Blood Glutathione as an Index of Radiation-Induced Oxidative Stress in Mice and Humans

The effect of x-rays on GSH and GSSG levels in blood was studied in mice and humans. An HPLC method that we recently developed was applied to accurately determine GSSG levels in blood. The glutathione redox status (GSH/GSSG) decreases after irradiation. This effect is mainly due to an increase in GSSG levels. Mice received single fraction radiotherapy, at total doses of 1.0 to 7.0 Gy. Changes in GSSG in mouse blood can be detected 10 min after irradiation and last for 6 h within a range of 2.0–7.0 Gy. The highest levels of GSSG (20.1 ± 2.9 ), a 4.7-fold increase as compared with controls) in mouse blood are found 2 h after radiation exposure (5 Gy). Breast and lung cancer patients received fractionated radiotherapy at total doses of 50.0 or 60.0 Gy, respectively. GSH/GSSG also decreases in humans in a dose–response fashion. Two reasons may explain the radiation-induced increase in blood GSSG: (a) the reaction of GSH with radiation-induced free radicals resulting in the formation of thyl radicals that react to produce GSSG; and (b) an increase of GSSG release from different organs (e.g., the liver) into the blood. Our results indicate that the glutathione redox ratio in blood can be used as an index of radiation-induced oxidative stress. © 1997 Elsevier Science Inc.     

Role of Oxidative Stress and the Glutathione System in Loss of Dopamine Neurons Due to Impairment of Energy Metabolism

Abstract: Alterations in the glutathione system and impairment in energy metabolism have both been implicated in the loss of dopamine neurons in Parkinson's disease. This study examined the importance of cellular glutathione and the involvement of oxidative stress in the loss of mesencephalic dopamine and GABA neurons due to inhibition of energy metabolism with malonate, the reversible, competitive inhibitor of succinate dehydrogenase. Consistent with previous findings, exposure to malonate for 24 h followed by 48 h of recovery caused a dose-dependent loss of the dopamine population with little effect on the GABA population. Toxicity was assessed by simultaneous measurement of the high-affinity uptake of [³H]dopamine and [¹⁴C]GABA. Total glutathione content in rat mesencephalic cultures was decreased by 65% with a 24-h pretreatment with 10 µM buthionine sulfoxamine. This reduction in glutathione level greatly potentiated damage to both the dopamine and GABA populations and removed the differential susceptibility between the two populations in response to malonate. These findings point to a role for oxidative stress occurring during energy impairment by malonate. Consistent with this, several spin-trapping agents, α-phenyl-tert-butyl nitrone and two cyclic nitrones, MDL 101,002 and MDL 102,832, completely prevented malonate-induced damage to the dopamine neurons in the absence of buthionine sulfoxamine. The spin-trapping agents also completely prevented toxicity to both the dopamine and GABA populations when cultures were exposed to malonate after pretreatment with buthionine sulfoxamine to reduce glutathione levels. Counts of tyrosine hydroxylase-positive neurons verified enhancement of cell loss by buthionine sulfoxamine plus malonate and protection against cell loss by the spin-trapping agents. NMDA receptors have also been shown to play a role in malonate-induced dopamine cell loss and are associated with the generation of free radicals. Consistent with this, toxicity to the dopamine neurons due to a 1-h exposure to 50 µM glutamate was attenuated by the nitrone spin traps. These findings provide evidence for an oxidative challenge occurring during inhibition of energy metabolism by malonate and show that glutathione is an important neuroprotectant for midbrain neurons during situations when energy metabolism is impaired.     

Low Glutathione and High Iron Govern the Susceptibility of Oligodendroglial Precursors to Oxidative Stress

Abstract: We have previously shown, using qualitative approaches, that oligodendroglial precursors are more readily damaged by free radicals than are astrocytes. In the present investigation we quantified the oxidative stress experienced by the cells using oxidation of dichlorofluorescin diacetate to dichlorofluorescein as a measure of oxidative stress; furthermore, we have delineated the physiological bases of the difference in susceptibility to oxidative stress found between oligodendroglial precursors and astrocytes. We demonstrate that (a) oligodendroglial precursors under normal culture conditions are under six times as much oxidative stress as astrocytes, (b) oxidative stress experienced by oligodendroglial precursors increases sixfold when exposed to 140 mW/m² of blue light, whereas astrocytic oxidative stress only doubles, (c) astrocytes have a three times higher concentration of GSH than oligodendroglial precursors, (d) oligodendroglial precursors have >20 times higher iron content than do astrocytes, and (e) oxidative stress in oligodendroglial precursors can be prevented either by chelating intracellular free iron or by raising intracellular GSH levels to astrocytic values. We conclude that GSH plays a central role in preventing free radical-mediated damage in glia.     

Glutathione as an Antioxidant and Regulatory Molecule in Plants Under Abiotic Stress Conditions

The glutathione (GSH)/glutathione disulfide (GSSG) redox couple is involved in several physiologic processes in plants under both optimal and stress conditions. It participates in the maintenance of redox homeostasis in the cells. The redox state of the GSH/GSSG couple is defined by its reducing capacity and the half-cell reduction potential, and differs in the various organs, tissues, cells, and compartments, changing during the growth and development of the plants. When characterizing this redox couple, the synthesis, degradation, oxidation, and transport of GSH and its conjugation with the sulfhydryl groups of other compounds should be considered. Under optimal growth conditions, the high GSH/GSSG ratio results in a reducing environment in the cells which maintains the appropriate structure and activity of protein molecules because of the inhibition of the formation of intermolecular disulfide bridges. In response to abiotic stresses, the GSH/GSSG ratio decreases due to the oxidation of GSH during the detoxification of reactive oxygen species (ROS) and changes in its metabolism. The lower GSH/GSSG ratio activates various defense mechanisms through a redox signalling pathway, which includes several oxidants, antioxidants, and stress hormones. In addition, GSH may control gene expression and the activity of proteins through glutathionylation and thiol-disulfide conversion. This review discusses the size and redox state of the GSH pool, including their regulation, their role in redox signalling and defense processes, and the changes caused by abiotic stress.     

Glutathione Depletion Due to Copper-Induced Phytochelatin Synthesis Causes Oxidative Stress in Silene cucubalus

The relation between loss of glutathione due to metal-induced phytochelatin synthesis and oxidative stress was studied in the roots of copper-sensitive and tolerant Silene cucubalus (L.) Wib., resistant to 1 and 40 micromolar Cu, respectively. The amount of nonprotein sulfhydryl compounds other than glutathione was taken as a measure of phytochelatins. At a supply of 20 micromolar Cu, which is toxic for sensitive plants only, phytochelatin synthesis and loss of total glutathione were observed only in sensitive plants within 6 h of exposure. When the plants were exposed to a range of copper concentrations for 3 d, a marked production of phytochelatins in sensitive plants was already observed at 0.5 micromolar Cu, whereas the production in tolerant plants was negligible at 40 micromolar or lower. The highest production in tolerant plants was only 40% of that in sensitive plants. In both varieties, the synthesis of phytochelatins was coupled to a loss of glutathione. Copper at toxic concentrations caused oxidative stress, as was evidenced by both the accumulation of lipid peroxidation products and a shift in the glutathione redox couple to a more oxidized state. Depletion of glutathione by pretreatment with buthionine sulfoximine significantly increased the oxidative damage by copper. At a comparably low glutathione level, cadmium had no effect on either lipid peroxidation or the glutathione redox couple in buthionine sulfoximine-treated plants. These results indicate that copper may specifically cause oxidative stress by depletion of the antioxidant glutathione due to phytochelatin synthesis. We conclude that copper tolerance in S. cucubalus does not depend on the production of phytochelatins but is related to the plant's ability to prevent glutathione depletion resulting from copper-induced phytochelatin production, e.g. by restricting its copper uptake.     

Cellular Mechanisms of Resistance to Chronic Oxidative Stress

Oxidative stress is implicated in several pathologies such as AIDS, Alzheimer’s disease, and Parkinson’s disease, as well as in normal aging. As a model system to study the response of cells to oxidative insults, glutamate toxicity on a mouse nerve cell line, HT-22, was examined. Glutamate exposure kills HT-22 via a nonreceptor-mediated oxidative pathway by blocking cystine uptake and causing depletion of intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species and, ultimately, apoptotic cell death. Several HT-22 subclones that are 10-fold resistant to exogenous glutamate were isolated and the mechanisms involved in resistance characterized. The expression levels of neither heat shock proteins nor apoptosis-related proteins are changed in the resistant cells. In contrast, the antioxidant enzyme catalase, but not glutathione peroxidase nor superoxide dismutase, is more highly expressed in the resistant than in the parental cells. In addition, the resistant cells have enhanced rates of GSH regeneration due to higher activities of the GSH metabolic enzymes γ-glutamylcysteine synthetase and GSH reductase, and GSH S-transferases activities are also elevated. As a consequence of these alterations, the glutamate resistant cells are also more resistant to organic hydroperoxides and anticancer drugs that affect these GSH enzymes. These results indicate that resistance to apoptotic oxidative stress may be acquired by coordinated changes in multiple antioxidant pathways.     

Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

Abstract: Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of β-amyloid is mediated via free radicals, and as the deposition of β-amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein τ that resemble those found in PHF-τ. Contrary to causing an increase in τ phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of τ and so we conclude that oxidative stress is not the direct cause of τ hyperphosphorylation and hence of PHF formation.     

Lipid Peroxidation Scavengers Prevent the Carbonylation of Cytoskeletal Brain Proteins Induced by Glutathione Depletion

In this study, we investigated the possible link between lipid peroxidation (LPO) and the formation of protein carbonyls (PCOs) during depletion of brain glutathione (GSH). To this end, rat brain slices were incubated with the GSH depletor diethyl maleate (DEM) in the absence or presence of classical LPO scavengers: trolox, caffeic acid phenethyl ester (CAPE), and butylated hydroxytoluene (BHT). All three scavengers reduced DEM-induced lipid oxidation and protein carbonylation, suggesting that intermediates/products of the LPO pathway such as lipid hydroperoxides, 4-hydroxynonenal and/or malondialdehyde are involved in the process. Additional in vitro experiments revealed that, among these products, lipid hydroperoxides are most likely responsible for protein oxidation. Interestingly, BHT prevented the carbonylation of cytoskeletal proteins but not that of soluble proteins, suggesting the existence of different mechanisms of PCO formation during GSH depletion. In pull-down experiments, beta-actin and alpha/beta-tubulin were identified as major carbonylation targets during GSH depletion, although other cytoskeletal proteins such as neurofilament proteins and glial fibrillary acidic protein were also carbonylated. These findings may be important in the context of neurological disorders that exhibit decreased GSH levels and increased protein carbonylation such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis.     

不同应激因子对小鼠肝脏金属硫蛋白诱导合成的影响

目的筛选出小鼠肝脏金属硫蛋白（MT）合成量最大的诱导方式。方法从时间-效应和剂量-效应两方面研究了重金属元素（Cd）、微量元素（Zn）、重金属与微量元素的组合（Cd＋Zn）、生理因子（饥饿）及创伤因子等五大类组合应激因子、19种诱导方式对小鼠肝脏中MT诱导合成的影响及效果。结果生理因子诱导MT量最小,饥饿诱导小鼠肝脏MT的量随饥饿程度的加重而增加,但各组间差异不显著（P〉0.05）;创伤因子诱导产生MT的量最高,其诱导量随创伤恢复时间的增加而降低,各组之间差异显著（P〈0.01）,本实验诱导峰值（9.0241±0.6441μmol/g）出现在创伤后6 h;重金属元素和微量元素诱导量居中,且两者混合诱导量比单独诱导量之和要大。结论成功筛选出诱导小鼠肝脏MT合成最有效的因子和最佳时间,为进一步大量合成MT及研究其功能等奠定基础。     

Functional Activity of Blood Polymorphonuclear Leukocytes as an Oxidative Stress Biomarker in Human Subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r = .946, p < .01).     

Effects of Nerve Growth Factor on Glutathione Peroxidase and Catalase in PC 12 Cells

Abstract: Nerve growth factor (NGF) is a member of the neuro- trophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione per- oxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC 12 cells in a time- and dose-dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.     

Paraquat-induced Oxidative Stress in Drosophila melanogaster: Effects of Melatonin, Glutathione, Serotonin, Minocycline, Lipoic Acid and Ascorbic Acid

The efficacy of melatonin, glutathione, serotonin, minocycline, lipoic acid and ascorbic acid in counteracting the toxicity of paraquat in Drosophila melanogaster was examined. Male Oregon wild strain flies were fed for 5 days with control food or food containing the test substance. They were transferred in groups of five to vials containing only filter paper soaked with 20 mM paraquat in 5% sucrose solution. Survival was determined 24 and 48 h later. All the substances assayed increased the survival of D. melanogaster. At equimolar concentrations (0.43 mM) melatonin was more effective than serotonin, lipoic acid and ascorbic acid. However, lower concentrations of glutathione (0.22 mM) and minocycline (0.05 mM) were as efficient as melatonin. The highest survival rate (38.6%) after 48 h of paraquat treatment was found with 2.15 mM of lipoic acid. No synergistic effect of melatonin with glutathione, serotonin, minocycline, lipoic acid and ascorbic acid was detected.     

Biomarkers of Oxidative Stress Study IV: ozone exposure of rats and its effect on antioxidants in plasma and bronchoalveolar lavage fluid

The objective of this study was to determine whether acutely exposing rats to ozone would result in the loss of antioxidants from plasma and bronchoalveolar lavage fluid (BALF). Additional goals were to compare analyses of the same antioxidant concentration between different laboratories, to investigate which methods have the sensitivity to detect decreased levels of antioxidants, and to identify a reliable measure of oxidative stress in ozone-exposed rats. Male Fisher rats were exposed to either 2.0 or 5.0 ppm ozone inhalation for 2 h. Blood plasma and BALF samples were collected 2, 7, and 16 h after the exposure. It was found that ascorbic acid in plasma collected from rats after the higher dose of ozone was lower at 2 h, but not later. BALF concentrations of ascorbic acid were decreased at both 2 and 7 h postexposure. Tocopherols (α, δ, γ), 5-nitro-γ-tocopherol, tocol, glutathione (GSH/GSSG), and cysteine (Cys/CySS) were not decreased, regardless of the dose or postexposure time point used for sample collection. Uric acid was significantly increased by the low dose at 2 h and the high dose at the 7 h point, probably because of the accumulation of blood plasma in the lung from ozone-increased alveolar capillary permeability. We conclude that measurements of antioxidants in plasma are not sensitive biomarkers for oxidative damage induced by ozone and are not a useful choice for the assessment of oxidative damage by ozone in vivo.     

Glutathione sulfinamide serves as a selective,endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors

Nitroxyl (HNO) donors exhibit promising pharmacological characteristics for treatment of cardiovascular disorders, cancer, and alcoholism. However, whether HNO also serves as an endogenous signaling agent is currently unknown, largely because of the inability to selectively and sensitively detect HNO in a cellular environment. Although a number of methods to detect HNO have been developed recently, sensitivity and selectivity against other nitrogen oxides or biological reductants remain problematic. To improve selectivity, the electrophilic nature of HNO has been harnessed to generate modifications of thiols and phosphines that are unique to HNO, especially compared to nitric oxide (NO). Given high bioavailability, glutathione (GSH) is expected to be a major target of HNO. As a result, the putative selective product glutathione sulfinamide (GS(O)NH₂) may serve as a high-yield biomarker of HNO production. In this work, the formation of GS(O)NH₂ after exposure to HNO donors was investigated. Fluorescent labeling followed by separation and detection using capillary zone electrophoresis with laser-induced fluorescence allowed quantitation of GS(O)NH₂ with nanomolar sensitivity, even in the presence of GSH and derivatives. Formation of GS(O)NH₂ was found to occur exclusively upon exposure of GSH to HNO donors, thus confirming selectivity. GS(O)NH₂ was detected in the lysate of cells treated with low-micromolar concentrations of HNO donors, verifying that this species has sufficient stability to server as a biomarker of HNO. Additionally, the concentration-dependent formation of GS(O)NH₂ in cells treated with an HNO donor suggests that the concentration of GS(O)NH₂ can be correlated to intracellular levels of HNO.     

Transition Metals, Ferritin, Glutathione, and Ascorbic Acid in Parkinsonian Brains

The regional distributions of iron, copper, zinc, magnesium, and calcium in parkinsonian brains were compared with those of matched controls. In mild Parkinson's disease (PD), there were no significant differences in the content of total iron between the two groups, whereas there was a significant increase in total iron and iron (III) in substantia nigra of severely affected patients. Although marked regional distributions of iron, magnesium, and calcium were present, there were no changes in magnesium, calcium, and copper in various brain areas of PD. The most notable finding was a shift in the iron (II)/iron (III) ratio in favor of iron (III) in substantia nigra and a significant increase in the iron (III)-binding, protein, ferritin. A significantly lower glutathione content was present in pooled samples of putamen, globus pallidus, substantia nigra, nucleus basalis of Meynert, amygdaloid nucleus, and frontal cortex of PD brains with severe damage to substantia nigra, whereas no significant changes were observed in clinicopathologically mild forms of PD. In all these regions, except the amygdaloid nucleus, ascorbic acid was not decreased. Reduced glutathione and the shift of the iron (II)/iron (III) ratio in favor of iron (III) suggest that these changes might contribute to pathophysiological processes underlying PD.     

Superoxide Dismutase, Catalase, and Glutathione Peroxidase Activities in Copper/Zinc-Superoxide Dismutase Transgenic Mice

Copper/zinc-superoxide dismutase (CuZn-SOD) transgenic mice overexpress the gene for human CuZn-SOD. To assess the effects of the overexpression of CuZn-SOD on the brain scavenging systems, we have measured the activities of manganese-SOD (Mn-SOD), catalase, and glutathione peroxidase (GSH-Px) in various regions of the mouse brain. In nontransgenic mice, cytosolic CuZn-SOD activity was highest in the caudate-putamen complex; this was followed by the brainstem and the hippocampus. The lowest activity was observed in the cerebellum. In transgenic mice, there were significant increases of cytosolic CuZn-SOD activity in all of these regions, with ratios varying from a twofold increase in the brainstem to 3.42-fold in the cerebellum in comparison with nontransgenic mice. Particulate Mn-SOD was similarly distributed in all brain regions, and its levels also were significantly increased in superoxide dismutase (SOD)-transgenic mice. In the brains of nontransgenic mice, cytosolic catalase activity was similar in all brain regions except the cortex, which showed less than 50% of the activity observed in the other regions. In transgenic mice, cytosolic catalase activity was significantly increased, with the cortex showing the greatest changes (133%) in comparison with nontransgenic mice. The smallest increases were observed in the hippocampus (34%). In contrast to what was observed for SOD and catalase, there were no significant changes in cytosolic GSH-Px activity in any of the brain regions examined. The present results indicate that, in addition to displaying marked increases in the levels of brain CuZn-SOD activity, SOD-transgenic mice also exhibit increases in other enzymes that scavenge oxygen-based radicals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repeated Restraint Stress Induces Oxidative Damage in Rat Hippocampus

It has been shown that emotional stress may induce oxidative damage, and considerably change the balance between pro-oxidant and antioxidant factors in the brain. The aim of this study was to verify the effect of repeated restraint stress (RRS; 1 h/day during 40 days) on several parameters of oxidative stress in the hippocampus of adult Wistar rats. We evaluated the lipid peroxide levels (assessed by TBARS levels), the production of free radicals (evaluated by the DCF test), the total radical-trapping potential (TRAP) and the total antioxidant reactivity (TAR) levels, and antioxidant enzyme activities (SOD, GPx and CAT) in hippocampus of rats. The results showed that RRS induced an increase in TBARS levels and in GPx activity, while TAR was reduced. We concluded that RRS induces oxidative stress in the rat hippocampus, and that these alterations may contribute to the deleterious effects observed after prolonged stress.     

Roles of sedentary aging and lifelong physical activity in exchange of glutathione across exercising human skeletal muscle

Reactive oxygen species (ROS) are important signaling molecules with regulatory functions, and in young and adult organisms, the formation of ROS is increased during skeletal muscle contractions. However, ROS can be deleterious to cells when not sufficiently counterbalanced by the antioxidant system. Aging is associated with accumulation of oxidative damage to lipids, DNA, and proteins. Given the pro-oxidant effect of skeletal muscle contractions, this effect of age could be a result of excessive ROS formation. We evaluated the effect of acute exercise on changes in blood redox state across the leg of young (23±1 years) and older (66±2 years) sedentary humans by measuring the whole blood concentration of the reduced (GSH) and oxidized (GSSG) forms of the antioxidant glutathione. To assess the role of physical activity, lifelong physically active older subjects (62±2 years) were included. Exercise increased the venous concentration of GSSG in an intensity-dependent manner in young sedentary subjects, suggesting an exercise-induced increase in ROS formation. In contrast, venous GSSG levels remained unaltered during exercise in the older sedentary and active groups despite a higher skeletal muscle expression of the superoxide-generating enzyme NADPH oxidase. Arterial concentration of GSH and expression of antioxidant enzymes in skeletal muscle of older active subjects were increased. The potential impairment in exercise-induced ROS formation may be an important mechanism underlying skeletal muscle and vascular dysfunction with sedentary aging. Lifelong physical activity upregulates antioxidant systems, which may be one of the mechanisms underlying the lack of exercise-induced increase in GSSG.