Depletion of intracellular glutathione mediates zinc-induced cell death in rat primary astrocytes

In the present study, we investigated the possible mechanisms of cellular injury induced by zinc in rat primary astrocytes and C6 glioma cells. Reactive oxygen species (ROS) production, cellular glutathione (GSH) level and mitochondrial transmembrane potential were examined. Exposure to 200-300 microM Zn2+ for 24 h resulted in significant lactate dehydrogenase (LDH) release in rat primary astrocytes and C6 glioma cells. An exposure of 200 microM Zn2+ resulted in profound morphological changes, for example, shrunken and fragmented nuclei. Pretreatment of a protein synthesis inhibitor, cycloheximide, did not attenuate cellular toxicity induced by Zn2+. Zn2+ exposure increased intracellular ROS levels by about 250%, and depleted cellular GSH within 2 h, which preceded observable LDH release from the cell. Addition of GSH, N-acetylcysteine (NAC) and ascorbic acid substantially attenuated cellular death induced by Zn+ in a concentration dependent manner. ROS production and morphological changes induced by zinc were also inhibited by co-treatment of GSH or NAC with Zn2+. Zn2+ significantly depolarized mitochondrial transmembrane potential, which was reversed by co-treatment of GSH or NAC with zinc. In summary, ROS generation, GSH depletion and mitochondrial dysfunction may be key factors in Zn2+-induced glial toxicity.     

Hepatoprotective properties of kombucha tea against TBHP-induced oxidative stress via suppression of mitochondria dependent apoptosis

Kombucha, a fermented tea (KT) is claimed to possess many beneficial properties. Recent studies have suggested that KT prevents paracetamol and carbon tetrachloride-induced hepatotoxicity. We investigated the beneficial role of KT was against tertiary butyl hydroperoxide (TBHP) induced cytotoxicity and cell death in murine hepatocytes. TBHP is a well known reactive oxygen species (ROS) inducer, and it induces oxidative stress in organ pathophysiology. In our experiments, TBHP caused a reduction in cell viability, enhanced the membrane leakage and disturbed the intra-cellular antioxidant machineries while simultaneous treatment of the cells with KT and this ROS inducer maintained membrane integrity and prevented the alterations in the cellular antioxidant status. These findings led us to explore the detailed molecular mechanisms involved in the protective effect of KT. TBHP introduced apoptosis as the primary phenomena of cell death as evidenced by flow cytometric analyses. In addition, ROS generation, changes in the mitochondrial membrane potential, cytochrome c release, activation of caspases (3 and 9) and Apaf-1 were detected confirming involvement of mitochondrial pathway in this pathophysiology. Simultaneous treatment of KT with TBHP, on the other hand, protected the cells against oxidative injury and maintained their normal physiology.In conclusion, KT was found to modulate the oxidative stress induced apoptosis in murine hepatocytes probably due to its antioxidant activity and functioning via mitochondria dependent pathways and could be beneficial against liver diseases, where oxidative stress is known to play a crucial role.     

Effect of lemon verbena supplementation on muscular damage markers, proinflammatory cytokines release and neutrophils�� oxidative stress in chronic exercise

Intense exercise is directly related to muscular damage and oxidative stress due to excessive reactive oxygen species (ROS) in both, plasma and white blood cells. Nevertheless, exercise-derived ROS are essential to regulate cellular adaptation to exercise. Studies on antioxidant supplements have provided controversial results. The purpose of this study was to determine the effect of moderate antioxidant supplementation (lemon verbena extract) in healthy male volunteers that followed a 90-min running eccentric exercise protocol for 21 days. Antioxidant enzymes activities and oxidative stress markers were measured in neutrophils. Besides, inflammatory cytokines and muscular damage were determined in whole blood and serum samples, respectively. Intense running exercise for 21 days induced antioxidant response in neutrophils of trained male through the increase of the antioxidant enzymes catalase, glutathione peroxidase and glutathione reductase. Supplementation with moderate levels of an antioxidant lemon verbena extract did not block this cellular adaptive response and also reduced exercise-induced oxidative damage of proteins and lipids in neutrophils and decreased myeloperoxidase activity. Moreover, lemon verbena supplementation maintained or decreased the level of serum transaminases activity indicating a protection of muscular tissue. Exercise induced a decrease of interleukin-6 and interleukin-1β levels after 21 days measured in basal conditions, which was not inhibited by antioxidant supplementation. Therefore, moderate antioxidant supplementation with lemon verbena extract protects neutrophils against oxidative damage, decreases the signs of muscular damage in chronic running exercise without blocking the cellular adaptation to exercise.     

Tertiary butyl hydroperoxide induced oxidative damage in mice erythrocytes: Protection by taurine

The present study was undertaken to investigate the protective role of taurine, against t-butyl hydroperoxide (TBHP) induced oxidative stress in murine erythrocytes. Erythrocytes were treated either with TBHP alone or with taurine, followed by TBHP exposure. TBHP-induced oxidative stress increased methemoglobin formation, lipid peroxidation and protein carbonylation in erythrocytes. The same exposure, however, depleted cellular GSH content and altered the activities of the antioxidant enzymes as well as of methemoglobin reductase; reduced activities of Ca(+) and Na(+)/K(+) ATPase and intracellular ATP levels. Taurine transport inhibitor, β-alanine, treated erythrocytes showed increased phosphatidylserine externalization and ROS formation on TBHP exposure and taurine could not revert the effect. TBHP exposure increased intracellular calcium and upregulated the level of calpain. Administration of taurine could, however, prevent the TBHP induced oxidative imbalance. Electron micrographs of erythrocytes showed changed morphology with an increase in the number of echinocytes. Taurine treatment could restore the normal levels of the antioxidant enzymes and metabolites of the erythrocytes. Results suggest that the oxidative insult introduced in erythrocytes by TBHP administration is prevented by taurine mainly via membrane stabilization.     

Skin aging and oxidative stress: Equol’s anti-aging effects via biochemical and molecular mechanisms

Oxygen in biology is essential for life. It comes at a cost during normal cellular function, where reactive oxygen species (ROS) are generated by oxidative metabolism. Human skin exposed to solar ultra-violet radiation (UVR) dramatically increases ROS production/oxidative stress. It is important to understand the characteristics of human skin and how chronological (intrinsic) aging and photo-aging (extrinsic aging) occur via the impact of ROS production by cascade signaling pathways. The goal is to oppose or neutralize ROS insults to maintain good dermal health. Botanicals, as active ingredients, represent one of the largest categories used in dermatology and cosmeceuticals to combat skin aging. An emerging botanical is equol, a polyphenolic/isoflavonoid molecule found in plants and food products and via gastrointestinal metabolism from precursor compounds. Introductory sections cover oxygen, free radicals (ROS), oxidative stress, antioxidants, human skin aging, cellular/molecular ROS events in skin, steroid enzymes/receptors/hormonal actions and genetic factors in aging skin. The main focus of this review covers the characteristics of equol (phytoestrogenic, antioxidant and enhancement of extracellular matrix properties) to reduce skin aging along with its anti-aging skin influences via reducing oxidative stress cascade events by a variety of biochemical/molecular actions and mechanisms to enhance human dermal health.     

Mitochondrial glutathione and oxidative stress: implications for pulmonary oxygen toxicity in premature infants

Administration of supplemental oxygen, despite being an important clinical therapy, can cause significant lung damage. Because they have underdeveloped lungs, prematurely born human infants frequently require supportive therapies that employ elevated oxygen concentrations, which put them at risk for developing pulmonary oxygen toxicity. This risk is made even greater by the immaturity of their cellular antioxidant defenses. Although the exact mechanisms of oxygen toxicity are still not fully defined, cellular damage is probably mediated by increased production of chemically reactive oxygen species (ROS) in the mitochondria. Cellular protection against ROS is provided by a variety of antioxidant molecules and enzymes, including the glutathione (GSH)-dependent antioxidant system. The GSH-dependent antioxidant enzyme system provides vital cellular protection against ROS, particularly hydrogen peroxide and certain organic hydroperoxides, under pathological and toxicological conditions, by using selenium-dependent and -independent peroxidases to reduce hydrogen peroxide or lipid peroxides to water or the respective alcohols, with the concurrent oxidation of GSH to glutathione disulfide (GSSG). In the mitochondria, limitations of GSH synthesis and transmembrane transport suggest that optimal functioning of the mitochondrial GSH system, and maintenance of adequate thiol-disulfide redox tone is essential to protect against the injurious effects of ROS. Manipulation of endogenous GSH concentrations can alter cellular responses to oxidant injury. Beneficial effects are evident when intracellular GSH concentrations are increased. In conditions that increase mitochondrial production of ROS, such as exposure to high concentrations of oxygen, therapies based on enhancing mitochondrial GSH concentrations could be highly beneficial.     

Is antioxidant potential of the mitochondrial targeted ubiquinone derivative MitoQ conserved in cells lacking mtDNA?

MitoQ has been developed as a mitochondrial targeted antioxidant for diseases associated with oxidative stress. Here we show that MitoQ blocks the generation of reactive oxygen species (ROS) and mitochondrial protein thiol oxidation, and preserves mitochondrial function and ultrastructure after glutathione (GSH) depletion. Furthermore, the antioxidant effect of MitoQ is conserved in cells lacking mitochondrial DNA, indicating that its antioxidant properties do not depend on a functional electron transport chain (ETC). Our results elucidate the antioxidant mechanism of MitoQ and suggest that it may be a useful therapeutic for disorders associated with a dysfunctional ETC and increased ROS production.     

Subcellular compartmentalization of glutathione: correlations with parameters of oxidative stress related to genotoxicity

Glutathione (GSH) is a major component of the antioxidant defence system of mammalian cells and is found in subcellular pools within the cytoplasm, nucleus and mitochondria. To evaluate the relationships between these pools and parameters of oxidative stress related to genotoxicity, wild type (WT) and 8-oxo-2'-deoxyguanosine glycosylase 1 (OGG1)-null (mOGG1(-/-)) mouse embryonic fibroblasts (MEF) were treated with buthionine sulphoximine (BSO; 0-1000 microM, 24 h), an inhibitor of GSH biosynthesis. BSO treatment resulted in a concentration-dependent depletion of GSH from the cytoplasm, but depletion of mitochondrial and nuclear GSH occurred only at concentrations > or =100 microM. GSH levels were correlated with reactive oxygen species (ROS), lipid peroxidation (measured as the increase in the genotoxic end-product malondialdehyde (MDA)) and oxidative DNA modifications, measured as both frank DNA strand-breaks (FSB) and oxidized purine lesions (OxP) using the alkaline comet assay with formamidopyrimidine DNA glycosylase (FPG) modification; this system allowed for the identification of BSO-induced DNA modifications as primarily mutagenic 8-oxo-2'-deoxyguanosine lesions. A number of significant correlations were observed. First, negative linear correlations were observed between mitochondrial GSH and ROS (r = -0.985 and r = -0.961 for WT and mOGG1(-/-) MEF, respectively), and mitochondrial GSH and MDA (r = -0.967 and r = -0.963 for WT and mOGG1(-/-) MEF, respectively). Second, positive linear correlations were observed between ROS and MDA (r = 0.996 and r = 0.935 for WT and mOGG1(-/-) MEF, respectively), and ROS and OxP (r = 0.938 and r = 0.981 for WT and mOGG1(-/-) MEF, respectively). Finally, oxidative DNA modifications displayed a negative linear correlation with nuclear GSH (r = -0.963 and -0.951 between nuclear GSH and FSB and OxP, respectively, for WT MEF and r = -0.960 between nuclear GSH and OxP in mOGG1(-/-) MEF), thus, demonstrating the genotoxic potential of compounds that deplete GSH. The findings highlight the critical roles of the mitochondrial and nuclear GSH pools in protecting cellular components, particularly DNA, from oxidative modification.     

Selective oxidative stress in cell nuclei by nuclear-targeted D-amino acid oxidase

The effects of nuclear-localized oxidative stress on both nuclear antioxidant systems, and the processes that they regulate, are not clearly understood. Here, we targeted a hydrogen peroxide (H(2)O(2))-producing enzyme, D-amino acid oxidase (DAAO), to the nucleus (NLS-DAAO) and used this to generate H(2)O(2) in the nuclei of cells. On addition of N-acetyl-D-alanine (NADA), a substrate of DAAO, to NLS-DAAO-transfected HeLa cells, a twofold increase in ROS production relative to untreated, transfected control was observed. Staining of cellular thiols confirmed that NLS-DAAO-induced ROS selectively modified the nuclear thiol pool, whereas the cytoplasmic pool remained unchanged. Furthermore, NLS-DAAO/NADA-induced ROS caused significant oxidation of the nuclear GSH pool, as measured by nuclear protein S-glutathionylation (Pr-SSG), but under the same conditions, nuclear Trx1 redox state was not altered significantly. NF-kappaB reporter activity was diminished by NLS-DAAO/NADA-stimulated nuclear oxidation. We conclude that nuclear GSH is more susceptible to localized oxidation than is nuclear Trx1. Furthermore, the attenuation of NF-kappaB reporter activity in the absence of nuclear Trx1 oxidation suggests that critical nuclear redox proteins are subject to control by S-glutathionylation during oxidative stress in the nucleus.     

Endothelial function and oxidative stress.

Increased oxidative stress impairs endothelial function and is thought to mediate vascular disease. Several pathological conditions increase the production of reactive oxygen species (ROS) in the vascular wall, including hypercholesterolemia, diabetes, and hypertension. These conditions are associated with endothelial dysfunction and cardiovascular disease. Thus, overall vascular function is dependent upon the balance of oxidant and antioxidant mechanisms, which determines endothelial function. Endothelial function is usually defined as nitric oxide (NO) production and/or bioavailability. Because ROS can interact and inactivate NO, vascular oxidative stress can lead to decrease NO bioavailability. This results in endothelial dysfunction and increased risk of cardiovascular diseases. Several pharmacological approaches have been used to improve endothelial function and decrease oxidative stress. These include treatment modalities that augment the antioxidant defense mechanisms, increase NO production, and inhibit ROS-generating enzymes. This review provides an overview of the relationship between endothelial function and oxidative stress.     

d-Amphetamine-induced cytotoxicity and oxidative stress in isolated rat hepatocytes

Amphetamines (AMP) are potent psychostimulants and commonly used drugs of abuse. Its chronic administration creates tolerance and addiction and also associated with neurotoxicity and hepatocellular damage through oxidative stress. The present study was designed to evaluate the cytotoxic effects as well as the oxidative stress induced by d-amphetamines in isolated rat hepatocytes. Hepatocytes were isolated by collagenase perfusion technique and were exposed to different concentrations of AMP (0.2, 0.4, 0.8 and 1.6 mM) in a time-course experiment for up to 2 h. AMP exposure induced a significant decrease in cell viability and a significant increase in the leakage of hepatic enzymes {lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and asparate aminotransferase (AST)} in a concentration and time-related manner. In the same experiment, GSH content and thiobarbituric acid reactive substances (TBARS) generation were determined as indices of oxidative stress and lipid peroxidation respectively. AMP exposure results in a significant decrease in cellular GSH content as well as a significant enhancement of TBARS accumulation in a concentration and time-related manners. The obtained results suggested that 2-h exposure of hepatocytes to AMP (0.8 mM) was accompanied by submaximal responses. Therefore, a subsequent dose–response experiment was designed to evaluate the role of GSH modulation and oxidative stress in AMP toxicity in hepatocytes at 2 h. LDH release and TBARS generation were used as indicators in this experiment. Pretreatment with the GSH-depleting agents, chlorodinitrobenzene (CDNB), buthionine sulfoximine (BSO), or bis(chloroethyl)-nitrosurea (BCNU) enhanced the cytotoxicity of AMP. Conversely, pretreatment with GSH or sulfhydryl compounds such as methionine (MT), cysteine (CYS) or dithiothreitol (DTT) attenuated AMP toxicity. Similarly, co-incubation with enzymatic antioxidants, superoxide dismutase (SOD) or catalase (CAT) or iron chelator, desferroxiamine (DFO) or the hydroxyl radical scavengers, dimethylsulfoxide (DMSO) exhibited significant protection against AMP cytotoxicity. The present results indicate that AMP has a potential cytotoxic effect in isolated rat hepatocytes. AMP cytotoxicity is concentration-dependent. GSH depletion and oxidative stress play an important role in enhancing hepatotoxic potential of AMP in isolated rat hepatocyte. Thiol group-donors, antioxidants, free radical scavengers and iron chelators can play a critical role against AMP-induced cellular damage.     

Oxygen, the lead actor in the pathophysiologic drama: enactment of the trinity of normoxia, hypoxia, and hyperoxia in disease and therapy

Aerobic life has evolved a dependence on molecular oxygen for its mere survival. Mitochondrial oxidative phosphorylation absolutely requires oxygen to generate the currency of energy in aerobes. The physiologic homeostasis of these organisms is strictly maintained by optimal cellular and tissue-oxygenation status through complex oxygen-sensing mechanisms, signaling cascades, and transport processes. In the event of fluctuating oxygen levels leading to either an increase (hyperoxia) or decrease (hypoxia) in cellular oxygen, the organism faces a crisis involving depletion of energy reserves, altered cell-signaling cascades, oxidative reactions/events, and cell death or tissue damage. Molecular oxygen is activated by both nonenzymatic and enzymatic mechanisms into highly reactive oxygen species (ROS). Aerobes have evolved effective antioxidant defenses to counteract the reactivity of ROS. Although the ROS are also required for many normal physiologic functions of the aerobes, overwhelming production of ROS coupled with their insufficient scavenging by endogenous antioxidants will lead to detrimental oxidative stress. Needless to say, molecular oxygen is at the center of oxygenation, oxidative phosphorylation, and oxidative stress. This review focuses on the biology and pathophysiology of oxygen, with an emphasis on transport, sensing, and activation of oxygen, oxidative phosphorylation, oxygenation, oxidative stress, and oxygen therapy.     

Histopathological aspects and local implications of oxidative stress in patients with oral lichen planus

Aerobic life is connected with continuous production of free radicals, particularly reactive oxygen species (ROS). Cells posses an enzymatic and non-enzymatic antioxidant system to maintain redox homeostasis. Oxidant-antioxidant imbalance resulting in excessive accumulation of ROS is defined as oxidative stress. Oral lichen planus (OLP) is a chronic inflammation of unknown etiology. Several researchers suggest that oxidative stress is implicated in the pathogenesis of this disorder. The aim of this study was to evaluate the histopathological alterations and the status of local oxidative stress and antioxidant defense system in patients with OLP. We evaluated and compared the local levels of oxidative stress biomarkers malondialdehyde (MDA) and glutathione (GSH) in patients with OLP with that of normal controls. Increased levels of MDA and decreased levels of GSH suggest the idea of oxidative stress implication in the pathogenesis of oral lichen planus.     

Combination therapy targeting cancer metabolism

Cancer cells undergo significant metabolic adaptation. Cellular transformation enhances both glycolysis and mitochondrial respiration efficiency through the induction of HIF-1α and HIF-2α. In this process, energy production and synthesis of macromolecules are maximized with minimal ROS accumulation. Furthermore, a series of antioxidant enzymes are induced to mitigate the damaging effects of ROS. Examination of these metabolic changes provides rationale for a synergistic approach to combination anti-cancer therapy; targeted inhibition of HIF and inhibition of cellular defenses against oxidative stress.     

Antioxidant effects of sulfur-containing amino acids

Sulfur is an essential element for the entire biological kingdom because of its incorporation into amino acids, proteins and other biomolecules. Sulfur atoms are also important in the iron-containing flavoenzymes. Unlike humans, plants can use inorganic sulfur to synthesize sulfur-containing amino acids. Therefore, plants are an important source of sulfur for humans. Sulfur-containing compounds are found in all body cells and are indispensable for life. Some of sulfur-containing antioxidant compounds are, cysteine, methionine, taurine, glutathione, lipoic acid, mercaptopropionylglycine, N-acetylcysteine, and the three major organosulfur compounds of garlic oil, diallylsulfide, diallyldisulfide and diallyltrisulfide. In a comparison of the structure-function relationship among these sulfur-containing antioxidant compounds, dihydrolipoic acid (the reduced form of LA) is the most effective antioxidant. Dihydrolipoic acid contains two sulfhydryl groups and can undergo further oxidation reaction to form lipoic acid. The antioxidative activities of sulfur-containing compounds follow a general trend, the more highly reduced forms are stronger antioxidants and the number of sulfur atoms determine, at least in part, their modulatory activites on the glutathione related antioxidant enzymes. In this article, the antioxidant effects and the antioxidative activities, of sulfur-containing amino acids, are reviewed. In addition, the general antioxidant effects and the structure-function relationship of some sulfur-containing compounds are also reviewed.     

Oxidative stress and haematological changes in immobilized rats

Immobilization stress induces formation of reactive oxygen species (ROS) and leads to the oxidative injury in various tissues. In this study, the effects of immobilization stress on peripheral blood cells distribution, plasma level of thiobarbituric acid reactive substances (TBARS), and activities of antioxidant enzymes in erythrocytes were investigated in male Fischer rats. A significant increase in plasma TBARS was observed during and after the stress. Dramatic increases of neutrophils and monocytes imply that ROS formation resulted from their activation. Furthermore, the antioxidant activities of catalase and superoxide dismutase (SOD) in erythrocytes were dramatically increased during and after the stress, while a large fall in erythrocyte number was observed. These findings suggest that the activation of immune cells can be a source of the immobilization-induced ROS production, and that antioxidant enzymes in erythrocytes play an important role in preventing the ROS-induced injuries.     

Differential effect of PMS777, a new type of acetylcholinesterase inhibitor, and galanthamine on oxidative injury induced in human neuroblastoma SK-N-SH cells

In the search for highly selective and potent cholinesterase inhibitors (AChEI) being able to improve oxidative injury, PMS777, a tetrahydrofuran derivative, was designed as a novel dual PAF and acetylcholinesterase inhibitor. The aim of this study was to investigate the modulatory effects of PMS777 and galanthamine, another AChEI, on the oxidative injury induced in neuronal cells. The SK-N-SH cells stimulated with LPS+IL-(1beta) were selected to investigate the direct inhibitory effect of PMS777 and galanthamine. LPS+IL-(1beta) induced oxidative injury as assessed by ROS production (29%), GSH depletion (11%) and loss of mitochondrial activity (22%). GSH depletion was never decreased by either drug. In contrast, ROS production and mitochondrial activity were totally prevented by addition of PMS777 but not galanthamine. PMS777 also inhibits butylcholinesterase and it shows selectivity for acetylcholinesterase. Thus, this PAF antagonist inaugurates a new type of AChEI, able to fight oxidative injury. Therefore, PMS777 could be of interest on patients with cognitive impairments and inflammatory damage, as in AD.