Enhancement of gamma-glutamylcysteine synthetase mRNA in rat kidney by methyl mercury.

Glutathione (GSH), a major cellular antioxidant, is elevated 2- to 3-fold in kidneys of rats during prolonged treatment with mercury as methyl mercury hydroxide (MMH). Increased renal GSH is accompanied by a dose- and time-related elevation in the relative abundance of mRNA hybridizable to a cDNA probe which encodes renal gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. Renal GCS mRNA is maximally elevated 4.4-fold at 3 weeks following initiation of MMH treatment. Enhancement of GSH and GCS mRNA content corresponds to a relative sparing of renal cells from oxidative tissue damage during MMH exposure. These observations suggest that increased synthesis of GSH at the genetic level occurs as an initial adaptive response to mercury-induced oxidative stress in kidney cells.     

Augmented biosynthesis of cadmium sulfide nanoparticles by genetically engineered Escherichia coli

Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: γ‐glutamylcysteine synthetase (γ‐GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over‐express either γ‐GCS or GS and found that γ‐GCS over‐expression resulted in inclusion body formation and impaired cell physiology, whereas GS over‐expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over‐expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild‐type cells in size (2–5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was ≈2.5 times that attained in the wild‐type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli‐based biosynthesis offers a more energy‐efficient and eco‐friendly method as opposed to chemical processes requiring high temperature and toxic solvents. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Altered metabolism of glutathione in cells transformed by oncogenes which cause resistance to ionizing radiations

We measured glutathione (GSH) metabolism in normal NIH/3T3 fibroblasts, and in cells transformed by the oncogenes sis, erbB, src, ras, dbl, and raf.erbB,src,ras and raf, but not sis and dbltransformants, showed increased level of total and reduced GSH as compared with normal NIH/3T3 fibroblasts; oxidized GSH was elevated only in src- and ras-transformed cells. Increased total GSH content was associated with decreased activity of the synthetic enzyme γ-glutamylcysteine synthetase, and oxidized GSH level with increased activity of GSH reductase. These data suggest that GSH synthesis was selectively enhanced in cells transformed by specific oncogenes, with resulting down-regulation of its synthetic enzyme; alterations of GSH metabolism appeared to be peculiar of transformation by specific oncogenes, and not trivial epiphenomena of neoplastic transformation. Oncogenic transformants that presented elevated level of GSH were also those reported to be resistant to antineoplastic drugs and ionizing radiations, thus confirming a possible link between altered GSH metabolism and resistance to antineoplastic treatment.     

Modulation of the transport of a lysosomal enzyme by PDGF

The major excreted protein (MEP) of transformed mouse fibroblasts is the lysosomal protease, cathepsin L. MEP is also secreted by untransformed mouse cells in response to growth factors and tumor promoters, and is thought to play a role in cell growth and transformation. To determine the relationship between MEP synthesis and MEP secretion, we have examined these events in PDGF-treated NIH 3T3 cells. PDGF enhanced MEP synthesis and caused the diversion of MEP from the lysosomal delivery pathway to a secretory pathway. These two effects were found to be regulated independently at various times after growth factor addition. Short PDGF treatments (0.5 or 1 h) resulted in quantitative secretion of MEP although synthesis was near the control level. High levels of both synthesis and secretion occurred between 2 and 14 h of PDGF treatment. Between 18 and 30 h, the amount of secreted MEP returned to the low control level even though synthesis remained elevated. The secretion was specific for MEP; other lysosomal enzymes were not found in the media from PDGF-treated cells. PDGF-induced secretion of MEP was inhibited 84% by cycloheximide, suggesting that protein synthesis is required to elicit this effect. PDGF also caused a time-dependent increase in mannose 6-phosphate (Man-6-P) receptor-mediated endocytosis. These data support a model in which PDGF alters the distribution of Man-6-P receptors such that the Golgi concentration of receptors becomes limiting, thereby causing the selective secretion of the low affinity ligand, MEP.     

Role of Nerve Growth Factor in Oxidanl Homeostasis: Glutathione Metabolism

Abstract— Free radicals are generated in the CNS by ongoing oxygen metabolism and biological events associated with injury and inflammation. Increased free radical levels may also persist in some chronic neurological diseases and in the aged. Nerve growth factor (NGF) is a member of the neurotrophin family of proteins that can regulate neuronal development, maintenance, and recovery from injury. NGF protected rat pheochromocytoma PC12 cells, an adrenal chromaffin-like NGF-responsive cell line, from the oxidant stress accompanying hydrogen peroxide treatment by stimulating GSH levels and enzymes in the GSH metabolism cycle and in the GSH/GSH peroxidase antioxidant redox system, a ubiquitous cellular antioxidant system. Specifically, NGF increased γ-glutamylcysteine synthetase (GCS) activity, the rate-limiting enzyme for GSH synthesis, by 50% after 9h and GSH levels by 100% after 24 h of treatment. NGF stimulated GSH peroxidase by 30% after 3 days and glucose 6-phosphate dehydroge-nase by 50% after 2 days. Treatment with NGF and cyclo-heximide, or actinomycin D, which inhibit protein and RNA synthesis, respectively, blocked the NGF stimulation of GCS and glucose 6-phosphate dehydrogenase. Increased GSH levels due to NGF treatment were responsible for the significant protection of PC12 cells from hydrogen peroxide-induced stress. Pretreatment of PC12 cells with NGF for 24 h rescued cells from the toxic effects of the extracellular hydrogen peroxide generated by the glucose/glucose oxidase system but did not rescue cells that were subjected to GSH deprivation due to treatment with 10 μMl -buthionine-(S,R)-sulfoximine, an inhibitor of GCS. However, treatment with 10 μMl -buthionine-(S,R)-sulfoximine alone did not affect PC12 cell viability, NGF stimulation of neurite extension, and NGF induction of GCS, GSH peroxidase, and glucose 6-phosphate dehydrogenase activity. When GSH levels were measured in PC12 cells that were treated for 24 h with other neurotrophins and growth factors, such as brain-derived neurotrophic factor, neurotro-phin-3, epidermal growth factor, insulin-like growth factor-I, and basic fibroblast growth factor, only epidermal growth factor was found to increase GSH levels by 30%. Whereas NGF increased GSH levels in the human neuro-blastoma SK-N-SH-SY5Y and the human melanoma A-875 in serum-free medium, addition of fetal calf serum to the medium abolished the NGF effects on GSH levels in the NGF-responsive cell lines, SK-N-SH-SY5Y, A-875, and the CNS C6 rat glioma subclone 2BD.     

Inducers of gamma-glutamylcysteine synthetase and their effects on glutathione synthetase expression

Synthesis of GSH occurs via two enzymatic steps, the first is catalyzed by gamma-glutamylcysteine synthetase (GCS) and the second is catalyzed by GSH synthetase (GS). A heavy (HS) and light subunit (LS) make up GCS; regulation of both subunits have been well characterized, whereas regulation of GS is largely unknown. In this study, we examined the effects of treatments known to influence the gene expression of GCS subunits on GS expression. Insulin and hydrocortisone treatment of rat hepatocytes or ethanol-feeding of rats for 9 weeks, which increased the expression of GCS-HS only, had no influence on GS expression. However, two-thirds partial hepatectomy in rats which increased the expression of GCS-HS only, also increased GS expression. Treatment of hepatocytes or rats with diethyl maleate, buthionine sulfoximine, tert-butylhydroquinone, or thioacetamide, which increased the expression of both GCS subunits, increased the expression of GS. The GSH synthesis capacity increased 50-100% by treatments that increased only the GCS-HS expression, whereas it increased 161-200% by treatments that increased both GCS-HS and GS expression. Thioacetamide treatment of Chang cells increased cell GSH and GS expression by 50%, but had minimal influence on GCS subunits. Thus, GS induction can further increase the cell's GSH synthetic capacity and in some cells may be as important as GCS in determining the rate of GSH synthesis.     

Increased glutathione synthesis associated with platelet-derived growth factor stimulation of NIH3T3 fibroblasts

Previous data show a relation between GSH content and proliferation of normal and tumour cells. We recently demonstrated a specific involvement of GSH in the autophosphorylation activity of the platelet-derived growth factor (PDGF) receptor in NIH3T3 fibroblasts. In this study we demonstrate that the stimulation by PDGF of serum-starved NIH3T3 cells increases cellular GSH content, while no change in oxidized GSH content was measured. Experiments performed with actinomycin, cycloheximide and buthionine sulfoximide, a specific inhibitor of the rate-limiting enzyme of the de novo synthesis of GSH gamma-glutamylcysteine synthetase (gamma-GCS), confirm PDGF induction of GSH synthesis. These results provide the first demonstration that PDGF mediated transduction signals seem strictly related to mechanisms involved in the increase of gamma-GCS activity associated with increased gamma-GCS heavy subunit mRNA levels. In fact, serum and epidermal growth factor (EGF) stimulation of quiescent NIH3T3 and NIH3T3, which overexpress EGF receptor, does not affect GSH content or its synthesis. These data may be related to a possible GSH role in the redox regulation of cell proliferation mediated by PDGF.     

Enhanced glutathione production by using low-pH stress coupled with cysteine addition in the treatment of high cell density culture of Candida utilis

Aims: To investigate the effects of pH stress coupled with cysteine addition on glutathione (GSH) production in the treatment of high cell density culture of Candida utilis. Methods and Results: We have previously observed that most Candida utilis cells remained viable after being subjected to pH at 1·2 for 3 h and that some intracellular GSH leaked into the medium. A cysteine addition strategy was applied in fed‐batch production of GSH. A single cysteine addition resulted in higher GSH yield than two separate additions without pH stress. An increase in intracellular GSH content triggered inhibition of γ‐glutamylcysteine synthetase (γ‐GCS). A strategy that combines cysteine addition with low‐pH stress was developed to relieve the inhibition of γ‐GCS. Conclusion: Without pH stress, single shot and double shot cysteine addition yielded a total GSH of 1423 and 1325 mg l^?1. In comparison, a low‐pH stress counterpart resulted in a total GSH of 1542 and 1730 mg l^?1, respectively. With low‐pH stress, we observed GSH secretion into the medium at 673 and 558 mg l^?1 and an increase in the γ‐GCS activity by 1·2‐ and 1·5‐fold, respectively. The specific GSH production yield increased from 1·76% to 1·91% (w/w) for single shot, and 1·64% to 2·14% for double shots. Significance and Impact of the Study: Low‐pH shift was applied to alleviate the feedback inhibition of intracellular GSH on γ‐GCS activity by secreting GSH into the medium. This strategy is coupled with cysteine addition to enhance GSH production in Candida utilis.     

GSH system in relation to redox state in dystrophic skin fibroblasts

Glutathione and GSH-related enzymes were determined in human Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) skin fibroblasts in order to relate muscular dystrophy to the redox state of the cell. The analysis of GSH, GSSG and total GSH levels in normal and dystrophic-cultured fibroblasts shows no differences in normal growth condition. However, the specific activity of two GSH-related enzymes, glutathione S-transferases (GST) and gamma-glutamylcysteine synthetase (gamma-GCS), shows significant variations between normal and both types of dystrophic skin fibroblasts. These results suggest that even in normal growth condition some components of GSH metabolism may be altered. A condition of sublethal oxidation obtained by H(2)O(2) treatment was able to show a difference in the cellular response of GSH system components between normal and dystrophic cells. While in DMD cells there is a decrease of roughly 55% in GSH and of 30% in total GSH concentration, no changes are measured in normal and BMD cells. The remarkable increase in glutathione peroxidase (GPx) activity and decrease in GSH-reductase (GR) activity measured in DMD cells can in part explain these changes. These results indicate a different capacity of DMD cells to support oxidative stress with respect to BMD and normal cells, and suggest a possible role of the GSH-antioxidant system in dystrophic pathology.     

Role and regulation of the fibroblast growth factor axis in human thyroid follicular cells

Thyroidal levels of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) are elevated in human thyroid hyperplasia. To understand the significance of this, effects of FGFR1 activation on normal human thyrocyte growth and function in vitro and the regulation of FGF-2 and FGFR1 expression have been examined. FGF-2 stimulated cell growth, as measured by cell counting, and inhibited thyroid function as measured by 125I uptake. Sensitivity to FGF-2 disappeared after 7 days, although FGFR1 expression was maintained. Thyroid-stimulating hormone (TSH, 300 mU/l) increased FGFR1 mRNA expression within 4 h and protein expression by 8 h. Exogenous FGF-2 decreased FGFR1 protein. Endogenous FGF-2 levels were low (approximately 1-2 pg/microg protein), and TSH treatment decreased these by 50%. Protein kinase C (PKC) activation increased FGF-2 mRNA and FGF-2 secretion within 2 h. This effect was enhanced (4.4-fold) when cells were cultured in TSH. We conclude that TSH stimulates FGFR1 but not FGF-2 expression. PKC activation stimulates FGF-2 synthesis and secretion, and TSH synergizes with PKC activators. Increases in FGFR1 or FGF-2 or in both may contribute to goitrogenesis.     

Cysteine control over glutathione homeostasis in Chinese hamster fibroblasts overexpressing a gamma-glutamylcysteine synthetase activity.

Gamma-glutamylcysteine synthetase (GCS) catalyses the first step of glutathione (GSH) biosynthesis and is considered to be the rate-limiting step of this pathway. In several experimental systems, GCS overexpression has been associated with GSH pool expansion and drug resistance. In this report, we describe a mutant line of Chinese hamster fibroblasts that overexpress this activity by 4-5 times, due to the amplification of the gene encoding the catalytic subunit of GCS. These mutant cells contained a wild-type steady-state level of GSH and, after depletion, synthesized GSH at the same rate as wild-type cells because their rate of endogenous production of cysteine was limiting. An exogenous supply of cysteine expanded the pool of GSH in mutant cells by 80% but did not increase that of wild-type cells, and, in GSH-depleted cells, increased the rate of GSH biosynthesis by eight and 35-times in wild-type and mutant cells, respectively. These experiments indicated that GCS overexpression had no consequence on the metabolism of GSH, unless a supply of cysteine was provided. Mutant cells were not resistant to cisplatin or nitrogen mustard.     

Contrasting Antioxidant and Cytotoxic Effects of Peroxiredoxin I and II in PC12 and NIH3T3 Cells

We examined the impact of peroxiredoxin-I (Prx-I) and peroxiredoxin-II (Prx-II) stable transduction on oxidative stress in PC12 neurons and NIH3T3 fibroblasts and found variability depending on cell type and Prx subtype. In PC12 neurons, Prx-II suppressed reactive oxygen species (ROS) generation by 36% (p < 0.01) relative to vector-infected control cells. However, in NIH3T3 fibroblasts, Prx-II overexpression resulted in a 97% (p < 0.01) increase in ROS generation. Prx-I transduction elevated ROS generation in PC12 cells. The effect of Prx-I on PC12 cells was potentiated in the presence of menadione, and suppressed by an inhibitor of nitric oxide synthetase. Prx-II transduction resulted in 25–35% lower levels of glutathione (GSH) in both cell types, while Prx-I transduction increased GSH levels in neurons and decreased GSH and caspase-3 activity in fibroblasts. Prx-I and Prx-II also had differing effects on cell viability. These results suggest that Prx-I and Prx-II can either increase or decrease intracellular oxidative stress depending on cell type or experimental conditions, particularly conditions affecting nitric oxide levels.Equivalent contributions were made by each author     

Permeable FGF-1 Nuclear Localization Signal Peptide Stimulates DNA Synthesis in Various Cell Types but Is Cell-Density Sensitive and Unable to Support Cell Proliferation

An earlier report indicated that a 26-amino-acid peptide (SA), comprised of the nuclear localization signal (NLS) of fibroblast growth factor-1 (FGF-1) and a membrane-permeable peptide, was able to stimulate DNA synthesis after it was taken up by NIH3T3 fibroblasts. Here, we report that SA, but not a mutant with the NLS motif destroyed, induced DNA synthesis in BALB/c3T3 murine fibroblasts, human vascular endothelial (HUVE) cells, and primary cultured hepatocytes, although the activity was weaker than that of FGF-1. The kinetics of SA-induced DNA synthesis and G₁cyclin expression were similar to those elicited by FGF-1, indicating that SA induces cell cycle progression. Kinetic analysis also suggested that SA stimulates only a fraction of the DNA replication in BALB/c3T3 cells. At high cell densities, SA-induced G₁cyclin expression and DNA synthesis were more strongly inhibited than those induced by FGF-1. SA did not induce cell division in HUVE and BALB/c3T3 cells and did not interfere with FGF-1-stimulated proliferation of HUVE cells. These results indicate that SA is able to partially induce cell cycle progression through a contact-inhibition sensitive signaling pathway, but it is insufficient to support cell mitosis. We also suggest that signaling by SA does not interfere with that of FGF-1.     

Control of glutathione and phytochelatin synthesis under cadmium stress. Pathway modeling for plants

Glutathione (GSH) plays several roles in cell metabolism such as redox state regulation, oxidative stress control, and protection against xenobiotics and heavy metals. GSH is synthesized in two steps catalysed by gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase. gamma-ECS is feedback inhibited by GSH, which has led to the proposal that this enzyme acts as the rate-limiting step in the pathway. Thus far, the study of GSH metabolism has been confined to GSH synthesis (GSH supply), without considering the GSH-consuming enzymes (GSH demand). Several works have shown that the demand block of enzymes may have a significant control on a pathway; therefore, we hypothesize that GSH-consuming enzymes may exert some control on GSH synthesis. A kinetic model of GSH and phytochelatin synthesis in plants was constructed using the software GEPASI and the kinetic data available in the literature. The main conclusions drawn by the model concerning metabolic control analysis are (1) gamma-ECS is indeed a rate-limiting step in GSH synthesis, but only if GSH-consuming enzymes are not taken into account. (2) At low demand, GSH-consuming enzymes exert significant flux-control on GSH synthesis whereas at high demand, supply and demand blocks share the control of flux. (3) In unstressed conditions, flux to GSH is controlled mainly by demand, so that gamma-ECS determines the degree of homeostasis of the GSH concentration. Under cadmium exposure, the GSH demand increases and flux-control is re-distributed almost equally between the supply and demand blocks. (4) To enhance phytochelatins synthesis without depleting the GSH pool, at least two enzymes (gamma-ECS and PCS) should be increased and/or, alternatively, a branching flux (GSH-S-transferases) could be partially diminished.     

Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG.