Expression Of Glutathione Synthetase Research Articles

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Foliar application of NaHS alleviates Cd toxicity in soybean plants through regulation of Glutathione metabolism

Hydrogen sulfide (H2S) regulates many critical processes of plants and their responses to abiotic stresses such as heavy metals. In the present study, the effect of sodium hydrogen sulfide (NaHS) as an H2S donor (0, and 50 μM) was investigated in soybean plants under Cd stress (0, and 50 μM). These experiments were done according to a factorial design based on a completely randomized design with three replications. Cd exposure reduced growth parameters and relative water content. Cd also caused a reduction in photosynthetic pigment, an increase in ion leakage and oxidative stress as indicated by lipid peroxidation and H2O2 production. These results demonstrated the toxicity of the applied Cd concentrations on soybean plants. However, foliar application of NaHS improved the shoot length and fresh weight on Cd-stressed plants. NaHS also alleviated the toxic effects of Cd on ion leakage, relative water content, and photosynthetic pigments of leaves. The activity of glutathione reductase and the expression of genes to the family of glutathione synthetase including glutathione synthetase, chloroplastic-like (LOC102663288) (GS288), glutathione synthetase, chloroplastic (LOC100778473) (GS473) and homoglutathione synthetase (Gene ID: 547,989) (HGS), were observed to be upregulated under the influence of NaHS treatment. However, the expression of glutathione synthetase, and chloroplastic-like glutathione synthetase (LOC100813419) (GS419)) were reduced by NaHS in Cd-stressed plants. These results confirm the regulatory effect of NaHS application on physiological parameters and glutathione metabolism at molecular levels on soybean plants.

Gss deficiency causes age-related fertility impairment via ROS-triggered ferroptosis in the testes of mice

Glutathione synthetase (GSS) catalyzes the final step in the synthesis of glutathione (GSH), a well-established antioxidant. Research on the specific roles of the Gss gene during spermatogenesis remains limited due to the intricate structure of testis. In this study, we identified pachytene spermatocytes as the primary site of GSS expression and generated a mouse model with postnatal deletion of Gss using Stra8-Cre (S8) to investigate the role of GSS in germ cells. The impact of Gss knockout on reducing male fertility is age-dependent and caused by ferroptosis in the testis. The 2-month-old S8/Gss−/− male mice exhibited normal fertility, due to a compensatory increase in GPX4, which prevented the accumulation of ROS. With aging, there was a decline in GPX4 and an increase in ALOX15 levels observed in 8-month-old S8/Gss−/− mice, resulting in the accumulation of ROS, lipid peroxidation, and ultimately testicular ferroptosis. We found that testicular ferroptosis did not affect spermatogonia, but caused meiosis disruption and acrosome heterotopia. Then the resulting aberrant sperm showed lower concentration and abnormal morphology, leading to reduced fertility. Furthermore, these injuries could be functionally rescued by inhibiting ferroptosis through intraperitoneal injection of GSH or Fer-1. In summary, Gss in germ cells play a crucial role in the resistance to oxidative stress injury in aged mice. Our findings deepen the understanding of ferroptosis during spermatogenesis and suggest that inhibiting ferroptosis may be a potential strategy for the treatment of male infertility.

Myocardial Glutathione Synthase and TRXIP Expression Are Significantly Elevated in Hypertension and Diabetes: Influence of Stress on Antioxidant Pathways.

Antioxidant protection is one of the key reactions of cardiomyocytes (CMCs) in response to myocardial damage of various origins. The thioredoxin interacting protein (TXNIP) is an inhibitor of thioredoxin (TXN). Over the recent few years, TXNIP has received significant attention due to its wide range of functions in energy metabolism. In the present work, we studied the features of the redox-thiol systems, in particular, the amount of TXNIP and glutathione synthetase (GS) as markers of oxidative damage to CMCs and antioxidant protection, respectively. This study was carried out on 38-week-old Wistar-Kyoto rats with insulin-dependent diabetes mellitus (DM) induced by streptozotocin, on 38- and 57-week-old hypertensive SHR rats and on a model of combined hypertension and DM (38-week-old SHR rats with DM). It was found that the amount of TXNIP increased in 57-week-old SHR rats, in diabetic rats and in SHR rats with DM. In 38-week-old SHR rats, the expression of TXNIP significantly decreased. The expression of GS was significantly higher compared with the controls in 57-week-old SHR rats, in DM rats and in the case of the combination of hypertension and DM. The obtained data show that myocardial damage caused by DM and hypertension are accompanied by the activation of oxidative stress and antioxidant protection.

Uric acid effects on glutathione metabolism estimated by induction of glutamate-cysteine ligase, glutathione reductase and glutathione synthetase in mouse J744A.1 macrophage cell line.

Elevated plasma levels of uric acid (UA) are considered an independent risk factor for hypertension, diabetes, cardiovascular disease, endothelial and vascular damage, obesity, and metabolic syndrome. Even physiological concentrations of soluble UA have been proved to induce gene expression of macrophage-secreted inflammatory cytokines and stimulate production of reactive oxygen species in mature adipocytes. UA is also described as a powerful endogenous plasma antioxidant, which reveals a paradox of duality for this parameter.

Mitochondria-Related Genes In Myoblasts Are Influenced By Age And Exercise Mimetic Stimulation

Skeletal muscle (SKM) health is impacted by the aging process and results in delayed regeneration and mitochondrial dysfunction. PURPOSE: To investigate the molecular effects of an in vitro SKM myoblast aging protocol in combination with a β-2 adrenergic receptor agonist treatment on the expression of genes related to mitochondrial health. METHODS: Human SKM myoblasts (n = 6 per group) were cultured (3 x 105 seeding density) and proliferated to 85% confluency upon extraction. Groups included control cells (CON), aged cells (AGED), CON plus formoterol stimulation (CON + FORM; 30 nM for 3 h), and AGED plus FORM stimulation (AGED + FORM; 30 nM for 3 h). AGED myoblasts were passaged 18 times to exhibit reduced proliferative properties experienced in SKM of older adults, whereas CON myoblasts were analyzed at passage 5. qPCR analyses included the following genes: Nuclear Respiratory Factor (NRF1), Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1 Alpha/Beta (PGC-1α/β), Superoxide Dismutase 2 (SOD2), Mitochondrial Transcription Factor A (TFAM), and Glutathione Synthetase (GSS). Data were analyzed via one-way ANOVA followed by Games-Howell post-hoc analyses with a Welch correction. Data are expressed as mean ± SEM. RESULTS: PGC1-β: CON + FORM (3.03 ± 0.43) was greater than CON (1.00 ± 0.14, p = 0.02); AGED + FORM (4.35 ± 0.51) was greater than CON (1.00 ± 0.14, p < 0.01) and AGED (1.49 ± 0.20, p = 0.01). TFAM: CON + FORM (2.92 ± 0.38) was greater than CON (1.00 ± 0.16, p = 0.01). NRF1, PGC1-α, SOD2, and GSS: No significance between groups. All reported differences are significant. CONCLUSION: Although the AGED and AGED + FORM groups were not significant, likely due to high inter-group variance, there is evidence of genetic dysregulation. Increases in SOD2 and GSS expression in the AGED group indicate increased oxidative stress, which potentially decreased in response to FORM stimulation. The potential presence of increased oxidative stress may influence the increase in mitochondrial biogenesis-related gene expression to combat this oxidative stress and reduce mitochondrial dysfunction. FORM appears to stabilize mitochondrial biogenesis-related genes in AGED myoblasts, indicating improved overall mitochondrial health. Supported by NIH T32 Grant HL007457

Effects of free and dipeptide forms of methionine supplementation on oxidative metabolism of broilers under high temperature

Our previous studies have shown that methionine supplementation could help to attenuate the effects of heat stress on the metabolism of broiler chickens. Here we investigated for the first time the effects of methionine supplementation in the form of DL-methionyl-DL-methionine on broilers subjected to heat stress during the growth phase. Broilers were divided into two groups; one group was reared under thermoneutral conditions and the other under continuous heat stress (30 ± 1 °C, 60% relative humidity). Both groups were subdivided into three dietary treatments: a methionine-deficient (MD) diet, a diet supplemented with free methionine (DL-M), and a diet supplemented with methionine dipeptide (DL-MM). Broilers raised under chronic heat stress had lower feed intake and weight gain than broilers raised under thermoneutral conditions (P < 0.05). There were no differences in animal performance between methionine-supplemented diets (DL-M and DL-MM). Heat-stressed birds had significantly higher heterophil/lymphocyte (H/L) ratio than thermoneutral birds. Under heat stress, broilers fed DL-M and DL-MM diets had lower H/L ratio than birds fed the MD diet. Higher concentrations of carbonylated proteins and lower concentration of reduced glutathione were observed in broilers raised under heat stress. In comparing heat-stressed broilers, we found that birds fed the DL-M diet had lower concentrations of thiobarbituric acid-reactive substances and carbonylated proteins than those fed the MD diet (P < 0.05). Higher expression of glutathione peroxidase (GPX) and glutathione synthetase (GSS) genes was observed in heat-stressed broilers (P < 0.05). Under heat stress, the MD diet increased GPX expression compared with other diets. Under thermoneutral conditions, the DL-M diet resulted in the highest GSS expression. There was a negative correlation between DNA methylation and GPX and GSS expression. Our results showed that supplementation of broiler diets with free methionine or methionine dipeptide may help attenuate the effects of heat stress through enhanced activation of genes related to the glutathione antioxidant system. Methionine effects were found for gene regulation, gene expression, and post-translational processing.

The Eruca sativa Genome and Transcriptome: A Targeted Analysis of Sulfur Metabolism and Glucosinolate Biosynthesis Pre and Postharvest.

Rocket (Eruca sativa) is a source of health-related metabolites called glucosinolates (GSLs) and isothiocyanates (ITCs) but little is known of the genetic and transcriptomic mechanisms responsible for regulating pre and postharvest accumulations. We present the first de novo reference genome assembly and annotation, with ontogenic and postharvest transcriptome data relating to sulfur assimilation, transport, and utilization. Diverse gene expression patterns related to sulfur metabolism, GSL biosynthesis, and glutathione biosynthesis are present between inbred lines of rocket. A clear pattern of differential expression determines GSL abundance and the formation of hydrolysis products. One breeding line sustained GSL accumulation and hydrolysis product formation throughout storage. Multiple copies of MYB28, SLIM1, SDI1, and ESM1 have increased and differential expression postharvest, and are associated with GSLs and hydrolysis product formation. Two glucosinolate transporter gene (GTR2) copies were found to be associated with increased GSL accumulations in leaves. Monosaccharides (which are essential for primary metabolism and GSL biosynthesis, and contribute to the taste of rocket) were also quantified in leaves, with glucose concentrations significantly correlated with the expression of numerous GSL-related genes. Significant negative correlations were observed between the expression of glutathione synthetase (GSH) genes and those involved in GSL metabolism. Breeding line “B” showed increased GSH gene expression and low GSL content compared to two other lines where the opposite was observed. Co-expression analysis revealed senescence (SEN1) and oxidative stress-related (OXS3) genes have higher expression in line B, suggesting that postharvest deterioration is associated with low GSL concentrations.

Effect of Sterigmatocystin or Aflatoxin Contaminated Feed on Lipid Peroxidation and Glutathione Redox System and Expression of Glutathione Redox System Regulatory Genes in Broiler Chicken.

Authors studied the effect of sterigmatocystin from infected corn (STC), purified sterigmatocystin (PSTC), and aflatoxin B1 from infected corn (AFB1) on lipid peroxidation and glutathione redox parameters, including the expression of their encoding genes in a sub-chronic (14 days) trial. A total of 144 three-week-old cockerels was divided into four experimental groups (n = 36 in each). Control feed was contaminated with STC or PSTC (1590 µg STC/kg or 1570.5 µg STC/kg feed), or with AFB1 (149.1 µg AFB1/kg feed). Six birds from each group were sampled at day 1, 2, 3, 7 and 14 of mycotoxin exposure. As parameters of lipid peroxidation, conjugated dienes (CD) and trienes (CT) were measured in the liver, while malondialdehyde (MDA) concentration was determined in blood plasma, red blood cell hemolysate and liver. Reduced glutathione (GSH) concentration and glutathione peroxidase (GPx) activity were determined in the same samples, and expression of glutathione peroxidase 4 (GPX4), glutathione synthetase (GSS) and glutathione reductase (GSR) genes was measured by RT-PCR in the liver. STC, PSTC or AFB1 caused a slight, but not significant, increase in CD and CT levels; however, in the case of MDA, no increase was found in the liver. Glutathione redox system was activated in the liver by AFB1, but less markedly by STC/PSTC. PSTC and AFB1 resulted in a higher expression of GPX4, while GSS expression was down-regulated by AFB1 on day 1, but up-regulated by STC on day 2 and by both mycotoxins on day 7. However, on day 14, GSS expression was down-regulated by PSTC. Expression of GSR was low on day 1 in AFB1 and PSTC groups, but later it was up-regulated by AFB1. The observed changes regarding gene expression strengthen the hypothesis that the mild oxidative stress, caused by the applied STC doses, activates the glutathione redox system of broiler chickens.

Link between cardiac function and the antioxidative defense mechanism in aged rats

Aging presents profound structural and physiological changes in the cardiovascular system. Oxidative stress, a major contributing factor during the aging process, has been involved in various age-related cardiovascular pathologies. Nevertheless, the underlying mechanism of oxidative stress in the aging heart is still unclear. This study was designed to determine whether changes in cardiac structure and function in aged rats were associated with decreases in the antioxidative defense mechanism. Young (3-month-old) and aged (24-month-old) rats were used in this study, and the differences in function, structure, antioxidative capacity and the expression of antioxidative-related proteins between the two groups were compared. By using echocardiography, we observed that compared to young rats, the left ventricular internal end-diastolic diameter (LVID; d) and left ventricular volume at diastole (LV Vol; d) were significantly increased in aged rats, while the MV E/A (E wave and A wave ratio, the ratio of peak velocity of early to late filling of mitral inflow), which represents heart diastolic function, was significantly decreased in aged rats. In addition, we observed degenerative histological modifications and an increased number of apoptotic cells in aged rats. We further detected the protein expression of catalase (CAT), glutathione synthetase (GSS), superoxide dismutase-1 (SOD-1), heme oxygenase-1 (HO-1) and NADPH: quinone oxidoreductase 1 (NQO1) in cardiac tissue. Western blot results showed that the expression of GSS was significantly decreased and that the expressions of CAT, SOD-1, and HO-1 were slightly decreased in aged rats. Immunohistochemistry results further confirmed the decreased expression of GSS, SOD-1 and NQO1 in cardiomyocytes in aged rats. Taken together, our data suggest that aging may affect the morphology and function of the heart by oxidative stress and the antioxidative defense mechanism.

Variations in bovine embryo production between individual donors for OPU-IVF are closely related to glutathione concentrations in oocytes during in vitro maturation

Variations in embryo production between individual oocyte donors represent a serious problem in cattle production, when implementing ovum pick-up (OPU) and in vitro maturation (IVM) of oocytes. However, the precise cause of this problem is unknown. Here, we aimed to investigate the relationship between the glutathione (GSH) concentration in IVM oocytes and embryo development to explore a potential cause of the variations between individual donors. First, we found a high positive correlation (r = 0.80) between the GSH concentration in IVM oocytes and the blastocyst development rate of oocytes collected in the same OPU session for each donor (N = 11). Second, we selected two donors with significantly different blastocyst development rates. In samples from these donors, we examined the dynamics of oocytes GSH concentrations, and the gene expression of the glutathione synthetase (GSS) and glutathione peroxidase (GPX) genes in cumulus-oocyte complexes (COCs) during IVM. At 0 and 24 h after IVM, oocytes from the donor with the highest blastocyst development rate (high donor) exhibited significantly higher oocyte GSH concentrations than oocytes from the donor with the lowest blastocyst development rate (low donor, P < 0.05). At 4 and 9 h after IVM, GSH concentrations gradually decreased in oocytes from both donors. The GSS expression levels at 0, 4, and 9 h after IVM were significantly higher in COCs from the high donor than in COCs from the low donor (P < 0.05). The expression levels of GPX, a marker of oxidative stress, at 4 and 9 h after IVM were significantly higher in COCs from the low donor than in COCs from the high donor. Finally, adding cysteine into the IVM medium of oocytes collected in the same OPU session from the low donor significantly increased oocytes GSH concentrations and blastocyst development rates (P < 0.05). In conclusion, we showed that oocyte GSH concentrations were related to the differences in embryo development between individual donors. Our results suggested that increasing the GSH concentration in IVM oocytes could reduce the individual differences in embryo production between donors.

Effect of alterations in apoptotic pathway on development of metabolic syndrome in patients with psoriasis vulgaris.

An increase in the incidence of metabolic syndrome (MetS) has been identified in patients with psoriasis. To evaluate the role of changes in expression of apoptosis activators [B-cell lymphoma (Bcl)-2-like protein 4 (BAX), cytochrome c (cytC) and caspase-3 (CASP3)] and apoptosis inhibitors [Bcl-2, survivin, cyclin D1 (CCND1), superoxide dismutase (SOD), catalase 3 (CAT), glutathione synthetase (GS), heat shock protein (Hsp)27, Hsp60, Hsp70 and Hsp90] on development of MetS in patients with psoriasis vulgaris. Fifty patients with psoriasis were enrolled; 25 had MetS. Twenty-five healthy people and 25 people with only MetS were included as a control group. Serum fasting blood glucose, urea, creatinine, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, thyroid-stimulating hormone, fraction of thyroxine, fasting insulin and highly sensitive C-reactive protein levels were measured. Expression of BAX, cytC, CASP3, Bcl-2, survivin, CCND1, SOD, CAT, GS, and Hsp27, Hsp60, Hsp70 and Hsp90 were measured in peripheral blood. Clinical activation of patients with psoriasis was calculated using Psoriasis Area and Severity Index scores. In patients with MetS there was an increase in expression of genes for cytC, survivin and Hsp27, Hsp60 and Hsp90, and a decrease in expression of CCND1. Furthermore, expression levels of CCND1 were identified to be an independent risk factor for MetS development in patients with psoriasis. The increase in expression of survivin and Hsp27, Hsp60 and Hsp90, and the decrease in CCND1 expression may be important mechanisms in the development of MetS in patients with psoriasis.

Fucoxanthin enhances the level of reduced glutathione via the Nrf2-mediated pathway in human keratinocytes.

Fucoxanthin, a natural carotenoid, is abundant in seaweed with antioxidant properties. This study investigated the role of fucoxanthin in the induction of antioxidant enzymes involved in the synthesis of reduced glutathione (GSH), synthesized by glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS), via Akt/nuclear factor-erythroid 2-related (Nrf2) pathway in human keratinocytes (HaCaT) and elucidated the underlying mechanism. Fucoxanthin treatment increased the mRNA and protein levels of GCLC and GSS in HaCaT cells. In addition, fucoxanthin treatment promoted the nuclear translocation and phosphorylation of Nrf2, a transcription factor for the genes encoding GCLC and GSS. Chromatin immune-precipitation and luciferase reporter gene assays revealed that fucoxanthin treatment increased the binding of Nrf2 to the antioxidant response element (ARE) sequence and transcriptional activity of Nrf2. Fucoxanthin treatment increased phosphorylation of Akt (active form), an up-regulator of Nrf2 and exposure to LY294002, a phosphoinositide 3-kinase (PI3K)/Akt inhibitor, suppressed the fucoxanthin-induced activation of Akt, Nrf2, resulting in decreased GCLC and GSS expression. In accordance with the effects on GCLC and GSS expression, fucoxanthin induced the level of GSH. In addition, fucoxanthin treatment recovered the level of GSH reduced by ultraviolet B irradiation. Taken together, these findings suggest that fucoxanthin treatment augments cellular antioxidant defense by inducing Nrf2-driven expression of enzymes involved in GSH synthesis via PI3K/Akt signaling.

Gene expression analysis of genes coding key enzymes of cadmium detoxification in garden pea symbiotic nodules

Background. Cadmium is one of the most wide-ranging and dangerous pollutants for all living organisms, including plants. Currently, the mechanisms of cadmium accumulation in plant tissues and plant tolerance to its toxic effect are intensively studied. Metal-binding ligands, such as glutathione and phytochelatins, are one of the most important components in cadmium homeostasis in plants. Materials and methods. The pea line SGE and mutant SGECdt differed by cadmium tolerance were used. Gene expression for γ-glutamylcysteine synthetase (GSH1), glutathione synthetase (GSHS), homoglutathione synthetase (hGSHS) and phytochelatin synthase (PsPCS) was measured in pea nodules using realtime PCR. Results. GSH1 expression was slightly influenced by cadmium cloride. GSHS expression was upregulated in SGE and slightly downregulated in SGECdt. Cadmium cloride caused increased expression of hGSHS and PsPCS in both pea line SGE and the mutant SGECdt. Conclusion. Increased tolerance to cadmium of symbiotic nodules in the mutant SGECdt is not linked with expression pattern of analyzed genes.

Major distinctions in the antioxidant responses in liver and kidney of Cd2 +-treated common carp (Cyprinus carpio)

This study is related to the accumulation of Cd2+, its effects on oxidative stress biomarkers and its role in macromolecule damage in liver and kidney of common carp. We present evidence of an increased ratio of reduced to oxidized glutathione (GSH/GSSG) in both organs after 10mg/L Cd2+ exposure, with different underlying biological mechanisms and consequences. In the liver, the expressions and/or activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase increased to cope with the Cd2+-generated toxic effects during the first 48h of treatment. In contrast, none of these selected antioxidant markers was significantly altered in the kidney, whereas the expression of glutathione synthetase was upregulated. These results suggest that the major defense mechanism provoked by Cd2+ exposure involves the regeneration of GSH in the liver, while its de novo synthesis predominates in the kidney. High levels of accumulation of Cd2+ and peroxynitrite anion (ONOO−) were detected in the kidney; the major consequences of ONOO− toxicity were enhanced lipid peroxidation and GSH depletion. The accumulation of ONOO− in the kidney suggests intensive production of NO and the development of nitrosative stress. In the liver the level of hydrogen peroxide was elevated.

Glutathione and glutathione peroxidase expression in breast cancer: An immunohistochemical and molecular study

The use of prognostic markers for breast cancer allows therapeutic strategies to be defined more efficiently. The expression of glutathione (GSH) and glutathione peroxidase (GPX) in tumor cells has been evaluated as a predictor of prognosis and response to cytotoxic treatments. Its immunoexpression was assessed in 63 women diagnosed with invasive ductal carcinoma in a retrospective study. The results showed that high GSH expression was associated with tumors negative for the estrogen receptor (ER) (P<0.05), and GPX expression was associated with tumors negative for the progesterone receptor (PR) and patient mortality. Focusing on the 37 patients who received adjuvant chemotherapy/radiotherapy (Group I), high expression of GPX was associated with a high rate of patient mortality (P<0.05). The 19 patients who received only adjuvant chemotherapy (Group II) showed high expression of GSH in relation to metastasis (P<0.05). In addition, high levels of GPX expression were significantly associated with a shorter overall survival (P<0.05). To confirm this, the expression of precursor genes of GSH [glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS)] and the GPX gene was analyzed using quantitative PCR in cultured neoplastic mammary cells treated with doxorubicin. Doxorubicin treatment was able to eliminate tumor cells without alterations in the gene expression of GSS, but led to underexpression of the GCLC and GPX genes. Our results suggest that high levels of GPX may be related to the development of resistance to chemotherapy in these tumors, response to treatment and the clinical course of the breast cancer patients.

Disruption of redox homeostasis and induction of apoptosis by suppression of glutathione synthetase expression in a mammalian cell line

The stable HepG2 transfectants anti-sensing expression of the glutathione synthetase (GS) gene exhibited delayed cell growth and increased reactive oxygen species (ROS) level. After the treatment with hydrogen peroxide, the intracellular ROS level was much higher in the stable transfectants than in the vector control cells. However, the GSH levels decreased more significantly in the stable transfectants than in the vector control cells, in the presence of hydrogen peroxide. Hydrogen peroxide-induced apoptosis of the stable transfectants was notably higher than that of the vector control cells. The GS anti-sense RNAs rendered the HepG2 cells more sensitive to growth arrest caused by glucose deprivation. They also sensitized the HepG2 cells to cadmium chloride (Cd) and nitric oxide (NO)-generating sodium nitroprusside (SNP). In brief, the results confirm that GS plays an important role in the defense of the human hepatoma cells against oxidative stress by reducing apoptosis and maintaining redox homeostasis.

Effects of Yiguanjian Decoction on liver cirrhosis formation: a differential proteomics study in rats

To investigate the effects of Yiguanjian Decoction, a compound traditional Chinese herbal medicine, on rats with cirrhosis based on the method of differential proteomics. Wistar male rats (n=48) were randomly divided into normal control group (n=12) and model-making group (n=36). Rat cirrhosis model was established by intraperitoneal injection of 50% carbon tetrachloride (CCl4) plus olive oil solution (1 mL/kg, twice weekly for 9 weeks). After 3- and 6-week injection, 6 rats each time were sacrificed for dynamic observation before medicine intervention, and the 24 remained rats were randomly divided into untreated group (n=12) and Yiguanjian Decoction group (n=12) at the first day of the 7th week. All animals were sacrificed by the end of the 9th week, and total protein of liver tissue was isolated by two-dimensional gel electrophoresis (2-DE); some differentially expressed protein spots were analyzed and identified by matrix-assisted laser desorption/ionization two-stage time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and database querying. Protein expressions of Cu/Zn superoxide dismutase (Cu/Zn SOD) and DJ-1 were validated by Western blot and immunohistochemical methods. 2-DE maps with high resolution and good repeatability were obtained. In all 50 protein spots identified by MALDI-TOF/TOF-MS and database querying, there were 5 protein spots related to oxidative stress named Cu/Zn SOD, DJ-1, glutathione synthetase, glutathione S-transferase Yb-1 subunit and aldo-keto reductase family 7, A2 respectively. Compared with the normal control group, expressions of Cu/Zn SOD, DJ-1, glutathione S-transferase Yb-1 subunit and aldo-keto reductase family 7, A2 in the untreated group were decreased significantly. Expressions of Cu/Zn SOD and aldo-keto reductase family 7, A2 were decreased time-dependently. Compared with the untreated group in 9th week, protein expressions of Cu/Zn SOD, DJ-1, glutathione S-transferase Yb-1 subunit and aldo-keto reductase family 7, A2 in the Yiguanjian Decoction groups were increased significantly while expression of glutathione synthetase was decreased notably. Western blot and immunohistochemical results of Cu/Zn SOD and DJ-1 expressions coincided with proteomics results. Anti-oxidative depression is a key pathological change of cirrhosis induced by CCl4 in rats, and increasing expression of proteins related to anti-oxidative stress may be a major mechanism of Yiguanjian Decoction in treating cirrhosis induced by CCl4 effectively.

Alternative RNA splicing in expression of the glutathione synthetase gene in human cells

Ubiquitous free radical production occurs continuously in cells and tissues. Glutathione is the most abundant mammalian antioxidant, and is synthesized by glutathione synthetase (GSS). Therefore, GSS plays an important role in defending the cell against reactive oxygen species. The expression of GSS has been studied in human cells; however, sequence information about alternative splicing variants of GSS mRNA has not been reported. In the present study, we identified a novel alternative splicing variant (ASV) of the GSS gene in 10 human normal tissues and five human cancer cell lines. The deleted transcript of GSS was characterized by an in-frame deletion of 333 bp, corresponding to the complete loss of exons 4 and 5. Thus this GSS ASV causes protein truncation. We quantified the mRNA of GSS ASV in human normal tissues using real-time PCR. The ASV was detected in colon, kidney, lung, liver, placenta, peripheral blood and uterus, but not in heart, skeletal muscle and spleen tissue. Our results provide a basis for more detailed studies on the regulation of GSS, and for further evaluation of this and other possible roles of GSS. Understanding the regulation of GSS expression is very important for the development of new strategies for controlling the development of GSH-based redox homeostasis.

HIF1 Induced Glutathione Dysregulation in Platelets Associated with Increased Risk of Vascular Disease in Chuvash Polycythemia.

The von Hippel-Lindau protein-hypoxia-inducible factor pathway is a transcriptional system controlling cellular responses to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of α and β subunits. Under normoxia, the prolyl residues of the α subunit are hydroxylated allowing the von Hippel-Lindau protein (pVHL) to bind, which targets HIFα for proteasomal degradation. During hypoxia, HIFα does not bind to pVHL and instead forms a transcriptional complex with HIFβ leading to increased expression of a broad range of hypoxia-regulated genes. Patients with Chuvash polycythemia (CP) are homozygous for a 598 C->T mutation in the VHL gene, resulting in a pVHL that causes ineffective degradation of HIFα. Clinically, CP patients suffer from premature mortality related to vascular thrombotic events - an increased risk that is not related to their elevated hematocrit, blood pressure, or known cardio or cerebrovascular risk factors. Lab abnormalities seen in CP include erythrocytosis and an elevated plasma glutathione level (Sergueeva et al, Haematologica. Feb 2008). Glutathione (GSH) plays an essential role in cellular antioxidant protection, and its levels are controlled by two mechanisms. GSH is oxidized to glutathione disulfide (GSSG) but is replenished by reduction of GSSG via glutathione reductase (GSR). GSH is also increased by de novo synthesis, which is regulated at several levels, including GSH feedback inhibition, and glutathione synthetase (GSS) and glutamate cysteine ligase (GCL) activity. To establish the molecular basis of elevated GSH, we examined expression of GSS, GSR, and GCL in the platelets of 11 CP patients and 8 Chuvash controls using qRT-PCR. Analysis revealed a 2-fold increased expression of GCL in CP. GSR and GSS were not statistically different. This data suggests that increased GCL activity might be the mechanism by which GSH is elevated in CP, but whether HIF directly regulates GCL or whether these differences reflect a more global process are presently unknown. Since the promoter of GCL contains an oxidative stressresponse element, transcriptional up regulation of GCL by increased oxidative stress secondary to HIF dysregulation might drive increased GSH synthesis. To examine this question, we measured GSSG in our samples. Although CP patients had elevated GSH (8.00 uM vs. 4.32 uM, p 0.002), the GSH/GSSG ratio (a widely used marker of redox state) showed no differences between CP and controls. Thus, overexpression of GCL and elevated GSH appear not to be compensatory responses to increased oxidative stress in CP. We also found increased GCL expression in VHL mutant mice. To determine if HIF1 might regulate this expression, we next measured GCL expression in HIF1 deficient embryos at embryonic day 9.5 and found decreased expression of GCL. In the homozygote knock-out embryos where HIF1 is absent, GCL expression is decreased, in contrast to CP patients, where HIF1 and GCL expression are both increased. Collectively, our data suggests that HIF1 dysregulates cellular redox homeostasis by upregulating GCL and increasing GSH synthesis in an oxidative-stress-independent manner. The significance of elevated GSH in CP and its possible relationship to increased thromboses remains to be defined. Reactive oxygen species mediate the vascular inflammation seen in the development of atherosclerotic disease, and GSH is an important intracellular scavenger that protects cells against oxidative damage. Yet CP patients have increased GSH as well as increased thrombosis, which is especially provoking since recently published evidence suggests that increased redox potential may be harmful to the cardiovascular system (Rajasekaran, et al Cell 2007). Perhaps the GSH elevation may contribute to the increased vascular disease that constitutes the major cause of mortality in this disorder of hypoxia sensing. The cellular antioxidant defense system is intimately linked to oxidative stress, hypoxia regulation, and vascular homeostasis. Our proposed future studies employing HIF2 knock out mice, model cell systems for HIF1 and HIF2, and the role of GSH in platelet function will be used to further explore the molecular mechanisms that regulate these complex pathways.