Changes In GSH Levels Research Articles

English

Arsenic is a major threat to a large part of the population due to its carcinogenic nature. The toxicity of arsenic emerges from glutathione (GSH) depletion caused by arsenic with unknown mechanism. GSH depletion leads to apoptosis, lipid peroxidation and eventual cell death. The present study was designed to provide insight into the extent of changes in GSH level by arsenic. Plasma and cytosolic fraction were investigated for determination of changes in GSH metabolic status caused by arsenic in the form of arsenic trioxide (ATO). The depletion of GSH level was found to be positively correlated with increasing parameters, that is, arsenic concentration and time of incubation. Our findings show that changes in GSH status produced by arsenic could be due to adduct (As-(SG)3) formation. This change in GSH metabolic status provides information regarding mechanism of toxicity of ATO. These findings are important for the rational design of antidote for the prevention of arsenic induced toxicity.   Key words: Arsenic trioxide (ATO), glutathione (GSH), dithiobisnitrobenzoic acid (DTNB), plasma, cytosolic fraction (C.F).

Effect of prostaglandin E1 (PGE1) and sildenafil on serotonin metabolism and some oxidative damage markers in rat prostate gland and brain

The aim of this study was to evaluate the effect of sildenafil and prostaglandin E1 (PGE1) (drugs used in erectile dysfunction) on production of free radicals in prostate and brain of rat. A single dose of sildenafil (10 mg kg(-1) ) and PGE1 (20 μg kg(-1) ) was given to Sprague-Dawley rats (300 g weight) intraperitoneally. The levels of testosterone were measured in blood. Their brains and prostate glands were separated to measure lipid peroxidation, Na(+) and K(+) ATPase activity, reduced glutathione (GSH) and serotonin levels, by means of validated methods. The levels of testosterone increased slightly in animals treated with sildenafil and PGE1. The activity of total ATPase was increased in the prostate of animals treated with sildenafil + PGE1 but decreased in those that received sildenafil alone. PGE1 caused significant diminution of GSH levels in both organs. Sildenafil increased the levels of serotonine in brain, whereas in prostate they decreased instead. Our results suggest that sildenafil induced changes in GSH levels as well as in the serotonergic metabolism, alone or with PGE1 in prostate and brain, respectively. Thus, the combination therapy may be ideal to sustain the biochemical balance due to biphasic stimulation on brain and prostate.

Treatment with p38 inhibitor partially prevents Calu-6 lung cancer cell death by a proteasome inhibitor, MG132

MG132 (carbobenzoxy-Leu-Leu-leucinal) as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). In this study, we investigated the effects of MEK (mitogen-activated protein [MAP] kinase or extracellular signal–regulated kinase [ERK] kinase) or p38 inhibitor on MG132-treated Calu-6 lung cancer cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. Treatment with 10 μmol/L MG132 inhibited the growth of Calu-6 cells at 24 hours. MG132 induced apoptosis in Calu-6 cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ m). ROS were increased in MG132-treated Calu-6 cells. MG132 also induced GSH depletion in Calu-6 cells. Treatment with MEK inhibitor did not significantly affect cell growth, cell death, ROS, and GSH levels in MG132-treated Calu-6 cells. Furthermore, MG132 increased the phosphorylation of p38 in Calu-6 cells at 1 and 24 hours. Treatment with p38 inhibitor significantly prevented cell growth inhibition, MMP (ΔΨ m) loss and apoptosis in MG132-treated Calu-6 cells. This inhibitor increased ROS level and decreased GSH depletion in these cells. In conclusion, p38 inhibitor partially prevented Calu-6 cell death by MG132, which might be affected by GSH level changes.

γ-Glutamyl transpeptidase null mice fail to develop tolerance to coumarin-induced Clara cell toxicity

Coumarin was used as a model Clara cell toxicant to test the hypothesis that tolerance to injury requires increased γ-glutamyl transpeptidase (GGT) activity. Wildtype (GGT+/+) and GGT-deficient (GGT−/−) mice on a C57BL/6/129SvEv hybrid background were dosed orally with corn oil (vehicle) or coumarin (200mg/kg). In vehicle-treated mice, Clara cell secretory protein (CC10) expression was distributed throughout the bronchiolar epithelium. After one dose of coumarin, CC10 expression was dramatically reduced and the bronchiolar epithelium was devoid of Clara cells in GGT+/+ and GGT−/− mice. In wildtype mice, 9 doses of coumarin produced tolerance, characterized as a renewed bronchiolar epithelium with Clara cells expressing CC10 along with a 40% increase in total glutathione (GSH) and a 7-fold increase in GGT activity in the lung. In contrast, tolerance was not observed in GGT−/− mice. To assess whether changes in whole lung levels of GSH and GGT activity reflect Clara cell specific changes an enriched population of cells was isolated from female wildtype B6C3F1 mice made tolerant to coumarin. Compared to Clara cells from control mice, GSH and GGT activity increased 3- and 13-fold, respectively. Collectively, these data suggest Clara cell tolerance to coumarin toxicity requires increased GGT activity favoring enhanced GSH synthesis.

The Attenuation of MG132, a Proteasome Inhibitor, Induced A549 Lung Cancer Cell Death by p38 Inhibitor in ROS-Independent Manner

MG132, as a proteasome inhibitor, can induce apoptotic cell death through formation of reactive oxygen species (ROS). In this study, we investigated the effects of MAPK (MEK, JNK, and p38) inhibitors on MG132-treated A549 lung cancer cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. Treatment with 10 microM MG132 inhibited the growth of A549 cells at 24 h. MG132 also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; deltapsi(m)). ROS were not increased in MG132-treated A549 cells. MG132 increased GSH-depleted cell numbers and decreased GSH levels. MEK and JNK inhibitors did not strongly affect cell growth, cell death, ROS, and GSH levels in MG132-treated A549 cells. In contrast, p38 inhibitor reduced cell growth inhibition, apoptosis, and MMP (deltapsi(m)) loss by MG132. However, p38 inhibitor did not change ROS level and GSH content. In conclusion, MG132 inhibited the growth of A549 cells via apoptosis without formation of ROS. Treatment with p38 inhibitor rescued some cells from MG132-induced apotposis, which was not affected by ROS and GSH level changes.

Gamma‐linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells

Various polyunsaturated fatty acids, especially gamma-linolenic acid (GLA), inhibit the growth of a variety of tumor cells. Some evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis. In the current study, we tested the apoptotic effect of GLA on human chronic myelogenous leukemia K562 cells. GLA induced K562 cell death in a dose-dependent manner. Typical apoptotic nuclei were shown by staining of K562 cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. Flow cytometric analysis also demonstrated that GLA caused dose-dependent apoptosis of K562 cells. The apoptosis could be inhibited by a pancaspase inhibitor (z-VAD-fmk), suggesting the involvement of caspases. Further, release of cytochrome c, activation of caspase-3 and cleavage of PARP were found in GLA-induced apoptosis. GLA treatment could also elevate lipid peroxidation in K562 cells, and antioxidant alpha-tocopherol could reverse the cytotoxicity of GLA. The saturated fatty acid SA, which did not exhibit significant increase in lipid peroxidation, also did not induce cytotoxicity. Intracellular GSH was also determined, and there was no marked change of GSH levels in cells after incubation with GLA compared with the control. These results demonstrate that GLA could induce apoptosis in K562 cells. Apoptosis is mediated by release of cytochrome c, activation of caspase-3. Lipid peroxidation may play a role in GLA cytotoxicity.

Toxicity of amorphous silica nanoparticles in mouse keratinocytes

The present study was designed to examine the uptake, localization, and the cytotoxic effects of well-dispersed amorphous silica nanoparticles in mouse keratinocytes (HEL-30). Mouse keratinocytes were exposed for 24 h to various concentrations of amorphous silica nanoparticles in homogeneous suspensions of average size distribution (30, 48, 118, and 535 nm SiO2) and then assessed for uptake and biochemical changes. Results of transmission electron microscopy revealed all sizes of silica were taken up into the cells and localized into the cytoplasm. The lactate dehydrogenase (LDH) assay shows LDH leakage was dose- and size-dependent with exposure to 30 and 48 nm nanoparticles. However, no LDH leakage was observed for either 118 or 535 nm nanoparticles. The mitochondrial viability assay (MTT) showed significant toxicity for 30 and 48 nm at high concentrations (100 μg/mL) compared to the 118 and 535 nm particles. Further studies were carried out to investigate if cellular reduced GSH and mitochondria membrane potential are involved in the mechanism of SiO2 toxicity. The redox potential of cells (GSH) was reduced significantly at concentrations of 50, 100, and 200 μg/mL at 30 nm nanoparticle exposures. However, silica nanoparticles larger than 30 nm showed no changes in GSH levels. Reactive oxygen species (ROS) formation did not show any significant change between controls and the exposed cells. In summary, amorphous silica nanoparticles below 100 nm induced cytotoxicity suggest size of the particles is critical to produce biological effects.

Glutathione level and glutathione-dependent enzyme activities in blood serum of patients with gastrointestinal tract tumors

Objectives Glutathione (GSH) and enzymes cooperating with it – glutathione peroxidase (GSHPx), glutathione S-transferase (GST) and glutathione reductase (GSHR) – play crucial role in cell defence against reactive oxygen species (ROS), which are implicated in tumor disease. The aim of this study was to determine if neoplastic diseases of gastrointestinal tract may influence blood GSH level and its dependent enzyme activity. Design and methods Blood serum was obtained before and after surgery from patients with gastric, liver and colorectal cancers, and colorectal cancer liver metastases. Lipid peroxidation and GSH levels, and GSH-dependent enzyme activities were determined by means of spectrophotometric methods. Results Increased level of lipid peroxidation and significant differences in GSH level and GSHPx, GST and GSHR activities were observed in serum taken before and after surgery from patients with gastrointestinal tract tumors compared to those in control serum (from healthy blood donors). Conclusions Increase of lipid peroxidation and changes in GSH level and related enzyme activities, suggest oxidative stress in serum of patients with gastrointestinal tract tumor causes, which probably arise as a result of enormous production of ROS in the system. These alterations reflect the presence of functional defence mechanism against oxidative stress related firmly to the glutathione metabolism.

In vitroeffects of alloxan/copper combinations on lipid peroxidation, protein oxidation and antioxidant enzymes

The in vitro effects of alloxan and the product of its reduction dialuric acid (alone or in combination with copper ions) on lipid peroxidation, carbonyl content, GSH level and antioxidant enzyme activities in rat liver and kidney have been studied. The effects of Cu2+/alloxan and Cu2+/dialuric acid were compared with those of Fe3+/alloxan and Fe3+/dialuric acid. Unlike alloxan, dialuric acid increased liver and kidney lipid peroxidation; similar effects were registered in the presence of Fe3+. In the presence of Cu2+/dialuric acid, the lipid peroxidation was strongly inhibited and vice versa--the liver protein oxidation was increased. Alloxan and dialuric acid, as well as their combinations with Fe3+ had no effect on the total GSH level. Both substances did not affect the Cu2+-induced changes in GSH level, glucose-6-phosphate dehydrogenase and gluthatione reductase activities. In contrast, Cu2+ had no effect on dialuric-acid induced changes in gluthatione peroxidase and superoxide dismutase activities. The present in vitro results, concerning the metal dependence of the effects of alloxan and dialuric acid, are a premise for in vivo study of alloxan effects in metal-loaded animals.

DNA Methyltransferases Modulate the Bystander Effect in Mouse Embryonic Stem Cells.

Cells exposed to radiation or other genotoxic agents can induce DNA damage and other stress responses in non-irradiated cells that are either cultured with the irradiated cells or have been exposed to culture medium from irradiated cells. This is called the bystander effect. In a previous study we found that the descendents of bystander cells exposed to Mitomycin C (MMC) are themselves capable of inducing homologous recombination in un-exposed cells. This suggests that MMC induces persistent and transmissible changes in expression in bystander cells. Bystander effects are likely caused by epigenetic mechanisms rather than “classic” mutations, i.e. changes in DNA sequence. One of the epigenetic mechanisms cells employ for changing expression is DNA methylation in which DNA methyltransferases (DNMTs) add a methyl group to the 5 carbon of cytosine. In this study we asked if ionizing radiation can induce transmissible DNA damage in bystander cells by examining if bystander cells exposed to irradiated cells were themselves able to induce damage in naive cells. Furthermore, we asked if this was dependent on DNMT activity in the irradiated cells. We irradiated wild-type (WT) and DNMT triple knockout (DNMT TKO) mouse embryonic stem cells (ESCs) and after two weeks of continuous culture, we collected conditioned medium (CM). CM was then added to cultures of naive WT ESCs (primary bystanders). Three weeks later, CM was collected from the primary bystanders and added to naïve WT cells (secondary bystanders). We assessed DNA damage by evaluating strand breaks using the alkaline Comet assay and sister chromatid exchange (SCE) analysis. As expected, we found that medium from cells irradiated with 5 Gy induced modest damage in bystander cells. The median Olive tail moment was 2.8 in bystander cells exposed to conditioned medium from irradiated cells compared to 1.0 in control bystander cells (p < 0.0001). Homologous recombination was 0.15 chromatid exchanges per chromosome compared to 0.092 in control bystanders (p < 0.0001). We also observed an increase in strand breaks in secondary bystanders of a similar magnitude to that found in primary bystanders, indicating that radiation-induced bystanders are themselves able to induce damage. In contrast to WT cells, the irradiated DNMT TKO cells did not induce strand breaks in bystander cells, as measured by the Comet assay, but did induce HR. Surprisingly, we also observed that un-irradiated DNMT TKO cells induce DNA damage in bystanders, and furthermore that the magnitude of the effect is similar to that induced by irradiated WT cells. These data suggest that methyltransferases have a complex role in bystander effects. Bystander effects may be mediated by free radicals. To see if the DNMT TKO cells had changes in antioxidant levels, glutathione (GSH) and glutathione peroxidase (GPX) activity were determined. There was no significant change in GSH levels between WT and DNMT TKO cells. However, DNMT TKO cells had significantly higher levels of GPX activity (275.4 + 19.8 mU/mg protein) compared to control cells (122.0 + 16.4 mU/mg, p= 0.0001). Taken together, these results show that radiation-induced bystander cells can themselves induce damage in un-irradiated cells and suggest that cells lacking DNA methylation activity can induce bystander effects.

Glutathione depletion activates mitogen-activated protein kinase (MAPK) pathways that display organ-specific responses and brain protection in mice

Because mitogen-activated protein kinases (MAPK) are downstream effectors of antioxidant responses, changes in GSH levels in an organism might induce organ-specific responses. To test our hypothesis, mice were treated intraperitoneally with L-buthionine- S-R-sulfoximine (BSO) to inhibit GSH synthesis. A time-related GSH depletion in the liver and kidney correlated with p38 MAPK phosphorylation and induction of thioredoxin 1 ( Tx-1) transcription. This positive regulation was associated with nuclear translocation of NF-κB and ATF-2 and c-Jun phosphorylation in the liver, but only c-Jun phosphorylation in the kidney. Increased levels of GSH were observed in the brain together with extracellular regulated kinase 2 (ERK2) activation, Nrf2 nuclear accumulation, and increases in transcription of Nrf2, xCT, γ-glutamylcysteine synthetase ( γGCSr), and Tx-1. Pretreatment with MAPK inhibitors SB203580 and U0126, or addition of the exogenous thiol N-acetylcysteine, abrogated both p38 MAPK and ERK2 activation as well as downstream effects on gene expression. No effect on γGCSr was observed. These results indicate that in mice, GSH depletion is associated with p38 MAPK phosphorylation in the liver and kidney and with ERK2 activation in the brain, in what could be considered part of the brain's protective response to thiol depletion.

A role for CFTR in the elevation of glutathione levels in the lung by oral glutathione administration

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is the only known apical glutathione (GSH) transporter in the lung. The purpose of these studies was to determine whether oral GSH or glutathione disulfide (GSSG) treatment could increase lung epithelial lining fluid (ELF) GSH levels and whether CFTR plays a role in this process. The pharmacokinetic profile of an oral bolus dose of GSH (300 mg/kg) was determined in mice. Plasma, ELF, bronchoalveolar lavage (BAL) cells, and lung tissue were analyzed for GSH content. There was a rapid elevation in the GSH levels that peaked at 30 min in the plasma and 60 min in the lung, ELF, and BAL cells after oral GSH dosing. Oral GSH treatment produced a selective increase in the reduced and active form of GSH in all lung compartments examined. Oral GSSG treatment (300 mg/kg) resulted in a smaller increase of GSH levels. To evaluate the role of CFTR in this process, Cftr knockout (KO) mice and gut-corrected Cftr KO-transgenic (Tg) mice were given an oral bolus dose of GSH (300 mg/kg) and compared with wild-type mice for changes in GSH levels in plasma, lung, ELF, and BAL cells. There was a twofold increase in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in wild-type mice; however, GSH levels only increased by 40% in the plasma, 60% in the lung, 50% in the ELF, and twofold in the BAL cells within the gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed in the uncorrected Cftr KO mice. These studies suggest that CFTR plays an important role in GSH uptake from the diet and transport processes in the lung.

1-Methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons

Decreased glutathione levels associated with increased oxidative stress are a hallmark of numerous neurodegenerative diseases, including Parkinson's disease. GSH is an important molecule that serves as an anti-oxidant and is also a major determinant of cellular redox environment. Previous studies have demonstrated that neurotoxins can cause changes in reduced and oxidized GSH levels; however, information regarding steady state levels remains unexplored. The goal of this study was to characterize changes in cellular GSH levels and its regulatory enzymes in a dopaminergic cell line (N27) following treatment with the Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP +). Cellular GSH levels were initially significantly decreased 12 h after treatment, but subsequently recovered to values greater than controls by 24 h. However, oxidized glutathione (GSSG) levels were increased 24 h following treatment, concomitant with a decrease in GSH/GSSG ratio prior to cell death. In accordance with these changes, ROS levels were also increased, confirming the presence of oxidative stress. Decreased enzymatic activities of glutathione reductase and glutamate–cysteine ligase by 20–25% were observed at early time points and partly account for changes in GSH levels after MPP + exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP + treatment was not associated with increased efflux of glutathione to the medium. These data further elucidate the mechanisms underlying GSH depletion in response to the Parkinsonian toxin, MPP +.

Molecular identification and characterisation of the glycine transporter (GLYT1) and the glutamine/glutamate transporter (ASCT2) in the rat lens

Glutathione (GSH) is an essential antioxidant required for the maintenance of lens transparency. In the lens, GSH is maintained at unusually high concentrations as a result of direct GSH uptake and/or intracellular de novo synthesis from its precursor amino acids; cysteine, glycine and glutamine/glutamate. With increasing age, the levels of GSH, particularly in the core of the lens, are significantly reduced. It has been proposed that alterations in the transport of GSH and/or its precursor amino acids may contribute to the changes in GSH levels in older lenses. As considerable uncertainty exists about the molecular identity of GSH transporters in the lens, we have focused on identifying transporters involved in the uptake of the precursor amino acids required for GSH synthesis. Previously, we identified an uptake system for cyst(e)ine mediated by the Xc − exchanger and the Excitatory Amino Acid Transporters (EAATs) in the rat lens. In this current study, we have identified and localised additional uptake systems that contribute to GSH synthesis. Transcripts for GLYT1 (glycine transporter) and ASCT2 (glutamine/glutamate transporter) were detected in rat lens fiber cells using the reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of both GLYT1 and ASCT2 at the protein level. Immunocytochemistry revealed GLYT1 expression to be restricted to cortical regions of the lens. Labelling was predominantly cytoplasmic with some labelling of the membrane. In contrast, ASCT2 was expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, ASCT2 expression was predominantly cytoplasmic. However, with deeper distance into the lens, labelling became more membraneous indicating insertion of ASCT2 into the membranes of mature fiber cells of the lens core. The molecular identification and localisation of GLYT1 and ASCT2 in the lens suggests that these transporters may be responsible for the uptake of the precursor amino acids, glycine and glutamine, which are involved in GSH synthesis. Moreover, the presence of ASCT2 in the centre of the lens raises the possibility that ASCT2 may work with the Xc − exchanger to accumulate cysteine where it can potentially act as a low molecular mass antioxidant.

Growth and antioxidant system of the cyanobacterium Synechococcus elongatus in response to microcystin-RR

Microcystins, one type of the cyanobacterial toxins, show a broad range of hazardous effects on other organisms. Most of the researches on the toxic effects of microcystins have involved in animals and higher plants. Little work, however, has been done on evaluating the mechanisms of microcystin toxicity on algae. In this study, the toxicological effects of microcystin-RR (MC-RR) on the cyanobacterium Synechococcus elongatus were investigated. For this purpose, six physio-biochemical parameters (cell optical density, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST)) were tested in algal cells when exposed to 100 μg−1 microcystin-RR. The results showed that the growth of Synechococcus elongatus (expressed as optical density) was significantly inhibited compared with the control. At the same time, the treated algae exhibited a pronounced increase in production of ROS and MDA after 6 days exposure to microcystin-RR. Significant changes in GSH levels and GSH-Px, GSH activities were also detected in algal cells, with higher values being observed in the toxin treated algae after 6 days exposure. GST activities in the treated algae exhibited a decline after exposure and rapid augmentation on day 3, thereafter, they kept at a high level when compared to the control group. GSH contents and GSH-Px activities were also significantly raised in the toxin-treated algae cells from day 3, but they showed a sharp decrease on day 4, which was the onward of cell proliferation. These results suggested that oxidative stress manifested by elevated ROS levels and MDA contents might be responsible for the toxicity of microcystin to Synechococcus elongatus and the algal cells could improve their antioxidant ability through the enhancement of enzymatic and non-enzymatic preventive substances.

Characterization of the Role of Glutathione in Repin-Induced Mitochondrial Dysfunction, Oxidative Stress and Dopaminergic Neurotoxicity in Rat Pheochromocytoma (PC12) Cells

Repin, a major constituent in extracts of the plant Centaurea repens is thought to be the active principal responsible for the development of equine nigropallidal encephalomalacia (ENE), a fatal Parkinson-like neurodegenerative disorder in horses. Although the exact mechanism by which ingestion of this weed causes ENE is uncertain, a limited body of experimental evidence suggests a critical role for the glutathione redox system. In the present study, the mechanism of repin neurotoxicity was examined in PC12 cells with a focus on determining the role of glutathione (GSH) in repin-induced mitochondrial dysfunction, oxidative stress and dopaminergic toxicity. The results demonstrate that repin reduced both cellular GSH levels and mitochondrial function in a manner that was time- and concentration-dependent. The repin-induced changes in GSH levels were found to precede the changes in mitochondrial function. Depletion of GSH with a potent GSH depletor (ethacrynic acid (EA)) and a GSH synthesis inhibitor (buthionine sulfoximine (BSO)) prior to repin treatment enhanced the repin-induced mitochondrial change. In addition, repin caused a concentration-dependent decrease in cellular dopamine levels in NGF-differentiated PC12 cells. Increases in intracellular GSH levels induced by pre-treatment with reducing agents ( N-acetyl- l-cysteine or reduced glutathione) completely protected the cells from repin-induced mitochondrial and dopaminergic toxicity. Antioxidants, coenzyme-Q and ascorbic acid completely blocked repin-induced dopaminergic toxicity. These data suggest that GSH plays a critical role in repin-induced neurotoxicity and that the maintenance of neuronal redox status may prove to be a useful strategy for the prevention and/or treatment of ENE. The results support the view that GSH depletion, leading to oxidative damage and subsequent mitochondrial dysfunction, may serve as a trigger for neuronal cell death.

Effects of ascorbic acid on high GSH and normal dog erythrocytes (I)

Dog erythrocytes with high GSH and normal GSH concentrations were compared under effects of ascorbic acid. In the presence of glucose, these two types of erythrocytes showed a similar increase in intracellular ascorbic acid without change in GSH levels. Addition of iron (Fe3+) to the incubation medium enhanced ascorbic acid accumulation. In the absence of glucose, GSH fell markedly in both types of erythrocyte and less ascorbic acid accumulated in normal GSH cells than high GSH cells. With iron, normal cells showed GSH depletion and marked methaemoglobin formation. Lipid peroxidation with ascorbic acid and iron increased at a similar rate in both types of erythrocyte and was inhibited by catalase. Ferricyanide reduction in both erythrocytes loaded with ascorbic acid were similar and increased with glucose or catalase. There was a high correlation between intracellular ascorbic acid content and ferricyanide reduction. These results suggest that high GSH and normal GSH dog erythrocytes have a similar capacity for accumulating ascorbic acid and for reducing ferricyanide. However, normal GSH erythrocytes are more susceptible to the oxidant effect of ascorbic acid than high GSH cells; this is probably due to a smaller GSH reserve, which is exhausted more rapidly under oxidative stress.

α-Tocopherol Increases the Intracellular Glutathione Level in HaCaT Keratinocytes

f -Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of f -tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with f -tocopherol for 24 h, the intracellular GSH was increased at every concentration of f -tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with f -tocopherol at 50 w M for 24 h exhibited resistance against H 2 O 2 . However, a short exposure of HaCaT keratinocytes to f -tocopherol for 1 h did not influence either the GSH level or the resistance to H 2 O 2 . These findings suggest that GSH, which is inductively synthesized by f -tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of f -tocopherol on the GSH level, BSO, which is a typical inhibitor of n -glutamylcysteine synthetase ( n -GCS), was used. When BSO was added to HaCaT keratinocytes, no action of f -tocopherol on the GSH level was observed. On the other hand, f -tocopherol resulted in the up-regulation of n -GCS-HS (heavy subunit) mRNA. In addition, water soluble f -tocopherol derivatives ( f -tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that f -tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of n -GCS-HS mRNA.

GSH Role on Platelet-Derived Growth Factor Receptor Tyrosine Phosphorylation Induced by H2O2

This study, conducted on NIH3T3 cells, demonstrates that GSH depletion obtained by buthionine sulfoximine (BSO) treatment does not affect platelet-derived growth-factor receptor (PDGFr) autophosphorylation or cell protein phosphorylation induced by exogenous addition of H2O2, while it does decrease tyrosine phosphorylation obtained by PDGF stimulation. This last effect seems due to the lack of H2O2 generation; for the first time a relation between intracellular GSH content and H2O2 production induced by PDGF has been demonstrated. Therefore, changes of GSH levels can affect the early events of the PDGFr signal pathways by redox regulation. It has also demonstrated that in NIH3T3 cells, H2O2 can directly activate tyrosine phosphorylation by a reversible effect with the involvement of SH-group. This H2O2 effect is increased by vanadate and by GSH depleting agent, diethylmaleate, which unlike BSO is able to produce H2O2 as the current study shows.

Long-standing hyperglycemia in C57BL/6J mice does not affect retinal glutathione levels or endothelial/pericyte ratio in retinal capillaries

Free radicals have been suggested to play a role in the development of diabetic retinopathy. The aim of the present study was to examine whether the metabolic perturbations caused by high-fat feeding of two strains of mice, the C57BL6/J mice and the NMRI mice, interfere with one of the free radical enzyme defense systems in the retina, i.e., glutathione (GSH), and whether morphological changes occur in the retinal vessels. C57BL/6J mice and NMRI mice were fed a high-fat diet (55%) for 18 months. High-fat fed mice of both strains developed overweight, hyperinsulinemia, and hyperlipidemia. In addition, the high-fat fed C57BL/6J mice also developed sustained hyperglycemia for at least 15 months. The C57BL/6J mice had lower retinal GSH levels than the NMRI mice, both when given a normal diet (29.6±1.2 vs. 37.1±1.4 nmol/mg protein; p<0.01) and when given a high-fat diet (27.0±1.6 vs. 34.7±2.6 nmol/mg protein; p<0.05). Despite the long-standing hyperglycemia, hyperinsulinemia and hyperlipidemia in the C57BL/6J mice, high-fat feeding did not cause any changes in the retinal tissue levels of GSH (27.0±1.6 vs. 29.6±1.2 nmol/mg protein) or cysteine (7.61±0.63 vs. 6.80±0.59 nmol/mg protein). Similarly, high-fat feeding did not affect retinal GSH or cysteine levels in NMRI mice. No light microscopical retinal vessel changes were seen, either in C57BL/6J or in NMRI mice. The study therefore shows that long-standing metabolic perturbations induced by dietary obesity do not induce signs of retinopathy in two different strains of mice. Further studies are needed to explore whether this is explained by increased expression of protecting systems making these strains of mice resistant to effects of oxidative stress.