compared with control. Application of H2O2 to DPI-inhibited wheat seedlings prevented the reduction in the activities of APX, GR, MDHAR, DHAR, GalLDH and γ-ECS, and the contents of AsA, GSH, total ascorbate and total glutathione induced by DPI under water stress. Meanwhile, pre-treatment with DPI increased the malondialdehyde content and electrolyte leakage. Application of H2O2 to DPI-inhibited wheat seedlings prevented the increases in MDA content and EL. Our results suggested that water stress-induced H2O2 is a signal that leads to the up-regulation of ascorbate and glutathione metabolism and has an important role for acquisition of water-stress tolerance in wheat.

Signaling molecule methylglyoxal ameliorates cadmium injury in wheat (Triticum aestivum L) by a coordinated induction of glutathione pool and glyoxalase system

Methylglyoxal (MG) now is found to be an emerging signaling molecule. It can relieve the toxicity of cadmium (Cd), however its alleviating mechanism still remains unknown. In this study, compared with the Cd-stressed seedlings without MG treatment, MG treatment could stimulate the activities of glutathione reductase (GR) and gamma-glutamylcysteine synthetase (γ-ECS) in Cd-stressed wheat seedlings, which in turn induced an increase of reduced glutathione (GSH). Adversely, the activated enzymes related to GSH biosynthesis and increased GSH were weakened by N-acetyl-L-cysteine (NAC, MG scavenger), 2,4-dihydroxy-benzylamine (DHBA) and 1,3-bischloroethyl-nitrosourea (BCNU, both are specific inhibitors of GR), buthionine sulfoximine (BSO, a specific inhibitors of GSH biosynthesis), and N-ethylmaleimide (NEM, GSH scavenger), respectively. In addition, MG increased the activities of glyoxalase I (Gly I) and glyoxalase II (Gly II) in Cd-treated seedlings, followed by declining an increase in endogenous MG as comparision to Cd-stressed seedlings alone. On the contrary, the increased glyoxalase activity and decreased endogenous MG level were reversed by NAC and specific inhibitors of Gly I (isoascorbate, IAS; squaric acid, SA). Furthermore, MG alleviated an increase in hydrogen peroxide (H2O2) and malondialdehyde (MDA) in Cd-treated wheat seedlings. These results indicated that MG could alleviate Cd toxicity and improve the growth of Cd-stressed wheat seedlings by a coordinated induction of glutathione pool and glyoxalase system.

Cerium improves the copper tolerance of turf grass Poa pratensis by affecting the regeneration and biosynthesis of ascorbate and glutathione in leaves

This study investigated the effects of cerium on the regeneration and biosynthesis of ascorbate and glutathione in the leaves of turf grass Poa pratensis seedlings under copper (Cu) stress. The results showed that Cu stress increased the activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), l-galactono-1,4-lactone dehydrogenase (GalLDH) and gamma-glutamylcysteine synthetase (γ-ECS), and the contents of reduced ascorbic acid (AsA), reduced glutathione (GSH), total ascorbate and total glutathione. However, Cu stress markedly increased the malondialdehyde content and electrolyte leakage and decreased the fresh and dry weights. Pretreatment with cerium (Ce(NO3)3) enhanced the activities of APX, GR, DHAR, GalLDH and γ-ECS, and the contents of AsA, GSH, total ascorbate and total glutathione under Cu stress. Meanwhile, pretreatment with Ce(NO3)3 decreased the malondialdehyde content and electrolyte leakage and increased the fresh and dry weights under Cu stress. Our results suggested that Ce(NO3)3 alleviated oxidative damage by regulating the metabolism of ascorbate and glutathione under Cu stress, and has important role in the acquisition of Cu tolerance in P. pratensis seedlings. Therefore, Ce(NO3)3 can be used as a regulator to improve the Cu tolerance of P. pratensis.

Roles of glutathione in antioxidant defense, inflammation, and neuron differentiation in the thalamus of HIV-1 transgenic rats.

Inflammation and oxidative stress in the brain are major causes of HIV-associated neurocognitive disorders. Previously we have reported high content of glutathione (GSH) in the thalamus of rats with F344 genetic background. In this study, we investigated the changes of GSH metabolism and GSH-dependent antioxidant enzymes in the rat thalamus in response to HIV-1 transgenesis, and their associations with oxidative stress, inflammation, and neuronal development. Male HIV-1 transgenic (HIV-1Tg) rats and wild type F344 rats at 10 months were used in this study, with 5 rats in each group. Parameters measured in this study included: total and oxidized GSH, glutathione peroxidase (GPx), glutathione-S-transferase (GST), gamma-glutamylcysteine synthetase (GCS), gamma-glutamyl transferase (GGT), cysteine/cystine transporters, 4-hydroxynonenal (HNE), interleukin 12 (IL12), neuronal nuclei (NeuN), microtubule-associated protein (MAP2), and glia fibrillary acidic protein (GFAP). The levels of total GSH, oxidized GSH (GSSG) and MAP2 protein, and enzymatic activities of GCS, GPx and GST were significantly higher in HIV-1Tg rats compared with F344 rats, but the ratio of GSSG/GSH, activity of GGT and levels of HNE, NeuN protein and GFAP protein did not change. HIV-1Tg rats showed a lower level of IL12 protein. GSH positively correlated with GCS, GST and MAP2, GSSG/GSH ratio positively correlated with HNE and IL12, the activities of GPx, GST and GCS positively correlated with each other, and negatively correlated with HNE. These findings suggest an important role of the GSH-centered system in reducing oxidative stress and neuroinflammation, and enhancing neuron differentiation in the thalamus of HIV-1Tg rats.

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn2+) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn2+ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn2+ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn2+ induced oxidative stress.

Effects of exogenous hydrogen sulfide on the ascorbate and glutathione metabolism in wheat seedlings leaves under water stress

This study investigated the effects of exogenous hydrogen sulfide on the ascorbate and glutathione metabolism in wheat seedlings leaves under water stress. The results showed that pretreatment with sodium hydrosulfide (NaHS), hydrogen sulfide donor, increased the activities of ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase and gamma-glutamylcysteine synthetase, and the contents of reduced ascorbic acid, reduced glutathione, total ascorbate and total glutathione under water stress, compared to control and water stress without NaHS. Meanwhile, pretreatment with NaHS decreased the malondialdehyde content and electrolyte leakage induced by water stress in plants, compared to control and water stress without NaHS. Our results suggested that exogenous hydrogen sulfide alleviated oxidative damage by regulating the ascorbate and glutathione metabolism in wheat seedlings under water stress.

Egg Yolk Peptides Up-regulate Glutathione Synthesis and Antioxidant Enzyme Activities in a Porcine Model of Intestinal Oxidative Stress

Long-term oxidative stress in the gastrointestinal tract can lead to the development of chronic intestinal disorders. Many food-derived antioxidants are effective in vitro, but the variable reports of in vivo efficacy and the pro-oxidant nature of some antioxidants necessitate alternative strategies for the reduction of in vivo oxidative stress. Compounds that up-regulate the production of endogenous antioxidants such as glutathione (GSH) and antioxidant enzymes provide novel approaches for the restoration of redox homeostatis. Egg yolk peptides (EYP) prepared from Alcalase and protease N digestion of delipidated egg yolk proteins were found to exhibit antioxidative stress properties. The effect of EYP supplementation was examined in a hydrogen peroxide-induced human colon cell line and in an animal model of intestinal oxidative stress. EYP significantly reduced the pro-inflammatory cytokine, IL-8, in Caco-2 cells. In piglets given intraperitoneal infusions of hydrogen peroxide, EYP treatment increased GSH and gamma-glutamylcysteine synthetase mRNA expression and activity, significantly increased antioxidant enzyme activities, in particular catalase and glutathione S-transferase activities, and reduced protein and lipid oxidation in the duodenum, jejunum, ileum, and colon. Furthermore, EYP boosted the systemic antioxidant status in blood by increasing the GSH concentration in red blood cells. These results suggest that EYP supplementation is a novel strategy for the reduction of intestinal oxidative stress.

Vitamin E prevents buthionine sulfoximine-induced biochemical disorders in the rat.

Antioxidant therapy can improve the protection and metabolic activity of cells and tissues. In this study, the effect of vitamin E administration on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion in the rat lung and liver was investigated. Hepatic GSH was depleted by intraperitoneal administration of BSO (4 mmol kg(-1)), twice a day, for 30 days to rats. We also investigated whether the lung and liver mitochondrial GSH contents were influenced by BSO administration and whether an extracellular supply of vitamin E could prevent the changes caused by BSO-mediated GSH depletion. Glutathione levels in lung and liver tissues were depleted by 47% and 60%, respectively. Depletion of hepatic and pulmonary GSH in turn causes decline in the levels of mitochondrial GSH, leading to impaired antioxidant defence function of mitochondria. Both the cytosolic and mitochondrial glutathione disulfides (GSSG) were altered during BSO treatment, and led to drastic increase in GSSG/GSH redox status. One of the experimental groups was given vitamin E (65 mg (kg diet)(-1)) mixed with rat feed. The rats fed with vitamin E were found to have partially restored GSH levels in liver and lung, diminished levels of TBARS and minimized tissue damage. The current findings suggest that the impaired glutathione and glutathione-dependent enzyme status may be correlated with the elevated lipid peroxidation and mitochondrial membrane damage and that vitamin E therapy to the BSO-administered rats prevents the above changes. However, vitamin E did not have any effect on the activity of gamma-glutamyl cysteine synthetase (gamma-GCS).

Hemin reduces cellular sensitivity to imatinib and anthracyclins via Nrf2

Heme plays an important biomodulating role in various cell functions. In this study, we examined the effects of hemin on cellular sensitivity to imatinib and other anti-leukemia reagents. Hemin treatment of human BCR/ABL-positive KCL22 leukemia cells increased IC(50) values of imatinib, that is, the drug resistance, in a dose-dependent manner without any change in the BCR/ABL kinase activity. Imatinib-induced apoptosis was also suppressed by hemin treatment in KCL22 cells. Hemin treatment increased the activity of gamma-glutamylcysteine synthetase (gamma-GCS) light subunit gene promoter, which contains a Maf recognition element (MARE). Protein levels of gamma-GCS and heme oxygenase-1 (HO-1), two MARE-containing genes, were also increased after hemin treatment. Knockdown of Nrf2 expression by RNA interference largely abolished the effect of hemin on imatinib-treated cells, suggesting that Nrf2 recognition of MARE is essential for the hemin-mediated protective effect. Similar to hemin, treatment of cells with delta-aminolevulinic acid (delta-ALA), the obligatory heme precursor, also increased IC(50) values of imatinib. In contrast, inhibition of cellular heme synthesis by succinylacetone increased the sensitivity of cells to imatinib in two imatinib-resistant cell lines, KCL22/SR and KU812/SR. Hemin treatment also decreased the sensitivity of cells to four anthracyclins, daunorubicin, idarubicin, doxorubicin, and mitoxantrone, in BCR/ABL-negative leukemia U937 and THP-1 cells, as well as in KCL22 cells. These findings thus indicate that cellular heme level plays an important role in determining the sensitivity of cells to imatinib and certain other anti-leukemia drugs and that the effect of heme may be mediated via its ability to upregulate Nrf2 activity.

Chronic non-spherocytic hemolytic anemia associated with severe neurological disease due to -glutamylcysteine synthetase deficiency in a patient of Moroccan origin

A previously undescribed mutation of hereditary gamma-glutamylcysteine synthetase (GCS) deficiency was found in a 5 year old boy of Moroccan origin. He presented with chronic haemolytic anaemia, delayed psychomotor development and progressive motor sensitive neuropathy of lower extremities. The parents were third degree relatives. The activity of glycolytic enzymes were found to be normal in the propositus, his parents and a sister, but and a complete lack of GSH was found in the propositus. Accordingly, the measurement of de novo GSH synthetic enzymes was undertaken, and severe GCS deficiency was found in the propositus. Both parents and his sister presented GCS activity ranging from 69% to 90% of normal. GCS gene sequencing showed that the propositus was homozygous for a 1241C>T mutation in exon 11 and both parents and his sister were heterozygous. This mutation predicts a Pro414Leu amino acid substitution. Even though the homology between GCS and crystallographically solved, functionally related proteins is not very high, a three-dimensional model of GCS was derived using Modeller Software. GCS deficiency is a very rare autosomal recessive disorder reported so far in only 8 unrelated probands with severe haemolytic anaemia. In only 3 of these was the anaemia associated with severe neurological dysfunction. We report here the fourth case of GCS deficiency presenting neuropathy, giving further support to the eventual relationship between this enzymopathy and neurological damage.

The glutathione antioxidant system is enhanced in growth hormone transgenic coho salmon (Oncorhynchus kisutch)

Insertion of a growth hormone (GH) transgene in coho salmon results in accelerated growth, and increased feeding and metabolic rates. Whether other physiological systems within the fish are adjusted to this accelerated growth has not been well explored. We examined the effects of a GH transgene and feeding level on the antioxidant glutathione and its associated enzymes in various tissues of coho salmon. When transgenic and control salmon were fed to satiation, transgenic fish had increased tissue glutathione, increased hepatic glutathione reductase activity, decreased hepatic activity of the glutathione synthesis enzyme gamma-glutamylcysteine synthetase, and increased intestinal activity of the glutathione catabolic enzyme gamma-glutamyltranspeptidase. However, these differences were mostly abolished by ration restriction and fasting, indicating that upregulation of the glutathione antioxidant system was due to accelerated growth, and not to intrinsic effects of the transgene. Increased food intake and ability to digest potential dietary glutathione, and not increased activity of glutathione synthesis enzymes, likely contributed to the higher levels of glutathione in transgenic fish. Components of the glutathione antioxidant system are likely upregulated to combat potentially higher reactive oxygen species production from increased metabolic rates in GH transgenic salmon.

Copper-induced changes in the growth, oxidative metabolism, and saponin production in suspension culture roots of Panax ginseng in bioreactors

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 microM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 microM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 muM but decreased at 50 microM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O2*-) accumulation, and H2O2 content at 5 and 10 microM Cu-treated roots were not increased but strongly increased at 50 microM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 microM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 microM Cu, but both G-POD and CAT activities were inhibited at 50 microM Cu. Glutathione metabolism enzymes such as gamma-glutamylcysteine synthetase (gamma-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 microM Cu but were strongly inhibited at 50 microM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 microM Cu was associated to the strong induction of gamma-glutamyltranspeptidase (gamma-GGT) activity. These results indicate that plant could grow under Cu stress (5-25 microM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.

Effect ofSemecarpus anacardiumLinn. Nut Milk Extract on Glutathione and Its Associated Enzymes in Experimentally Induced Mammary Carcinoma

Reduced glutathione (GSH) is a ubiquitous thiol-containing tripeptide that plays a key role in the etiology of many diseases and, in particular, cancer. GSH, the foremost internal protective system, participates directly in the destruction of free radical compounds and detoxification of carcinogens. The effect of Semecarpus anacardium nut milk extract was studied for gaining insight into the disease relationship to GSH and its metabolizing enzymes. Mammary carcinoma was induced by giving 7,12-dimethylbenz[a]anthracene (DMBA) (25 mg/mL of olive oil) perorally by gastric intubation, and nut milk extract of S. anacardium was administered orally (200 mg/kg of body weight/day) for 14 days to mammary carcinoma-bearing rats. The levels of GSH and its metabolizing enzyme activities were determined in liver and kidney homogenates. Significant decreases in GSH, glutathione peroxidase, glutathione S-transferase, glutathione reductase, and gamma-glutamylcysteine synthetase and a concomitant increase in oxidized glutathione, gamma-glutamyl transpeptidase, and glucose 6-phosphate dehydrogenase were observed in DMBA-induced mammary carcinoma in rats, while drug treatment reversed the conditions to near normal levels. There was a marked increase in GSH level and gamma-glutamylcysteine synthetase activity in drug control rats. These findings suggest that S. anacardium can exert its protective effect in maintaining the glutathione redox status by restoring the associated enzymes against oxidative stress in experimental mammary carcinoma.

Enhanced γ‐glutamylcysteine synthetase activity decreases drug‐induced oxidative stress levels and cytotoxicity

Multidrug resistance of cancer cells can be intrinsic or acquired and occurs due to various reasons, including increased repair of genotoxic damage, an enhanced ability to remove/detoxify chemical agents, or reactive oxygen species (ROS), and repression of apoptosis. Human A2780/100 ovarian carcinoma cells exhibit resistance to DNA cross-linking agents, chlorambucil (Cbl), cisplatin (Cpl), melphalan (Mel), and ionizing radiation (IR) compared to the parental cell line, A2780. In the present study, we show that when A2780/100 and A2780 cells were treated with Cbl, GSH was extruded via methionine or cystathionine-inhibitable transporters of intact plasma membrane. GSH loss was followed by a rapid increase in ROS levels. The resistant, but not drug-sensitive cells normalized the intracellular GSH concentration along with ROS levels within 4-6 h after Cbl addition, and survived drug treatment. Normalization of GSH and ROS levels in A2780/100 cells correlated well with elevated gamma-glutamylcysteine synthetase (gamma-GCS) activity (10 +/- 1.8-fold over A2780 cells). Ectopic overexpression of the gamma-GCS heavy subunit in drug-sensitive cells nearly restored GSH and ROS to pre-treatment levels consequently increased cellular resistance to genotoxic agents (Cbl, Cpl, and IR), while overexpression of gamma-GCS light subunit had no such effects. Thus, in our model system, drug-resistant cells have the inherent ability to maintain increased gamma-GCS activity, reestablish physiological GSH, and cellular redox state and maintain increased cellular resistance to DNA cross-linking agents and IR.

Melatonin attenuates diabetes‐induced oxidative stress in rabbits

Oxidative stress is considered to be the main cause of diabetic complications. As the role of antioxidants in diabetes therapy is still underestimated, the aim of the present investigation was to study the antioxidative action of melatonin in comparison with N-acetylcysteine (NAC) under diabetic conditions. Alloxan-diabetic rabbits were treated daily with either melatonin (1 mg/kg, i.p.), NAC (10 mg/kg, i.p.) or saline. Blood glutathione redox state and serum hydroxyl free radicals (HFR), creatinine and urea levels were monitored. After 3 wk of treatment animals were killed and HFR content, reduced glutathione/oxidized glutathione (GSH/GSSG) ratio as well as the activities of glutathione reductase, glutathione peroxidase and gamma-glutamylcysteine synthetase were estimated in both liver and kidney cortex. Diabetes evoked a several-fold increase in HFR levels accompanied by a significant decline in GSH/GSSG ratio in serum and the examined organs. In contrast to NAC, melatonin (at 1/10 the dose of NAC) attenuated diabetes-induced alterations in glutathione redox state and HFR levels, normalized creatinine concentration and diminished urea content in serum. Moreover, the indole resulted in an increase in glutathione reductase activity in both studied organs and in a rise in glutathione peroxidase and gamma-glutamylcysteine synthetase activities in the liver. In contrast to NAC, melatonin seems to be beneficial for diabetes therapy because of its potent antioxidative and nephroprotective action. The indole-induced increase in the activities of the enzymes of glutathione metabolism might be of importance for antioxidative action of melatonin under diabetic conditions.

Time-course development of the Cd2+ hyper-accumulating phenotype in Euglena gracilis

To determine the onset of the Cd2+-hyperaccumulating phenotype in Euglena gracilis, induced by Hg2+ pretreatment (Avilés et al. in Arch Microbiol 180:1-10, 2003), the changes in cellular growth, Cd2+ uptake, and intracellular contents of sulfide, cysteine, gamma-glutamylcysteine, glutathione and phytochelatins during the progress of the culture were analyzed. In cells exposed to 0.2 mM CdCl2, the Cd2+-hyperaccumulating phenotype was apparent only after 48 h of culture, as indicated by the significant increase in cell growth and higher internal contents of sulfide and thiol-compounds, along with a higher gamma-glutamylcysteine synthetase activity. However, the stiochiometry of thiol-compounds/Cd2+ accumulated was similar for both control and Hg2+-pretreated cells. Moreover, the value for this ratio was 2.1 or lower after 48-h culture, which does not suffice to fully inactivate Cd2+. It is concluded that, although the glutathione and phytochelatin synthesis pathway is involved in the development of the Cd2+-hyperaccumulating phenotype in E. gracilis, apparently other pathways and sub-cellular mechanisms are also involved. These may be an increase in other Cd2+ chelating molecules such as di- and tricarboxylic acids, phosphate and polyphosphates, as well as Cd2+ compartmentation into organelles.