Epilepsy Research 66 (2005) 185–194

�-Tocopherol protects against pentylenetetrazol- andmethylmalonate-induced convulsions

Marinei Cristina Pereira Ribeiroa, Daiana Silva deAvila a,Carmen Yolanda Matiauda Schneidera, Fernando Stahl Hermesa, Ana Flavia Furiana,

Mauro Schneider Oliveiraa, Maribel Antonello Rubinb, Martina Lehmannc,Josef Krieglsteinc, Carlos Fernando Melloa,∗

a Departamento de Fisiologia e Farmacologia, Centro de Ciencias da Saude, Universidade Federal de Santa Maria,97105-900 Santa Maria, RS, Brazil

b Departamento de Quımica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazilc Philipps-Universitat Marburg, Institut fur Pharmakologie und Toxikologie, Ketzerbach 63, 35032 Marburg, Germany

Received 25 May 2005; received in revised form 8 August 2005; accepted 10 August 2005Available online 12 September 2005

anismsrefore,weage

ionsetrazol

th the

t ofn ofions,

Abstract

Increased excitatory amino acid transmission and decreased GABAergic inhibitory responses seem to be important mechin the genesis of convulsions, where reactive oxygen species (ROS) have recently been suggested to play a critical role. Theadministration of antioxidants may be potentially beneficial for the treatment of convulsive states. In the current studyinvestigated the effect of the systemic Vitamin E administration, an antioxidant, on the convulsions and oxidative daminduced by two convulsant agents with different mechanisms of action: methylmalonic acid (MMA), which induces convulsthrough energy depletion and secondary activation of glutamatergic mechanisms and ROS production and pentylenet(PTZ), which is a chemical convulsant that causes convulsions by blocking the GABAA receptor-coupled chloride ionophore.Adult male Wistar rats (270–300 g) were injected with vehicle (5% Tween 80 in 0.9% NaCl; 1 ml/kg, i.p.) or�-tocopherol (25,75 or 225 mg/kg, i.p.), once a day for 7 days. On the seventh day of antioxidant treatment, the animals were injected wiantioxidant (or vehicle) and, 30 min later, they were intrastriatally injected with NaCl (9�mol/2�l) or with MMA (6 �mol/2�l)or PTZ (3.26�mol/2�l). The animals were observed for the appearance of convulsive behavior and the striatal contenthiobarbituric acid-reactive substances (TBARS) and total protein carbonylation were determined. Intrastriatal injectioincreasing amounts of PTZ and of MMA caused the appearance of convulsive behavior. PTZ- and MMA-induced convulsTBARS production and total protein carbonylation were attenuated by�-tocopherol in a dose-dependent manner.© 2005 Elsevier B.V. All rights reserved.

Keywords: �-Tocopherol; Pentylenetetrazol; Seizure; TBARS; Protein carbonyl; Striatum

∗ Corresponding author. Fax: +55 3220 8241.E-mail address: cf.mello@smail.ufsm.br (C.F. Mello).

0920-1211/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.eplepsyres.2005.08.005

186 M.C.P. Ribeiro et al. / Epilepsy Research 66 (2005) 185–194

1. Introduction

The role of reactive nitrogen and oxygen speciesin the generation and spreading of convulsions is notestablished. While a significant number of studieshave suggested that reactive species may play a role inthe genesis and/or maintenance of convulsive episodes(Ogunmekan, 1979; Ogunmekan and Hwang, 1989;Hiramatsu et al., 1996; Fighera et al., 1999; Rauca etal., 1999; Abe et al., 2000; Bashkatova et al., 2003;Mori et al., 2004; Patel, 2004; Patsoukis et al., 2004a,b),there are also various studies in which such a role hasbeen questioned (Levy et al., 1992; Milatovic et al.,2001, 2002; Rossato et al., 2002; Oliveira et al., 2004).Although the determining factors for such a discrep-ancy are not clear, it is possible that methodologicalpeculiarities may account for it. First, it is worthnoting that most of the protocols used to determinethe involvement of reactive species in the genesisand/or maintenance of convulsive episodes rely on theability of antioxidants to prevent or attenuate seizuresinduced by different convulsants. Considering thisassumption, one must also take into account thatanticonvulsant effect may vary not only with supposedantioxidant activity, but with the nature and doses ofthe convulsant and antioxidant agents employed, andw sucha eats,a d byi ffec-t ,1 orie t al.,1 llelstd s pri-me rr nvul-s ated ;T -m n asmie anta tsa

Accordingly, MMA-induced convulsions are exac-erbated by ammonia (Marisco et al., 2003), whichalso increases tissue lipoperoxidation. These data, tosome extent, are corroborated by the findings thatMMA induces dose-dependent lipoperoxidation invitro (Fontella et al., 2000) and ex vivo (Fontella et al.,2000; Marisco et al., 2003; Malfatti et al., 2003) as wellas concomitant impairment of Na+,K+-ATPase activity(Wyse et al., 2000; Malfatti et al., 2003), a key enzymeactivity in the maintenance of ionic gradients. Althoughbiochemical and pharmacological evidence suggest arole for reactive species in the convulsions induced byMMA, the reasons why antioxidants tested so far affordonly partial protection against MMA-induced convul-sions, and if antioxidants cause a parallel decrease inoxidative markers ex vivo, remain to be determined.

The role of reactive species in the convulsant effectof pentylenetetrazol (PTZ), a well known GABAAantagonist, has also been investigated based on thesame assumption that if reactive species were involvedin the convulsant effect of PTZ, convulsions shouldbe prevented or attenuated by antioxidants or reactivespecies scavengers in a dose-dependent manner. Infact, PTZ-induced convulsions are attenuated byphenyl-butyl-nitrone (Rauca et al., 2004), tocopherol(Bashkatova et al., 2003), mexidol (Bashkatova et al.,2(d ft arkerso andf iedb taa .,2 ofa ivitya orted( isi instb1 antd roleo e ofd oseso ns),w oseso ed

ith the experimental approach used to determinen involvement. Despite these methodological cavrole for reactive species in the convulsions elicite

ron has been suggested, since convulsions are eively prevented by antioxidants (Willmore and Rubin981; Levy et al., 1990; Thomas et al., 1997; Mt al., 1998; Samuelsson et al., 2003; Kabuto e998) and tissue reactive species generation para

o increased excitability (Hiramatsu et al., 1996). Theseata agree with the demonstration that iron causeary reactive species generation in the brain (Willmoret al., 1983; Kabuto et al., 1998). Accordingly, a role foeactive species has also been proposed in the coant action of methylmalonic acid (MMA), a succinehydrogenase (SDH) inhibitor (Dutra et al., 1993oyoshima et al., 1995; Fleck et al., 2004) that accuulates in an inborn error of the metabolism knowethylmalonic acidemia (Fenton et al., 2001). MMA-

nduced convulsions are attenuated by creatine (Royest al., 2003), an ergogenic compound with antioxidctivity (Maulik et al., 1993), and by the antioxidanscorbate and alpha-tocopherol (Fighera et al., 1999).

003), glutathione (Abe et al., 1999, 2000), melatoninSugden, 1983; Bikjdaouene et al., 2003) and highoses of ascorbate (Oliveira et al., 2004). Some o

hese studies have also assessed biochemical mf oxidative damage or reactive species formation,

ound that anticonvulsant activity was accompany a decrease in total lipoperoxidation (Bashkatova el., 2003), protein carbonylation (Oliveira et al., 2004),nd 2,3- and 2,5-DHBA production (Rauca et al004). Conversely, a significant antioxidant effectscorbate without concomitant anticonvulsant actgainst PTZ-induced seizures has also been repOliveira et al., 2004). To further complicate thssue, lack of effect of full doses of tocopherol agaicuculline- and PTZ-induced seizures (Levy et al.,990, 1992), were also reported. Since the discrepata of the literature regarding the anticonvulsantf antioxidants may have emerged due to the usifferent convulsants, or the use of subeffective df antioxidants (or inappropriate dosing regimee decided to investigate whether increasing df tocopherol decrease PTZ- and MMA-induc

M.C.P. Ribeiro et al. / Epilepsy Research 66 (2005) 185–194 187

seizures. The suitability of the antioxidant dosesemployed in this study was assessed by measuring theformation of thiobarbituric acid reactive substancesand total protein carbonylation in cerebral tissue.

2. Experimental procedures

2.1. Animals

Adult male Wistar rats (270–300 g) maintainedunder controlled light and environment (12:12 hlight–dark cycle, 24± 1◦C, 55% relative humidity)with free access to standard lab chow with a nom-inal Vitamin E content of 15 mg/kg (Guabi, SantaMaria, Brazil) and water were used. Animal utiliza-tion protocols followed the Official Government Ethicsguidelines and were approved by the University EthicsCommittee.

All reagents were purchased from Sigma (St. Louis,MO), except thiobarbituric acid (TBA), which wasobtained from Merck (Darmstadt, Germany).

2.2. �-Tocopherol treatment and surgicalprocedure

in0 cf r 7d thea /kg,i ) wasu ders mf asa ce-d untile dantt

2

thea le)a tedw ts( .4w

(2�l) were performed over a 2 min period using a10�l Hamilton syringe attached to a 30 gauge needle,whose tip protruded 2 mm from the cannula, allowinginjection into the dorsal striatum. Immediately afterthe injections the animals were transferred to a roundopen field (54.7 cm in diameter) with a floor dividedinto 11 equal areas. The animals were observed for theappearance of convulsive behavior for 15 min. Duringthis period the number and duration of convulsiveepisodes were recorded. Immediately after the behav-ioral evaluation the animals were sacrificed for cannulaplacement determination or biochemical evaluation.

2.4. TBARS assay ex vivo

Immediately after the behavioral evaluation, the ani-mals were sacrificed by decapitation and their brainswere exposed by the removal of the parietal bone.Both the injected and the contralateral striata wererapidly removed using a stainless steel puncher (5 mmin internal diameter). Tissues were homogenized in 10volumes (w/v) of 10 mM Tris–HCl (pH 7.4), containingsodium dodecyl sulfate (SDS, 0.10%—final concentra-tion) using a glass homogenizer and TBARS contentwas estimated in a medium containing 0.2 ml of brainhomogenate, 0.1 ml of 8.1% SDS, 0.4 ml of acetic acidb ar-b uptf con-d ancew ta

2

riataw stri-a /v)o ssh eter-mw ered e,a ,4-d Cl( ina M

Animals were injected with vehicle (5% Tween 80.9% NaCl; 1 ml/kg, i.p.) or�-tocopherol—alcoholi

orm—(25, 75 or 225 mg/kg, i.p.), once a day, foays. On the fourth day of antioxidant treatmentnimals were anesthetized with thiopental (40 mg

.p.) and a 9 mm stainless steel cannula (gauge 27nilaterally inserted 2 mm above the striatum untereotaxic guidance (AP 0 mm, ML 3 mm, V 1.5 mrom the dura). Chloramphenicol (200 mg/kg, i.p.) wdministered immediately before the surgical proure and once a day in the subsequent days,xperiment was carried out (seventh day of antioxireatment), in order to prevent infections.

.3. Behavioral evaluation

On the seventh day of antioxidant treatment,nimals were injected with the antioxidant (or vehicnd, 30 min thereafter, they were intrastriatally injecith NaCl (11�mol) or with one of the convulsan

6�mol methylmalonic acid—pH adjusted to 7ith 4 M NaOH or 3.25�mol of PTZ). Injections

uffer (500 mM, pH 3.4), and 0.75 ml of 0.81% thiobituric acid (TBA). The mixture was finally made

o 2 ml with type I ultrapure water, and heated at 95◦Cor 90 min in a water bath using a glass ball as aenser. After cooling to room temperature, absorbas measured in the supernatant at 532 nm (Ohkawa el., 1979).

.5. Protein carbonyl assay ex vivo

Punches of the injected and contralateral stere rapidly removed as described above, andtal tissue was homogenized in 10 volumes (wf 10 mM Tris–HCl buffer pH 7.4 using a glaomogenizer. The protein carbonyl content was dined by the method described byYan et al. (1995),ith some modifications. Briefly, homogenates wiluted to 750–800�g/ml of protein in each samplnd 1 ml aliquots were mixed with 0.2 ml of 2initrophenylhydrazine (10 mM DNPH) or 0.2 ml H2 M). After incubation at room temperature for 1 hdark ambient, 0.6 ml of denaturing buffer (150 m

188 M.C.P. Ribeiro et al. / Epilepsy Research 66 (2005) 185–194

sodium phosphate buffer, pH 6.8, containing 3% SDS),1.8 ml of heptane (99.5%) and 1.8 ml of ethanol(99.8%) were added sequentially, and mixed with vor-tex agitation for 40 s and centrifuged for 15 min. Next,the protein isolated from the interface was washed twotimes with 1 ml of ethyl acetate/ethanol 1:1 (v/v) andsuspended in 1 ml of denaturing buffer. Each DNPHsample was read at 370 nm in a Hitachi U-2001 spec-trophotometer against the corresponding HCl sam-ple (blank), and total carbonylation calculated usinga molar extinction coefficient of 22,000 M−1 cm−1

according toLevine et al. (1990). Protein content wasmeasured by method ofBradford (1976)and bovineserum albumin was used as standard.

2.6. Statistical analyses

Data from ex vivo TBARS and total carbonyldeterminations were analyzed by a 2 (saline or�-tocopherol)× 2 (saline or convulsant agent)× 2(injected or non-injected hemisphere) factorialANOVA, with the hemisphere factor treated as awithin-subject factor. Post hoc analyses were carriedout by theF-test for simple effect, when appropriate.P < 0.05 was considered significant. Behavioral datawere analyzed by a factorial 4 (saline, 25, 75 or2 t)A d byp ear,q sseda

3

c-t ;F al.,2 03ads t ofV

redP Sta-t tiono ons,a

the duration of convulsive episodes [F(3,40) = 6.83;P < 0.001], which increased linearly with PTZ doses:significant linear trend for number [F(1,40) = 21.07;P < 0.001] and duration [F(1,40) = 20.32;P < 0.001] ofconvulsive episodes. Based on these results, the effec-tive convulsant dose of 3.26�mol of PTZ was deter-mined and used throughout the study.

The administration of�-tocopherol attenuatedMMA-induced seizures in a dose-dependent manner,confirming and extending previous data from our group(Fighera et al., 1999). The statistical analysis (one-wayANOVA) revealed a significant effect of�-tocopherolon the number [F(3,32) = 5.13;P < 0.005; Fig. 1A]and duration [F(3,32) = 7.39;P < 0.0001;Fig. 1B] ofMMA-induced convulsive episodes. Post hoc analysisrevealed that�-tocopherol decreased the number andduration of convulsive episodes linearly with its dose[significant linear trend:F(1,32) = 10.37;P < 0.005 andF(1,32) = 17.67;P < 0.001 for the number and durationof convulsive episodes, respectively].

�-Tocopherol treatment also attenuated the convul-sions induced by PTZ in a dose-dependent manner.Statistical analysis (one-way ANOVA) revealed that�-tocopherol decreased the number [F(3,32) = 3.48;P < 0.05; Fig. 2A] and duration [F(3,32) = 8.43;P < 0.0001;Fig. 2B] of convulsive episodes elicitedb art ;P ivee

ona idedt A-a ringt la-t id-e ) ofT iatar 5 or2 )b tionf -t ca thats g)i nt(i

25 mg/kg Vitamin E)× 2 (saline or convulsant agenNOVA. Dose–effect relationships were assesseartitioning the total sum of squares into trend (linuadratic or cubic) components. All data are expres mean± S.E.M.

. Results

Since it is well known that the intrastriatal injeion of MMA induces behavioral (Fighera et al., 1999leck et al., 2004; Royes et al., 2003; Marisco et003; de Mello et al., 1996; Fighera et al., 20)nd electrographic seizures (Malfatti et al., 2003) in aose-dependent manner, we chose the dose of 6�mol/triatum to evaluate the putative protective effecitamin E against MMA-induced seizures.

The convulsant doses of intrastriatally administeTZ were determined by a dose–effect experiment.

istical analysis revealed that the intrastriatal injecf PTZ induced the appearance of clonic convulsissessed by the number [F(3,40) = 7.82;P < 0.001] and

y PTZ linearly with its dose [significant linerend:F(1,32) = 10.28;P < 0.005 andF(1,32) = 14.84< 0.001 for the number and duration of convulspisodes, respectively].

Since�-tocopherol afforded significant protectigainst MMA- and PTZ-induced seizures, we dec

o test whether�-tocopherol protected against MMnd PTZ-induced oxidative damage by measu

otal striatal lipoperoxidation and protein carbonyion. Statistical analysis (three-way ANOVA, consring the hemispheres as a within subject factorBARS and total protein carbonyl content in the strevealed a significant pretreatment (vehicle or 25, 725 mg/kg�-tocopherol) by treatment (NaCl or MMAy hemisphere (contralateral or injected) interac

or TBARS content [F(3,60) = 4.85;P < 0.05] and proein carbonylation [F(3,45) = 2.96;P < 0.05]. Post honalysis (Student–Newman–Keuls test) revealedystemic�-tocopherol administration (25–225 mg/knhibited MMA-induced increase of TBARS conteFig. 3A) and protein carbonylation (Fig. 3B) in thenjected striatum.

M.C.P. Ribeiro et al. / Epilepsy Research 66 (2005) 185–194 189

Fig. 1. Effect of�-tocopherol (25, 75 and 225 mg/kg, i.p.) on thenumber (A) and duration (B) of convulsive episodes induced byMMA (6 �mol/2�l). Data are mean± S.E.M. forn = 9 in each group.* P < 0.05 compared with control group (Student–Newman–Keulstest).

�-Tocopherol also prevented PTZ-induced oxidati-ve damage. Statistical analysis revealed a significantpretreatment (0, 25, 75 or 225 mg/kg Vitamin E) bytreatment (NaCl or PTZ) by hemisphere (contralat-

Fig. 2. Effect of�-tocopherol (25, 75 and 225 mg/kg, i.p.) on thenumber (A) and duration (B) of convulsive episodes induced by PTZ(3.26�mol/2�l). Data are mean± S.E.M. forn = 8–9 in each group* P < 0.05 compared with control group (Student–Newman–Keulstest).

eral or injected) interaction for total striatal TBARSand protein carbonyl content [F(3,51) = 4.58;P < 0.05and F(3,51) = 3.47;P < 0.05, respectively]. Post hocanalysis (Student–Newman–Keuls test) revealed that

190 M.C.P. Ribeiro et al. / Epilepsy Research 66 (2005) 185–194

Fig. 3. Effect of �-tocopherol (25, 75 or 225 mg/kg, i.p.) andof MMA (6 �mol/2�l) on striatal TBARS (A) and protein car-bonyl (B) content. Data mean± S.E.M. for n = 8–9 in each group.* P < 0.05 compared with saline–saline group.#P < 0.05 comparedwith saline–MMA group (Student–Newman–Keuls test).

systemic�-tocopherol administration (25–225 mg/kg)inhibited PTZ-induced increase of TBARS (Fig. 4A)and protein carbonylation (Fig. 4B) in the injected stria-tum.

4. Discussion

In the present study we show that�-tocopheroldecreases PTZ- and MMA-induced convulsive behav-ior, TBARS formation and protein carbonylation exvivo. These results confirm and extend previous stud-ies from our group, which have suggested a role forreactive species in the convulsive behavior elicited byMMA ( Fighera et al., 1999, 2003; Marisco et al., 2003;Malfatti et al., 2003; Royes et al., 2005), and constitutenew evidence for a role for reactive species in the con-vulsions elicited by PTZ.

Accumulating evidence of differing nature hassuggested that mitochondria-derived reactive speciesare involved in the deleterious effects of MMA.This dicarboxylic acid increases lipoperoxidationand decreases total antioxidant capacity in cerebralhomogenates in vitro (Fontella et al., 2000) and exvivo (Royes et al., 2005). Accordingly, the intrac-erebral and systemic administration of MMA alsoi ;M zzoe ,2 A-i niedb exh lhi tiono edo thea tralr A( n,M m-p -dw to-c .,1 ata am-a per-

ncreases TBARS production (Marisco et al., 2003alfatti et al., 2003; Fighera et al., 2003; Pettenut al., 2003) and protein carbonylation (Royes et al.005), which are oxidative damage markers. MM

nduced oxidative damage, in turn, is accompay Na+,K+-ATPase inhibition in cerebral cortomogenates in vitro (Wyse et al., 2000) and in striataomogenates ex vivo (Malfatti et al., 2003). It is

nteresting that MMA does not cause lipoperoxidar Na+,K+-ATPase inhibition if the assay is carriut with purified synaptic plasma membranes (inbsence of mitochondria), further indicating a cenole for mitochondria in the deleterious effect of MMWyse et al., 2000). In fact, besides SDH inhibitioMA causes mitochondrial dysfunction due to colex I inhibition (Brusque et al., 2002) and mitochonria transition pore formation (Maciel et al., 2004),hich are experimental conditions linked to mihondrial reactive species production (Cassarino et al998). Regarding this point, it is worth pointing out thmmonia, a compound known to induce oxidative dge, increases MMA-induced convulsions and lipo

M.C.P. Ribeiro et al. / Epilepsy Research 66 (2005) 185–194 191

Fig. 4. Effect of �-tocopherol (25, 75 or 225 mg/kg, i.p.) andof PTZ (3.26�mol/2�l) on striatal TBARS (A) and protein car-bonyl (B) content. Data mean± S.E.M. for n = 8–9 in each group.* P < 0.05 compared with saline–saline group.#P < 0.05 comparedwith saline–MMA group (Student–Newman–Keuls test).

oxidation without altering SDH activity (Marisco et al.,2003).

These findings, together with the finding thatMMA-induced Na+,K+-ATPase inhibition in cerebralcortex homogenates is fully prevented by the antiox-idant glutathione (Wyse et al., 2000), strongly supporta role for mitochondria-derived reactive species inMMA-induced neurotoxicity. Antioxidants have beenused as a tool to infer the participation of reactivespecies in the neurochemical and behavioral effectsof MMA. Accordingly, GM1 ganglioside (50 mg/kg)(Fighera et al., 2003), ascorbic acid (100 mg/kg)(Fighera et al., 1999) and �-tocopherol (40 mg/kg)(Fighera et al., 1999) attenuate MMA-induced convul-sive behavior, and ascorbic acid (50 mg/kg) attenuatesMMA-induced learning deficits (Pettenuzzo et al.,2003). The studies ofFighera et al. (1999)andPettenuzzo et al. (2003), however, have not inves-tigated dose–effect relationships or measures ofoxidative damage. In this study we showed that�-tocopherol decreases both MMA-induced oxidativedamage and convulsive behavior. In addition, we showthat increasing�-tocopherol doses afford increasingprotection against MMA-induced seizures. Such alinear relationship between tocopherol doses andanticonvulsive activity: (a) further supports a rolef ns;( enc van-t lonica

t par-t tt ucedb odelo -n ultsah Z-i duc-t cere-bR ticc hana� ther-a le-

or reactive species in MMA-induced convulsiob) indicates that this vitamin, which has beonsidered devoid of major side effects, may be adageously used as add-on therapy in methylmacidemia.

The currently reported protective effect of�-ocopherol against PTZ-induced seizures is oficular interest because it supports the view tha�-ocopherol may be useful to prevent seizures indy different agents. Though the currently used mf intracerebral injection of a GABAA agent canot be regarded a model of epilepsy, our resgree with those fromBashkatova et al. (2003), whoave demonstrated that�-tocopherol, attenuates PT

nduced seizures and cerebral TBARS and NO proion. In fact, acute PTZ-induced seizures decreaseral content of�-tocopherol in rats (Rauca et al., 2004).egarding this point it is worth noting that epilephildren present plasma Vitamin E levels lower tge-matched controls (Ogunmekan, 1979), and thatd--tocopheryl acetate proved to be useful as add-onpy for epilepsy in children in a randomized, doub

192 M.C.P. Ribeiro et al. / Epilepsy Research 66 (2005) 185–194

blind, placebo-controlled clinical trial (Ogunmekanand Hwang, 1989).

On the other hand, there are also studies reportingno protective effect of�-tocopherol against the convul-sions induced by PTZ (Levy et al., 1990) or by otherchemical convulsants (Levy et al., 1992; Milatovic etal., 2002; Willmore et al., 1986; Gupta et al., 2001;Monahan and Yamamoto, 2002).

The reason for these discrepant results is unknown,but it is possible that methodological differences, suchas the convulsive agent used, may account for them.There is evidence indicating that antioxidants otherthan tocopherol have little or no effect on the convul-sions elicited by kainic acid (Milatovic et al., 2002;MacGregor et al., 1993), quinolinic acid (Rossato etal., 2002) and NMDA (Abe et al., 1999). Therefore, itwould not be surprising to find tocopherol ineffectiveagainst the convulsions induced by these agents. In fact,tocopherol (and other antioxidants) is effective againstthe convulsions elicited by agents whose mechanismof action seems to involve reactive species generation,such as ferrous chloride (Mori et al., 2004), hyperbaricoxygen (Jerrett et al., 1973), penicillin (Kryzhanovskiiet al., 1980) and MMA (see above).

The discussion above may explain why tocopherolpresents anticonvulsive activity against selected con-v on-fl Z-i oto-c imi-l used.T osesm ility.O lab-o erolo thea caused mayr erols co-p wasn ions

min-i t rea-s ucedb eemt r PTZ

threshold (Levy et al., 1990), a test in which a lowconcentration of PTZ is continuously infused to theanimal. Lastly, rats from different strains were usedin these studies, and this fact may have contributed tothe discrepant findings. It has been reported that nor-motensive and hypertensive Wistar Kyoto and SpragueDawley rats show differences in growth rate, myopathy,testes degeneration, and immunological responses inresponse to Vitamin E deficiency (Gabriel et al., 1984).Moreover, other strain differences in antioxidant statushave been reported (Binda et al., 2001; Abılio et al.,2004) that might render a given strain more sensitivethan other to the protective effects of tocopherol. Thisfits well with the recent finding that superoxide dismu-tase 2 deficient mice, which are more susceptible tothe convulsant effects of kainate, develop mitochon-drial oxidative stress and spontaneous seizures as afunction of advancing age (Liang and Patel, 2004), andfurther supports the view that inherited dysfunctions,which may pass unnoticed at first, may render an animalnot only more susceptible to a given stimulus (Seto-Ohshima et al., 2003), but also more responsive to aprotective agent (Murashima et al., 1998). Consideringthese studies, we may speculate that the use of differ-ent rat strains may have contributed to the discrepantfindings regarding the protection of tocopherol againstP therc ecifics

A

S.C in( ),A ira( fel-l

R

A ct of

A oge-854,

ulsive agents, but it still does not explain the cicting findings regarding its ability to prevent PTnduced seizures. A careful examination of the prols of these studies reveals that, in all of them, sar tocopherol doses and dosage regimens wereherefore, the use of tocopherol subeffective day be ruled out as a possible source of variabne also has to consider that differences betweenratories may be found in the amount of tocophf the lab chow used, and this may contribute forppearance of discrepant results. This occurs beietary tocopherol can saturate neural tissue, andender the animal irresponsive to additional tocophupplementation. Unfortunately, the amount of toherol in the lab chow used by most of the labsot informed by the authors, making this discusspeculative, in nature.

On the other hand, PTZ doses and routes of adstration varied between the studies, and one mighon that tocopherol could not prevent seizures indy high doses of PTZ. This, however, does not s

o be the case, because tocopherol does not alte

TZ-induced seizures in the literature. However, oauses cannot be ruled out at the moment, and sptudies have to be performed to clear this point.

cknowledgements

Work supported by CNPq, FAPERGS, CAPE.F. Mello (grant: 500120/2003-0), M.A. Rub

500096/2003-1), D.S.Avila (grant: 500120/2003-0.F. Furian (grant: 500120/2003-0) and M.S. Olive

grant: 500120/2003-0) are the recipients of CNPqowships.

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