Changes in GSH-antioxidant system induced by daunorubicin in human normal and diabetic fibroblasts

We investigated the effect of daunorubicin on glutathione content and activity of GSH-related enzymes in cultured normal and diabetic human fibroblasts. Cells were incubated with 4 microM daunorubicin (DNR) for 2 h followed by culture in drug-free medium for up to 72 h. Treatment of diabetic cells with the drug caused a time-dependent depletion of intracellular GSH and a decrease of the GSH to total glutathione ratio. GSH depletion was accompanied by apoptotic changes in morphology of the nucleus. Analysis of GSH-related enzymes showed a significant increase of the activities of Se-dependent and Se-independent peroxidases and glutathione S-transferase. In contrast, glutathione reductase activity was reduced by 50%. Significant differences between normal and diabetic cells exposed to DNR were observed in the level of GST and Se-dependent glutathione peroxidase activities. These findings indicated that daunorubicin efficiently affects the GSH antioxidant defense system both in normal and diabetic fibroblasts leading to disturbances in glutathione content as well as in the activity of GSH-related enzymes.     

Glutathione metabolism and antioxidant responses during Eleutherococcus senticosus somatic embryo development in a bioreactor

Compared to non-embryogenic callus, proembryonic mass, globular, and heart-shaped embryos of Eleutherococcus senticosus had higher levels of endogenous reduced glutathione (GSH). GSH content declined during the course of the embryo development (torpedo and cotyledon). Similarly, glutathione reductase that is involved in the recycling of GSH providing a constant intracellular level of GSH was also higher in globular and heart-shaped embryos. The transient increase in GSH contents also correlated with the changes in measured γ-glutamylcysteine synthetase activity over the same period. The endogenous levels of oxidized glutathione showed similar trend during development of the somatic embryos, whereas it declined in maturing somatic embryos. A pronounced increase in glutathione-S-transferase, glutathione peroxidase, catalase, and guaiacol peroxidase activity was observed during somatic embryo maturation. Ascorbate-glutathione cycle enzymes (ascorbate peroxidase; dehydroascorbate reductase and monodehydroascorbate reductase) activities also induced indicated that antioxidant enzymes played an important role during embryo development. These results suggested that the coordinated up-regulations of the antioxidant enzymes and glutathione redox system provide protection during somatic embryo development in E. senticosus. Antioxidant responses through alterations of the glutathione redox systems, have been described in the present studies have a significant role in somatic embryo development.     

Cytotoxicity of daunorubicin in trisomic (+21) human fibroblasts: relation to drug uptake and cell membrane fluidity

The influence of daunorubicin (DNR) on survival of human normal (S-126) and trisomic, with respect to chromosome 21 (T-164; S-240), skin fibroblasts and some parameters related to it, such as intracellular drug accumulation, distribution and interaction with cell membrane, were studied. The in vitro growth-inhibition assay indicated that DNR was less cytotoxic for trisomic than for normal cells. Comparison of kinetic parameters and intracellular distribution of this compound showed that the uptake and the amount of intracellular free DNR were greater in normal than in trisomic cells. Contrary to this, there were no significant differences between the amount of DNA-bound drug in both types of cells. TMA-DPH and 12-AS fluorescence anisotropy measurements demonstrated that DNR decreased lipid fluidity in the inner hydrophobic region of plasma membrane in both cell types, but did not influence the fluidity of the outer surface of membrane. We conclude that fibroblasts derived from individuals affected with Down's syndrome are better protected from the damage induced by DNR than normal cells.     

GSH system in relation to redox state in dystrophic skin fibroblasts

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

Role of antioxidant defense during different stages of preadult life cycle in European corn borer (Ostrinia nubilalis, Hubn.): Diapause and metamorphosis

Antioxidant enzymes, total glutathione (GSH), and ascorbic acid (ASA) were determined in whole body homogenates of nondiapausing larvae, diapausing larvae during the diapausing period (October, December, and February), and in pupae emerged from both diapausing and nondiapausing larvae of the European corn borer (Ostrinia nubilalis, Hubn., Lepidoptera: Pyralidae). The activities of catalase, selenium nondependent glutathione peroxidase (GPx), and glutathione-S-transferase (GST), as well as the content of GSH and ASA, were found to vary throughout the larval diapause. Compared to diapausing larvae, nondiapausing larvae were higher in levels of catalase, GPx, GST, and dehydroascorbate reductase (DHAR) activity. GSH content was also increased. However, nondiapausing larvae contained less ASA than diapausing ones. Pupae had higher GPx and GST activity and an increased ASA content compared to larvae. The pupae emerged from nondiapausing larvae had higher GST, glutathione reductase (GR), and DHAR activities, but lower GPx activity and ASA content than those emerged from diapausing larvae. Correlation analysis revealed differences in the way the antioxidant level is equilibrated for a particular stage and developmental pattern. The results suggest that cellular antioxidants are involved in both the protection of cells and the regulation of redox levels during the pre-adult stages of Ostrinia nubilalis. Arch. Insect Biochem. Physiol. 55:79-89, 2004.     

Subcellular localization and modification with ageing of glutathione, glutathione peroxidase and glutathione reductase activities in human fibroblasts

Differential centrifugation and isopycnic equilibration in density gradients were used to localize glutathione (GSH), glutathione peroxidase and glutathione reductase in the subcellular organelles of WI-38 fibroblasts. GSH was present in all the subcellular fractions, whereas the glutathione peroxidase and reductase activities were restrained to the cytoplasm and the mitochondrial fractions. After equilibration in density gradients, the results showed the presence of GSH, glutathione peroxidase and glutathione reductase in both the cytoplasm and mitochondria. GSH was also located in plasma membranes and probably in peroxisomes, endoplasmic reticulum and lysosomal membranes. Evolution of GSH in ageing fibroblasts showed a sudden increase of its concentration just before cell death. The glutathione peroxidase activity already decreases in the early passages, while the decrease of the glutathione reductase activity was constant and reached a drastic low level at the end of the culture. In conclusion, GSH is probably involved in the cell degeneration associated with ageing but because of its multiple functions and its ubiquitous localization, it is difficult to assert to which extent this metabolite is implicated in the ageing process.     

Some aspects of glutathione metabolism in ataxia-telangiectasia fibroblasts

Levels of glutathione (GSH) and two enzymes involved in GSH metabolism, glutathione reductase (GR) and glutathione-S-transferase(s) (GST), were measured in four SV40-transformed human fibroblast cell lines. MRC5-V1 and GM0637, derived from normal individuals, had mean GSH levels of 4.2 and 6.5 nmoles/10(6) cells, respectively. TAT2SF and AT5BIVA, both from ataxia-telangiectasia (A-T) patients, respectively had 6.5 and 4.2 nmol/10(6) cells, indicating that basal GSH levels were similar in A-T and normal cells. There was some variation in GST activity among the four cell lines but deficiency in this enzyme cannot be associated with radiosensitivity in A-T. When GR activity was measured, A-T cells had approximately 82 per cent of the mean normal activity. Though statistically significant, (P = 0.05), this small deficiency could be due to chance and is unlikely to be responsible for the radiosensitive phenotype of A-T.     

Hepatoprotective and antioxidant property of Andrographis paniculata (Nees) in BHC induced liver damage in mice

Andrographis paniculata (AP) treatment prevents BHC induced increase in the activities of enzymes y-Glutamyl transpeptidase, glutathione-S-transferase and lipid peroxidation. The activities of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and the levels of glutathione were decreased following BHC effect. Administration of AP showed protective effects in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase as well the level of glutathione. The activity of lipid peroxidase was also decreased. The result indicate antioxidant and hepatoprotective action of A. paniculata.     

Influence of estrogen on glutathione levels and glutathione-metabolizing enzymes in uteri and R3230AC mammary tumors of rats

The glutathione content and the activities of several enzymes in its metabolism, glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase, were assayed in uteri obtained from estrogen-treated rats and in R3230AC mammary adenocarcinomas obtained from ovariectomized, intact and estrogen-treated hosts. Normal mammary glands, obtained 10–12 days post-partum, were also examined for these parameters.A daily pharmacological dose of 0.4 μg of estradiol-17β induced a maximal increase in uterine weight and in reduced glutathione (GSH); higher doses of estrogen did not significantly increase either of these parameters. Levels of oxidized glutathione (GSSG) were comparable in both estrogen-treated and untreated rats. The time course of the estrogen-induced uterotrophic response was associated with increases in glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase activities with the increased GSH level preceding the increase in uterine weight. Compared to neoplasms from intact or ovariectomized animals, tumors from estrogen-treated hosts exhibited significant decreases in levels of GSSG and GSH, as well as in glutathione reductase and glutathione peroxidase activities, but demonstrated a significant elevation of γ-glutamyl transpeptidase activity. Normal glands from lactating rats had decreased GSH levels, lower activities of glutathione reductase and glutathione peroxidase, but elevated γ-glutamyl transpeptidase activity versus tumors from intact rats. Tumors from estrogen-treated rats more closely resembled mammary glands during lactation. The divergent growth responses elicited by estrogen in the uterus and mammary tumor are correlated with the observed changes in GSH levels and enzymes involved in glutathione metabolism.     

Oxygen toxicity in the nervous tissue: Comparison of the antioxidant defense of rat brain and sciatic nerve

Nervous tissue, central and peripheral, is, as any other, subject to variations in oxygen tension, and to the attack of different xenobiotics; these situations may promote the generation of activated oxygen species of free radical character. Results are presented showing that the content of total glutathione (GSH) in brain is 10-fold that found in the sciatic nerve of the rat (2620 vs. 261 nmol/g wet weight, respectively). The existence of a relatively high superoxide dismutase activity in peripheral nervous tissue, when compared with brain or liver, in combination with the DT-diaphorase activity detected in the sciatic nerve might represent an effective defense mechanism against quinone toxicity, as is also discussed. Nervous tissue, both central and peripheral lack Se-independent GSH peroxidase activity. Finally, the activities of other glutathione-related enzymes studied in the sciatic nerve are very low, when compared with the central nervous tissue, thus suggesting a higher susceptibility of peripheral tissue to oxidative stress damage, since GSH concentration and/or any GSH-related enzymatic activities, e.g. GSH peroxidase or glutathione disulfide reductase, might become limiting.     

Coordinate induction of glutathione biosynthesis and glutathione-metabolizing enzymes is correlated with salt tolerance in tomato

The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that differ in stress tolerance. Salt increased GSH content and GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant Lycopersicon pennellii (Lpa) but not in Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of gamma-glutamylcysteine synthetase protein, an effect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.     

Activity of glutathione-dependent enzymes, catalase and superoxide dismutase in the liver and myocardium of rats with vitamin A deficiency

The alimentary deficiency of vitamin A causes marked shifts in the metabolism of GSH: the levels of GSH, GSSG and cysteine in the liver increase, while the activities of glutathione-S-transferase (using glycerol as substrate) and gamma-glutamyltransferase in the liver show a rise. At the same time, vitamin A deficiency causes a decrease of the glutathione peroxidase and catalase activity in the liver. The data obtained are discussed in terms of the role of GSH and enzymes of GSH metabolism in the protection of cells against the damaging influence of lipid peroxidation.     

Hepatoprotective effects of saponarin, isolated from Gypsophila trichotoma Wend. on cocaine-induced oxidative stress in rats

The antioxidant effect of saponarin, which is the main flavone isolated from Gypsophila trichotoma Wend., and its protection against cocaine hepatotoxicity were investigated in male Wistar rats. The animals were treated with cocaine (40 mg/kg i.p.) alone and also after 3 consecutive days of pretreatment with saponarin (80 mg/kg p.o.). After 18 hours the rats were sacrificed by decapitation. The production of thiobarbituric acid reactive substances, reduced glutathione (GSH) and the activity of the following antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase were assessed in liver homogenate. Administered alone, cocaine induced significant hepatotoxicity manifested with GSH depletion and reduced antioxidant defences. Saponarin pretreatment, however, decreased cocaine toxicity both by increasing GSH levels and antioxidant enzyme activities. The results of this study proved the antioxidant activity of saponarin and its protective effect against cocaine-induced oxidative stress and hepatotoxicity.     

Glutathione system adaptation to acute stress in the heart of rats during different regimes of hypoxia training

The intensity of lipid peroxidation (LPO), reduced and oxidized glutathione (GSH and GSSG) contents, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase (G-6-PDH), and NADP-isocitrate dehydrogenase (NADP-IDH) activities were studied in the heart of male rats exposed to two modes of intermittent hypoxic training (IHT): I-breathing in normobaric chamber with 7% O2 gas mixture for 5 min with 15 min normoxic intervals 4 times daily during 3 weeks; II-breathing by 12% O2 gas mixture in the same manner). After adaptation to hypoxia, the rats were subjected to 6h-immobilization stress. It has been shown that stress action after IHT (regime I) caused the increase in LPO and the shift of GSH/GSSG to disulfides. A disbalance in antioxidative defense system was determined by the decrease in glutatione peroxidase, G-6-PDH activities, and GSH content. The support of glutathione reductase activity under stress in this group with simultaneous decrease of enzyme activity in the pentose phosphate pathway was realized through the participation of NADP-IDH. Hypoxic training in regime II induced LPO decrease in the heart tissue after stress. The increase in the heart GSH content, optimal balance of glutathione-related enzymes in this group evidences for the dependence of adaptation effects on the vigor of hypoxic exposition. Our results suggest the active participation of glutathione system in the formation of adaptation reactions under the extreme factor influences through the action on intracellular red/ox potential as well as effectiveness of antioxidant defense.     

Curative effect of methionine on certain enzymes of chick kidney cortex under lanthanum toxicity situation.

Acute single dose administration of lanthanum chloride (250 mg/kg body wt, ip) to chicks have been found to alter the levels of enzymes of the antioxidant defence system of chick renal cortex fractions. Such changes involved significant decrease in activities of glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase and catalase of kidney epithelial cells. However glutathione-S-transferase activity was not altered. Glutathione and total thiol contents were decreased while lipoperoxidative reactions in kidney-cortex was significantly enhanced. The data indicate that amelioration of lanthanum toxicity condition by methionine supplementation may be due to the methionine serving as a precursor of glutathione.     

Effect of saffron on thymocyte proliferation, intracellular glutathione levels and its antitumor activity.

Liposome encapsulation of saffron effectively enhanced its antitumor activity towards Sarcoma-180 (S-180) and Ehrlich ascites carcinoma solid tumors in mice. Significant inhibition (P < 0.001) in the growth of these tumors was observed as compared with vehicle (control) mice. In the presence of phytohemagglutinin (PHA), a T cell mitogen, saffron stimulated non-specific proliferation of lymphocytes in vitro. The intracellular reduced glutathione and related enzymes, i.e. glutathione reductase and glutathione-S-transferase, of S-180 tumor cells were significantly elevated when incubated with saffron, possibly acting to maintain functional levels of other antioxidants. Our studies indicate the antioxidant activity of saffron.     

Effects of resveratrol and 4-hexylresorcinol on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

The protective effects of resveratrol and 4-hexylresorcinol against oxidative DNA damage in human lymphocytes induced by hydrogen peroxide were investigated. Resveratrol and 4-hexylresorcinol showed no cytotoxicity to human lymphocytes at the tested concentration (10-100 μM). In addition, DNA damage in human lymphocytes induced by H 2 O 2 was inhibited by resveratrol and 4-hexylresorcinol. Resveratrol and 4-hexylresorcinol at concentrations of 10-100 μM induced an increase in glutathione (GSH) levels in a concentration-dependent manner. Moreover, these two compounds also induced activity of glutathione peroxidase (GPX) and glutathione reductase (GR). The activity of glutathione-S-transferase (GST) in human lymphocytes was induced by resveratrol. Resveratrol and 4-hexylresorcinol inhibited the activity of catalase (CAT). These data indicate that the inhibition of resveratrol and 4-hexylresorcinol on oxidative DNA damage in human lymphocytes induced by H 2 O 2 might be attributed to increase levels of GSH and modulation of antioxidant enzymes (GPX, GR and GST).     

Antioxidant Enzymatic System in Neuronal and Glial Cells Enriched Fractions of Rat Brain After Aluminum Exposure

The aim of this work was to investigate as to how neurons and glial cells separated from the brain cortex respond to oxidative stress induced by aluminum. Female SD rats were exposed to aluminum at the dose level of 100 mg/kg b.w. for 8 weeks. Neuronal and glial cell-enriched fractions were obtained from rat cerebral cortex by sieving the trypsinated homogenate through a series of nylon meshes, followed by centrifugation on ficoll density gradient. Total glutathione content, glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-s-transferase (GST) along with antioxidant enzymes superoxide dismutase (SOD), catalase were estimated in neuronal and glial-enriched fractions in both control (N-c and G-c) and aluminum exposed animals (N-a and G-a). Secondary products of lipid peroxidation that is MDA levels were estimated by measuring the (TBARS) levels. Our results indicate that TBARS levels were significantly higher in glial cell fraction of unexposed controls (Gc) than the neuronal cells (Nc). Correspondingly the glial cells had higher levels of GSH, GSSG, GPx and GST where as neurons had higher levels of catalase, SOD and GR. Following aluminum exposures significant increase in the TBARS levels was observed in neurons as compared to glial cells which also showed a significant decrease in SOD and catalase activity. The decrease in the TBARS levels in the glial cells could be related to the increase in the GSH levels, GR activity, and GST activity which were found to be increased in glial enriched fractions following aluminum exposure. The increase in activity of various enzymes viz GR, GST in glial cells as compared to neurons suggests that glial cells are actively involved in glutathione homeostasis. Our conclusion is that glial and neurons isolated from rat cerebral cortex show a varied pattern of important antioxidant enzymes and glial cells are more capable of handling the oxidative stress conditions.     

Assay of free-radical toxicity and antioxidant effect on the Hep 3B cell line: a test survey using lindane

The content of reduced glutathione and of glutathione disulfide as well as the activities of glutathione reductase, glutathione peroxidase, glutathione S-transferases, catalase and superoxide dismutases were determined in human hepatoma Hep 3B cells in relation to free-radical toxicity in order to appreciate the defense capacities of these cells compared to data on normal hepatocytes. When Hep 3B cells were exposed to lindane, a known inducer of free-radical production, superoxide dismutase activity appeared as the best-adapted cellular parameter for early detection of the resulting free-radical toxicity.Abbreviations AAS atomic absorption spectrometry - CDNB 1-chloro-2,4-dinitrobenzene - DMEM Dulbecco's modified Eagle medium - GPx glutathione peroxidase - G.Red glutathione reductase - GSH reduced glutathione - GSSG glutathione disulfide - GST glutathione S-transferases - Prot proteins - SOD superoxide dismutase