Effect of glutathione reductase knockdown in response to UVB-induced oxidative stress in human lung adenocarcinoma

Background

Glutathione reductase (GR) plays a critical role in the maintenance of physiological redox status in cells. However, the comprehensive investigations of GR-modulated oxidative stress have not been reported.

Methods

In the present study, we cultured a human lung adenocarcinoma line CL1-0 and its GR-knockdown derivative CL1-0ΔGR to evaluate their differential responses to UVB-irradiation.

Results

We identified 18 proteins that showed significant changes under UVB-irradiation in CL1-0ΔGR cells rather than in CL1-0 cells. Several proteins involving protein folding, metabolism, protein biosynthesis and redox regulation showed significant changes in expression.

Conclusions

In summary, the current study used a comprehensive lung adenocarcinoma-based proteomic approach for the identification of GR-modulated protein expression in response to UVB-irradiation. To our knowledge, this is the first global proteomic analysis to investigate the role of GR under UVB-irradiation in mammalian cell model.     

Supplementary selenium influences the response to fatty acid-induced oxidative stress in humans

The mutual influences of wheat selenium (Se) andn-3 polyunsaturated fatty acids(n- 3 PUFA) on plasma Se and indicators of increased oxidative stress were investigated in a randomized, doubleblind study with 31 women (23.5 ±3.4 yr). Groups 1 and 2 ingested 5.4 g n-3 PUFA daily (as ethyl esters), whereas groups 3 and 4 received placebo capsules. Groups 2 and 3 received 3 slices of high Se bread daily, providing 115 Μg Se, in addition to the 77± 26 ug Se in the diet. Groups 1 and 4 received placebo slices. Blood samples were drawn at baseline and at 3 and 6 wk. Serum Se concentrations increased in both groups given Seenriched bread, but significantly less in subjects givenn-3 PUFA (group 2). There were no changes in the plasma ratio α-tocopherol:mg cholesterol or plasma ascorbic acid levels. In group1, plasmaconjugated dienes and thiobarbituric acid-reactive substances (TBARS) rose by 130% (p < 0.005) and 126% (p < 0.005), respectively. Two-way ANOVA showed significant interaction effects of Se andn-3 PUFA on changes in conjugated dienes (p = 0.03) and TBARS (p = 0.015), Se treatment apparently modifying the peroxidative effects ofn-3 PUFA. In subjects receivingn-3 PUFA, changes in conjugated dienes and TBARS were negatively correlated with changes in serum Se. In summary,n-3 PUFA modified the effect of Se supplementation, whereas Se seemed to modify the peroxidative effects ofn-3 PUFA.     

ATM deficiency and oxidative stress: a new dimension of defective response to DNA damage

ATM is one of the sentries at the gate of genome stability. This multifunctional protein kinase orchestrates the intricate array of cellular responses to DNA double-strand breaks. Absence or inactivation of ATM leads to the pleiotropic genetic disorder ataxia-telangiectasia (A-T), whose hallmarks are neuronal degeneration, immunodeficiency, genomic instability, premature aging and cancer predisposition. Several features of the complex clinical and cellular phenotype of A-T are reminiscent of other syndromes involving neurodegeneration, premature aging or genomic instability. A common denominator of many of these conditions is the perturbation of the cellular balance of reactive oxygen species, which leads to constant oxidative stress. Of these disorders, ATM deficiency is one of the most extensively studied with regard to the genome instability-oxidative stress connection. This connection may provide new insights into the phenotypes associated with genetic deficiencies of DNA damage responses, and point to new strategies to alleviate some of their clinical symptoms.     

Effect of selenium and protein deficiency on selenium and glutathione peroxidase in rats

Twenty-four weanling male Wistar rats were divided into four groups fed diets containing adequate or deficient levels of selenium (0.5 ppm [+ Se] or <0.02 ppm [−Se] and protein (15% [+Pro] or 5% [−Pro]), but adequate levels of all other nutrients for 4 wk to determine the effects of Se deficiency and protein deficiency on tissue Se and glutathione peroxidase (GSHPx) activity in rats. Plasma, heart, liver, and kidney Se and GSHPx were significantly lower in Se-deficient groups in relation to Se-sufficient groups. In Se-deficient groups, Se and GSHPx were significantly higher in −Se−Pro rats in heart, liver, and kidney. Data analysis showed that there were significant interaction effects between dietary Se and protein on Se and GSHPx of rats. It is assumed that under the condition of Se deficiency. a low level of protein may decrease Se and GSHPx utilization, increase GSHPx synthesis, and result in Se redistribution. This could account for high levels of Se and GSHPx in the −Se−Pro rats compared to −Se+Pro rats.     

Tumor-suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione S-transferase

Maspin, a novel serine protease inhibitor, suppresses tumor progression in several cancer models, including an in vivo model for prostate cancer bone metastasis. However, the molecular mechanism of maspin remains illusive, primarily because its molecular targets are unknown. To this end, we used a full-length maspin cDNA bait to screen against both a primary prostate tumor cDNA prey library and a HeLa cDNA prey library by the yeast two-hybrid method. We found that heat shock protein 90, glutathione S-transferase (GST), and heat shock protein 70 interacted with maspin with the highest frequencies. We confirmed the maspin/GST interaction using purified proteins, human epithelial cell lines, and human prostate tissues. A maspin variant that has a point mutation of Arg(340) to Ala (Mas(R340A)) showed a significantly decreased affinity for GST. Although purified maspin had no effect on the activity of purified GST in vitro, intracellular interaction between endogenous maspin and GST correlated with an elevated total GST activity in both MDA-MB-435- and DU145-derived stably transfected cells. Consistently, tumor cells treated with purified wild type maspin, but not Mas(R340A), enhanced cellular GST activity. Maspin expression in cancer cell lines also correlated with decreased basal levels of reactive oxygen species (ROS). Furthermore, H(2)O(2) treatment not only induced GST expression but also increased intracellular maspin/GST interaction, which was inversely correlated with the level of ROS generation. Conversely, maspin knockdown by small interfering RNA increased the basal, as well as H(2)O(2)-induced, ROS generation. Furthermore, the maspin effect on ROS generation was completely abolished by a GST inhibitor, indicating an essential role of GST in maspin-mediated cellular response to oxidative stress. Consistently, oxidative stress-induced vascular endothelial growth factor A expression was significantly inhibited in maspin-expressing cells. Together, our data suggest a new mechanism by which maspin, through its direct interaction with GST, may inhibit oxidative stress-induced ROS generation and vascular endothelial growth factor A induction, thus preventing further adverse effects on tumor genetics and stromal reactivity.     

The response to oxidative stress induced by magnesium deficiency in kidney bean plants

To understand the plant response to oxidative stresses, we studied the influence of magnesium (Mg⁺⁺) deficiency on the formation of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and protease activity in kidney bean plants. The expression pattern of proteins under Mg⁺⁺ deficiency also was examined via two-dimensional electrophoresis. The formation of H₂O₂ and MDA increased in the primary leaves of plants grown in a nutrient solution deficient in Mg⁺⁺. Protease activity in Mg⁺⁺-deficient plants was also higher than in those grown with sufficient Mg⁺⁺. The expression pattern of the proteins showed that 25 new proteins were generated and 64 proteins disappeared under Mg⁺⁺-deficient conditions. Therefore, a deficiency in Mg⁺⁺ may cause oxidative stress and a change in protein expression. Some of these proteins may be related to the oxidative stress induced by Mg⁺⁺ deficiency.     

Capillary electrophoresis of glutathione to monitor oxidative stress and response to antioxidant treatments in an animal model

Glutathione plays a central role in metabolism and antioxidant defence. Several factors can influence the analytical efficiency and rapidity of the quantitative determination of glutathione. Procedures in sample pre-treatment have been compared in order to minimize analytical errors. Capillary electrophoresis has been chosen as a more adequate technique for obtaining a rapid and simple method for glutathione and glutathione disulfide determination in the blood and liver of the rat. The methods, once optimised, have been validated and applied for monitoring the oxidative stress in an animal model, such as the rat made diabetic by streptozotocin injection, when the animals are treated with antioxidants and compared with the corresponding controls.     

Mycothiol-dependent mycobacterial response to oxidative stress

The effect of exogenous oxidative stress on mycothiol (MSH) levels and redox balance was investigated in mycobacteria. Both the thiol-specific oxidant diamide and hydrogen peroxide induced up to 75% depletion of MSH to form the disulfide form, mycothione (MSSM), in Mycobacterium bovis BCG. In comparison, Mycobacterium smegmatis, a saprophytic mycobacterium, displays a greater tolerance towards these oxidants, reflected by the lack of fluxes in MSH levels and redox ratios upon oxidative stress treatments. The basal ratio of MSH to MSSM was established to be 50:1 in M. bovis BCG and 200:1 in M. smegmatis.     

Interaction between vitamin B6 and source of selenium on the response of the selenium-dependent glutathione peroxidase system to oxidative stress induced by oestrus in pubertal pig

This study aimed to assess the interaction between vitamin B₆ and selenium (Se) for the flow of Se towards the Se-dependent glutathione peroxidase (GPX) system in response to oxidative stress naturally induced by oestrus in a pubertal pig model. At first oestrus, forty-five gilts were randomly assigned to the experimental diets (n = 9/group): basal diet (CONT); CONT + 0.3 mg/kg of Na-selenite (MSeB₆0); MSeB₆0 + 10 mg/kg of HCl-B₆ (MSeB₆10); CONT + 0.3 mg/kg of Se-enriched yeast (OSeB₆0); and OSeB₆0 + 10 mg/kg of HCl-B₆ (OSeB₆10). Blood samples were collected at each oestrus (long-term profiles), and daily from day −4 to +3 (slaughter) of the fourth oestrus (peri-oestrus profiles) after which liver, kidneys, and ovaries were collected. For long-term profiles, CONT had lower blood Se than Se-supplemented gilts (p < 0.01) and OSe was higher than MSe (p < 0.01). Lower erythrocyte pyridoxal-5-phosphate was found in B₆0 than B₆10 (p < 0.01). No treatment effect was observed on GPX activity. For peri-oestrus profiles, treatment effects were similar to long-term profiles. Treatment effects on liver Se were similar to those for long-term blood Se profiles and OSe had higher renal Se concentrations than MSe gilts (p < 0.01). Gene expressions of GPX1, GPX3, GPX4, and selenocysteine lyase in liver and kidney were greatest in OSeB₆10 gilts (p < 0.05). These results suggest that dietary B₆ modulate the metabolic pathway of OSe towards the GPX system during the peri-oestrus period in pubertal pigs.     

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.     

AMP-activated protein kinase deficiency reduces ozone-induced lung injury and oxidative stress in mice

Background

Acute ozone exposure causes lung oxidative stress and inflammation leading to lung injury. At least one mechanism underlying the lung toxicity of ozone involves excessive production of reactive oxygen and nitrogen intermediates such as peroxynitrite. In addition and beyond its major prooxidant properties, peroxynitrite may nitrate tyrosine residues altering phosphorylation of many protein kinases involved in cell signalling. It was recently proposed that peroxynitrite activates 5''-AMP-activated kinase (AMPK), which regulates metabolic pathways and the response to cell stress. AMPK activation as a consequence of ozone exposure has not been previously evaluated. First, we tested whether acute ozone exposure in mice would impair alveolar fluid clearance, increase lung tissue peroxynitrite production and activate AMPK. Second, we tested whether loss of AMP-activated protein kinase alpha1 subunit in mouse would prevent enhanced oxidative stress and lung injury induced by ozone exposure.

Methods

Control and AMPKα1 deficient mice were exposed to ozone at a concentration of 2.0 ppm for 3 h in glass cages. Evaluation was performed 24 h after ozone exposure. Alveolar fluid clearance (AFC) was evaluated using fluorescein isothiocyanate tagged albumin. Differential cell counts, total protein levels, cytokine concentrations, myeloperoxidase activity and markers of oxidative stress, i.e. malondialdehyde and peroxynitrite, were determined in bronchoalveolar lavage (BAL) and lung homogenates (LH). Levels of AMPK-Thr¹⁷² phosphorylation and basolateral membrane Na(+)-K(+)-ATPase abundance were determined by Western blot.

Results

In control mice, ozone exposure induced lung inflammation as evidence by increased leukocyte count, protein concentration in BAL and myeloperoxidase activity, pro-inflammatory cytokine levels in LH. Increases in peroxynitrite levels (3 vs 4.4 nM, p = 0.02) and malondialdehyde concentrations (110 vs 230 μmole/g wet tissue) were detected in LH obtained from ozone-exposed control mice. Ozone exposure consistently increased phosphorylated AMPK-Thr¹⁷² to total AMPK ratio by 80% in control mice. Ozone exposure causes increases in AFC and basolateral membrane Na(+)-K(+)-ATPase abundance in control mice which did not occur in AMPKα1 deficient mice.

Conclusions

Our results collectively suggest that AMPK activation participates in ozone-induced increases in AFC, inflammation and oxidative stress. Further studies are needed to understand how the AMPK pathway may provide a novel approach for the prevention of ozone-induced lung injury.     

Synaptosomal response to oxidative stress: Effect of vinpocetine

It has been suggested that reactive oxygen species (ROS) play a role in the neuronal damage occurring in ischemic injury and neurodegenerative disorders and that their neutralization by antioxidant drugs may delay or minimize neurodegeneration. In the present study we examine whether vinpocetine can act as an antioxidant and prevent the formation of ROS and lipid peroxidation in rat brain synaptosomes. After ascorbate/Fe²⁺ treatment a significant increase in oxygen consumption (about 5-fold) and thiobarbituric acid reactive substances (TBARS) formation (about 7-fold) occurred as compared to control conditions. Vinpocetine inhibited the ascorbate/Fe²⁺ stimulated consumption of oxygen and TBARS accumulation, an indicator of lipid peroxidation, in a concentration-dependent manner. The ROS formation was also prevented by vinpocetine. Oxidative stress increased significantly the fluorescence of the probes 2′,7′-dichlorodihydrofluorescein (DCFH₂-DA) (about 6-fold) and dihydrorhodamine (DHR) 123 (about 10-fold), which is indicative of intrasynaptosomal ROS generation. Vinpocetine at 100 μM concentration decreased the fluorescence of DCFH₂-DA and DHR 123 by about 50% and 83%, respectively. We conclude that the antioxidant effect of vinpocetine might contribute to the protective role exerted by the drug in reducing neuronal damage in pathological situations.     

Distinct roles for glutathione S-transferases in the oxidative stress response in Schizosaccharomyces pombe

We have identified three genes, gst1(+), gst2(+), and gst3(+), encoding theta-class glutathione S-transferases (GSTs) in Schizosaccharomyces pombe. The gst1(+) and gst2(+) genes encode closely related proteins (79% identical). Our analysis suggests that Gst1, Gst2, and Gst3 all have GST activity with the substrate 1-chloro-2,4-dinitrobenzene and that Gst3 has glutathione peroxidase activity. Although Gst1 and Gst2 have no detectable peroxidase activity, all three gst genes are required for normal cellular resistance to peroxides. In contrast, each mutant is more resistant to diamide than wild-type cells. The gst1Delta, gst2Delta, and gst3Delta mutants are also more sensitive to fluconazole, suggesting that GSTs may be involved in anti-fungal drug detoxification. Both gst2(+) and gst3(+) mRNA levels increase in stationary phase, and all three gst genes are induced by hydrogen peroxide. Indeed, gst1(+), gst2(+), and gst3(+) are regulated by the stress-activated protein kinase Sty1. The Gst1 and Gst2 proteins are distributed throughout the cell and can form homodimers and Gst1-Gst2 heterodimers. In contrast, Gst3 is excluded from the nucleus and forms homodimers but not complexes with either Gst1 or Gst2. Collectively, our data suggest that GSTs have separate and overlapping roles in oxidative stress and drug responses in fission yeast.     

Synaptosomal response to oxidative stress: effect of vinpocetine

It has been suggested that reactive oxygen species (ROS) play a role in the neuronal damage occurring in ischemic injury and neurodegenerative disorders and that their neutralization by antioxidant drugs may delay or minimize neurodegeneration. In the present study we examine whether vinpocetine can act as an antioxidant and prevent the formation of ROS and lipid peroxidation in rat brain synaptosomes. After ascorbate/Fe²⁺ treatment a significant increase in oxygen consumption (about 5-fold) and thiobarbituric acid reactive substances (TBARS) formation (about 7-fold) occurred as compared to control conditions. Vinpocetine inhibited the ascorbate/Fe²⁺ stimulated consumption of oxygen and TBARS accumulation, an indicator of lipid peroxidation, in a concentration-dependent manner. The ROS formation was also prevented by vinpocetine. Oxidative stress increased significantly the fluorescence of the probes 2',7'-dichlorodihydrofluorescein (DCFH₂-DA) (about 6-fold) and dihydrorhodamine (DHR) 123 (about 10-fold), which is indicative of intrasynaptosomal ROS generation. Vinpocetine at 100 μM concentration decreased the fluorescence of DCFH₂-DA and DHR 123 by about 50% and 83%, respectively. We conclude that the antioxidant effect of vinpocetine might contribute to the protective role exerted by the drug in reducing neuronal damage in pathological situations.     

Selenium modifies glutathione peroxidase activity and glutathione concentration in mice exposed to ozone-provoked oxidative stress

The aim of this study was to show the direct effect of selenium on glutathione peroxidase (GSH-Px) activity and GSH/GSSG concentrations in 3- and 6-month-old mice. An ozone-oxygen mixture was used to provoke an oxygen stress. To measure the Se-effect mice were gavaged with sodium selenite. GSH-Px activity and total glutathione concentrations were determined in serum and in the postnuclear fraction of liver and lungs. Additionally glutathione concentrations were determined in whole blood. Both ozone and selenium, administered separately, reduced GSH-Px activity in lungs of 6-month-old animals, while in young mice an opposite effect of Se was observed. Ozone administered jointly with Se did not influence GSH-Px activity in 6-month-old mice, while in young, 3-month-old mice, a stimulatory effect in lungs was observed. There were no significant changes in GSH-Px activity in the liver of 6-month-old mice, but the stimulatory effect occurred in young mice treated with Se and Se & ozone jointly. In young mice, ozone (also ozone with Se) augmented glutathione concentrations. The response to ozone and selenium strictly depended on age and the antagonism between selenium and ozone was observed only in a few cases.     

Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae.

Three glutathione peroxidase homologs (YKL026C, YBR244W, and YIR037W/HYR1) were found in the Saccharomyces Genome Database. We named them GPX1, GPX2, and GPX3, respectively, and we investigated the function of each gene product. The gpx3Delta mutant was hypersensitive to peroxides, whereas null mutants of the GPX1 and GPX2 did not show any obvious phenotypes. Glutathione peroxidase activity decreased approximately 57 and 93% in the gpx3Delta and gpx1Delta/gpx2Delta/gpx3Delta mutants, respectively, compared with that of wild type. Expression of the GPX3 gene was not induced by any stresses tested, whereas that of the GPX1 gene was induced by glucose starvation. The GPX2 gene expression was induced by oxidative stress, which was dependent upon the Yap1p. The TSA1 (thiol-specific antioxidant) gene encodes thioredoxin peroxidase that can reduce peroxides by using thioredoxin as a reducing power. Disruption of the TSA1 gene enhanced the basal expression level of the Yap1p target genes such as GSH1, GLR1, and GPX2 and that resulted in increases of total glutathione level and activities of glutathione reductase and glutathione peroxidase. However, expression of the TSA1 gene did not increase in the gpx1Delta/gpx2Delta/gpx3Delta mutant. Therefore, de novo synthesis and recycling of glutathione were increased in the tsa1Delta mutant to maintain the catalytic cycle of glutathione peroxidase reaction efficiently as a backup system for thioredoxin peroxidase.     

Oxidative stress during selenium deficiency in seedlings ofTrigonella foenum-graecum and mitigation by mimosine part II. glutathione metabolism

Adaptive alterations in glutathione (GSH) metabolism were studied during oxidative stress induced by selenium (Se) deficiency in germinating seedlings of Trigonella foenum-graecum grown for 72 h and the response to supplementation individually of Se or mimosine was explored. Growth enhancement with improved mitochondrial efficiency was elicited by supplementation of Se at 0.5-0.75 ppm or mimosine at 0.1-0.2 mM. Total thiol and protein levels of mitochondrial and soluble fractions, in general, did not vary significantly with supplementation of either Se or mimosine except that the mitochondrial protein levels in mimosine groups (0.1-0.2 mM) decreased by 20-30%. Mitochondrial glutathione peroxidase (GSH-Px) increased by twofold in activity toward H2O2, cumene hydroperoxide (CHP), and t-butyl hydroperoxide (tBHP) in Se groups, and by 50-60% increase toward H2O2 and CHP but by a twofold enhancement in enzyme activity with tBHP in mimosine groups. Soluble GSH-Px activity increased by 30-40% only in mimosine groups and remained unaltered in Se groups. Glutathione S-transferase activity (GST) in the soluble fraction of both Se and mimosine groups increased dramatically by fivefold to sixfold. Distinct differences were noted in the response of the stressed seedlings toward exposure to Se or mimosine and included a decline in glutathione reductase (GR) activity by 50-60% in both mitochondria and soluble fractions of Se groups and an increase in GR activity of the mitochondria by twofold and of the soluble enzyme activity by 30% in the mimosine groups. Mimosine exposure resulted in a dose-dependent decrease in the gamma-glutamyl transpeptidase levels, but, in contrast, a significant enhancement by 50% was noted in the Se group at 0.75 ppm. The results including the differential response of GR activity to Se or mimosine supplementation are reflective of an effective reductive environment in Se groups and increased turnover of GSH in the presence of mimosine.