The multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of glutathione and glutathione disulfide from brain astrocytes

Astrocytes play an important role in the glutathione (GSH) metabolism of the brain. To test for an involvement of multidrug resistance protein (Mrp) 1 and 5 in the release of GSH and glutathione disulfide (GSSG) from astrocytes, we used astrocyte cultures from wild-type, Mrp1-deficient [Mrp1(-/-)] and Mrp5-deficient [Mrp5(-/-)] mice. During incubation of wild-type or Mrp5(-/-) astrocytes, GSH accumulated in the medium at a rate of about 3 nmol/(h.mg), whereas the export of GSH from Mrp1(-/-) astrocytes was only one-third of that. In addition, Mrp1(-/-) astrocytes had a 50% higher specific GSH content than wild-type or Mrp5(-/-) cells. The presence of 50 microm of the Mrp inhibitor MK571 inhibited the rate of GSH release from wild-type and Mrp5(-/-) astrocytes by 60%, but stimulated at the low concentration of 1 microm GSH release by 40%. In contrast, both concentrations of MK571 did not affect GSH export from Mrp1(-/-) astrocytes. Moreover, in contrast to wild-type and Mrp5(-/-) cells, GSSG export during H(2)O(2) stress was not observed for Mrp1(-/-) astrocytes. These data demonstrate that in astrocytes Mrp1 mediates 60% of the GSH export, that Mrp1 is exclusively responsible for GSSG export and that Mrp5 does not contribute to these transport processes.     

Ethanol-induced oxidative stress in rat astrocytes: role of HSP70

Ethanol intake is associated with increase in lipid peroxidation and formation of reactive oxygen species in different cerebral areas, in neurons as well as in astrocytes. The latter's integrity is essential for the normal growth of neurons. In previous studies we observed, in different cerebral areas of both acutely and chronically ethanol-treated rats, correlation between ethanol-induced oxidative stress and the increased expression of HSP70 (70 kDa heat shock proteins), chaperonins having a protective and stabilizing effect on stress–induced cell injury. In this study we examined, in vitro, the role of HSP70 on chronically ethanol-treated rat astrocytes by transfection with an anti-HSP70 antisense oligonucleotide. The results show that treatment with ethanol, from 50 to 100 mmol/L, induces a dose-dependent increase in the production of reactive oxygen species and of HSP70 levels, together with an impairment of the respiratory chain activity and a decrease in cell viability. In addition, our data indicate a drastic reduction of cellular metabolism in HSP70-deprived astrocytes, particularly when these cells were also ethanol-treated. In fact, transfection with HSP70 antisense induced moderate oxidative damage in control astrocytes and, consequently, a drastic decrease in the viability of ethanol-treated cells, with the mitochondrial functionality being particularly affected. Our results confirm that heat shock proteins confer a survival advantage to the astrocytes, preventing oxidative damage and nuclear DNA damage as well, and suggest the development of new drugs exerting a cytoprotective role either in physiological, or pathological conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

HIV-1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein-1 (Mrp1) in glial cells

Brain human immunodeficiency virus type-1 (HIV-1) infection is associated with oxidative stress, which may lead to HIV-1 encephalitis, a chronic neurodegenerative condition. In vitro , oxidative stress can be induced in glial cells by exposure to HIV-1 envelope protein glycoprotein (gp120). Multidrug resistance proteins (Mrps) are known to efflux endogenous substrates (i.e. GSH and GSSG) involved in cellular defense against oxidative stress. Altered GSH/GSSG export may contribute to oxidative damage during HIV-1 encephalitis. At present, it is unknown if gp120 exposure can alter the functional expression of Mrp isoforms. Heat-shock protein 70, inducible nitric oxide synthase, intracellular GSSG, 2',7'-dichlorofluorescein fluorescence, and extracellular nitrite were increased in primary cultures of rat astrocytes triggered with gp120, suggesting an oxidative stress response. RT-PCR and immunoblot analysis demonstrated increased Mrp1 mRNA (2.3-fold) and protein (2.2-fold), respectively, in gp120 treated astrocytes while Mrp4 mRNA or protein expression was not changed. Cellular retention of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, an established Mrp substrate, was reduced (twofold) in gp120-treated astrocytes, suggesting increased Mrp-mediated transport. In addition, GSH and GSSG export were enhanced in gp120-triggered cells. These data suggest that gp120 can up-regulate Mrp1, but not Mrp4, functional expression in cultured astrocytes. Our observation of increased GSH/GSSG efflux in response to gp120 treatment implies that Mrp isoforms may be involved in regulating the oxidative stress response in glial cells.     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 M) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50–60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.     

Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by approximately 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2*- generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4-6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.     

Formaldehyde stimulates Mrp1-mediated glutathione deprivation of cultured astrocytes

Ionizing radiations can induce oxidative stress on target tissues, acting mainly through reactive oxygen species (ROS). The aim of this work was to investigate if 17-β-estradiol (βE) was able to prevent hippocampal-related behavioral and biochemical changes induced by neonatal ionizing radiation exposure and to elucidate a potential neuroprotective mechanism. Male Wistar rats were irradiated with 5 Gy of X-rays between 24 and 48 h after birth. A subset of rats was subcutaneously administered with successive injections of βE or 17-α-estradiol (αE), prior and after irradiation. Rats were subjected to different behavioral tasks to evaluate habituation and associative memory as well as anxiety levels. Hippocampal ROS levels and protein kinase C (PKC) activity were also assessed. Results show that although βE was unable to prevent radiation-induced hippocampal PKC activity changes, most behavioral abnormalities were reversed. Moreover, hippocampal ROS levels in βE-treated irradiated rats approached control values. In addition, αE administered to irradiated animals was effective in preventing radiation-induced alterations. In conclusion, βE was able to counteract behavioral and biochemical changes induced in irradiated animals, probably acting through an antioxidant mechanism.     

星形胶质细胞通过谷胱甘肽保护MN9D细胞抵抗鱼藤酮所致氧化应激

星形胶质细胞维持神经元微环境,给予营养和代谢支持,并调节其对损伤的反应。鱼藤酮特异阻断线粒体复合物Ⅰ,长期暴露于鱼藤酮可能增加患帕金森病的几率,并引起帕金森综合征。然而,星形胶质细胞在鱼藤酮所致多巴胺能神经元损伤过程中的作用尚无报道。本研究采用多巴胺能神经元细胞系MN9D细胞模型,将经过或未经过星形胶质细胞条件培养基处理的MN9D细胞暴露于不同浓度的鱼藤酮中,用计数法测生长曲线,MTT法测细胞活性,DCFH染色流式细胞仪测氧化应激水平,比色法测还原型谷胱甘肽含量。结果显示,MN9D细胞在条件和普通培养基培养条件下生长曲线无明显差别;鱼藤酮浓度依赖性地降低细胞活性;不同浓度鱼藤酮作用24、48h后,经条件培养基处理的细胞其活性显著高于普通培养基培养的细胞：不同浓度的条件培养基都有保护作用,纯的条件培养基保护作用稍弱：预先24h条件培养基处理或同时给予鱼藤酮和条件培养基处理都有保护作用,鱼藤酮作用12h后再给予条件培养基则无保护作用;经条件培养基处理的细胞氧化应激水平降低：另外,条件培养基提高了细胞内还原型谷胱甘肽含量,缓解了鱼藤酮所致的谷胱甘肽耗竭。结果提示,星形胶质细胞可保护MN9D细胞抵抗鱼藤酮所致的氧化应激,还原型谷胱甘肽可能参与了该保护过程。     

Mitochondrial glutathione protects against cell death induced by oxidative and nitrative stress in astrocytes

The major cellular antioxidant, glutathione, is mostly localized in the cytosol but a small portion is found in mitochondria. We have recently shown that highly selective depletion of mitochondrial glutathione in astrocytes in culture markedly increased cell death induced by the peroxynitrite donor, 3-morpholino-syndnonimine. The present study was aimed at characterizing the increase in susceptibility arising from mitochondrial glutathione loss and testing the possibility that elevating this metabolite pool above normal values could be protective. The increased vulnerability of astrocytes with depleted mitochondrial glutathione to Sin-1 was confirmed. Furthermore, these cells showed marked increases in sensitivity to hydrogen peroxide and also to high concentrations of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine. The increase in cell death was mostly due to necrosis as indicated by substantially increased release of lactate dehydrogenase and staining of nuclei with propidium iodide but little change in annexin V staining and caspase 3 activation. The enhanced cell loss was blocked by prior restoration of the mitochondrial glutathione content. It was also essentially fully inhibited by treatment with cyclosporin A, consistent with a role for the mitochondrial permeability transition in the development of cell death. Susceptibility to the classical apoptosis inducer, staurosporine, was only affected to a small extent in contrast to the response to the other substances tested. Incubation of normal astrocytes with glutathione monoethylester produced large and long-lasting increases in mitochondrial glutathione content with much smaller effects on the cytosolic glutathione pool. This treatment reduced cell death on exposure to 3-morpholino-syndnonimine or hydrogen peroxide but not S-nitroso-N-acetyl-pencillamine or staurosporine. These findings provide evidence for an important role for mitochondrial glutathione in preserving cell viability during periods of oxidative or nitrative stress and indicate that increases in this glutathione pool can confer protection against some of these stressors.     

Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents

Recent evidence has been provided for astrocyte degeneration in experimental models of neurodegenerative insults associated with glutamate transport alteration. To determine whether astrocyte death can directly result from altered glutamate transport, we here investigated the effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC) on undifferentiated or differentiated cultured rat striatal astrocytes. PDC induced death of differentiated astrocytes without affecting undifferentiated astrocyte viability. Death of differentiated astrocytes was also triggered by another substrate inhibitor but not by blockers of glutamate transporters. The PDC-induced death was delayed and apoptotic, and death rate was dose and treatment duration-dependent. Although preceded by extracellular glutamate increase, this death was not mediated through glutamate receptor stimulation, as antagonists did not provide protection. It involves oxidative stress, as a decrease in glutathione contents and a dramatic raise in reactive oxygen species preceded cell loss, and as protection was provided by antioxidants. PDC induced a similar percentage of GSH depletion in the undifferentiated astrocytes, but only a slight increase in reactive oxygen species. Interestingly, undifferentiated astrocytes exhibited twofold higher basal GSH content compared with the differentiated ones, and depleting their GSH content was found to render them susceptible to PDC. Altogether, these data demonstrate that basal GSH content is a critical factor of astrocyte vulnerability to glutamate transport alteration with possible insights onto concurrent death of astrocytes and gliosis in neurodegenerative insults.     

氧应力在产朊假丝酵母发酵生产谷胱甘肽过程中的作用

为了提高产朊假丝酵母合成GSH的能力,采用外界氧应力刺激的方式对细胞进行处理。稳定期之前添加H2O2对细胞生长有抑制作用,但稳定期添加H2O2对GSH的合成有促进作用,当H2O2添加总浓度为30 mmol.L-1时,无论采用一次性添加还是补加策略,都可以提高GSH的合成能力,胞内GSH质量分数提高幅度最大接近于20%,GSH产量最多提高17%。GSH分批发酵结果表明,稳定期补加H2O2对于产朊假丝酵母细胞来说,要比一次性添加H2O2对提高胞内GSH含量并最终增加GSH产量更为有效,该结果为实现氧应力刺激下GSH的过量合成提供了条件。     

Up-regulation of P-glycoprotein expression by glutathione depletion-induced oxidative stress in rat brain microvessel endothelial cells

Glutathione (GSH) depletion has been implicated in the pathogenesis of neurological diseases. During GSH depletion, cells of the blood-brain barrier (BBB) are subjected to chronic oxidative stress. In this study, we investigated the effect of such stress, produced with the GSH synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO), on expression of P-glycoprotein (Pgp) in primary cultured rat brain microvessel endothelial cells that comprise the blood-brain barrier (BBB). Application of BSO to cell monolayers at concentrations up to 800 microm caused increases in expression of Pgp. Concentrations >or= 400 microm BSO decreased cell viability. Application of 200 microm BSO caused a significant increase in Pgp function activity, as assessed by rhodamine 123 (Rh123) accumulation experiments. At this concentration, BSO produced time-dependent decreases in levels of intracellular GSH and increases in levels of intracellular reactive oxygen species (iROS). The increases were also observed within 48 h following BSO treatment in mdr1a and mdr1b mRNA. Exposure of cells to BSO for 24 h produced maximal effects in the accumulation of iROS, and in expression and function of Pgp. The ROS scavenger N-acetylcysteine prevented ROS generation and attenuated the changes of both expression and activity of Pgp induced by BSO. Therefore, the transport of Pgp substrates may be affected by changing Pgp expression under conditions of chronic oxidative stress induced by GSH depletion.     

Cyclohexyl-linked tricyclic isoxazoles are potent and selective modulators of the multidrug resistance protein (MRP1)

Structure–activity relationship (SAR) studies on the tricyclic isoxazole series of MRP1 modulators have resulted in the identification of potent and selective inhibitors containing cyclohexyl-based linkers. These studies ultimately identified compound 21b, which reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC₅₀ = 0.093 μM), while showing no inherent cytotoxicity. Additionally, 21b inhibits ATP-dependent, MRP1-mediated LTC₄ uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC₅₀ = 0.064 μM) and shows selectivity (1115-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, 21b showed tumor regression and growth delay in MRP1-overexpressing tumors in vivo.     

Reactive oxygen intermediates and glutathione regulate the expression of cytosolic ascorbate peroxidase during iron-mediated oxidative stress in bean

Excess of free iron is thought to harm plant cells by enhancing the intracellular production of reactive oxygen intermediates (ROI). Cytosolic ascorbate peroxidase (cAPX) is an iron-containing, ROI-detoxifying enzyme induced in response to iron overload or oxidative stress. We studied the expression of cAPX in leaves of de-rooted bean plants in response to iron overload. cAPX expression, i.e., mRNA and protein, was rapidly induced in response to iron overload. This induction correlated with the increase in iron content in leaves and occurred in the light as well as in the dark. Reduced glutathione (GSH), which plays an important role in activating the ROI signal transduction pathway as well as in ROI detoxification, was found to enhance the induction of APX mRNA by iron. To determine whether cAPX induction during iron overload was due to an increase in the amount of free iron, which serves as a co-factor for cAPX synthesis, or due to iron-mediated increase in ROI production, we tested the expression of APX in leaves under low oxygen pressure. This treatment, which suppresses the formation of ROI, completely abolished the induction of cAPX mRNA during iron overload, without affecting the rate of iron uptake by plants. Taken together, our results suggest that high intracellular levels of free iron in plants lead to the enhanced production of ROI, which in turn induces the expression of cAPX, possibly using GSH as an intermediate signal. We further show, using cAPX-antisense transgenic plants, that cAPX expression is essential to prevent iron-mediated tissue damage in tobacco.     

Osmotic stress induces loss of glutathione and increases the sensitivity to oxidative stress in H9c2 cardiac myocytes

It has been observed that H9c2 cardiac cells cultured in physiologic solutions exhibit delayed cell death after repeated medium replacements, of which the cause was the relatively mild osmotic challenges during the renewal of the culture medium. Interestingly, the cell damage was associated with altered intracellular GSH homeostasis. Therefore, this study attempted to elucidate the effects of osmotic stress on GSH metabolism. In cells subjected to osmotic stress by lowering the NaCl concentration of the medium, the cell swelling was rapidly counterbalanced, but the intracellular GSH content was significantly lower in 3 h. Meanwhile, the ratio of GSH-to-GSSG was not affected. As expected, osmotic stress also increased the sensitivity to H₂O₂, which was attributable to the decrease of GSH content. The decrease of GSH content was similarly evident when the synthetic pathways of GSH were blocked by BSO or acivicin. It was concluded that osmotic stress induced the decrease of intracellular GSH content by increased consumption and this loss of GSH rendered the cells susceptible to a subsequent oxidative stress.     

Protection of cells from oxidative stress by microsomal glutathione transferase 1

Rat liver microsomal glutathione transferase 1 (MGST1) is a membrane-bound enzyme that displays both glutathione transferase and glutathione peroxidase activities. We hypothesized that physiologically relevant levels of MGST1 is able to protect cells from oxidative damage by lowering intracellular hydroperoxide levels. Such a role of MGST1 was studied in human MCF7 cell line transfected with rat liver mgst1 (sense cell) and with antisense mgst1 (antisense cell). Cytotoxicities of two hydroperoxides (cumene hydroperoxide (CuOOH) and hydrogen peroxide) were determined in both cell types using short-term and long-term cytotoxicity assays. MGST1 significantly protected against CuOOH and against hydrogen peroxide (although less pronounced and only in short-term tests). These results demonstrate that MGST1 can protect cells from both lipophilic and hydrophilic hydroperoxides, of which only the former is a substrate. After CuOOH exposure MGST1 significantly lowered intracellular ROS as determined by FACS analysis.     

Molecular modeling of the human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr³²⁴ in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated.     

Foc4 2个谷胱甘肽S-转移酶基因的克隆、鉴定及其在外源氧化胁迫下的表达

为鉴定香蕉枯萎病菌(尖孢镰刀菌古巴专化型4号生理小种,Fusarium oxysporum f.sp.cubense race 4,Foc4)中的2个假想谷胱甘肽S转移酶(GSTs),采用RT-PCR方法克隆了这2个GSTs基因cDNA编码序列,随后分别将2个基因定名为Fogst1和Fogst2。其中,Fogst1的开放阅读框长609 bp,编码202个氨基酸残基,Fogst2的开放阅读框长693 bp,编码230个氨基酸残基。进化树分析表明:Fogst1属于GSTs超家族的sigma(σ)亚型成员,Fogst2属于GSTs超家族中目前未知的亚家族成员。为了验证Fogst1和Fogst2的表达,分别构建了Fogst1和Fogst2的原核表达重组载体pET28a-Fogst1和pET28a-Fogst2,并将pET28a-Fogst1和pET28a-Fogst2转化到大肠杆菌表达菌株BL21,经IPTG诱导后获得以可溶形式表达的重组蛋白Fogst1和Fogst2。GSTs活性分析表明,以CDNB为底物检测,2个重组蛋白均具有GSTs酶活性。分别取外源氧化胁迫处理后1、5、12、24 h菌丝样品进行相对荧光定量PCR分析,结果表明:Fogst1和Fogst2在前5 h表达量均大幅上调,表达量随后下调并恢复正常水平。这些结果均暗示Fogst1和Fogst2可能参与了Foc4抗外源氧化胁迫过程。     

The role of multidrug resistance protein 1 (MRP1) in transport of fluorescent anions across the human erythrocyte membrane

We employed human red blood cells as a model system to check the affinity of MRP1 (Multidrug Resistance-associated Protein 1) towards fluorescein and a set of its carboxyl derivatives: 5/6-carboxyfluorescein (CF), 2,7-bis-(2-carboxyethyl)-5/6-carboxyfluorescein (BCECF) and calcein (CAL). We found significant differences in the characteristics of transport of the dyes tested across the erythrocyte membrane. Fluorescein is transported mainly in a passive way, while active efflux systems at least partially contribute to the transport of the other compounds. Inside-out vesicle studies revealed that active transport of calcein is masked by another, ATP-independent, transport activity. Inhibitor profiles of CF and BCECF transport are typical for substrates of organic anion transporters. BCECF is transported mainly via MRP1, as proven by the use of QCRL3, a monoclonal antibody known to specifically inhibit MRP1-mediated transport. Lack of effect of QCRL3 on CF uptake excludes the possibility of MRP1 being a transporter of this dye. No inhibition of CF accumulation by cGMP, thioguanine and 6-mercaptopurine suggests also that this fluorescent marker is not a substrate for MRP5, another ABC transporter identified in the human erythrocyte membrane.     

Age-related effect induced by oxidative stress on the cerebral glutathione system

In the forebrain from male Wistar rats aged 5, 15 and 25 months, age-related putative alterations in the glutathione system (reduced and oxidized glutathione; redox index) were chronically induced by the administration in drinking water of free radical generators (hydrogen peroxide, ferrous chloride) or of inhibitors of endogenous free radical defenses (diethyl-dithio-carbamate, an inhibitor of superoxide dismutase activity). In hydrogen peroxide administered rats, both reduced glutathione and the cerebral glutathione redox index markedly declined as a function of aging, whereas oxidized glutathione consistently increased. In contrast, chronic iron intake failed to modify the reduced glutathione in forebrain from the rats of the different ages tested, whereas the oxidized glutathione was increased in the older brains. The chronic intake of diethyl-dithio-carbamate enhanced the concentrations of reduced glutathione in the forebrains from the rats of the different ages tested, the oxidized glutathione being unchanged. In 15-month-old rats submitted to chronic oxidative stress, ergot alkaloids (and particularly dihydroergocriptine) interfered with cerebral glutathione system, while papaverine was always ineffective. The comprehensive analysis of the data indicates that: (a) both the type of oxidative stress and the age of the animals modulate the cerebral responsiveness to the putative modifiers in the level of tissue free radicals; (b) aging magnifies the cerebral alterations induced by oxidative stress; the (c) cerebral glutathione system may be modified by metabolic rather than by circulatory interferences; (d) a balance between the various cerebral antioxidant defenses is present, the perturbation of an antioxidant system resulting in the compensatory modified activity of component(s) of another system.