Glucose Deprivation Increases HydrogenPeroxide Level in Immunostimulated RatPrimary Astrocytes

Ji Woong Choi,1 Chan Young Shin,1 Byoung Kwon Yoo,1 Min Sik Choi,1

Woo Jong Lee,1 Byoung Hee Han,1 Won-Ki Kim,2 Hyoung-Chun Kim,3

and Kwang Ho Ko1*1Department of Pharmacology, College of Pharmacy, Seoul National University, Seoul, Republic of Korea2Department of Pharmacology, College of Medicine, Ewha Institute of Neuroscience,Ewha Womans University, Seoul, Republic of Korea3College of Pharmacy, Kangwon National University, Chunchon, Kangwon-do, Republic of Korea

Activated astrocytes produce a large amount of bioactivemolecules, including reactive oxygen and nitrogen spe-cies. Astrocytes are in general resistant to those reactivespecies. However, we previously reported that immuno-stimulated astrocytes became highly vulnerable to met-abolic insults, such as glucose deprivation. In this study,we investigated whether H2O2 production was associ-ated with the increased vulnerability. Glucose deprivationfor up to 8 hr did not change the intracellular level ofH2O2 in astrocytes. Treatment with lipopolysaccharideplus interferon-� for 48 hr evoked astroglial H2O2 pro-duction; however, no apparent death or injury was ob-served in immunostimulated astrocytes. Glucose depri-vation after 48 hr of immunostimulation markedlyincreased H2O2 level, depleted adenosine triphosphate(ATP), and enhanced lactate dehydrogenase (LDH) re-lease. The ATP depletion and LDH release were in partprevented by catalase, mannitol, and N-acetyl-L-cysteine. The enhanced level of H2O2 in glucose-deprived immunostimulated astrocytes appeared to besecondary to the depletion of reduced glutathione. 4-(2-Aminoethyl)bebzenesulfonyl fluoride (AEBSF), an inhibi-tor of NADPH oxidase, reduced H2O2 level and LDHrelease in glucose-deprived immunostimulated astro-cytes. H2O2, either endogenously produced or exog-enously added, depolarized mitochondrial transmem-brane potential in glucose-deprived astrocytes, leadingto their ATP depletion and death. The present resultsstrongly indicate that glucose deprivation causes deteri-oration of immunostimulated astrocytes by increasingthe intracellular concentration of H2O2.© 2004 Wiley-Liss, Inc.

Key words: immunostimulation; glucose deprivation;H2O2; ATP; rat primary astrocyte

Astroglial cells play an integral role in brain ho-meostasis by regulating ion and neurotransmitter transportand also providing trophic supports and energy substrates

for neuron. In various pathological conditions, such asneurodegenerative diseases, astroglial cells are activated bycytokines (Griffin et al., 1998; Stoll et al., 1998). Activatedastroglial cells produce neurotoxic molecules, such asproinflammatory cytokines and reactive nitrogen and ox-ygen radicals (Johnstone et al., 1999; Murphy, 2000).

We and other researchers have reported that acti-vated astroglial cells produce large amounts of reactivenitrogen species (RNS) via activation of inducible nitricoxide synthase (iNOS; Murphy, 2000; Shin et al., 2001).Nitric oxide (NO) and its reaction product with O2

–,ONOO–, have direct neurotoxic effects by mechanismsinvolving tyrosine nitrosylation, S-nitrosylation, mito-chondrial dysfunction, and activation of apoptotic cascade(Viner et al., 1999; Brown and Borutaite, 2001). Not onlyRNS but also reactive oxygen species (ROS), includingH2O2, are generated in activated glial cells (Robinson etal., 1999; Le et al., 2001). Hydroxyl radical, which isproduced by Fe2�-catalyzed reaction from H2O2, ishighly reactive and damages cells by disrupting DNA andmembrane lipid and protein (Stohs and Bagchi, 1995).H2O2 produced from activated astroglial cells may inducean alteration of cellular antioxidative system, such as man-ganese superoxide dismutase (Mn-SOD; Pinteaux et al.,1996). Previously, we reported that treatment withinterferon-� (IFN-�) and lipopolysaccharide (LPS) did notcause any apparent toxicity in cultured astrocytes (Choi etal., 2000; Shin et al., 2002). However, immunostimulated

Contract grant sponsor: Korea Health 21 R&D Project of the Ministry ofHealth and Welfare, Republic of Korea; Contract grant number: 02-PJ1-PG3-21304-0012.

*Correspondence to: Kwang Ho Ko, PhD, Department of Pharmacology,College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong,Kwanak-Gu, Seoul 151-742, Korea. E-mail: khk123@plaza.snu.ac.kr

Received 3 July 2003; Revised 6 November 2003; Accepted 11 November2003

Journal of Neuroscience Research 75:722–731 (2004)

© 2004 Wiley-Liss, Inc.

astroglial cells became highly vulnerable to secondary in-sults, such as glucose deprivation (Choi et al., 2000; Shinet al., 2002). The increased susceptibility of immuno-stimulated astroglial cells to glucose deprivation was attrib-uted to the abrupt depolarization of mitochondrial trans-membrane potential (MTP) and depletion of intracellularadenosine triphosphate (ATP; Choi et al., 2000; Shin etal., 2002). In this study, we investigated whether H2O2production was associated with the potentiated death ofimmunostimulated rat primary astrocytes by glucose dep-rivation.

MATERIALS AND METHODS

Materials

H2O2 was purchased from Merck (Darmstadt, Germany).Recombinant rat IFN-�, glucose-free Dulbecco’s modified Ea-gle medium (DMEM), DMEM/F12, and fetal bovine serum(FBS) were obtained from Gibco BRL (Grand Island, NY).Lipopolysaccharide (LPS) and 4-(2-aminoethyl)bebzenesulfonylfluoride (AEBSF) were from Sigma (St. Louis, MO). NG-nitro-L-arginine (NNA) was from Research Biochemical Interna-tional (Natick, MA). 2�,7�-Dichlorodihydrofluorescein diac-etate acetyl ester (H2DCFDA), tetramethylrhodamine ethylester (TMRE), and monochlorobimane (mBCl) were from Mo-lecular Probes (Eugene, OR). All other chemicals, includingN�-nitro-L-arginine methyl ester (L-NAME), were fromSigma.

Culture of Rat Primary Astrocytes andImmunostimulation

Primary astrocytes were cultured from the prefrontal cor-tices of 2–4-day-old Sprague-Dawley rat pups as describedpreviously (Shin et al., 2001, 2002). Briefly, cells were dissoci-ated for 10 min by mild trypsinization at 37°C and passedthrough sterile nylon sieves (80-�m pore size) into DMEM/F12containing 10% heat-inactivated FBS. Cells (5 � 104 cells/ml)were then plated onto poly-L-lysine (20 �g/ml)-coated 75-cm2

culture flasks and maintained for 1 week. Cells were thentrypsinized, replated onto 24-well culture plates, and maintainedin DMEM/F12 supplemented with 10% FBS. Seven days later,cells were used for experiments. The cultures contained mainlyastrocytes (�95%) and microglial cells (�5%), as identified byusing antibodies against glial fibrillary acidic protein (GFAP;Santa Cruz Biotechnology, Santa Cruz, CA) for astrocytes andantiisolectin B4 (Sigma) for microglia. For immunostimulation,cells were pretreated for 48 hr with IFN-� (100 U/ml) and LPS(1 �g/ml). After immunostimulation, glucose deprivation wasachieved by repeated rinsing and incubation in glucose-freeDMEM that was not supplemented with serum, which inter-fered with the lactate dehydrogenase (LDH) assay.

Measurement of Intracellular H2O2

According to the method described by Rota et al. (1999),intracellular H2O2 generation was assessed using the probe 2,7-dichlorofluorescein (DCF; Molecular Probes). DCF fluores-cence was measured by using a microscope with a �40, 1.4-numerical-aperture, Plan Apo oil-immersion objective (DMIRB; Leica) equipped with confocal attachment (excitation,

488 nm; emission, 515–540 nm; TCS NT system; Leica). Fivedifferent cultures were used in this study, and groups of 10–20subconfluent cells or 20–30 confluent cells were randomlyselected from the image for each sample. The fluorescence ofDCF can be photoactivated by the lasers used for confocalmicroscopy. Thus, laser power was minimized (�8%) by ad-justing the acoustical potical transmission filter (AOTF), andthen the photomultiplier (PMT) value was adjusted. Also, flu-orescence intensity was acquired within 3 sec to minimize thephotoactivation by laser and was corrected for autofluorescence(i.e., fluorescence of cells not loaded with DCF). In controlexperiments, no photoactivation was observed during the wholeperiod of fluorescence monitoring. The laser intensity, photo-multiplier sensitivity, and offset were kept constant for every setof experiments.

Determination of H2O2 Release

The rate of H2O2 formation was measured by monitoringthe changes in fluorescence of scopoletin in the presence ofhorseradish peroxidase (HRP), as described previously (De laHarpe and Nathan, 1985). Cells were rinsed and incubated withKrebs Ringer-buffered solution (129 mM NaCl, 4.86 mM KCl,0.54 mM CaCl2, 1.22 mM MgSO4, 15.8 mM Na2HPO4, pH7.4) containing 20 �M scopoletin and 10 U/ml HRP for 2 hr.Fluorescence was monitored at an excitation wavelength of350 nm and emission wavelength of 460 nm with a fluorescencemicroplate reader (Tecan). The amount of H2O2 was calculatedfrom the standard curve with known concentrations (10–100 nmol/liter) of H2O2.

Measurement of Cell Death

Cell death was assessed by morphological examination ofcells using phase-contrast microscopy and quantified by measur-ing LDH release into the medium at various time points afterstarting glucose deprivation (Shin et al., 2001, 2002). The LDHamount corresponding to complete glial damage/death wasmeasured in sister cultures treated with 0.1% Triton X-100 for30 min at 37°C. Basal LDH levels (generally less than 3% of totalLDH release) were determined in sister cultures subjected tosham wash with 5 mM glucose containing DMEM and sub-tracted from the levels in experimental conditions to yield theLDH signal specific to experimental injury.

Measurement of GSH

GSH was measured as described by Fernandez-Checa andKaplowitz (1990), with some modifications. Astrocytes werewashed with phosphate-buffered saline solution (PBS) and thenincubated at 37°C for 10 min with 50 �M mBCl. Cells werewashed with PBS and lysed with PBS containing 0.2% TritonX-100. The fluorescence of the mBCl-GSH conduct was mea-sured with a fluorescence microplate reader at an excitationwavelength of 360 nm and an emission wavelength of 465 nm.The GSH content of the samples was calculated from thestandard curve prepared from GSH standards incubated in thepresence of mBCl and glutathione transferase (Sigma).

Measurement of NADPH Oxdiase Activity

NADPH oxidase acivity was measured by using lucigeninchemiluminescence as described previously (Lee et al., 2003).

Increased H2O2 Level by GD in Activated Astrocytes 723

Cells were washed with ice-cold PBS, collected by scraping, andcentrifuged at 1,000g for 1 min. Cell pellets were homogenizedwith lysis buffer containing various protease inhibitors (20 mMmonobasic potassium phosphate, pH 7, 1 mM EGTA, 10 �Maprotinin, 0.5 �M leupeptin, and 0.5 mM phenylmethylsulfonylfluoride) and incubated for 20 min on ice. The homogenate wascentrifuged at 1,000g for 15 min, and protein content wasdetermined by using Bradford protein assay kit (Bio-Rad, Her-cules, CA). NADPH oxidase activity was determined in aKrebs/HEPES buffer with 25 �M lucigenin as the electronacceptor and NADPH (100 �M) as the substrate. The reactionwas started by addition of 25 �g protein, and photon emissionwas measured by using LB953 luminometer (Beckman). Abuffer blank containing lucigenin was subtracted from eachreading, and the data were calculated by comparison with astandard curve generated with xanthine/xanthine oxidase.

Mesurement of ATP Contents

The level of intracellular ATP was measured with a lu-minescence detection kit (Molecular Probes), as described pre-viously (Shin et al., 2001, 2002). Briefly, cells were lysed with10% trichloroacetic acid (TCA) and sonicated for 15 min on ice.To the lysates were added 2 mM EDTA and 2 mg/ml BSA.After centrifugation, the supernatant was neutralized with 4 MKOH and the ATP content was determined.

Fluorescence Imaging of MTP

To evaluate the changes in MTP, cells were loaded withTMRE (100 nM) for 20 min and exposed to various condition.TMRE fluorescence at an excitation wavelength of 568 nm andan emission wavelength of 590 nm in cells was measured with amicroscope (Leica) equipped with a confocal attachment (TCSNT system). To minimize photobleaching and other free radicaldye reactions, laser intensity and exposure time (�3 sec) wereminimized. Several fields per sample were selected randomly byphase-contrast optics, and then fluorescence images were cap-tured with MetaMorph imaging and analysis software. Also, thefluorescence intensity was corrected for autofluorescence. Theintensity of autofluorescence (i.e., fluorescence of cells notloaded with TMRE) was unchanged during the whole experi-mental period. As a positive control, the potent mitochondrialinhibitor carbonyl cyanide 4-trifluoromethoxyphenylhydrazone(FCCP; 5 �M) was applied at the end of the experiment.

Statistical Analysis

Data are expressed as the mean SEM and analyzed forstatistical significance by using one-way analysis of variance(ANOVA), followed by Neuman-Keul’s test as a post hoc test,and P � .05 was considered significant.

RESULTSRat primary astrocytes were immunostimulated with

LPS (1 �g/ml) plus IFN-� (100 U/ml) for 48 hr. Inimmunostimulated cells, the H2O2 level was significantlyincreased at 48 hr compared with that in control (unstimu-lated) cells. Cotreatment with catalase (500 U/ml) com-pletely prevented the increase of DCF fluorescence (Fig.1A,B). Peroxides other than H2O2 also oxidize reducedDCF. However, the decreased fluorescence signal from

catalase indicates that the majority of the fluorescencesignal evoked by stimulated cells is derived from H2O2.Other H2O2 scavengers—mannitol (2 mM) and NAC(20 mM)—prevented the increase in DCF fluorescence(Fig. 1A,B). As shown previously (Choi et al., 2000; Shinet al., 2002), immunostimulated astrocytess produced alarge amount of NO and peroxynitrite (data not shown).However, the reagents employed to inhibit H2O2 pro-duction in this study did not alter the production of NOunder the same experimental conditions (data not shown).

Fig. 1. Production of H2O2 in immunostimulated astrocytes.A,B: Astrocytes were immunostimulated with LPS (1 �g/ml) andIFN-� (100 U/ml) in the presence or absence of catalase (500 U/ml),NAC (20 mM), or mannitol (2 mM). Forty-eight hours later, cells wereincubated with H2DCF-DA to determine ROS production with aconfocal microscope (A), and the fluorescence intensities per cells werequantified (B). C: Astrocytes were similarly treated with LPS andIFN-� in the presence or absence of catalase, NAC, or mannitol, asdescribed for A and B. Forty-eight hours later, astrocytes were incu-bated with scopoletin (20 �M) and horseradish peroxidase (10 U/ml).Two hours later, the amount of H2O2 was determined. Data aremean SEM (n � 4). P � .05, significantly different from control (*)or untreated (#) cells.

724 Choi et al.

H2O2 release was further determined by using anotherH2O2 detector, scopoletin. In immunostimulated astro-cytes, H2O2 level was increased by 3.4-fold, and thisincrease was largely inhibited by catalase, NAC, and man-nitol (Fig. 1C).

We previously reported that immunostimulated as-troglial cells are highly vulnerable to glucose deprivation(Choi et al., 2000; Ju et al., 2000; Shin et al., 2002).Prolonged glucose deprivation was reported to cause ametabolic oxidative stress (Lee et al., 1998). Thus, wehypothesized that increased H2O2 level in the immuno-stimulated and glucose-deprived astrocytes mediated thepotentiated cell death. To test this hypothesis, immuno-stimulated astrocytes were challenged by glucose depriva-tion, and DCF fluorescence (H2O2 production) and LDHrelease (cell death) were measured at 2 and 8 hr afterglucose deprivation, respectively. Although glucose depri-vation alone did not significantly increase the level ofH2O2 (data not shown), it increased H2O2 level (Fig.2A,B) and cell death in immunostimulated astrocytes (Fig.2C). We also found that intracellular levels of reducedglutathione (GSH), a well-known antioxidant system inastrocytes, were markedly decreased in glucose-deprivedimmunostimulated astrocytes (Fig. 2D). Treatment withcatalase, NAC, or mannitol during glucose deprivationperiod prevented the increase in H2O2 level (Fig. 2A,B),cell death (Fig. 2C), and GSH depletion (Fig. 2D) inimmunostimulated rat primary astrocytes. These resultssuggest that increased level of H2O2 in glucose-deprivedimmunostimulated astrocytes may mediate the increasedcell death.

Previously, H2O2 itself was reported to induce oxi-dative stress (Li et al., 2001). Thus, we further examinedwhether treatment of H2O2 increased DCF fluorescenceand cell death in glucose-deprived astrocytes. Exogenousaddition of 20 �M H2O2 increased DCF fluorescenceabout 2.2-fold 2 hr after H2O2 exposure (Fig. 3). Com-bined exposure to H2O2 and glucose deprivation for 2 hrincreased DCF fluorescence about 3.8-fold (Fig. 3). Theincrease of DCF fluorescence by H2O2 and glucose dep-rivation in combination was completely blocked by cata-lase, NAC, and mannitol (Fig. 3). Although glucose dep-rivation alone did not cause LDH release up to 8 hr, thelongest time observed in this study, an initial brief expo-sure to 20 �M H2O2 caused significant LDH release 4 hrafter glucose deprivation (Fig. 4A). H2O2 alone (5–100 �M) did not induce LDH release in glucose-supplemented condition but synergistically increased LDHrelease in glucose-deprived astrocytes (Fig. 4B). Catalase,but not SOD, inhibited the potentiated cell death causedby H2O2 and glucose deprivation (Fig. 4C). Similar resultswere also obtained with NAC and mannitol (data notshown).

To assess the mechanism underlying generation ofH2O2 in immunostimulated astrocytes, NADPH oxidaseactivity was measured using lucigenin. NADPH oxidaseactivity was increased about 2-fold in immunostimulatedcells compared with unstimulated cells (Fig. 5A). To de-

termine whether NADPH oxidase is involved in H2O2generation and cytotoxicity, immunostimulated cells weredeprived of glucose for 2 hr (for DCF; Fig. 5B) or 8 hr (forLDH release; Fig. 5C) in the presence of the NADPHoxidase inhibitor AEBSF (20 �M). We found that AEBSFsignificantly reduced the increase of H2O2 level and celldeath in immunostimulated glucose-deprived astrocytes.

We previously reported that the increased death ofimmunostimulated and glucose-deprived astrocytes waspreceded by a collapse of MTP and loss of intracellularATP (Choi et al., 2000; Shin et al., 2002). We measuredthe changes of MTP by using TMRE, an indicator ofMTP depolarization (Scaduto and Grotyohann, 1999). Aswe previously showed (Choi et al., 2000), neither immu-nostimulation nor glucose deprivation alone caused dis-ruption of MTP in astrocytes (Fig. 6A,B). In contrast,glucose deprivation in immunostimulated astrocytes sig-nificantly depolarized MTP to 70% of control value,which is comparable to that observed after treatment witha potent mitochondrial uncoupler, FCCP (5 �M; Fig.6A,B). Cotreatment with catalase, NAC, or mannitol pre-vented the glucose deprivation-induced MTP disruptionin immunostimulated astrocytes (Fig. 6A,B). Exogenouslyadded H2O2 (20 �M) during glucose deprivation alsoinduced rapid collapse of MTP, which was also largelyinhibited by catalase, NAC, or mannitol (Fig. 6A,B). At ahigher concentration (100 �M), however, H2O2 alonedid not affect MTP under glucose-supplemented condi-tions: The TMRE fluorescence intensities in control and100 �M H2O2-treated cells were 155 5.4 and 158 8.2 arbitrary units, respectively. Furthermore, immuno-stimulation and glucose deprivation decreased ATP levelto 15% of control value (Fig. 6C). Treatment of catalase,NAC, or mannitol during the glucose deprivation periodcompletely inhibited ATP depletion (Fig. 6C). ATP de-pletion and LDH release caused by cotreatment of H2O2and glucose deprivation were also completely inhibited bycatalase, NAC, or mannitol (data not shown).

DISCUSSIONActivated glial cells produce various kinds of bioac-

tive molecules, such as cytokines and reactive oxygen ornitrogen species, including O2

–, H2O2, NO, and per-oxynitrite (Johnstone et al., 1999; Murphy, 2000). Thosemolecules play important roles in pathological processes ofvarious neurodegenerative diseases. The role of H2O2 inthe synergistic cell death in glucose-deprived immuno-stimulated astrocytes was supported by several lines ofevidence. 1) Immunostimulated astrocytes produced a sig-nificant amount of H2O2. 2) Glucose deprivation did notitself significantly change cellular H2O2 level. However,H2O2 was accumulated in glucose-deprived immuno-stimulated astrocytes. 3) The potentiated death of glucose-deprived immunostimulated astrocytes was attenuated byH2O2 removal with the treatment of antioxidants, such ascatalase, NAC, or mannitol. H2O2 readily permeates thecell membrane. Thus, although catalase and mannitol donot diffuse into cells, they can scavenge the H2O2 diffusingout of the cells (Cheng et al., 1999).

Increased H2O2 Level by GD in Activated Astrocytes 725

Fig. 2. Glucose deprivation (GD) increases H2O2 level, GSH deple-tion, and LDH release in immunostimulated astrocytes. A,B: Astro-cytes were stimulated for 48 hr with LPS and IFN-�, followed by GDin the presence or absence of catalase (500 U/ml), NAC (20 mM), ormannitol (2 mM). Two hours later, cells were incubated withH2DCF-DA to determine ROS production by using a confocal mi-croscope (A), and the fluorescence intensities per cells were quantified

(B). C,D: The level of LDH (C) released into the culture media and theamount of intracellular GSH (D) were determined at 8 hr and 3 hr afterstarting glucose deprivation, respectively. Inset indicates the change ofGSH content in a time-dependent manner. Data are mean SEM(n � 4). P � .05, significantly different from immunostimulated (*) orglucose-deprived immunostimulated (#) cells.

726 Choi et al.

Astrocytes have a potential role in glycogen storageand utilization in the brain. Because NADPH is an essen-tial cosubstrate in GSH redox cycling (Kussmaul et al.,1999; Garcia-Nogales et al., 1999), astroglial glycogenmust be mobilized for the rapid generation of NADPH viathe pentose phosphate pathway (PPP) during H2O2 dis-posal. Therefore, it is likely that glucose deprivation in-duces astroglial cell death via depletion of NADPH andGSH (Papadopuolos et al., 1997; Dringen et al., 1999;Lord-Fontaine and Averill-Bates, 2002).

H2O2 immediately induces oxidation of GSH incultured neurons (Dringen et al., 1999) and astroglial cells(Dringen et al., 1998). However, H2O2 causes glial celldeath only at high concentrations (�200 �M; Papado-poulos et al., 1998; Takuma et al., 1999; Bhat and Zhang,1999). Astroglial cell death also occurs only after long(�18 hr) deprivation of glucose (Papadopoulos et al.,

1997). The survival of astroglial cells against the low levelof oxidative stress is due to the high antioxidative capacityof astrocytes (Peuchen et al., 1997). However, the situa-tion seems to be different in immunostimulated glucose-deprived astrocytes. Although there is no apparent toxicitywith immunostimulation alone (Ju et al., 2000; presentstudy), the antioxidative capacity of immunostimulatedastrocytes appears to be severely impaired by minor oxi-dative challenge, such as glucose deprivation. This is ingood agreement with our previous reports showing severeand synergistic depletion of GSH content, decrease inATP level, and deterioration of MTP in glucose-deprivedimmunostimulated astrocytes (Ju et al., 2000; Shin et al.,2002). Sustained oxidative stress may induce the imbal-ance between the cellular antioxidative capacity to scav-enge ROS and the rate of ROS production resulting froma lowered efficacy to detoxify H2O2 (O’Connor et al.,1995; Dobashi et al., 2000). The capacity of astrocytes todetoxify H2O2 has previously been reported to be signif-icantly decreased in the absence of glucose (Dringen andHamprecht, 1997; Papadopoulos et al., 1997). Taken to-gether, these results suggest that glucose deprivation mark-edly increases the level of H2O2 in immunostimulatedastrocytes by decreasing the capacity to remove cellularH2O2 via perturbation of the cellular antioxidant system,such as GSH.

Astrocytes have been shown to produce H2O2 undervarious experimental conditions both in vitro and in vivo(Hyslop et al., 1995; Kondo et al., 1996). Moreover,excessive H2O2 and O2

– production was also reportedafter middle cerebral artery occlusion–reperfusion injury(Peters et al., 1998). The mechanisms of H2O2 productionhave been proposed to include 1) activation of NADPHoxidase, an important generator of ROS in diverse cellsystems (Le et al., 2001); in the present study, we showedthat treatment with the NADPH inhibitor AEBSF par-tially blocked the increase of H2O2 level in immunostimu-lated astrocytes; 2) generation of ROS, including H2O2,by the mitochondrial respiratory chain under oxidativestress (Starkov et al., 2002); and 3) H2O2 production bythe stimulation of NOS activity (Heinzel et al., 1992).

Inability to maintain ATP level by the inhibition ofglycolysis is one of the main causes of cell death in neuronsand glial cells (Almeida et al., 2001). In many cases,intracellular energy metabolism and related processes havebeen regarded as one of the main targets for H2O2-induced toxicity (Tsai et al., 1997; Danshina et al., 2001;Chinopoulos and Adam-Vizi, 2001). In addition, disrup-tion of MTP has been reported in various models of celldeath (Petit et al., 1997). Oxidants, including H2O2, havebeen shown to induce MTP disruption via inhibiting themitochondrial respiratory chain and mitochondrial respi-ration (Cadenas and Davies, 2000; Heales and Bolanos,2002). In this study, the synergistic ATP loss and MTPdisruption by glucose deprivation in immunostimulated orlow-H2O2-challenged astrocytes were inhibited by co-treatment with catalase, NAC, or mannitol, implying apossible involvement of H2O2 in the synergistic ATP loss

Fig. 3. Exogenous H2O2 increases intracellular H2O2 production inglucose-deprived astrocytes. Astrocytes were initially and briefly treatedwith 20 �M H2O2 in glucose-free media in the presence or absence ofcatalase (500 U/ml), NAC (20 mM), or mannitol (2 mM). Two hourslater, cells were incubated with H2DCF-DA to determine ROS pro-duction by using a confocal microscope (A), and the fluorescenceintensities per cells were quantified (B). Data are mean SEM (n � 4).P � .05, significantly different from H2O2-treated (*) or glucose-deprived/H2O2-treated (#) cells.

Increased H2O2 Level by GD in Activated Astrocytes 727

Fig. 4. Exogenous H2O2 potentiates cell death caused by glucose depri-vation (GD). A: Astrocytes were initially and briefly treated with 20 �MH2O2 under the glucose-deprived condition for the indicated times.B: Cells were treated with various concentrations of H2O2 for 6 hr in thepresence or absence of glucose (5 mM). C: Cells were treated with catalase(500 U/ml) and superoxide dismutase (SOD; 100 U/ml) during glucosedeprivation, and the amount of LDH release was determined at 6 hr afterglucose deprivation. Data are mean SEM (n � 4). *P � .05, signifi-cantly different from the LDH levels measured at time zero (A), in theabsence of H2O2 (B), or with GD and H2O2 treatment.

Fig. 5. Involvement of NADPH oxidase in H2O2 production in im-munostimulated astrocytes. A: After immunostimulation by LPS andIFN-� for 24 hr, NADPH oxidase activity was assessed by measuringNADPH-induced production of superoxide in astrocyte homognates.*P � .05, significantly different from control (n � 4). B: Afterimmunostimulation by LPS and IFN-� for 48 hr, astrocytes weredeprived of glucose in the presence of AEBSF (20 �M) for 2 hr andthen incubated with H2DCF-DA to determine ROS production byusing a confocal microscope. C: The level of LDH released into theculture media was determined 8 hr after starting glucose deprivation.Values are means SEM (n � 4). For B and C, *P � .05, significantlydifferent between the indicated groups.

728 Choi et al.

and MTP disruption. We recently reported that adenosineand purine nucleotides protect against the peroxynitrite-induced astroglial death via conservation of cellular ATPlevel (Shin et al., 2002).

In summary, our study shows that nontoxic, mildmetabolic oxidative stress, such as glucose deprivation, can

lead activated astrocytes to death by increasing the intra-cellular level of H2O2 and subsequent mitochondrial dys-function and cellular energy failure. The increased vulner-ability of immunostimulated astrocytes to oxidative stressmay be associated with the neuropathophysiology of ce-rebral ischemic and/or other neurodegenerative diseases.

Fig. 6. Synergistic MTP depolarization and ATP loss by H2O2 andglucose deprivation in astrocytes. A,B: Astrocytes were treated withglucose deprivation and/or H2O2 (20 �M). Cells were also treated withLPS and IFN-� for 48 hr and further deprived of glucose for 2 hr in theabsence or presence of catalase (500 U/ml), NAC (20 mM), andmannitol (2 mM). For the control experiments, cells were treated with5 �M FCCP. Two hours after the start of glucose deprivation, cells

were incubated with TMRE (100 nM) for 20 min, and the fluores-cence intensity was determined by using a confocal microscope (A), andthe fluorescence intensity per cells was quantified (B). C: ATP contentwas determined 4 hr after glucose deprivation. Data are mean SEM(n � 4). *P � .05, significantly different from glucose-deprived im-munostimulated cells.

Increased H2O2 Level by GD in Activated Astrocytes 729

Thus, defining the role of H2O2 in immunostimulatedastrocytes deprived of energy sources, including glucose,should provide the basis for therapeutic strategies in vari-ous kinds of neurodegenerative diseases.

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