GSH Biosynthesis Research Articles

Exogenous Glutathione Enhances Mercury Tolerance by Inhibiting Mercury Entry into Plant Cells.

Despite the increasing understanding of the crucial roles of glutathione (GSH) in cellular defense against heavy metal stress as well as oxidative stress, little is known about the functional role of exogenous GSH in mercury (Hg) tolerance in plants. Here, we provide compelling evidence that GSH contributes to Hg tolerance in diverse plants. Exogenous GSH did not mitigate the toxicity of cadmium (Cd), copper (Cu), or zinc (Zn), whereas application of exogenous GSH significantly promoted Hg tolerance during seed germination and seedling growth of Arabidopsis thaliana, tobacco, and pepper. By contrast, addition of buthionine sulfoximine, an inhibitor of GSH biosynthesis, severely retarded seed germination and seedling growth of the plants in the presence of Hg. The effect of exogenous GSH on Hg specific tolerance was also evident in the presence of other heavy metals, such as Cd, Cu, and Zn, together with Hg. GSH treatment significantly decreased H2O2 and O2- levels and lipid peroxidation, but increased chlorophyll content in the presence of Hg. Importantly, GSH treatment resulted in significantly less accumulation of Hg in Arabidopsis plants, and thin layer chromatography and nuclear magnetic resonance analysis revealed that GSH had much stronger binding affinity to Hg than to Cd, Cu, or Zn, suggesting that tight binding of GSH to Hg impedes Hg uptake, leading to low Hg accumulation in plant cells. Collectively, the present findings reveal that GSH is a potent molecule capable of conferring Hg tolerance by inhibiting Hg accumulation in plants.

Cadmium and copper induced changes in growth, oxidative metabolism and terpenoids of Tanacetum parthenium

Morphological and biochemical responses of feverfew plants exposed to low (5μM) and high (35 and 70μM) levels of Cd or Cu were investigated. Increasing metal supply notably reduced the plant biomass. Elevated Cd and Cu levels also resulted in an increase in the leaf proline content. Besides, decrease in ascorbic acid (AsA) and glutathione (GSH) contents was similar in the leaves of Cd- and Cu-treated plants, indicating altered biosynthesis of AsA and GSH under metal excess. High metal doses stimulated increase in antioxidative enzyme activities that could be related to elevated hydrogen peroxide (H2O2) content and subsequent lipid peroxidation. Cd was typically more accumulated in shoots and roots than Cu, leading to higher translocation factor at high Cd doses. In terms of essential oil content, it seems that Cd had an inhibitory effect during the experiment, whereas Cu was found to stimulate it only at 5μM. Furthermore, high Cd supply enhanced the relative proportion of monoterpene hydrocarbons, while Cu increased the proportion of sesquiterpenes, especially at 5μM. This result provides the first evidence of the response of feverfew plants to Cd or Cu by associating stress-related responses with changes in terpenoids.

Glutathione in plants: biosynthesis and physiological role in environmental stress tolerance.

Glutathione (GSH; γ-glutamyl-cysteinyl-glycine) is a small intracellular thiol molecule which is considered as a strong non-enzymatic antioxidant. Glutathione regulates multiple metabolic functions; for example, it protects membranes by maintaining the reduced state of both α-tocopherol and zeaxanthin, it prevents the oxidative denaturation of proteins under stress conditions by protecting their thiol groups, and it serves as a substrate for both glutathione peroxidase and glutathione S-transferase. By acting as a precursor of phytochelatins, GSH helps in the chelating of toxic metals/metalloids which are then transported and sequestered in the vacuole. The glyoxalase pathway (consisting of glyoxalase I and glyoxalase II enzymes) for detoxification of methylglyoxal, a cytotoxic molecule, also requires GSH in the first reaction step. For these reasons, much attention has recently been directed to elucidation of the role of this molecule in conferring tolerance to abiotic stress. Recently, this molecule has drawn much attention because of its interaction with other signaling molecules and phytohormones. In this review, we have discussed the recent progress in GSH biosynthesis, metabolism and its role in abiotic stress tolerance.

Cadmium and α-lipoic acid activate similar de novo synthesis and recycling pathways for glutathione balance.

Glutathione (GSH) protects cells against oxidative stress. Redox modifiers induce GSH biosynthesis and recycling to maintain reduced environment inside cells. Cadmium (Cd2+) is a heavy metal that activates redox-sensitive transcriptional factors. The antioxidant α-lipoic acid (ALA) has shown to modulate GSH pathways. This study aimed to investigate de novo synthesis and recycling pathways for GSH balance by different Cd2+ concentrations and ALA in HepG2 cells. ALA activates Nrf2 pathway leading to GSH increment. Pre-treatment with 1μM Cd2+ or ALA produces tolerance to 5μM Cd2+ toxic effects. 5μM Cd2+ exposure significantly augmented nuclear Nrf2, GSH and GCLC, GCLM, HMOX1, TNFα and IL-6 mRNA expression but not GSR, however these upsurges were significantly abrogated by ALA or 1μM Cd2+ pre-treatments. Exposure to low Cd2+ concentration generate timely protective responses, similar to that elicited by ALA, maintaining a normal redox balance inside the cell due to GSH replenishment.

Post-translational Activation of Glutamate Cysteine Ligase with Dimercaprol: A NOVEL MECHANISM OF INHIBITING NEUROINFLAMMATION IN VITRO

Neuroinflammation and oxidative stress are hallmarks of various neurological diseases. However, whether and how the redox processes control neuroinflammation is incompletely understood. We hypothesized that increasing cellular glutathione (GSH) levels would inhibit neuroinflammation. A series of thiol compounds were identified to elevate cellular GSH levels by a novel approach (i.e. post-translational activation of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH biosynthesis). These small thiol-containing compounds were examined for their ability to increase intracellular GSH levels in a murine microglial cell line (BV2), of which dimercaprol (2,3-dimercapto-1-propanol (DMP)) was found to be the most effective compound. DMP increased GCL activity and decreased LPS-induced production of pro-inflammatory cytokines and inducible nitric-oxide synthase induction in BV2 cells in a concentration-dependent manner. The ability of DMP to elevate GSH levels and attenuate LPS-induced pro-inflammatory cytokine production was inhibited by buthionine sulfoximine, an inhibitor of GCL. DMP increased the expression of GCL holoenzyme without altering the expression of its subunits or Nrf2 target proteins (NQO1 and HO-1), suggesting a post-translational mechanism. DMP attenuated LPS-induced MAPK activation in BV2 cells, suggesting the MAPK pathway as the signaling mechanism underlying the effect of DMP. Finally, the ability of DMP to increase GSH via GCL activation was observed in mixed cerebrocortical cultures and N27 dopaminergic cells. Together, the data demonstrate a novel mechanism of GSH elevation by post-translational activation of GCL. Post-translational activation of GCL offers a novel targeted approach to control inflammation in chronic neuronal disorders associated with impaired adaptive responses.

Glutathione metabolic status in the aged rabbit aorta

It is not known whether aging alters glutathione metabolic status of the mammalian arterial tissue favoring vascular oxidative stress and dysfunction. Thus we assessed total, reduced and oxidized glutathione (TG, GSH and GSSG, respectively), the glutathione redox ratio (GRR, namely [GSSG]/[GSH+2GSSG]×100), and the activities of the glutathione status-regulating enzymes glutathione reductase (GSSG-Red), γ-glutamylcysteine synthetase (γ-GCS) and γ-glutamyl transpeptidase (γ-GT) in the aortic tissue of 9 young adult control rabbits (YACR, about 4months old) and 9 aged rabbits (AR, about 4.5years old); aortic lipid and protein oxidation and H2O2 were also determined as oxidative stress indicators. Vascular function was assessed on aortic ring preparations. TG and GSH concentrations, together with γ-GCS and γ-GT activities, were significantly lower, while GSSG content and the GRR higher, in the AR than in the YACR aortas; GSSG-Red activity did not differ significantly between the two groups. Heightened levels of lipid and protein oxidation and H2O2 occurred in the AR aortas, indicating age-dependent vascular oxidative stress. Moreover, in the whole population of 18 rabbits, the aortic values of GSH and related enzyme activities were inversely and significantly correlated with those of lipid and protein oxidation and H2O2, highlighting the antioxidant role of GSH and related enzymes in the vascular tissue. Aortic endothelium-dependent vasodilation was lower in the AR than in the YACR. In conclusion, glutathione metabolic status is altered in the aged rabbit aorta reflecting depressed γ-GCS- and γ-GT-related GSH biosynthesis and GSSG burden eventually favoring vascular oxidative stress and dysfunction.

RTA-408 Protects Kidney from Ischemia-Reperfusion Injury in Mice via Activating Nrf2 and Downstream GSH Biosynthesis Gene.

Acute kidney injury (AKI) induced by ischemia-reperfusion is a critical conundrum in many clinical settings. Here, this study aimed to determine whether and how RTA-408, a novel oleanane triterpenoid, could confer protection against renal ischemia-reperfusion injury (IRI) in male mice. Mice treated with RTA-408 undergoing unilateral ischemia followed by contralateral nephrectomy had improved renal function and histological outcome, as well as decreased apoptosis, ROS production, and oxidative injury marker compared with vehicle-treated mice. Also, we had found that RTA-408 could strengthen the total antioxidant capacity by increasing Nrf2 nuclear translocation and subsequently increased Nrf2 downstream GSH-related antioxidant gene expression and activity. In vitro study demonstrated that GSH biosynthesis enzyme GCLc could be an important target of RTA-408. Furthermore, Nrf2-deficient mice treated with RTA-408 had no significant improvement in renal function, histology, ROS production, and GSH-related gene expression. Thus, by upregulating Nrf2 and its downstream antioxidant genes, RTA-408 presents a novel and potential approach to renal IRI prevention and therapy.

Nitric oxide improves S-assimilation and GSH production to prevent inhibitory effects of cadmium stress on photosynthesis in mustard (Brassica juncea L.)

Nitric oxide (NO) is an important gaseous signalling molecule that participates in many developmental and physiological processes, including defense responses against toxic metals in plants. The role of NO in cadmium (Cd)-induced toxic effects on photosynthesis was examined in mustard (Brassica juncea L.) plants. Exposure of plants to 50 μM Cd significantly enhanced oxidative stress (H2O2 content and lipid peroxidation) and impaired plant growth and photosynthesis, chlorophyll fluorescence and reduced chlorophyll content and stomatal conductance. However, the exogenous application of 100 μM sodium nitroprusside (SNP, a donor of NO) reversed the effects of Cd through its stimulation of ROS-scavenging compounds (superoxide dismutase, ascorbate peroxidase, glutathione reductase and reduced glutathione). Exogenous SNP significantly increased plant growth, photosynthesis and chlorophyll content and diminished the accumulation of reactive oxygen species (H2O2) and TBARS content. It also reduced the effects of Cd on thylakoid membrane of the chloroplasts. Application of SNP together with 1.0 mM SO42- showed better responses than SNP alone. The protective effect of NO was achieved through enhanced production of reduced glutathione (GSH). GSH biosynthesis in plants treated with buthionine sulfoximine (BSO), a GSH biosynthetic inhibitor, was not completely inhibited in presence of NO and S, suggesting that NO stimulated S-assimilation and GSH production of Cd exposed plants. This study concludes that NO counteracts Cd toxicity in B. juncea strongly by regulating S-assimilation and GSH production.

Nanotoxicity of cobalt induced by oxidant generation and glutathione depletion in MCF-7 cells

There are very few studies regarding the biological activity of cobalt-based nanoparticles (NPs) and, therefore, the possible mechanism behind the biological response of cobalt NPs has not been fully explored. The present study was designed to explore the potential mechanisms of the cytotoxicity of cobalt NPs in human breast cancer (MCF-7) cells. The shape and size of cobalt NPs were characterized by scanning and transmission electron microscopy (SEM and TEM). The crystallinity of NPs was determined by X-ray diffraction (XRD). The dissolution of NPs was measured in phosphate-buffered saline (PBS) and culture media by atomic absorption spectroscopy (AAS). Cytotoxicity parameters, such as [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), neutral red uptake (NRU), and lactate dehydrogenase (LDH) release suggested that cobalt NPs were toxic to MCF-7 cells in a dose-dependent manner (50–200μg/ml). Cobalt NPs also significantly induced reactive oxygen species (ROS) generation, lipid peroxidation (LPO), mitochondrial outer membrane potential loss (MOMP), and activity of caspase-3 enzymes in MCF-7 cells. Moreover, cobalt NPs decreased intracellular antioxidant glutathione (GSH) molecules. The exogenous supply of antioxidant N-acetyl cysteine in cobalt NP-treated cells restored the cellular GSH level and prevented cytotoxicity that was also confirmed by microscopy. Similarly, the addition of buthionine-[S, R]-sulfoximine, which interferes with GSH biosynthesis, potentiated cobalt NP-mediated toxicity. Our data suggested that low solubility cobalt NPs could exert toxicity in MCF-7 cells mainly through cobalt NP dissolution to Co2+.

Non-linear impact of glutathione depletion on C. elegans life span and stress resistance

The redox environment in cells and organisms is set by low-molecular mass and protein-bound thiols, with glutathione (GSH) representing a major intracellular redox buffer. Subtle thiol oxidation elicits signal transduction processes and adaptive responses to cope with stressors, whereas highly oxidizing conditions may provoke cell death. We here tested how thiol depletion affects life span, stress resistance and stress signaling in the model organism Caenorhabditis elegans. Diethyl maleate (DEM), an α,β-unsaturated carbonyl compound that conjugates to GSH and other thiols, decreased C. elegans life span at a concentration of 1mM. In contrast, low and moderate doses of DEM (10–100µM) increased mean and maximum life span and improved resistance against oxidative stress. DEM-induced life span extension was not detectable in worms deficient in either the FoxO orthologue, DAF-16, or the Nrf2 orthologue, SKN-1, pointing to a collaborative role of the two transcription factors in life span extension induced by thiol depletion. Cytoprotective target genes of DAF-16 and SKN-1 were upregulated after at least 3 days of exposure to 100µM DEM, but not 1mM DEM, whereas only 1mM DEM caused upregulation of egl-1, a gene controlled by a p53-orthologue, CEP-1. In order to test whether depletion of GSH may elicit effects similar to DEM, we suppressed GSH biosynthesis in worms by attenuating γ-glutamylcysteine synthetase (gcs-1) expression through RNAi. The decline in GSH levels elicited by gcs-1 knockdown starting at young adult stage did not impair viability, but increased both stress resistance and life expectancy of the worms. In contrast, gcs-1 knockdown commencing right after hatching impaired nematode stress resistance and rendered young adult worms prone to vulval ruptures during egg-laying. Thus, modest decrease in GSH levels in young adult worms may promote stress resistance and life span, whereas depletion of GSH is detrimental to freshly hatched and developing worms.

Development of a Human Therapeutic L-Cyst(e)Ine-Degrading Enzyme for the Treatment of Hematological Malignancies

Cancer cells experience higher intrinsic oxidative stress than their normal counterparts and acquire adaptive antioxidant mechanisms to maintain redox balance. This increased antioxidant capacity has been correlated to malignant transformation, metastasis and resistance to standard anticancer drugs. This enhanced antioxidant state also correlates with cancer cells being more vulnerable to additional oxidative insults, therefore disruption of adaptive antioxidant mechanisms may have significant therapeutic implications. Hematological malignancies including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) are critically dependent on the cellular antioxidant glutathione (GSH), consistent with the higher intrinsic oxidative stress. L-cysteine is the rate-limiting substrate for GSH biosynthesis and adequate levels of cysteine are critical to maintain the intracellular homeostasis of GSH. CLL and a subset of ALL cells have been reported to rely on the stromal supply of cysteine to increase the synthesis of GSH in order to maintain redox balance, which in turn promotes cell survival and fosters drug resistance. One approach to target this cancer specific dependency is by therapeutic depletion of amino acids via enzyme administration; a clinically validated strategy for the treatment of ALL. Aeglea BioTherapeutics Inc. has developed a bioengineered cysteine and cystine degrading enzyme (Cyst(e)inase, AEB3103) and evaluated its therapeutic efficacy against hematological malignancies in in vitro, ex vivo and in vivo pre-clinical studies.The TCL1-TG:p53 -/- mouse model exhibits a drug resistant phenotype resembling human CLL with unfavorable cytogenetic alterations and highly aggressive disease progression. AEB3103 greatly decreased the viability of TCL1-TG:p53 -/- cells cultured in vitro, whereas the CLL therapeutic, fludarabine, showed minimal cytotoxic effects. In vivo treatment of TCL1-TG:p53 -/- mice with AEB3103 resulted in an increase in median survival time (7 months, p<0.0001) compared to the untreated control group (3.5 months, p<0.001) and a fludarabine treated group (5.3 months, p<0.001). These results indicate a superior therapeutic effect of AEB3103 compared to fludarabine. Additionally, evaluation of AEB3103 in in vitro 2D cultures of patient-derived CLL and MM cells, and in ex vivo 3D cultures of cells derived from ALL and AML PDx models resulted in significant cell growth inhibition with therapeutically relevant IC50 values. Collectively these results demonstrate the sensitivity of hematological malignancies to modulation of GSH levels via AEB3103-mediated cyst(e)ine depletion. DisclosuresAgnello:Aeglea BioTherapeutics: Employment. Alters:Aeglea BioTherapeutics: Employment, Equity Ownership. Tyler:Aeglea BioTherapeutics: Employment, Equity Ownership. Huang:Aeglea BioTherapeutics: Research Funding. Stone:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Research Funding; University of Texas at Austin: Employment, Patents & Royalties: I am an inventor of technology related to this abstract. Georgiou:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Lowe:Aeglea BioTherapeutics: Employment, Equity Ownership. Rowlinson:Aeglea BioTherapeutics: Employment, Equity Ownership.

The mechanism of improved intracellular organic selenium and glutathione contents in selenium-enriched Candida utilis by acid stress.

Batch culture of Candida utilis CCTCC M 209298 for the preparation of selenium (Se)-enriched yeast was carried out under different pH conditions, and maximal intracellular organic Se and glutathione (GSH) contents were obtained in a moderate acid stress environment (pH3.5). In order to elucidate the physiological mechanism of improved performance of Se-enriched yeast by acid stress, assays of the key enzymes involved in GSH biosynthesis and determinations of energy supply and regeneration were performed. The results indicated that moderate acid stress increased the activity of γ-glutamylcysteine synthetase and the ratios of NADH/NAD+ and ATP/ADP, although no significant changes in intracellular pH were observed. In addition, the molecular mechanism of moderate acid stress favoring the improvement of Se-yeast performance was revealed by comparing whole transcriptomes of yeast cells cultured at pH3.5 and 5.5. Comparative analysis of RNA-Seq data indicated that 882 genes were significantly up-regulated by moderate acid stress. Functional annotation of the up-regulated genes based on gene ontology and the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway showed that these genes are involved in ATP synthesis and sulfur metabolism, including the biosynthesis of methionine, cysteine, and GSH in yeast cells. Increased intracellular ATP supply and more amounts of sulfur-containing substances in turn contributed to Na2SeO3 assimilation and biotransformation, which ultimately improved the performance of the Se-enriched C. utilis.

236 - Hydrogen Sulfide Increases GSH Biosynthesis, Glucose Uptake and Utilization in Mouse Myotubes

Diabetes is a leading cause of chronic diseases, such as heart disease. Diabetic patients have lower blood concentrations of H 2 S and L-Cysteine (LC). LC undergoes enzymatic breakdown to produce hydrogen sulphide (H 2 S), a gasotransmitter that regulates glucose and lipid homeostasis. Using C2C12 mouse myoblast (differentiated into myotubes) as a model, this study investigates the hypothesis that beneficial effects of LC supplementation are mediated by the upregulation of H 2 S status in diabetic patients. Results show that exogenous administration of Sodium hydrosulfide (NaHS 10µM; 6 hours), a H 2 S donor, significantly ( p 2 S production induced by using CSE siRNA caused an increase in ROS, and down regulation of potent antioxidant GSH biosynthesis enzymes (GCLC, Glutathione Synthetase) but not GGLM. Further, CSE knockdown downregulate Glucose Transporter Type 4 (GLUT4) mRNA level and reduces glucose uptake in C2C12 myotubes. Taken together, these data suggest that the upregulation of physiological levels of H 2 S can have beneficial effects on glucose homeostasis and redox balance via GSH pathway. Further mechanistic studies are under investigation on how H 2 S regulate GSH biosynthesis and L-Cysteine transporter (SLC7A11) in the maintenance of glucose homeostasis. Supported by NIH/NCCIH RO1 AT007442

Humanin Protects RPE Cells from Endoplasmic Reticulum Stress-Induced Apoptosis by Upregulation of Mitochondrial Glutathione.

Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1–20 μg/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress.

Transcriptional reprogramming of genes related to ascorbate and glutathione biosynthesis, turnover and translocation in aphid-challenged maize seedlings

Aphids are pierce-sucking insects that severely affect redox homeostasis in the host tissues. Reduced ascorbate (AsA) and glutathione (GSH) serve as multifunctional molecules involved in constituting sophisticated plant defense responses to aphids' herbivory. This survey aimed to decipher transcriptional responses of forty-one genes associated with AsA and GSH biosynthesis, turnover and translocation in maize (Zea mays L.) seedlings infested with two hemipterans' species: bird cherry-oat aphid (Rhopalosiphum padi L.) and grain aphid (Sitobion avenae F). Cereal aphids' attack substantially stimulated the expression of fifteen maize genes – encoding l-galactose dehydrogenase (GalDH), l-galactono-1,4-lactone dehydrogenase (GLDH), ascorbate oxidase (AO), glutamate–cysteine ligase (GCL), glutathione synthetase (GS), two isozymes of glutathione peroxidase (GPX1, GPX3), five isoforms of glutathione transferase (GST9, GST11, GST16, GST31, GST38) and three GSH transporters (ABCC2, ABCC6, GT1). Transcriptional alternations of these genes were dependent on maize genotype, insect species, duration of infestation and aphids' abundance. Specific patterns of genes' expression as well as total activity of the corresponding enzymes in insect-challenged maize seedlings (cvs Ambrozja and Tasty Sweet, which are relatively resistant and susceptible, respectively) highlighted the molecular background of crucial part of the complex antioxidative responses of maize model plant toward cereal aphids' colonization.

Thymol Mitigates Cadmium Stress by Regulating Glutathione Levels and Reactive Oxygen Species Homeostasis in Tobacco Seedlings.

Thymol is a famous plant-derived compound that has been widely used in pharmacy due to its antioxidant and antimicrobial properties. However, the modulation of intrinsic plant physiology by thymol remains unclear. It is a significant challenge to confer plant tolerance to Cd (cadmium) stress. In the present study physiological, histochemical, and biochemical methods were applied to investigate thymol-induced Cd tolerance in tobacco (Nicotiana tabacum) seedlings. Thymol was able to alleviate Cd-induced growth inhibition of tobacco seedlings in both dose- and time-dependent manners. Both histochemical detection and in-tube assays suggested that thymol treatment blocked Cd-induced over-generation of reactive oxygen species (ROS), lipid peroxidation, and loss of membrane integrity in both leaves and roots. Thymol decreased Cd-induced cell death that was indicated in vivo by propidium iodide (PI) and trypan blue, respectively. Thymol stimulated glutathione (GSH) biosynthesis by upregulating the expression of γ-glutamylcysteine synthetase 1 (GSH1) in Cd-treated seedlings, which may contribute to the alleviation of Cd-induced oxidative injury. In situ fluorescent detection of intracellular Cd2+ revealed that thymol significantly decreased free Cd2+ in roots, which could be explained by the thymol-stimulated GSH biosynthesis and upregulation of the expression of phyochelatin synthase 1 (PCS1). Taken together, these results suggested that thymol has great potential to trigger plant resistant responses to combat heavy metal toxicity, which may help our understanding of the mechanism for thymol-modulated cell metabolic pathways in response to environmental stimuli.

Cisplatin-induced regulation of signal transduction pathways and transcription factors in p53-mutated subclone variants of hepatoma cells: Potential application for therapeutic targeting.

Cisplatin is commonly recognized as a DNA-damaging drug; however, its versatile antitumor effects have been demonstrated to extend beyond this narrow functional attribute. The present study determined how cisplatin regulates alternative pathways and transcription factors to exert its additional antitumor actions. Cisplatin was observed to be able to trigger an endoplasmic reticulum stress response through aggravated nitrosative stress coupled to perturbed mitochondrial calcium (Ca2+) homeostasis, which substantially downregulated glucose-regulated protein (GRP) 78 expression by suppressing the cleavage of activating transcription factor (ATF) 6α (90 kDa) to its active 50 kDa subunit. Concomitantly, the ATF4-ATF3-C/emopamil binding protein homologous protein axis was activated by cisplatin, which triggered cellular glutathione (GSH) depletion by strongly inhibiting γ-glutamylcysteine synthetase heavy chain (γ-GCSh), a key enzyme in GSH biosynthesis. The present study also demonstrated that cisplatin substantially inhibited β-catenin, causing a marked downregulation of survivin and B-cell lymphoma (Bcl)-2. Taken together, the present results uncovered a novel mechanism of cisplatin that could simultaneously trigger the inhibition of three prominent antiapoptotic effector molecules (Bcl-2, survivin and GRP78) and effectively promote GSH depletion by inhibiting γ-GCSh. These newly discovered functional attributes of cisplatin can provide an avenue for novel combined therapeutic strategies to kill hepatocellular carcinoma cells effectively.

Glutathione-mediated antioxidant response and aerobic metabolism: two crucial factors involved in determining the multi-drug resistance of high-risk neuroblastoma.

Neuroblastoma, a paediatric malignant tumor, is initially sensitive to etoposide, a drug to which many patients develop chemoresistance. In order to investigate the molecular mechanisms responsible for etoposide chemoresistance, HTLA-230, a human MYCN-amplified neuroblastoma cell line, was chronically treated with etoposide at a concentration that in vitro mimics the clinically-used dose. The selected cells (HTLA-Chr) acquire multi-drug resistance (MDR), becoming less sensitive than parental cells to high doses of etoposide or doxorubicin. MDR is due to several mechanisms that together contribute to maintaining non-toxic levels of H2O2. In fact, HTLA-Chr cells, while having an efficient aerobic metabolism, are also characterized by an up-regulation of catalase activity and higher levels of reduced glutathione (GSH), a thiol antioxidant compound. The combination of such mechanisms contributes to prevent membrane lipoperoxidation and cell death. Treatment of HTLA-Chr cells with L-Buthionine-sulfoximine, an inhibitor of GSH biosynthesis, markedly reduces their tumorigenic potential that is instead enhanced by the exposure to N-Acetylcysteine, able to promote GSH synthesis.Collectively, these results demonstrate that GSH and GSH-related responses play a crucial role in the acquisition of MDR and suggest that GSH level monitoring is an efficient strategy to early identify the onset of drug resistance and to control the patient's response to therapy.

Nrf2-dysregulation correlates with reduced synthesis and low glutathione levels in experimental autoimmune encephalomyelitis.

This study investigates the possible mechanism(s) underlying glutathione (GSH) deficiency in the mouse spinal cord during the course of myelin oligodendrocyte glycoprotein35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis. Using the classical enzymatic recycling method and a newly developed immunodot assay, we first demonstrated that total GSH levels (i.e. free GSH plus all its adducts) are reduced in EAE, suggesting an impaired synthesis. The decline in the levels of this essential antioxidant tripeptide in EAE coincides temporally and in magnitude with a reduction in the amount of γ-glutamylcysteine ligase, the rate-limiting enzyme in GSH synthesis. Other enzymes involved in GSH biosynthesis, whose genes also contain antioxidant-response elements, including glutathione synthetase, cystine/glutamate antiporter, and γ-glutamyl transpeptidase (γ-GT) are diminished in EAE as well. Low levels of γ-glutamylcysteine ligase, glutathione synthetase, and γ-GT are the consequence of reduced mRNA expression, which correlates with diminished expression of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in both the cytosol and nucleus. Interestingly, the low Nrf2 expression does not seem to be caused by increased degradation via Kelch-like ECH-associated protein 1-dependent or Kelch-like ECH-associated protein 1-independent mechanisms (such as glycogen synthetase kinase-3β activation), or by reduced levels of Nrf2 mRNA. This suggests that translation of this important transcription factor and/or other still unidentified post-translational processes are altered in EAE. These novel findings are central toward understanding how critical antioxidant and protective responses are lost in inflammatory demyelinating disorders.

106 Glutathione (GSH) distribution by quantitative MALDI imaging in reconstructed human skin upon activation of GSH biosynthesis by Nrf2 pathway activator

106 Glutathione (GSH) distribution by quantitative MALDI imaging in reconstructed human skin upon activation of GSH biosynthesis by Nrf2 pathway activator