Astrocyte-rich Primary Cultures Research Articles

Modulation of Cellular Levels of Adenosine Phosphates and Creatine Phosphate in Cultured Primary Astrocytes

Adenosine triphosphate (ATP) is the main energy currency of all cells, while creatine phosphate (CrP) is considered as a buffer of high energy-bond phosphate that facilitates rapid regeneration of ATP from adenosine diphosphate (ADP). Astrocyte-rich primary cultures contain ATP, ADP and adenosine monophosphate (AMP) in average specific contents of 36.0 ± 6.4 nmol/mg, 2.9 ± 2.1 nmol/mg and 1.7 ± 2.1 nmol/mg, respectively, which establish an adenylate energy charge of 0.92 ± 0.04. The average specific cellular CrP level was found to be 25.9 ± 10.8 nmol/mg and the CrP/ATP ratio was 0.74 ± 0.28. The specific cellular CrP content, but not the ATP content, declined with the age of the culture. Absence of fetal calf serum for 24 h caused a partial loss in the cellular contents of both CrP and ATP, while application of creatine for 24 h doubled the cellular CrP content and the CrP/ATP ratio, but did not affect ATP levels. In glucose-deprived astrocytes, the high cellular ATP and CrP contents were rapidly depleted within minutes after application of the glycolysis inhibitor 2-deoxyglucose and the respiratory chain inhibitor antimycin A. For those conditions, the decline in CrP levels always preceded that of ATP contents. In contrast, incubation of glucose-fed astrocytes for up to 30 min with antimycin A had little effect on the high cellular ATP content, while the CrP level was significantly lowered. These data demonstrate the importance of cellular CrP for maintaining a high cellular ATP content in astrocytes during episodes of impaired ATP regeneration.

The Antidiabetic Drug Metformin Stimulates Glycolytic Lactate Production in Cultured Primary Rat Astrocytes.

Metformin is the most frequently used drug for the treatment of type 2 diabetes in humans. However, only little is known about effects of metformin on brain metabolism. To investigate potential metabolic consequences of an exposure of brain cells to metformin, we incubated rat astrocyte-rich primary cultures with this compound. Metformin in concentrations of up to 30mM did not acutely compromise the viability of astrocytes, but caused a time- and concentration-dependent increase in cellular glucose consumption and lactate production. For acute incubations in the hour range, the presence of 10mM metformin doubled the glycolytic flux, while already 1mM metformin doubled glycolytic flux during incubation for 24h. In addition to metformin, also other guanidino compounds increased astrocytic lactate production. After 4h of incubation, half-maximal stimulation of glycolysis was observed for metformin, guanidine and phenformin at concentrations of around 3mM, 3mM and 30µM, respectively. The acute stimulation of glycolytic lactate production by metformin was persistent after removal of extracellular metformin and was also observed, if glucose was absent from the incubation medium or replaced by other hexoses. The metformin-induced stimulation of glycolytic flux was not prevented by compound C, an inhibitor of AMP-dependent protein kinase, nor was it additive to the stimulation of glycolytic flux caused by respiratory chain inhibitors. These data demonstrate that the antidiabetic drug metformin has the potential to strongly activate glycolytic lactate production in brain astrocytes.

Comparison of Primary and Secondary Rat Astrocyte Cultures Regarding Glucose and Glutathione Metabolism and the Accumulation of Iron Oxide Nanoparticles

Astrocyte-rich primary cultures (APCs) are frequently used as a model system for the investigation of properties of brain astrocytes. However, as APCs contain a substantial number of microglial and oligodendroglial cells, biochemical parameters determined for such cultures may at least in part reflect also the presence of the contaminating cell types. To lower the potential contributions of microglial and oligodendroglial cells on properties of the astrocytes in APCs we prepared rat astrocyte-rich secondary cultures (ASCs) by subculturing of APCs and compared these ASCs with APCs regarding basal metabolic parameters, specific enzyme activities and the accumulation of iron oxide nanoparticles. Immunocytochemical characterization revealed that ASCs contained only minute amounts of microglial and oligodendroglial cells. ASCs and APCs did not significantly differ in their specific glucose consumption and lactate production rates, in their specific iron and glutathione contents, in their specific activities of various enzymes involved in glucose and glutathione metabolism nor in their accumulation of iron oxide nanoparticles. Thus, the absence or presence of some contaminating microglial and oligodendroglial cells appears not to substantially modulate the investigated metabolic parameters of astrocyte cultures.

8-Hydroxy-efavirenz, the Primary Metabolite of the Antiretroviral Drug Efavirenz, Stimulates the Glycolytic Flux in Cultured Rat Astrocytes

In active antiretroviral therapy antiretroviral drugs are employed for the restoration of a functional immune system in patients suffering from the acquired immunodeficiency syndrome. However, potential adverse effects of such compounds to brain cells are discussed in connection with the development of neurocognitive impairments in patients. To investigate potential effects of antiretroviral drugs on cell viability and the glycolytic flux of brain cells, astrocyte-rich primary cultures were exposed to various antiretroviral compounds, including the non-nucleoside reverse transcriptase inhibitor efavirenz. In a concentration of 10μM, neither efavirenz nor any of the other investigated antiretroviral compounds acutely compromised the cell viability nor altered glucose consumption or lactate production. In contrast, the primary metabolite of efavirenz, 8-hydroxy-efavirenz, stimulated the glycolytic flux in viable astrocytes in a time- and concentration-dependent manner with half-maximal and maximal effects at concentrations of 5 and 10μM, respectively. The stimulation of glycolytic flux by 8-hydroxy-efavirenz was not additive to that obtained for astrocytes that were treated with the respiratory chain inhibitor rotenone and was abolished by removal of extracellular 8-hydroxy-efavirenz. In a concentration of 10μM, 8-hydroxy-efavirenz and efavirenz did not affect mitochondrial respiration, while both compounds lowered in a concentration of 60μM significantly the oxygen consumption by mitochondria that had been isolated form cultured astrocytes, suggesting that the stimulation of glycolytic flux by 8-hydroxy-efavrienz is not caused by direct inhibition of respiration. The observed alteration of astrocytic glucose metabolism by 8-hydroxy-efavirenz could contribute to the adverse neurological side effects reported for patients that are chronically treated with efavirenz-containing medications.

Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures

Arsenate is a toxic compound that has been connected with neuropathies and impaired cognitive functions. To test whether arsenate affects the viability and the GSH metabolism of brain astrocytes, we have used primary astrocyte cultures as model system. Incubation of astrocytes for 2h with arsenate in concentrations of up to 10mM caused an almost linear increase in the cellular arsenic content, but did not acutely compromise cell viability. The presence of moderate concentrations of arsenate caused a time- and concentration-dependent loss of GSH from viable astrocytes which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for arsenate in a concentration of about 0.3mM. The arsenate-induced stimulated GSH export from astrocytes was prevented by MK571, an inhibitor of the multidrug resistance protein 1. Exposure of astrocytes to arsenite increased the specific cellular arsenic content and stimulated GSH export to values that were similar to those observed for arsenate-treated cells, while dimethylarsinic acid was less efficiently accumulated by the cells and did not modulate cellular and extracellular GSH levels. The observed strong stimulation of GSH export from astrocytes by arsenate suggests that disturbances of the astrocytic GSH metabolism may contribute to the observed arsenic-induced neurotoxicity.

Copper export from cultured astrocytes

Copper is an essential trace metal that is required as a catalytic co-factor or a structural component of several important enzymes. However, since excess of copper can also harm cells due to its potential to catalyse the generation of toxic reactive oxygen species, transport of copper and the cellular copper content are tightly regulated. Astrocytes are known to efficiently take up copper ions, but it was not known whether these cells are also able to export copper. Treatment of astrocyte-rich primary cultures for 24h with copper chloride caused a concentration-dependent increase in the specific cellular copper content. During further 24h incubation in the absence of copper chloride, the copper-loaded astrocytes remained viable and released up to 45% of the accumulated copper. The rate of copper export was proportional to the amount of cellular copper, was almost completely prevented by lowering the incubation temperature to 4°C and was partly prevented by the endocytosis inhibitor amiloride. Copper export is most likely mediated by the copper ATPase ATP7A, since this transporter is expressed in astrocyte cultures and its cellular location is strongly affected by the absence or the presence of extracellular copper. The potential of cultured astrocytes to export copper suggests that astrocytes provide neighbouring cells in brain with this essential trace element.

The antiretroviral protease inhibitors indinavir and nelfinavir stimulate Mrp1‐mediated GSH export from cultured brain astrocytes

Combinations of antiretroviral drugs are successfully used for the treatment of acquired immune deficiency syndrome and reduce the incidence of severe human immunodeficiency virus (HIV)-associated dementia. To test whether such drugs affect the GSH metabolism of brain cells, we have exposed astrocyte-rich primary cultures to various antiretroviral compounds. Treatment of the cultures with the protease inhibitors indinavir or nelfinavir in low micromolar concentrations resulted in a time- and concentration-dependent depletion of cellular GSH from viable cells which was accompanied by a matching increase in the extracellular GSH content. In contrast, the reverse transcriptase inhibitors zidovudine, lamivudine, efavirenz or nevirapine did not alter cellular or extracellular GSH levels. Removal of indinavir from the medium by washing the cells terminated the stimulated GSH export immediately, while the nelfinavir-induced accelerated GSH export was maintained even after removal of nelfinavir. The stimulation of the GSH export from viable astrocytes by indinavir or nelfinavir was completely prevented by the application of MK571, an inhibitor of the multidrug resistance protein 1. These data demonstrate that indinavir and nelfinavir stimulate multidrug resistance protein 1-mediated GSH export from viable astrocytes and suggest that treatment of patients with such inhibitors may affect the GSH homeostasis in brain.

Accumulation of silver nanoparticles by cultured primary brain astrocytes

Silver nanoparticles (AgNP) are components of various food industry products and arefrequently used for medical equipment and materials. Although such particles enter thevertebrate brain, little is known on their biocompatibility for brain cells. To study theconsequences of an AgNP exposure of brain cells we have treated astrocyte-richprimary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubationof cultured astrocytes with micromolar concentrations of AgNP for up to 24 hresulted in a time- and concentration-dependent accumulation of silver, but didnot compromise the cell viability nor lower the cellular glutathione content. Incontrast, the incubation of astrocytes for 4 h with identical amounts of silver asAgNO3 already severely compromised the cell viability and completely deprived the cells ofglutathione. The accumulation of AgNP by astrocytes was proportional to theconcentration of AgNP applied and significantly lowered by about 30% in thepresence of the endocytosis inhibitors chloroquine or amiloride. Incubation at4 °C reducedthe accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed toAgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulatePVP-coated AgNP in a temperature-dependent process that most likely involvesendocytotic pathways.

Copper-treatment increases the cellular GSH content and accelerates GSH export from cultured rat astrocytes

To test whether copper exposure affects astroglial glutathione (GSH) metabolism, we have exposed astrocyte-rich primary cultures with copper chloride in concentrations of up to 30μM and investigated cellular and extracellular GSH contents. Cultured astrocytes accumulated copper in a concentration-dependent manner thereby increasing the specific cellular copper content within 24h up to sevenfold. The increase in the cellular copper content was accompanied by a proportional increase in the specific cellular GSH content that reached up to 165% of the values of cells that had been incubated without copper, while the low cellular content of GSH disulfide (GSSG) remained unaltered in copper-treated cells. Also the rate of GSH export was significantly increased after copper exposure reaching up to 177% of control values. The export of GSH from control and copper-treated astrocytes was lowered by more than 70%, if cells were incubated in presence of the multidrug-resistance protein (Mrp) 1 inhibitor MK571 or at a low incubation temperature of 4°C. These data demonstrate that copper accumulation stimulates GSH synthesis and accelerates Mrp1-mediated GSH export from cultured astrocytes. These processes are likely to contribute to the resistance of astrocytes against copper toxicity and could improve the supply of GSH precursors from astrocytes to neurons.

Copper Accelerates Glycolytic Flux in Cultured Astrocytes

Astrocyte-rich primary cultures were used to investigate the consequences of a copper exposure on the glucose metabolism of astrocytes. After application of CuCl(2) (30μM) the specific cellular copper content increased from initial 1.5±0.2nmol/mg to a steady state level of 7.9±0.9nmol/mg within about 12h. The copper accumulation was accompanied by a significant increase in the extracellular lactate concentration. The stimulating effect of copper on the lactate production remained after removal of extracellular copper. Copper treatment accelerated the rates of both glucose consumption and lactate production by about 60%. The copper induced acceleration of glycolytic flux was prevented by inhibition of protein synthesis, and additive to the stimulation of glycolysis observed for inhibitors of respiration or prolyl hydroxylases. A copper induced stimulation of glycolytic flux in astrocytes could have severe consequences for the glucose metabolism of the brain in conditions of copper overload.

Formaldehyde stimulates Mrp1-mediated glutathione deprivation of cultured astrocytes

Formaldehyde (Fal) is an environmental neurotoxin that is also endogenously produced in brain. Since the tripeptide glutathione (GSH) plays an important role in detoxification processes in brain cells, we have investigated the consequences of a Fal exposure on the GSH metabolism of brain cells, using astrocyte-rich primary cultures as model system. Treatment of these cultures with Fal resulted in a rapid time- and concentration-dependent depletion of cellular GSH and a matching increase in the extracellular GSH content. Exposure of astrocytes to 1mm Fal for 3h did not compromise cell viability but almost completely deprived the cells of GSH. Half-maximal deprivation of cellular GSH was observed after application of 0.3mm Fal. This effect was rather specific for Fal, since methanol, formate or acetaldehyde did not affect cellular GSH levels. The Fal-stimulated GSH loss from viable astrocytes was completely prevented by semicarbazide-mediated chemical removal of Fal or by the application of MK571, an inhibitor of the multidrug resistance protein 1. These data demonstrate that Fal deprives astrocytes of cellular GSH by a multidrug resistance protein 1-mediated process.

2-Deoxyribose Deprives Cultured Astrocytes of their Glutathione

High concentrations of 2-deoxy-D-ribose (2dRib) have been reported to cause oxidative stress and to disturb the glutathione (GSH) metabolism of various cell types. Exposure of astrocyte-rich primary cultures to millimolar concentrations of 2dRib or its stereoisomer 2-deoxy-L-ribose, but not the incubation with ribose, 2-deoxyglucose, glucose, fructose or saccharose, lowered the cellular GSH content in a time and concentration dependent manner. After exposure for 4 h to 30 mM 2dRib the cells contained 2dRib in a concentration of about 24 mM. Under these conditions 2dRib did not compromise cell viability and the ability of the cells to synthesise GSH, nor were the cellular ratio of glutathione disulfide (GSSG) to GSH and the extracellular concentrations of GSH or GSSG increased. These data demonstrate that 2dRib deprives viable cultured astrocytes of GSH and suggest that a cellular reaction of GSH with 2dRib or its metabolites is involved in the deprivation of astrocytic GSH.

Zinc prevents the copper-induced damage of cultured astrocytes

Copper is essential for several cellular processes, but an excess of cellular copper is known to be cell toxic. To study the consequences of a copper treatment of astrocytes, we have used astrocyte-rich primary cultures as model system to investigate cellular functions and cellular integrity of these cells after application of micromolar concentrations of copper chloride. After exposure of the cells to copper, the cell-associated copper content increased strongly in a time and concentration dependent manner. While incubation of cultured astrocytes with 3 μM copper hardly affected the cells during incubation for up to 4 h, presence of 10 μM or 30 μM copper severly compromised cellular functions as demonstrated by a loss in total and soluble protein contents, a lowered MTT reduction capacity, lowered activities of the enzymes lactate dehydrogenase, glucose-6-phosphate dehydrogenase and glutathione reductase, a lowered cellular glutathione content, an increased lipid peroxidation, and an elevated membrane permeability for propidium iodide. Presence of an excess of zinc inhibited cellular copper accumulation and prevented most of the detrimental consequences of a copper exposure, suggesting that the beneficial effect of zinc against the copper-induced impairment of cultured astrocytes is mediated by inhibition of the cellular copper accumulation.

Copper accumulation by cultured astrocytes

To study copper transport in brain astrocytes, we have used astrocyte-rich primary cultures as model system. Cells in these cultures contained a basal copper content of 1.1+/-0.4 nmol per mg protein. The cellular copper content increased strongly after application of copper chloride in a time and concentration-dependent manner. Analysis of the linear copper accumulation during the first 5 min of copper exposure revealed that cultured astrocytes accumulated copper with saturable kinetics with apparent K(M)- and V(max)-values of 9.4+/-1.8 microM and 0.76+/-0.13 nmol/(min x mg protein), respectively. In contrast, incubation of astrocytes with copper in the presence of ascorbate caused a linear increase of the copper accumulation rates for copper concentrations of up to 30 microM. In addition, copper accumulation was strongly inhibited by the presence of an excess of zinc or of various other divalent metal ions. The presence of mRNA and of immunoreactivity of the copper transport protein Ctr1 in astrocyte cultures suggests that Ctr1 contributes to the observed copper accumulation. However, since some characteristics of the observed copper accumulation are not consistent with Ctr1-mediated copper transport, additional Ctr1-independent mechanism(s) are likely to be involved in astrocytic copper accumulation.

Uptake of ferrous iron by cultured rat astrocytes

Astrocytes are considered to play an important role in iron homeostasis of the brain, yet the mechanisms involved in the uptake of iron into astrocytes remain elusive. To investigate the uptake of iron into astrocytes, we have applied ferric ammonium citrate (FAC) to rat astrocyte-rich primary cultures. These cultures express the mRNAs of two membrane-bound ferric reductases, Dcytb and SDR2, and reduce extracellular ferric iron (100 muM) with a rate of 3.2 +/- 0.4 nmol/(hr x mg). This reduction rate is substantially lower than the rate of cellular iron accumulation from 100 muM FAC [24.7 +/- 8.9 nmol/(hr x mg)], which suggests that iron accumulation from FAC does at best partially depend on extracellular ferric reduction. Nonetheless, when the iron in FAC was almost completely reduced by an excess of exogenous ascorbate, astrocytes accumulated iron in a time- and concentration-dependent manner with specific iron accumulation rates that increased linearly for concentrations of up to 100 muM ferrous iron. This accumulation was attenuated by lowering the incubation temperature, by the presence of ferrous iron chelators, or by lowering the pH from 7.4 to 6.8. These data indicate that, in addition to the DMT1-mediated uptake of ferrous iron, astrocytes can accumulate ferric and ferrous iron by mechanisms that are independent of DMT1 or transferrin.

Accumulation of Iron Oxide Nanoparticles by Cultured Brain Astrocytes

Magnetic iron oxide nanoparticles are considered for various diagnostic and therapeutic applications in brain including their use as contrast agent for magnetic resonance imaging or as tool for magnetic drug delivery. However, little is known so far on the consequences of a treatment of brain cells with such nanoparticles. In order to study the biocompatibility of iron oxide nanoparticles and the accumulation of iron from such particles in brain cells, we have used astrocyte-rich primary cultures as model system. Iron oxide nanoparticles were chemically synthesized with a yield of about 80% regarding the iron content. Transmission electron microscopy revealed that the synthesized iron oxide nanoparticles had a diameter of about 10 nm. Exposure of astrocyte cultures to 100 microM iron that were supplied as citrate-coated iron oxide nanoparticles did not cause any acute loss in cell viability but resulted in an almost linear increase in the cellular iron content that reached after 6 h of incubation at 37 degrees C a total cellular iron content of about 300 nmol/mg protein. The rate of iron accumulation from iron oxide nanoparticles was significantly higher than that from the low molecular weight iron complex ferric ammonium citrate (FAC). Lowering the incubation temperature from 37 degrees C to 4 degrees C reduced the iron accumulation rate from iron oxide nanoparticles or FAC by about 60%. In addition, presence of ferric or ferrous iron chelators did not affect the cellular iron accumulation from iron oxide nanoparticles. These data suggest that cultured astrocytes are able to accumulate intact iron oxide nanoparticles.

Regulation of astrocyte inflammatory responses by the Parkinson's disease‐associated gene DJ–1

The Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulates proinflammatory responses in mouse astrocyte-rich primary cultures. When treated with a Toll-like receptor 4 agonist, the bacterial endotoxin lipopolysaccharide (LPS), Dj-1-knockout astrocytes generated >10 times more nitric oxide (NO) than littermate controls. Lentiviral reintroduction of DJ-1 restored the NO response to LPS. The enhanced NO production in Dj-1(-/-) astrocytes was mediated by a signaling pathway involving reactive oxygen species leading to specific hyperinduction of type II NO synthase [inducible NO synthase (iNOS)]. These effects coincided with significantly increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and p38(MAPK) inhibition suppressed NO production and iNOS mRNA and protein induction. Dj-1(-/-) astrocytes also induced the proinflammatory mediators cyclooxygenase-2 and interleukin-6 significantly more strongly, but not nerve growth factor. Finally, primary neuron cultures grown on Dj-1(-/-) astrocytes became apoptotic in response to LPS in an iNOS-dependent manner, directly demonstrating the neurotoxic potential of astrocytic DJ-1 deficiency. These findings identify DJ-1 as a regulator of proinflammatory responses and suggest that loss of DJ-1 contributes to PD pathogenesis by deregulation of astrocytic neuroinflammatory damage.

Gap junction hemichannel-mediated release of glutathione from cultured rat astrocytes

Release of glutathione (GSH) from astrocytes is essential for the supply of neurons with the GSH precursor cysteine. In order to test whether gap junction hemichannels could contribute to GSH release from astrocytes, we incubated astrocyte-rich primary cultures from neonatal rat brain in the absence of divalent cations, a condition that is known to increase the opening probability of hemichannels. During incubation in divalent cation free incubation solution (DCFS) the cells remained viable and released about 50% of the initial cellular GSH within 15 min. This extracellular GSH accumulation in DCFS was lowered by the presence of Ca 2+ in a concentration dependent manner with a half-maximal inhibition at a Ca 2+ concentration of 107 ± 46 μM. Extracellular GSH accumulation in DCFS was also blocked by the divalent cations Mg 2+, Ba 2+ and Sr 2+ as well as by the known gap junction inhibitors carbenoxolone (CBX), flufenamic acid (FFA) and lanthanum chloride. In contrast, the P2X 7 receptor blocker brilliant blue G (BBG) did not affect GSH release in divalent cation free solution. This pharmacological profile strongly suggests that astrocytes are able to release GSH via open hemichannels. This release of GSH may have severe consequences for the antioxidative defense and for the GSH homeostasis in pathological brain.

Formation and Rapid Export of the Monochlorobimane–Glutathione Conjugate in Cultured Rat Astrocytes

Monochlorobimane (MCB) is often used to visualize glutathione (GSH) levels in cultured cells, since it is quickly converted to a fluorescent GSH conjugate (GS-MCB). To test for consequences of MCB application on the GSH metabolism of astrocytes, we have studied rat astrocyte-rich primary cultures as model system. MCB caused a concentration dependent rapid decrease in the cellular GSH content. Simultaneously, a transient accumulation of GS-MCB in the cells was observed with a maximal content 5 min after MCB application. The cellular accumulation was followed by a rapid release of GS-MCB into the medium with a maximal initial export rate of 27.9 +/- 6.5 nmol h(-1) mg protein(-1). Transporters of the family of multidrug resistance proteins (Mrps) are likely to be involved in this export, since the Mrp inhibitor MK571 lowered the export rate by 60%. These data demonstrate that, due to its rapid export from astrocytes, GS-MCB is only under well-defined conditions a reliable indicator of the cellular GSH concentration and that MK571 can be used to maintain maximal GS-MCB levels in astrocytes.

Glutathione peroxidase 1 and glutathione are required to protect mouse astrocytes from iron‐mediated hydrogen peroxide toxicity

The enzyme glutathione peroxidase 1 (GPx1) is involved in the cellular detoxification of peroxides. To test for the consequences of GPx deficiency in astrocytes, astrocyte-rich primary cultures from wild-type and GPx1-deficient [GPx1(-/-)] mice were exposed to H(2)O(2). In GPx1(-/-) astrocytes, the clearance rate of H(2)O(2) was slower than in wild-type cells. In contrast to GPx1-deficient astrocytes, wild-type cells exhibited, within 2 min of H(2)O(2) application, a rapid and transient accumulation of cellular glutathione disulfide that amounted to 60% of total glutathione. The peroxide treatment did not affect the viability of wild-type astrocytes, whereas 45% of the GPx1(-/-) cells died within 8 hr. However, the viability of both types of astrocytes was strongly compromised by lowering cellular glutathione content before peroxide application. In contrast, inactivation of catalase caused substantial cell death only in GPx1(-/-) cells but not in wild-type astrocytes. The cell death observed was prevented by the iron chelators deferoxamine, 1,10-phenathroline, or 2,2'-dipyridyl, whereas preincubation with ferric ammonium citrate increased the toxicity of peroxide treatments. These results demonstrate that GPx1 contributes to the rapid clearance of H(2)O(2) by mouse astrocytes and that both GPx1 and a high concentration of glutathione are required to protect these cells from iron-dependent peroxide damage.