Changes In Glutathione Content Research Articles

Pseudobombax parvifolium Hydroalcoholic Bark Extract: Chemical Characterisation and Cytotoxic, Mutagenic, and Preclinical Aspects Associated with a Protective Effect on Oxidative Stress.

Plants have long been used in traditional medicine to treat illnesses. Nevertheless, their chemical diversity requires studies to establish the extract dosage and its safe use. Pseudobombax parvifolium, an endemic species of the Brazilian Caatinga biome, is commonly used in folk medicine, due to its anti-inflammatory properties related to cellular oxidative stress; however, its biological properties have scarcely been studied. In this study, we chemically characterized the P. parvifolium hydroalcoholic bark extract (EBHE) and evaluated its cytotoxic, mutagenic, and preclinical aspects, as well as its antioxidant effect. Our phytochemical analysis revealed a significative total polyphenol content and identified loliolide for the first time in this species. Cytotoxicity, mutagenicity, and acute oral and repeated dose indicated no toxic effects on cell culture, Drosophila melanogaster, and Wistar rat exposure to different EBHE concentrations, respectively. Furthermore, we observed a significant decrease in lipid peroxidation and a mild hypoglycemic and hypolipidemic effect with repeated oral dosing of EBHE. Although there were no significant changes in glutathione content, we did observe a significant increase in superoxide dismutase at a dose of 400 mg/kg and in glutathione peroxidase at doses of 100, 200, and 400 mg/kg. These findings suggest that EBHE has potential as a source of bioactive molecules, and it can be used safely in traditional medicine and in the development of herbal medicines for application in the public health system.

Antiviral Effect of Selenomethionine on Porcine Deltacoronavirus in Pig Kidney Epithelial Cells.

Porcine deltacoronavirus (PDCoV) is an emerging porcine intestinal coronavirus in recent years, which mainly causes different degrees of vomiting and diarrhea in piglets and has caused great harm to the swine husbandry worldwide since its report. Selenium is an essential trace element for organisms and has been demonstrated to have antiviral effects. In this study, pig kidney epithelial (LLC-PK) cells were used to study the antiviral activity of selenomethionine (Se-Met) (2, 4, 8, and 16 μM) against PDCoV by detecting the replication of the virus, the expression of the mitochondrial antiviral signal protein (MAVS) protein, and the phosphorylation of interferon regulatory factor-3 (IRF-3), IFN-α, and IFN-β, and the changes in glutathione content, glutathione peroxidase, superoxide dismutase activity, and hydrogen peroxide content in the cells. The results showed that Se-Met at higher than physiological concentrations (16 μM) could significantly inhibit the replication of PDCoV in LLC-PK cells and enhance the expression of MAVS protein and the phosphorylation of IRF-3. In addition, Se-Met also improved the intracellular production of IFNα/β and antioxidant capacity with increasing doses. These data suggest that the availability of selenium through selenomethionine supports the antiviral response in porcine kidney cells, and the specific mechanism is attributed to the improved cellular antioxidant capacity and activation of the MAVS pathway by Se-Met.

Evaluation of caffeine effects on biochemical and genotoxic biomarkers in the neotropical freshwater teleost Prochilodus lineatus

Caffeine is often found in aquatic environments, leading to concerns regarding its adverse consequences for aquatic biota. Biochemical and genotoxic biomarkers were analysed in juveniles of Prochilodus lineatus to evaluate the effects of caffeine. Fish were exposed to caffeine (0.3, 3 and 30 μg L−1) for either 24 h or 168 h. Longer exposure to caffeine resulted in a significant reduction in the activity of the phase I biotransformation enzyme ethoxyresorufin-O-deethylase (EROD) in the brain but a significant increase in the liver. Changes in glutathione content (GSH), glutathione S-transferase (GST) activity, and lipid peroxidation were not found in the liver and brain of fish exposed to caffeine. DNA damage in erythrocytes were also not found. These results show that caffeine may interfere with the biotransformation mechanism of P. lineatus after 168 h exposure, but it does not generate sufficient changes to trigger a state of oxidative stress.

Diurnal changes of subcellular glutathione content in Arabidopsis thaliana

The aim of this study was to investigate diurnal changes of subcellular glutathione content in leaves of Arabidopsis thaliana by immunohistochemistry and quantitative transmission electron microscopy. The results revealed major diurnal changes in glutathione content. The highest content was found within 2 - 3 h of exposure to light (increase of 489 % in mitochondria, 318 % in plastids, 857 % in nuclei, 511 % in peroxisomes, and 900 % in the cytosol when compared to that during darkness), followed by a strong drop (49 % in mitochondria and the cytosol, 53 % in plastids, 68 % in nuclei, 38 % in peroxisomes) within the next 1 - 2 h. Glutathione content was 67 % lower in mitochondria, 76 % in plastids, 73 % in nuclei, 50 % in peroxisomes, and 68 % in the cytosol at the end of the light period which lasted for 8 h when compared to highest content. Lowest glutathione content was found in most cell compartments at the very end of the dark period. In context with previous studies it could be concluded that low glutathione content at the end of the dark period was caused by lack of glycine and cysteine due to lack of photorespiration and decreased sulfur uptake and assimilation during darkness. The rapid increase of glutathione content observed within 3 h of light was most probably caused by a strong increase in glutathione synthesis triggered by increased glycine and cysteine content. The strong drop of glutathione after that period was most probably due to elevated glutathione degradation rate or increased use of glutathione for phytochelatin synthesis.

Role of glutathione in functioning of the system of antioxidant protection in fish (review)

Oxidative stress causes damage to cell components by reactive oxygen species (ROS) originating from the effect of various chemical pollutants, such as heavy metal cations, polyaromatic hydrocarbons, organochlorine and phosphororganic pesticides, polychlorine biphenyls, dioxins, and other xenobiotics. To avoid pathological consequences of interaction between biological molecules and high-reactive ROS, cells have a detoxification mechanism that uses glutathione, a nonprotein thiol. Glutathione is a component of cellular protection against the toxic action of xenobiotics and metal cations. In the last decade, the effect of environmental pollutants on changes in glutathione concentrations in tissues of aquatic organisms has been touched on in a large number of works. This review summarizes data of the up-to-date studies on glutathione variability in fish tissues under the effect of biogenic and industrial xenobiotics.

Potential Role of Endoplasmic Reticulum Stress in Pathogenesis of Diabetic Retinopathy.

Diabetes mellitus is a metabolic disease that leads to several complications which include retinopathy. Multiple biochemical abnormalities have been proposed to explain the development of retinopathy, including oxidative stress. Although the existence of oxidative stress has been established in the retina from long standing diabetic animals, pathogenesis and progression of retinopathy remain unclear. In order to gain insight into the pathogenesis of diabetic retinopathy, we analyzed the levels of different oxidative stress biomarkers in the retina at early stages during the progress of streptozotocin-induced diabetes. No significant changes in glutathione content, expression of NADPH-oxidase, levels of lipid peroxidation, nor production of free radicals were observed in the retina up to 45days of diabetes induction. Likewise, a transient decrease in aconitase activity, parallel to an increase in the superoxide dismutase activity was observed at 20days of hyperglycemia, suggesting a high capacity of retina to maintain its redox homeostasis, at least at early stages of diabetes. Nonetheless, we found an early and time-dependent increase in the levels of oxidized proteins, which was not affected by the administration of the antioxidant quercetin. Also, positive immunoreactivity to the reticulum stress protein CHOP was found in glial Müller cells of diabetic rat retinas. These findings suggest the occurrence of endoplasmic reticulum stress as a primary event in retina pathogenesis in diabetes.

Compartment-specific importance of glutathione during abiotic and biotic stress.

The tripeptide thiol glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is the most important sulfur containing antioxidant in plants and essential for plant defense against abiotic and biotic stress conditions. It is involved in the detoxification of reactive oxygen species (ROS), redox signaling, the modulation of defense gene expression, and the regulation of enzymatic activities. Even though changes in glutathione contents are well documented in plants and its roles in plant defense are well established, still too little is known about its compartment-specific importance during abiotic and biotic stress conditions. Due to technical advances in the visualization of glutathione and the redox state through microscopical methods some progress was made in the last few years in studying the importance of subcellular glutathione contents during stress conditions in plants. This review summarizes the data available on compartment-specific importance of glutathione in the protection against abiotic and biotic stress conditions such as high light stress, exposure to cadmium, drought, and pathogen attack (Pseudomonas, Botrytis, tobacco mosaic virus). The data will be discussed in connection with the subcellular accumulation of ROS during these conditions and glutathione synthesis which are both highly compartment specific (e.g., glutathione synthesis takes place in chloroplasts and the cytosol). Thus this review will reveal the compartment-specific importance of glutathione during abiotic and biotic stress conditions.

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It has been previously observed that reperfusion of ischemic kidneys produces much more damage than that caused by the period of ischemia alone. However, the nature of the cellular insult that produces these changes is unknown. In this study, 30 male domestic native dogs were selected and randomly divided into five groups (n=6): G1:sham-operated(sham)(n=6), G2:ischemic-reperfusion induced group(I/R); about 45 min ischemia and 3 h reperfusion (n=6) , G3: I/R+melatonin(3 mg/kg) (n=6), G4: I/R + allopurinol (10 mg/kg)(n=6), G5: I/R +allopurinol + melatonin (n=6). Unilateral ureteral obstruction by ventral midline celiotomy in the left kidney was performed and after 14 days, renal samples were taken by ultrasound guided biopsy and samples were fixed in 10% buffered formalin and sent to pathology for grading. Data were analyzed by Mann-Whitney U test. P<0.05 was considered statistically significant. The degree of protection against renal dysfunction was less in comparison to animals treated with melatonin; we would suggest that melatonin-evoked changes in glutathione content and redox state results from increased activities of the enzymes of glutathione metabolism. There are a number of possible explanations for this finding, the most likely being that factors other than oxygen free radicals (for example, irreversible loss of mitochondrial function and ATP depletion and/or activation of proteolytic enzymes) contribute to renal dysfunction as the period of ischemia is extended.   Key words: Allopurinol, melatonin, renal ischemic reperfusion, dog.

Growth, Oxidative Damage and Antioxidant Responses in Greengram (Vigna radiata L.) under Short‐term Salinity Stress and its Recovery

AbstractSpatio‐temporal differences under short‐term NaCl‐salinity (0, 50, 100, 150 mm l−1) stress for 24 h and post‐NaCl recovery after 24 h on the growth, water relations, ionic composition, proline and antioxidants of 12‐day‐old roots, stem and leaves of Vigna radiata were observed. Fresh and dry weight, relative water content and K+ ion decreased, whereas, Na+ ion and Na+/K+ ratio increased significantly in roots, stem and leaves. Post‐NaCl recovered roots, stem and leaves showed similar results with lower values. Chlorophyll and carotenoid pigments decreased significantly in the leaves. Peroxide and lipid peroxidation level increased significantly with higher ratio in stressed compared to recovered leaves. Ascorbate and proline content increased significantly with no significant change in glutathione content in stressed roots, stem and leaves. Significant decrease in protein content, SOD, CAT, POX, APX and GR activities was observed in roots with no significant change in stem and leaves. The recovered parts showed similar results except increased POX and GR activities showing a tissue‐specific response to NaCl‐salinity stress. Improved tolerance to salt stress may be accomplished by increased capacity of antioxidative defence system and by lower level of lipid peroxidation and improvement in the plant water status, activities of some of the antioxidants in the recovered parts suggest that significant tissue differences in response to salt stress in V. radiata is closely related to differences in the activities of antioxidants, ion and proline content.

Prevention of Alloxan-induced Diabetes by Se-Methylselenocysteine Pretreatment in Rats: The Effect on Antioxidant System in Pancreas

In this study, we assessed the effects of Se-methylselenocysteine (MSC) pretreatment on the antioxidant system in the pancreas and the development of alloxan-induced diabetes in rats. The rats were treated with MSC at a dose of 0.75 ㎎/rat/day for 2 weeks. The MSC-treated rats evidenced significantly increased glutathione content, GSH/GSSG ratio, and glutathione peroxidase (GPx) and glutathione reductase (GRd) activities in the pancreas. Diabetes was induced via alloxan injection. The alloxan-diabetic rats evidenced significantly reduced glutathione content and glucose 6-phosphate dehydrogenase (G6PD) activity and increased catalase activity in the pancreas, when measured 3 days after the alloxan injection. 2-week MSC pretreatment was shown to prevent the alloxan-induced hyperglycemia as well as changes in glutathione content, G6PD activity, and catalase activity. The results of this study indicate that the prevention of alloxan-diabetes by MSC pretreatment is associated with its effects on antioxidants in the pancreas, namely, the increase in cellular content and the reduction of glutathione by the facilitation of glutathione recycling induced via increased GPx, GRd, and G6PD activities.

Origanum Vulgare Induces Apoptosis in Human Colon Cancer Caco2 Cells

Oregano spice is widely used in the Mediterranean diet, which is associated with a low risk for colon cancer. Although the medicinal benefits of oregano, such as the anti-inflammatory and antimicrobial activities, are well known; nonetheless, only few data are available on its effect in cancer prevention, especially concerning the mechanism of action. Here, we investigated the effect of Origanum vulgare ethanolic extracts on redox balance, cell proliferation, and cell death in colon adenocarcinoma Caco2 cells. Oregano extract leads to growth arrest and cell death in a dose- and time-dependent manner. Changes in glutathione content, as well as the increase in its oxidized form, may be involved in oregano-triggered death. Both extrinsic and intrinsic apoptotic pathways appear to be activated by spice extract. Our findings suggest that oregano amounts found in the Mediterranean diet can exert proapoptotic effects, which are selective for cancer cells. Moreover, whole extract, instead of a specific component, can be responsible for the observed cytotoxic effects.

Virus‐Induced Changes in the Subcellular Distribution of Glutathione Precursors in Cucurbita pepo (L.)

Changes in glutathione contents occur in plants during environmental stress situations, such as pathogen attack, as the formation of reactive oxygen species leads to the activation of the antioxidative defence system. As glutathione is synthesized out of its constituents cysteine, glycine, and glutamate the availability of these components will limit glutathione synthesis in plants especially during stress situations and therefore the ability of the plant to fight oxidative stress. To gain a deeper insight into possible limitations of glutathione synthesis during pathogen attack the present investigations were aimed to study how the subcellular distribution of glutathione precursors correlates with the subcellular distribution of glutathione during virus attack in plants. Selective antibodies against cysteine, glutamate, and glycine were used to study the impact of Zucchini yellow mosaic virus (ZYMV) infection on glutathione precursor contents within different cell compartments of cells from Cucurbita pepo (L.) plants with the transmission electron microscope (TEM). Generally, levels of cysteine and glutamate were found to be strongly decreased in most cell compartments of younger and older leaves including glutathione-producing cell compartments such as plastids and the cytosol. The strongest decrease of cysteine was found in plastids (- 54 %) and mitochondria (- 51 %) of younger leaves and in vacuoles (- 37 %) and plastids (- 29 %) of older leaves. The strongest decrease of glutamate in younger leaves occurred in peroxisomes (- 67 %) and nuclei (- 58 %) and in peroxisomes (- 64 %) and plastids (- 52 %) of the older ones. Glycine levels were found to be strongly decreased (- 63 % in mitochondria and - 53 % in plastids) in most cell compartments of older leaves and strongly increased (about 50 % in plastids and peroxisomes) in all cell compartments of the younger ones. These results indicate that low glycine contents in the older leaves were responsible for low levels of glutathione in these organs during ZYMV infection rather than limited amounts of cysteine or glutamate. Glutathione precursors were virtually absent in cell walls and intercellular spaces and play therefore no important role during ZYMV attack in the apoplast. While glutamate was absent in vacuoles, elevated levels of glycine (up to 30 %) and decreased cysteine contents (up to - 37 %) were observed in vacuoles during ZYMV infection. The impact of the present results on the current knowledge about glutathione synthesis and degradation on the cellular level during ZYMV infection are discussed.

Tissue-specific changes in glutathione content of hypoxic newborn pigs reoxygenated with 21% or 100% oxygen

We compared the responses towards oxidative stress in the liver, lung, brain, heart, kidney and small intestine of hypoxic newborn animals resuscitated with 21% or 100% oxygen. After stabilization, piglets (1–3 days, 1.6–2.0 kg, n = 8/group) were randomized to receive 2 h of alveolar hypoxia (FiO 2 = 0.10–0.14) followed by reoxygenation with 21% or 100% oxygen for 1 h and then another hour with 21% oxygen. Controls were sham-operated without hypoxia–reoxygenation. At the end of the experiment, tissues from liver, lung, brain, heart, kidney and small intestine were collected and tested for GSH, GSSG and lipid peroxidation levels and histological examination. In normoxic controls, liver had the highest GSH level, followed by brain, heart, lung, small intestine and kidney which had the highest level of oxidative stress markers (GSSG level and GSSG:GSH ratio). Hypoxic–reoxygenated piglets had the highest GSSG levels and GSSG:GSH ratio in the kidney. Hypoxic piglets resuscitated with 100% oxygen had higher GSSG:GSH ratios in the lung and liver, but not in the kidney, brain, heart and small intestine, than controls, which were not different from the 21% group. No significant differences in peroxidation and histological tissue damage were found between groups in the liver and lung. We concluded that although hypoxic piglets resuscitated with 100% oxygen have higher oxidative stress in the liver and lung than with 21% oxygen, there are no significant differences in peroxidation and histological tissue damage acutely.

Evoked Alterations In Some Biochemical Parameters And Protein Electrophoretic Pattern Of Some Tissues Of Broiler Chicken Treated With Coumarin

Coumarin compounds are used as dormant substances in agriculture. Physiologically, they are highly active, for example, act as inhibitor of growth of microorganisms. Moreover, coumarin is a maturally occuring substance most frequently used as a fragrance enhancer (in cosmetics, perfumes and soap) and stabilizer. In this study, chemical analysis of chicken tissues (brain, liver and kidney) as lipid constituents, cholesterol, liver, glycogen, glutathione, lipid per oxidase and protein electrophoresis (fractions) were tested after intermuscular (i.m.) injection with doses of 100mg/kg.b.wt. and 200mg/kg.b.wt. of coumarin for 10 days (every other day). The present study revealed that coumarin at a dose of 100mg/kg.b.wt. induced insignificant changes in the total lipid (T.L) of brain, liver and kidney tissues of broiler chicken. Otherwise, the high dose of coumarin (200 mg/ kg.b.wt.) caused a significant decrease in the T.L. of brain (P < 0.01) tissues, while insignificant change of kidney T.L. was recorded at a dose of 200 mg/kg.b.wt.. of coumarin. Also, insignificant changes of triglycerides (T.G.) and cholesterol (Chol.) content of brain, liver and kidney tissues of chicken group (G2) treated with coumarin (100mg/kg) were demonstrated. While, high dose (200 mg/kg coumarin) resulted in a significant decrease in the T.G. and Chol. of brain (P < 0.001 & P < 0.01 respectively) and liver (P < 0.001 & P < 0.001), the same dose showed insignificant changes of kidney T.G. an Chol. contents. Administration of coumarin (100mg/kg. G2) showed insignificant changes in glutathione content (GSH) of liver and kidney tissues, while significant decrease (P < 0.01) of brain GSH content was recorded compared with the control group. Besides, a dose of 100mg/kg.coumarin caused insignificant changes in lipid peroxides (TABrs) of brain and kidney tissues of chicken and significant increase (P < 0.001) of (TABrs) content of liver tissue. High dose of coumarin (200mg./kg. G3) showed significant increase of TABrs content of brain, liver, and kidney tissues (P < 0.01, P < 0.001 and P < 0.001 respectively) of broiler chicken compared with the control group. Both doses of administered coumarin (G2 & G3) caused significant decrease (P < 0.001) in the liver glycogen content. The present data revealed that coumarin caused qualitative and quantitative changes in tissues (brain, liver and kidney) protein fractionation pattern of chicken compared with that of controls. Sixteen bands were separated using polycrylamide gel electrophoresis (PAGE) – as protein fractions in both the control and coumarin treated groups. The changes (decrease or increase) in particular protein fractions may be related to the effect of xenobiotic (coumarin) on the specific genes encoding for these fractions. Thus, this work revealed that inspite of the benefit of coumarin substance, the xenobiotic effect and signs of intoxications were attained spacially at high doses of treatment.

Evoked Alterations In Some Biochemical Parameters And Protein Electrophoretic Pattern Of Some Tissues Of Broiler Chicken Treated With Coumarin

Coumarin compounds are used as dormant substances in agriculture. Physiologically, they are highly active, for example, act as inhibitor of growth of microorganisms. Moreover, coumarin is a maturally occuring substance most frequently used as a fragrance enhancer (in cosmetics, perfumes and soap) and stabilizer. In this study, chemical analysis of chicken tissues (brain, liver and kidney) as lipid constituents, cholesterol, liver, glycogen, glutathione, lipid per oxidase and protein electrophoresis (fractions) were tested after intermuscular (i.m.) injection with doses of 100mg/kg.b.wt. and 200mg/kg.b.wt. of coumarin for 10 days (every other day). The present study revealed that coumarin at a dose of 100mg/kg.b.wt. induced insignificant changes in the total lipid (T.L) of brain, liver and kidney tissues of broiler chicken. Otherwise, the high dose of coumarin (200 mg/ kg.b.wt.) caused a significant decrease in the T.L. of brain (P < 0.01) tissues, while insignificant change of kidney T.L. was recorded at a dose of 200 mg/kg.b.wt.. of coumarin. Also, insignificant changes of triglycerides (T.G.) and cholesterol (Chol.) content of brain, liver and kidney tissues of chicken group (G2) treated with coumarin (100mg/kg) were demonstrated. While, high dose (200 mg/kg coumarin) resulted in a significant decrease in the T.G. and Chol. of brain (P < 0.001 & P < 0.01 respectively) and liver (P < 0.001 & P < 0.001), the same dose showed insignificant changes of kidney T.G. an Chol. contents. Administration of coumarin (100mg/kg. G2) showed insignificant changes in glutathione content (GSH) of liver and kidney tissues, while significant decrease (P < 0.01) of brain GSH content was recorded compared with the control group. Besides, a dose of 100mg/kg.coumarin caused insignificant changes in lipid peroxides (TABrs) of brain and kidney tissues of chicken and significant increase (P < 0.001) of (TABrs) content of liver tissue. High dose of coumarin (200mg./kg. G3) showed significant increase of TABrs content of brain, liver, and kidney tissues (P < 0.01, P < 0.001 and P < 0.001 respectively) of broiler chicken compared with the control group. Both doses of administered coumarin (G2 & G3) caused significant decrease (P < 0.001) in the liver glycogen content. The present data revealed that coumarin caused qualitative and quantitative changes in tissues (brain, liver and kidney) protein fractionation pattern of chicken compared with that of controls. Sixteen bands were separated using polycrylamide gel electrophoresis (PAGE) – as protein fractions in both the control and coumarin treated groups. The changes (decrease or increase) in particular protein fractions may be related to the effect of xenobiotic (coumarin) on the specific genes encoding for these fractions. Thus, this work revealed that inspite of the benefit of coumarin substance, the xenobiotic effect and signs of intoxications were attained spacially at high doses of treatment.

Recycling processes of cellular ascorbate generate oxidative stress in pancreatic tissues in in vitro system.

Ascorbate is a reducing agent, which is also known to oxidize cellular components. Our proposed mechanism of the oxidative action is as follows: Ascorbate is concentrated in the pancreas and is leaked in adverse conditions, and oxidized to dehydroascorbate. The dehydroascorbate is carried into cells by a glucose transporter (GLUT) and reduced back to ascorbate. The reduction processes take electrons from other cellular components. Ascorbate or dehydroascorbate treatment elevated thiobarbituric acid-reactive substance (TBARS) concentrations in pancreas. The elevations in TBARS concentrations were blocked by cytochalasin B, a GLUT inhibitor. To confirm further the prooxidative action, changes in glutathione content were quantified. Glutathione concentrations were lower in ascorbate- or dehydroascorbate-treated groups. The ascorbate-induced decrease in glutathione was blocked by cytochalasin B. To prevent oxidation of ascorbate to dehydroascorbate, glutathione was added to the medium. The ascorbate plus glutathione and dehydroascorbate plus glutathione groups showed lower TBARS concentrations than those of the ascorbate and dehydroascorbate groups, respectively. There were changes in the morphology of Langerhans islets following ascorbate treatment, which disappeared following treatment with ascorbate plus cyto-chalasin B. The observations indicate that ascorbate generates oxidative stress and affects the structure of islets.

Changes in glutathione-related enzymes in tumor-bearing mice after cisplatin treatment.

The effect of cisplatin on five glutathione-related enzymes was studied in liver, kidney, and Dalton lymphoma cells of tumor-bearing mice. In liver, the activities of glutathione S-transferase, glutathione peroxidase, catalase, and superoxide dismutase decreased approximately 30-40%, 60-67%, 35-50% and 70-80% respectively, while glutathione reductase increased about 36-45% after cisplatin treatment. In kidney, catalase activity decreased by 47-82% at all time points (24-96 h) of cisplatin treatment, while glutathione S-transferase activity decreased significantly (approximately 24%) mainly at 72 h of treatment. An increase in glutathione reductase (approximately 1.5-2.5 times), glutathione peroxidase (significant at 24 h, 47%), and superoxide dismutase (approximately 15-60%) was noted in kidney after the treatment. In Dalton lymphoma cells, the activities of glutathione S-transferase, glutathione peroxidase, and catalase decreased very distinctly (approximately 2-5, 2-5 and 5-11 times, respectively) at all time points, but glutathione reductase decreased significantly only at 72 h of cisplatin treatment. Interestingly, the superoxide dismutase activity in Dalton lymphoma cells increased initially at 24-48 h and then decreased (approximately 60%) during later periods (72-96 h) of treatment. Cisplatin treatment caused a decrease in glutathione level in Dalton lymphoma cells (approximately 14-20%) and kidney (approximately 18-28%) but no change in liver. In view of the results, a definite correlation with the changes in glutathione concentrations and enzymatic activities in a tissue could not be firmly derived. It is suggested that the changes in various glutathione-related enzymes and glutathione levels in the tissues of the host during cisplatin-mediated chemotherapy could affect cellular antioxidant defense potential, which may play an important contributory role in cisplatin-mediated toxicity, particularly nephrotoxicity, and anticancer activity in the host.

Radiation effects on soluble metabolites in cultured HeLa cells examined by 1H MRS: changes in concentration of glutathione and of lipid catabolites induced by gamma rays and proton beams.

Cultured HeLa cells were irradiated with a single acute dose of either gamma rays (40 Gy) or low-energy proton beams (20 Gy). (1)H magnetic resonance spectra of intact cells harvested at different times after irradiation and of the correspondent perchloric acid (PCA) extracts prepared at different times after irradiation were run. Selected signals from glutathione and lactate were examined with the aim of investigating effects of irradiation on antioxidative stores and on mitochondrial activity. An increase of signal intensity of glutathione (GSH) takes place at 15 and 24 hr after irradiation, while a decrease of its signal intensity, accompanied by an increase of that of free glutamate, starts appearing 48 hr after irradiation. Lactate signal increases 48 hr after irradiation. Signals from lipid catabolites were also examined to explore their sensitivity in predicting the response to radiotherapy. Intensity ratios of signals of glycerophosphorylcholine and choline to that of phosphorylcholine increase with time after irradiation. Irradiating cells with gamma rays or proton beams at half a dose produces effects comparable to the metabolic variations presented here. The present experiments allow more insight into the complex pattern of the changes of GSH by irradiation and indicate that magnetic resonance spectroscopy signals from GSH, glutamate, lactate, and lipid catabolites are affected by irradiation. Finally, these data represent a first indication that the relative biological efficiency for some metabolic damage of low-energy proton beams with respect to gamma rays can reach a value of 2.

Induction of glucose-6-phosphate dehydrogenase by lipopolysaccharide contributes to preventing nitric oxide-mediated glutathione depletion in cultured rat astrocytes.

Treatment of cultured rat astrocytes with lipopolysaccharide (LPS; 1 microg/ml) increased mRNA expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting step in the pentose phosphate pathway (PPP), in a time-dependent fashion (0-24 h). This effect was accompanied by an increase in G6PD activity (1.74-fold) and in the rate of glucose oxidation through the PPP (6.32-fold). Inhibition of inducible nitric oxide synthase (iNOS) activity by 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; 50 microM) did not alter the LPS-mediated enhancement of G6PD mRNA expression or PPP activity. Blockade of nuclear factor-kappaB (NF-kappaB) activation by N-benzyloxycarbonyl-Ile-Glu-(O-tert-butyl)-Ala-leucinal (1 microM) prevented the expression of both iNOS mRNA and G6PD mRNA, suggesting that iNOS and G6PD are co-induced by LPS through a common transcriptional pathway involving NF-kappaB activation. Incubation of cells with LPS for 24 h increased intracellular NADPH concentrations (1.63-fold) as compared with untreated cells, but GSH concentrations were not modified by LPS treatment up to 60 h of incubation. However, inhibition of G6PD activity by dehydroepiandrosterone (DHEA; 100 microM), which prevented LPS-mediated enhancements in PPP activity and NADPH concentrations, caused a 50% decrease in the GSH/GSSG ratio after 24-36 h and in GSH concentrations after 60 h of incubation. Furthermore, the changes in glutathione concentrations caused by DHEA were abolished by AMT, suggesting that nitric oxide and/or its reactive derivatives would be involved in this process. From these results, we conclude that LPS-mediated G6PD expression prevents GSH depletion due to nitric oxide and suggest that this phenomenon may be a contributing factor in the defense mechanisms that protect astrocytes against nitric oxide-mediated cell injury.

Depletion of glutathione from brain cells in hyponatremia

In response to hyponatremia, brain cells extrude electrolytes and organic osmolytes, thereby minimizing brain edema. We demonstrate that rat brain is depleted of the antioxidant glutathione in response to hyponatremia and that osmotically-induced loss of glutathione makes neuronal cells more susceptible to oxidative injury. Total glutathione content of brain tissue decreased from 6.80 +/- 0.14 mumol/g dry wt in normonatremic controls to 5.00 +/- 0.31 mumol/g dry wt after 72 hours of hyponatremia. Following slow correction of hyponatremia, brain glutathione content returned to control values (6.77 +/- 0.34 mumol/g dry wt). Brain content of taurine, a beta-amino acid with antioxidant properties, similarly decreased in hyponatremia (29.6 +/- 0.9 to 17.1 +/- 1.2 mumol/g dry wt), then increased with slow correction (24.8 +/- 1.3 mumol/g dry wt). Although taurine served as an osmolyte in rat heart, liver and brain, osmotically-induced changes in glutathione content were found only in brain. We also studied osmotically-induced changes in glutathione and taurine content in C6 glioma and SK-N-SH neuroblastoma cells. In both cell lines, adaptive decreases in glutathione and taurine content were found in response to lowering medium sodium concentration from 140 mM to 100 mM. The cell content of these solutes increased after returning to media containing 140 mM sodium. Following exposure of both cell lines to hypoosmolar media, there was no increase in media content of glutathione. This suggest that osmotic depletion of glutathione is not due to cellular efflux of intact glutathione. We questioned if osmotic depletion of glutathione and taurine renders brain cells more susceptible to oxidative stress. Incubation of SK-N-SH cells with 1.0 mM H2O2 for four hours induced greater cytolytic injury in cells adapted to hypoosmolar media than in isoosmolar controls. Hypoosmolar C6 glioma cells were not significantly more sensitive to cytolytic injury from H2O2 than were cells grown in isosmolar media. We conclude that hypoosmolality induces glutathione depletion in rat brain in vivo and in cultured brain cells in vitro. Osmotic depletion of this antioxidant renders SK-N-SH neuronal cells more susceptible to oxidative injury.