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Molecular and Cellular Biochemistry 243: 139–145, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Prolonged glutamate excitotoxicity: Effects onmitochondrial antioxidants and antioxidantenzymes

Puneet Singh,1 Karun Arora Mann,2 Harjit Kaur Mangat2 andGurcharan Kaur1

1Neurochemistry and Neuroendocrinology Laboratory, Department of Biotechnology; 2Department of Zoology, Guru NanakDev University, Amritsar, India

Received 6 May 2002; accepted 7 August 2002

Abstract

Glutamate, a major excitatory amino acid neurotransmitter is also an endogenous excitotoxin. The present study examined theprolonged and delayed effects of glutamate excitotoxicity on mitochondrial lipid peroxidation and antioxidant parameters indifferent brain regions, namely, cerebral hemisphere, cerebellum, brain stem and diencephalon. Wistar rats (male) were ex-posed to monosodium glutamate (MSG) (4 mg × g body wt–1, i.p.) for 6 consecutive days and sacrificed on 30th and 45th dayafter last MSG dose. MSG treatment markedly decreased the mitochondrial manganese superoxide-dismutase (Mn-SOD),catalase and reduced glutathione (GSH) content, and increased the lipid peroxidation (LPx), uric acid and glutathione peroxi-dase (GPx) activity. These results indicate that oxidative stress produced by glutamate in vulnerable brain regions may persistfor longer periods and mitochondrial function impairment is an important mechanism of excitatory amino acid mediated neu-rotoxicity in chronic neurodegeneration. (Mol Cell Biochem 243: 139–145, 2003)

Key words: excitotoxicity, glutamate, membrane damage, mitochondrial free radical scavengers

Introduction

Glutamate, a major excitatory amino acid neurotransmitteris also an endogenous excitotoxin, the excitotoxicity beingdue to excessive stimulation of glutamate receptors [1]. Theexcessive stimulation results in an increase of intracellularcalcium ion concentration [Ca2+]

i [2]. Although, mitochon-

dria can buffer changes in [Ca2+]i after glutamate receptor ac-

tivation [3, 4], studies have revealed that prolonged activationof glutamate receptor (sensitive to NMDA) can result in adelayed Ca2+ deregulation (DCD) [5–7]. Unlike the imme-diate Ca2+ deregulation (ICD), which is a result of ‘low dose/short-term’ glutamate exposure and is associated with instan-taneous and short-term elevation of [Ca2+]

i, DCD results in a

sustained [8] or a prolonged [9] elevation in [Ca2+]i. This

delayed Ca2+ deregulation precedes and reliably predicts thesubsequent necrosis of the cell [10]. Further, a large body ofevidence reveals that deregulation of Ca2+ homeostasis resultsin mitochondrial overload with a subsequent production ofreactive oxygen species (ROS) [11–15], a phenomenon calledoxidative stress. These ROS attack virtually all major classesof biomolecules and result in membrane breakdown, cyto-toxicity, mutagenicity and most important the enzyme modi-fications.

Given that mitochondrial oxidative stress plays a criticalrole in excitotoxicity, anti-oxidant enzymes that scavengemitochondrial ROS would be predicted to protect againstexcitotoxicity. In this regard, studies have been carried out,which deal with the effects of glutamate on the lipid peroxi-dation and antioxidant levels in different brain regions of

Address for offprints: G. Kaur, Neurochemistry and Neuroendocrinology Laboratory, Department of Biotechnology, Guru Nanak Dev University, Amritsar143 005, India (E-mail: gurcharank@yahoo.com)

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neonates [16] and adults [17]. But, to date, there is no suchstudy, which specifically deals with the prolonged effects ofrepeated glutamate exposure (given to adult rats) on the mi-tochondrial anti-oxidants and anti-oxidant enzymes. Thisstudy was planned to establish a link between the prolongedglutamate excitotoxicity and the mitochondrial free radicalsscavengers, and the consequential mitochondrial membranedamage in brain tissue.

An enhanced GPx activity along with a significant increasein mitochondrial lipid peroxidation and uric acid levels, anda significant decline in catalase, manganese-superoxide dis-mutase (Mn-SOD) and reduced glutathione (GSH) levelsobserved in present study are in agreement with the hypoth-esis that excitotoxins may generate oxidative damage by af-fecting the oxidative stress scavengers. These results indicatethat oxidative stress produced by glutamate in vulnerablebrain regions may persist for longer periods and mitochon-drial function impairment is an important mechanism ofexcitatory amino acid mediated neurotoxicity in chronicneurodegeneration.

Materials and methods

Animals

Wistar rats (male) in the age group of 3–4 months, weighing160–180 g were used in this study. Approval by the Institu-tional Animal Ethical Committee for conducting experimentsin rats was obtained. Animals were maintained with food andwater ad libitum and under a 12-h light/12-h dark cycle.

Chemicals

Catalase, cytochrome c, dithiothreitol (DTT), glutathione per-oxidase, glutathione reductase (GR), manganese-superoxidedismutase, thiobarbituric acid, Tris and xanthine oxidase wereprocured from Sigma (St. Louis, MO, USA). All other chemi-cals and reagents were the purest, available commerciallyfrom local suppliers.

Induction of L-glutamate excitotoxicity

Test groups of animals were injected (i.p.) with mono-sodiumglutamate (MSG) suspension (4 mg × g body wt–1) and theirrespective controls with equimolar quantity of sodium chlo-ride for 6 consecutive days. The day following the last gluta-mate dose was regarded as day one, and accordingly, animalswere sacrificed on 30th and 45th day.

Preparation of mitochondrial fraction

After cervical dislocation, the animals were decapitated andthe brains were removed immediately. Brains were dissected,and brain-regions, cerebral hemisphere (CH), cerebellum(CB), brain stem (BS) and diencephalon (DC) were separated.Different brain regions were homogenized in 10 volumes ofchilled homogenizing buffer containing 250 mM Sucrose,12 mM Tris-HCl, 0.1 mM DTT, at pH 7.4. Homogenates(1 ml each) were centrifuged at 1000 g for 5 min at 4°C, thecontents were transferred to chilled Eppendorf tubes and thencentrifuged at 9000 g for 30 min at 4°C. The pellet so obtainedwas suspended in 0.5 ml homogenizing buffer with hand ho-mogenizer. Finally, the volume was made to 1 ml with thehomogenizing buffer.

Estimation of manganese superoxide dismutase (Mn-SOD)and catalase

For Mn-SOD assay, the rate of cytochrome C reduction bysuperoxide radical was monitored at 550 nm using the xan-thine/xanthine oxidase system as a source of superoxide radi-cals [18]. The cross-reactivity of Cu/Zn-SOD was checkedby using potassium cyanide. Mitochondrial fraction (50 µl)was mixed with 150 nM xanthine, 6 nM potassium cyanide,30 nM cytochrome C and 0.2 U × ml–1 xanthine oxidase in afinal volume of 3 ml and the change in absorbancy was ob-served. One unit of Mn-SOD inhibits the rate of reductionof cytochrome c by 50% in a coupled reaction with xanthine-xanthine oxidase at pH 7.8 at 25°C, as determined with astandard curve of purified Mn-SOD enzyme.

Catalase activity was determined according to method ofAebi [19]. Summarily, mitochondrial fraction (50 µl) wasadded to phosphate buffer (0.2 M, pH 7) containing 12 mMH

2O

2 as substrate and the change in absorbancy was noted at

240 nm. Enzyme activities were determined from the stand-ard curve of purified catalase and one unit of catalase equalsto the decomposition of 1 µmole of H

2O

2 per min at pH 7.0

at 25°C.

Assay of glutathione (Reduced) content (GSH) andglutathione peroxidase (GPx)

Total glutathione content was measured as described bySedlak and Lindsay [20]. In brief, mitochondrial fraction(100 µl) was mixed with 4.4 ml of 10 mM EDTA and 500 µltrichloroacetic acid (50% w/v). Contents were centrifuged at3000 g for 15 min. The supernatant so obtained was mixedwith 50 µl of 5,5′-dithiobis(2-nitrobenzoic acid) (10 mM) andthe absorbancy measured at 540 nm. Standard curve was pre-pared using pure glutathione.

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Glutathione peroxidase activity was measured indirectlyby monitoring the oxidation of NADPH [21]. The reactionmixture (1 ml) containing 100 nM GSH, 15 nM NADPHand 15 nM H

2O

2 in potassium phosphate buffer (50 mM, pH

7.5) was mixed with mitochondrial fraction (50 µl) and thechange in absorbancy was monitored at 340 nm. Glutath-ione peroxidase activity is defined as 1 µmole of NADPHoxidized per min at pH 7.5 at 25°C using purified GPx en-zyme.

Estimation of lipid peroxidation (LPx) and uric acidcontent

Method of Beug and Aust [22] was followed for the estima-tion of lipid peroxidation. In brief, FeSO

4 (1 mM) was incu-

bated with ascorbic acid (1.5 mM) in 1 ml Tris-HCl buffer(150 mM, pH 7.1) for 15 min at 37°C. The reaction wasstopped with 1 ml trichloroacetic acid (10% w/v). This wasfollowed by addition of 2 ml thiobarbituric acid (0.375% w/v). After keeping in boiling water-bath for 15 min, contentswere cooled off and then centrifuged. The absorbancy of thesupernatant so obtained was measured at 532 nm. The extentof lipid peroxidation was expressed as nanomoles of mel-ondialdehyde consumed per min at 25°C.

Phosphotungstate method was used for the determinationof uric acid content using BDH kit. Mitochondrial fraction(1 ml) was mixed with 0.5 ml each of sulfuric acid (0.6 N)and phosphotungstic acid (10% w/v). The contents were in-cubated at 30°C for 10 min, followed by filtration and the ab-sorbancy of the filtrate so obtained was measured at 710 nm.

Statistical analysis

Data are expressed as means ± S.E.M. Statistical analysis wascarried out using a two-way analysis of variance (ANOVA)and student’s t-test on different antioxidants and antioxidant

enzymes in ‘control’ and ‘MSG treated’ groups. The valueswere considered significantly different if p-values were lessthan 0.05.

Results

Effects of glutamate treatment on manganese superoxidedismutase and catalase

Mn-SOD activity was decreased in all the brain regions ofglutamate treated animals at 30th and 45th days after MSGtreatment. As compared to control animals, Mn-SOD activ-ity was significantly declined in CH, CB, BS and DC regions(p < 0.001) at 30th day, and in CH, CB and BS regions (p <0.001) at 45th day after glutamate treatment. The results arepresented in Table 1.

Like Mn-SOD, mitochondrial catalase activity in gluta-mate treated animals also showed a decrease in all the brainregions as compared to their respective controls. The decreasewas significant in case of CH (p < 0.001) and DC (p < 0.001)regions at 30th day after MSG treatment (Table 1). Further,the trend in decrease in catalase activity continued, with theeffects becoming more pronounced at 45th day after gluta-mate administration, thus establishing the long-term effectsof glutamate on the mitochondrial catalase activity in differ-ent brain regions.

Effects of glutamate treatment on glutathione (reduced)content and glutathione peroxidase

The reduced glutathione (GSH) content of the treated animalswas decreased in different brain regions as compared to theirrespective controls (Table 2). A significant decrease wasobserved in CH, DC (p < 0.001) and BS (p < 0.02) regionsat 30th day after glutamate administration. Further, GSH con-tent showed a recovery at 45th day, significantly, in CH (p <

Table 1. Levels of manganese-superoxide dismutase and catalase activity in different brain regions of glutamate treated animals

Enzyme Group CH CB BS DC

Mn-SOD Control 61.12 ± 1.49 53.22 ± 1.61 59.87 ± 1.29 62.08 ± 2.34(U·g tissue–1) T-30 41.76 ± 0.01d 38.99 ± 1.12d 44.54 ± 1.10d 46.94 ± 1.29d

T-45 43.11 ± 0.01d 41.95 ± 1.18d 41.95 ± 1.18d 52.64 ± 4.75

Catalase Control 1.10 ± 0.02 1.17 ± 0.07 1.258 ± 0.03 1.63 ± 0.07(U·g tissue–1) T-30 0.825 ± 0.01d 1.15 ± 0.07 1.35 ± 0.03 0.986 ± 0.03d

T-45 0.832 ± 0.01d 1.03 ± 0.03c 1.00 ± 0.01d,d′ 0.698 ± 0.02d,d′

Values are mean ± S.E.M. of 5 experiments with 3 rats (Control, T-30 and T-45) in each group. aa′p < 0.05; bb′p < 0.02; cc′p < 0.01; dd′p < 0.001. abcdstatisticallysignificant change in glutamate treated rats with respect to control rats; a′b′c′d′statistically significant change in rats sacrificed at 45th day after glutamate ad-ministration with respect to rats sacrificed at 30th day after glutamate administration. Abbreviations: T-30 – Test animals sacrificed at 30th day after gluta-mate administration; T-45 – Test animals sacrificed at 45th day after glutamate administration; CH – Cerebral hemisphere; CB – Cerebellum; BS – Brainstem; DC – Diencephalon.

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0.001) and DC (p < 0.05) regions. Exceptionally, CB regiondid not show any change in the GSH content.

GPx activity in 30th day test group was comparable to thatof control group animals in CH, CB and BS regions, whereas,DC region (p < 0.02) showed a significant increase. Further,the enzyme activity in 45th day test group also remained closeto its baseline in CB region, while it increased significantlyin CH (p < 0.005) and BS (p < 0.05) regions, and the DC re-gion showed elevated level of enzyme at 45th day also. Theresults are shown in Table 2.

Effects of glutamate treatment on lipid peroxidation anduric acid content

Lipid peroxidation was found to increase in all the discretebrain regions at 30th and 45th days after glutamate adminis-tration. As compared to controls, a significant increase wasobserved in CB, DC (p < 0.01) and BS (p < 0.001) regions at30th day, and CH, CB, BS (p < 0.01) and DC (p < 0.05) re-gions at 45th day after glutamate treatment. Further, among

the treated groups, a significant increase was observed onlyin CH (p < 0.02) region of 45th day treated groups (Table 3).

Except DC region, a slight increase in uric acid content wasobserved in different brain regions at 30th day of glutamateadministration. Further, the increase became significant in CHregion (p < 0.05) at 45th day. Results are presented in Table 3.

Discussion

The data in this paper shows the prolonged and delayed ef-fects of repeated glutamate doses on the mitochondrial freeradical scavenger system and the consequential membranedamage as inferred from altered levels of Mn-SOD, catalase,GPx, GSH, lipid peroxidation and uric acid content in mito-chondria in different brain regions of male rats. Male as com-pared to female rats were chosen in this study; firstly, becauseof a quantitatively higher expression of NMDA receptors inmales, and secondly, because the preoptic neurons of maleas compared to female rats shows a significant elevation in[Ca2+]

i after glutamate exposure [23].

Table 3. Lipid peroxidase and uric acid content in different brain regions of glutamate treated animals

Enzyme Group CH CB BS DC

Lipid peroxidation Control 25.88 ± 1.07 24.3 ± 2.05 28.57 ± 1.5 28.34 ± 0.49(nM·g tissue–1) T-30 31.45 ± 1.87 45.81 ± 1.61c 45.64 ± 1.35d 36.75 ± 1.46c

T-45 64.57 ± 4.93cb′ 60.94 ± 3.49c 40.25 ± 0.88c 42.56 ± 0.8d

Uric acid Control 0.41 ± 0.03 0.37 ± 0.03 0.36 ± 0.01 0.42 ± 0.004(mg·dl–1) T-30 0.34 ± 0.02 0.42 ± 0.02 0.39 ± 0.01 0.63 ± 0.02c′

T-45 0.5 ± 0.03a′ 0.52 ± 0.04 0.52 ± 0.04a 0.48 ± 0.02

Values are mean ± S.E.M. of 5 experiments with 3 rats (Control, T-30 and T-45) in each group. aa′p < 0.05; bb′p < 0.02; cc′p < 0.01; dd′p < 0.001. abcdstatisticallysignificant change in glutamate treated rats with respect to control rats; a′b′c′d′statistically significant change in rats sacrificed at 45th day after glutamate ad-ministration with respect to rats sacrificed at 30th day after glutamate administration. Abbreviations: T-30 – Test animals sacrificed at 30th day after gluta-mate administration; T-45 – Test animals sacrificed at 45th day after glutamate administration; CH – Cerebral hemisphere; CB – Cerebellum; BS – Brainstem; DC – Diencephalon.

Table 2. Effects of glutamate on the glutathione peroxidase and glutathione (reduced) contents in different brain regions of treated animals

Enzyme Group CH CB BS DC

GSH Control 8.44 ± 0.11 8.59 ± 0.28 9.21 ± 0.47 9.77 ± 0.29(mg·g tissue–1)

T-30 6.84 ± 0.06d 8.27 ± 0.11 8.39 ± 0.27b 8.29 ± 0.1d

T-45 8.39 ± 0.46d′ 8.43 ± 0.27 7.23 ± 0.87a 8.77 ± 0.37ba¢

Glutathione peroxidase Control 22.25 ± 0.32 22.02 ± 0.28 22.58 ± 0.63 22.24 ± 0.51(U·g tissue–1) T-30 22.58 ± 0.27 22.58 ± 0.25 23.14 ± 0.03 35.45 ± 0.45a

T-45 34.91 ± 1.46a 21.43 ± 1.01 38.96 ± 0.92ad′ 35.98 ± 1.34b

Values are mean ± S.E.M. of 5 experiments with 3 rats (Control, T-30 and T-45) in each group. aa′p < 0.05; bb′p < 0.02; cc′p < 0.01; dd′p < 0.001. abcdstatisticallysignificant change in glutamate treated rats with respect to control rats; a′b′c′d′statistically significant change in rats sacrificed at 45th day after glutamate ad-ministration with respect to rats sacrificed at 30th day after glutamate administration. Abbreviations: T-30 – Test animals sacrificed at 30th day after gluta-mate administration; T-45 – Test animals sacrificed at 45th day after glutamate administration; CH – Cerebral hemisphere; CB – Cerebellum; BS – Brainstem; DC – Diencephalon.

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Kubo et al. [24] have revealed that neonates injected withrepeated doses of glutamate showed degeneration of pyrami-dal cells of hippocampus and impaired discrimination-learn-ing in 10 week old rats. Similarly, in newborn mice injectedwith repeated doses of glutamate, brain lesions, obesity,stunted skeletal development, female sterility and pathologi-cal conditions of endocrine functions were manifested inadulthood [25–28]. The prolonged effects of glutamate, par-ticularly, in case of chronic neurodegenerative diseases havealso been discussed by Beal [29, 30]. Working on the similarlines, we have studied the prolonged glutamate excitotoxicityelicited by repeated doses of glutamate in adult male rats.

Preliminary studies in our laboratory revealed that therewere no significant changes in the mitochondrial antioxidantenzyme levels at 15th day after glutamate treatment (data notshown). Further studies at 30th and 45th days revealed altera-tions in the activities of different mitochondrial antioxidantenzymes and in LPx, GSH and uric acid content. The ob-served prolonged effects of glutamate are supported by theprevious reports on studies of free radical scavengers inneonate [16] and adult [17] rat brain.

In this study, a decrease in the mitochondrial Mn-SODactivity at 30th and 45th day after MSG treatment was ob-served. Low level of this primary antioxidant enzyme ob-served in the different brain regions make the brain tissuesusceptible to oxidative attack. Due to oxidative stress, changesin cellular redox status can occur, which can alter the DNAbinding and transcription activation of many transcriptionactivators. Recently, Yoneda et al. [31] has reported that ex-tracellular glutamate signals may be differentially transducedinto the nucleus to express AP-1 transcription factor, whichin turn modulates de novo synthesis of individual target pro-teins. Besides, the activation of a transcription activatorprotein, AP-2, has been shown to play a distinct role in thesuppression of Mn-SOD gene [32] and an inverse correla-tion of AP-2 transcription factors and Mn-SOD expressionhas been observed in normal human fibroblasts and their SV-40 transformed counterparts. Accordingly, the ROS produc-tion during glutamate induced DCD may lead to activationof AP-2, which in turn causes repression of Mn-SOD gene.

Like Mn-SOD, catalase also shows a decrease in activityat 30th and 45th day after MSG treatment. The decrease incatalase activity may be due to depletion or inactivation ofthe enzyme by increased production of free radicals [33]. Adecrease in Mn-SOD activity may be responsible for the in-crease in superoxide radicals besides their enhanced pro-duction in glutamate induced excitotoxicity. Indeed, manyprevious reports have shown that mitochondria generate su-peroxide and related ROS during glutamate receptor over-activation [11, 34]. During oxidative stress, the superoxidesdestroy the iron-sulfur (Fe-S) centers and thereby irrevers-ibly deactivate the iron containing enzymes [35,36]. In thesame way, there is a possibility that superoxide radicals can

destroy the Fe-S centers of catalase, leading to its inactiva-tion.

Glutathione peroxidase activity was significantly increasedat 45th day after glutamate treatment in all brain regions ex-cept CB. This observation is in sharp contrast to mitochon-drial activity of catalase and Mn-SOD. Doroshow et al. [37]have suggested that in tissues having insufficient catalaseactivity, H

2O

2 detoxification may be critically dependent upon

GPx activity. Given that, activation of GPx activity is com-monly associated with oxidative stress under metabolic stresscondition [38, 39], the variation in GPx activity as seen inthese experiments may reflect multiple disturbances, includ-ing direct manipulation by tissue ROS levels. Further, GSHcan function as an antioxidant in many ways. It can reactchemically with singlet oxygen, superoxides and hydroxylradicals, which themselves are the result of elevated [Ca2+]

i,

and therefore function directly as free radical scavenger. Inthis study, GSH showed an initial decrease, which recoveredin different brain regions except in diencephalon at 45 days.The inability of the DC region to recover its GSH contentshows its higher metabolic activity that makes this regionmore prone to oxidative stress.

Brain contains large amounts of polyunsaturated fatty ac-ids (PUFA), which are particularly vulnerable to free radicalattacks resulting in the formation of carbon and hydroxylradicals [40]. The latter initiates a chain reaction ultimatelygenerating numerous toxic reactants that rigidifies mem-branes by cross-linking, disrupting membrane integrity andchanging membrane proteins [2, 40]. A marked increase inlipid peroxidation was observed in the present and previousstudies [16, 17, 41]. Another study from our lab on hypo-glycemia associated oxidative stress revealed markedly lowlevels of catalase, GPx and Mn-SOD in brain as comparedto liver and kidney tissues [42]. A decrease in the activity ofcatalase and Mn-SOD enzyme may lead to an excessive lev-els of superoxide and H

2O

2 in brain tissue, which in turn

generate (·OH) radicals, resulting in initiation and propaga-tion of lipid peroxidation.

Uric acid, a product of purine metabolism has been re-ported to be a moderately effective antioxidant, and a sig-nificant decrease in its level results only after a substantialdecrease in the concentration of other antioxidants [43]. Thismight be the reason for the small alteration observed in mi-tochondrial uric acid content in the different brain regionsafter glutamate administration. The present results also sup-port the finding of Retsky and Frei [44] that vitamin C sparesother antioxidants from being used up at its own cost.

Detailed mechanism(s) by which delayed neuronal injuryoccurs are presently unclear. Three broadly accepted mecha-nisms include (i) involvement of many cytoplasmic calcium-dependent enzymes, such as nitric oxide (NO) synthase andprotease calpain [45], (ii) ROS generated as a consequenceof calcium deregulation, and (iii) the impaired bioenergetic

144

state that occurs because of calcium load. Stout et al. [14]have demonstrated that ultimate determinant of glutamatetoxicity is the mitochondrial formation of ROS rather thanNO. Also, the brain cells exposed to high glutamate concen-trations predominantly show necrotic cell death while thoseexposed to low concentrations show apoptosis [46], so it willbe inappropriate to assume that protease calpain and/or cy-tochrome C release and caspase activation can occur in ourcase. Further, Budd and Nicholls [47] have revealed thatprimary ATP-generation function of mitochondria are sepa-rate from the other functions dependent on Ca2+ accumula-tion and generation of ROS, thus ruling out any involvementof impaired bioenergetic state mechanism in cell death. Theseleaves out ROS as the predominant factor in the glutamateexcitotoxicity as hypothesized by Castilho et al. [10].

Finally, mitochondrially generated ROS cause oxidativedamage to plasma membrane inhabiting Ca2+ extrusion-path-ways leading to calcium ion accumulation in cytoplasm. Ini-tial Ca2+ entry in the cytoplasm and an elevated level ofcytoplasmic calcium ion concentration [Ca2+]

c is not per se

neurotoxic. Indeed, cells can maintain an elevated [Ca2+]c for

considerable periods [14]. Any further uptake of Ca2+ into mi-tochondria, as must have occurred due to repeated doses ofglutamate, creates a condition that results in an irreversiblerise in [Ca2+]

c, which ultimately presage the cell death. Thus,

mitochondrially produced ROS are the ultimate determinantof high glutamate toxicity.

The present study supports the role of delayed mitochon-drial superoxide production by chronic glutamate exposure.The excitotoxicity, particularly of delayed type, can interferewith normal gene expression of different enzymes or with theactivity of the repository of the enzymes already present.

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