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Environmental Tobacco Smoke Induces Oxidative Stressin Distinct Brain Regions of Infant MiceLarissa Helena Lobo Torres a , Wallace Luiz Moreira a , Raphael Caio Tamborelli Garcia a ,Raquel Annoni b , Ana Laura Nicoletti Carvalho b , Simone Aparecida Teixeira c , MaurílioPacheco-Neto d , Marcelo Nicolas Muscará c , Rosana Camarini c , Ana Paula de Melo Loureiroa , Mauricio Yonamine a , Thais Mauad b & Tania Marcourakis aa Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences,University of São Paulo, São Paulo, Brazilb Department of Pathology, School of Medicine, University of São Paulo, São Paulo, Brazilc Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, SãoPaulo, Brazild Department of Clinical Pathology, School of Medicine, University of São Paulo, São Paulo,Brazil

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To cite this article: Larissa Helena Lobo Torres, Wallace Luiz Moreira, Raphael Caio Tamborelli Garcia, Raquel Annoni, AnaLaura Nicoletti Carvalho, Simone Aparecida Teixeira, Maurílio Pacheco-Neto, Marcelo Nicolas Muscará, Rosana Camarini, AnaPaula de Melo Loureiro, Mauricio Yonamine, Thais Mauad & Tania Marcourakis (2012): Environmental Tobacco Smoke InducesOxidative Stress in Distinct Brain Regions of Infant Mice, Journal of Toxicology and Environmental Health, Part A: CurrentIssues, 75:16-17, 971-980

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Journal of Toxicology and Environmental Health, Part A, 75:971–980, 2012Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 onlineDOI: 10.1080/15287394.2012.695985

ENVIRONMENTAL TOBACCO SMOKE INDUCES OXIDATIVE STRESSIN DISTINCT BRAIN REGIONS OF INFANT MICE

Larissa Helena Lobo Torres1, Wallace Luiz Moreira1, Raphael Caio Tamborelli Garcia1,Raquel Annoni2, Ana Laura Nicoletti Carvalho2, Simone Aparecida Teixeira3, MaurílioPacheco-Neto4, Marcelo Nicolas Muscará3, Rosana Camarini3, Ana Paula de Melo Loureiro1,Mauricio Yonamine1, Thais Mauad2, Tania Marcourakis1∗

1Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, Universityof São Paulo, São Paulo, Brazil2Department of Pathology, School of Medicine, University of São Paulo, São Paulo, Brazil3Department of Pharmacology, Institute of Biomedical Science, University of São Paulo,São Paulo, Brazil4Department of Clinical Pathology, School of Medicine, University of São Paulo, São Paulo, Brazil

Environmental tobacco smoke (ETS) leads to the death of 600,000 nonsmokers annually andis associated with disturbances in antioxidant enzyme capacity in the adult rodent brain.However, little is known regarding the influence of ETS on brain development. The aim of thisstudy was to determine levels of malonaldehyde (MDA) and 3-nitrotyrosine (3-NT), as well asenzymatic antioxidant activities of glutathione peroxidase (GPx), glutathione reductase (GR),glutathione S-transferase (GST), and superoxide dismutase (SOD), in distinct brain structures.BALB/c mice were exposed to ETS twice daily for 1 h from postnatal day 5 through postnatalday 18. Acute exposure was performed for 1 h on postnatal day 18. Mice were euthanizedeither immediately (0) or 3 h after the last exposure. Immediately after an acute exposurethere were higher GR and GST activities and MDA levels in the hippocampus, higher GPx andSOD activities in the prefrontal cortex, and higher GST activity and MDA levels in the stria-tum and cerebellum. Three hours later there was an increase in SOD activity and MDA levelsin the hippocampus and a decrease in the activity of all enzymes in the prefrontal cortex.Immediately after final repeated exposure there were elevated levels of GST and GR activityand decreased GPx activity in the hippocampus. Moreover, a rise was found in GPx and GSTactivities in the prefrontal cortex and increased GST and GPx activity in the striatum and cere-bellum, respectively. After 3 h the prefrontal cortex showed elevated GR and GST activities,and the striatum displayed enhanced GST activity. Data showed that enzymatic antioxidantsystem in the central nervous system responds to ETS differently in different regions of thebrain and that a form of adaptation occurs after several days of exposure.

Exposure to environmental tobacco smoke(ETS) is associated with a significantly increasedrisk of developing cardiovascular diseasesincluding angina, myocardial infarction, andcongestive heart failure, as well as pulmonarydiseases, such as asthma, chronic obstruc-tive airway disease, and cancer (Bhalla et al.

The authors gratefully acknowledge the support of Prof. Dr. Milton de Arruda Martins and Dr. Fernanda D. T. Quirino dos SantosLopes and the financial support of FAPESP and CAPES.

Address correspondence to Tania Marcourakis, Faculdade de Ciências Farmacêuticas da Universidade de São Paulo, Av Prof LineuPrestes, 580 Bl 13B, CEP-05508-900, São Paulo-SP, Brazil. E-mail: tmarcour@usp.br

2009; Comhair et al. 2011; Eisner et al.2005), and annually leads to the deaths of600,000 nonsmokers globally (Öberg et al.2011). Environmental tobacco smoke, which iscomposed of both mainstream and sidestreamsmoke, is one of the most common indoor pol-lutants and affects approximately 40% children,

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35% women, and 33% men. Moreover, chil-dren who live in socioeconomically disadvan-taged conditions are regularly exposed to ETS(Sexton et al. 2011).

It is noteworthy that exposure to ETS isalso associated with an increased incidenceof deleterious effects on the brain. In a studyinvolving humans, Musso et al. (2007) showedthat active smokers have significantly reducedprefrontal attention network activity comparedwith nonsmokers, and this deficit is directlyrelated to the duration of smoking-years. In pri-mates, exposure to ETS induced morphologicalchanges in brain development that potentiallydamaged neuronal projections (Slotkin et al.2006). Sidestream smoke (ETS) was found toinduce reactive astrogliosis in distinct brainregions (Fuller et al. 2010) and apoptotic celldeath in whole brain of adult rats (Anbarasiet al. 2006a).

Oxidative stress is one of the mechanismspostulated to be associated with deleteriouseffects of ETS, which acts in a manner compara-ble to that noted with several other toxic com-pounds, such as formaldehyde, diesel exhaustparticles, and chloroform (Burke et al. 2007;Manzo et al. 2010; Zhang et al. 2010). Thequinone–hydroquinone complex is the princi-pal compound of the tar fraction of cigarettesmoke and participates in the redox reactionof O2 to O2

•–. Both the particulate phase andtar in cigarette smoke are characterized by thepresence of reactive species with a long half-life, whereas species in the gas phase havea relatively short half-life. The gas phase iscomposed of simple reactive oxygen species(ROS) and highly reactive substances, such asaldehydes and isoprene, which increase theproduction of intracellular reactive oxygen andnitrogen species by interacting with thiol groups(Church and Pryor 1985; Sobczak et al. 2004).

An increase in ROS leads to the mobi-lization of antioxidant defenses. Alberg et al.(2002) showed that active smokers pos-sess more than 25% reduction in solubleantioxidants, such as ascorbic acid, alpha-carotene, beta-carotene, and cryptoxanthin,compared with nonsmokers. In agreementwith these findings, several studies showedthat ETS promoted changes in markers of

oxidative stress in the brain (Anbarasi et al.2006b; Delibas et al. 2003; Manna et al.2006; Stangherlin et al. 2009). Stangherlinet al. (2009) demonstrated that passive tobaccosmoke produced changes in enzymatic andnonenzymatic antioxidant status in brains ofrat pups. Anabarasi et al. (2006b) found thatETS decreased the levels of nonenzymaticantioxidants. Manna et al. (2006) observedthat long-term exposure to ETS induced oxida-tive stress and activated NF-κB, COX-2, andmitogen-activated protein kinase, which aremediators of inflammation, in distinct brainregions of adult rats.

It is important to emphasize that theadult brain responds differently than imma-ture brain following an oxidant insult, such ashypoxia–ischemia. In the adult brain, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is overex-pressed, while catalase (CAT) and glutathioneperoxidase (GPx), activities increase, therebyreducing tissue damage. Although Cu,Zn-SODis also overexpressed in the immature brain,hydrogen peroxide (H2O2) accumulates, dueto decreases in CAT and GPx activities, andthis accumulation enhances tissue damage(Blomgren and Hagberg 2006; Ditelberg et al.1996; Yang et al. 1994). Thus, these studiessuggest that the immature brain is highly sus-ceptible to injury from free radicals because itsdefense systems are not fully developed.

Thus, the aim of the present study was tomeasure (1) lipid peroxidation by measuringmalonaldehyde (MDA) levels and protein nitra-tion by measuring 3-nitrotyrosine (3-NT) levelsand (2) the activities of the antioxidant enzymesGPx, glutathione reductase (GR), glutathioneS-transferase (GST), and SOD following ETSexposure in mice. In particular, our investiga-tion focused on the hippocampus, cerebellum,prefrontal cortex, and striatum in brains ofinfant mice.

MATERIALS AND METHODS

AnimalsBALB/c mice were obtained from the ani-

mal facility of the University of São Paulo,School of Medicine. This study was approved

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by the Ethics Committee of the School ofMedicine and the School of PharmaceuticalSciences, University of São Paulo. All of the ani-mals received care that was in compliance withthe ‘‘Principles of Laboratory Animal Care’’published by the National Institutes of Health.The animals were housed at 22–26◦C with a12-h light/dark cycle and received ad libitumwater and commercial pelleted food for smallrodents from Nuvital (Nuvilab CR-1; Colombo,Brazil).

Exposure to Environmental TobaccoSmoke (ETS)The size of each litter was adjusted to seven

pups within the first day after delivery. TheBALB/c pups were exposed, together with theirmothers, to a mixture of ETS and mainstreamsmoke from one of the most highly consumedBrazilian filtered cigarettes (containing 0.8 mgnicotine, 10 mg tar, and 10 mg carbon monox-ide [CO] per cigarette) for 14 d beginning onpostnatal day 5 at a rate of 2 h/d (1 h at 8 a.m.and 1 h at 5 p.m.). Acute exposure was per-formed for 1 h on postnatal day 18. Controlgroups inhaled compressed air only. The lev-els of CO in the chamber was measured every10 min using a gas detector brand-specificBiosystems (ToxiPro, Biosystems, USA).

At postnatal day 18, both male and femalepups were randomly selected from each litterand euthanized by cervical dislocation eitherimmediately (0 h) or 3 h after the last exposure,thereby forming the following groups (n = 5):groups I, acute (0 h); II, acute (3 h); III, 14 d(0 h); and IV, 14 d (3 h). Because no previouslypublished study mentioned the time intervalbetween the last exposure and euthanasia, theeffects were determined both immediately and3 h after the last exposure (Figure 1).

The brain was excised; various regionsincluding cerebellum, prefrontal cortex,striatum, and hippocampus were dissectedand stored at –80◦C until preparation ofhomogenate (1:10 g/ml in 0.1 M phosphatebuffer solution, pH 7.3). Each preparationcomprised of tissue that was pooled from fourmice for a total of five samples per group.

The biological exposure markers includedplasma nicotine and cotinine, which weremeasured by gas chromatography with anitrogen/phosphorus (Man et al. 2006) andcarboxyhemoglobin (COHb) detector and ana-lyzed using the spectrophotometric methodthat was previously optimized in our laboratory(Beutler and West 1984).

Determination of Enzymatic ActivitiesGPx activity was measured using the

procedure described by Flohé and Günzler(1984). tert-Butyl hydroperoxide was used asthe substrate, and the formation of oxidizedglutathione (GSSG) was indirectly measuredspectrophotometrically as NADPH consump-tion at 340 nm (Power Wave x 340, Bio-TekInstruments, Inc., software KC4 v3.0, USA)for 5 min. GR activity assay was performedaccording to Carlberg and Mannervik (1975).The reduction of GSSG to GSH was measuredas NADPH consumption and monitored spec-trophotometrically at 37◦C for 5 min at 340 nm(Power Wave x 340, Bio-Tek Instruments,Inc., software KC4 v3.0, USA). GST activityassay measured the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reducedglutathione (GSH) and based on the Habig et al.(1974) method. The formation of the complexwas monitored at 25◦C for 5 min at 340 nm ina spectrophotometer (Power Wave x 340, Bio-Tek Instruments, Inc., software KC4 v3.0, USA).

FIGURE 1. Experimental design during exposure period.

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SOD activity was determined usingxanthine and xanthine oxidase (XOD) togenerate superoxide radicals, which reactedwith 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to form a redformazan dye. SOD activity was measured asthe degree of inhibition of this reaction. Oneunit of SOD is defined as a 50% inhibition ofthe rate of INT reduction under the conditionsof the assay. A Randox kit assay (Crumlin,United Kingdom) was used to determine SODactivity. All enzyme activities are expressed asunits per microgram of protein. Protein wasdetermined by the Bradford method usinga commercial kit. All enzymatic assays wereconducted in triplicate.

Lipid PeroxidationLipid peroxidation was assessed by mea-

suring MDA production (Sim et al. 2003;van Hoorn et al. 2003). Tissue MDA wasreacted with thiobarbituric acid in an acidicmedium at 90◦C for 1 h. After centrifuga-tion to remove the proteins, the supernatantwas filtered through a 0.2-μm membrane, andthe colored complex was analyzed by reverse-phase high-performance liquid chromatogra-phy (HPLC/PDA, Shimadzu, Kyoto, Japan)using a C-18 analytical column (Phenomenex150 mm × 4.6 mm, 10 μm), eluted witha 50 mM phosphate buffer (pH 7):methanol(65:35, v/v) mixture at 1 ml/min and detectedspectrophotometrically at 532 nm.

Western Blot Analysis of 3-NTThe brain regions were homogenized in

ice-cold buffer containing 50 mM Tris-HCl(pH 7.4), 1.0 mM PMFS, 10 μg/ml leu-peptin, and 1.0 mM L-cit. Equal quantitiesof protein from each sample were resolvedby sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE; 10% poly-acrylamide) and transferred onto a nitrocel-lulose membrane. After blocking the non-specific sites with 0.2% (w/v) casein, mem-branes were incubated overnight at 18◦Cwith the primary mouse monoclonal antibody

(1:2000; 500 ng/ml, Upstate). The membraneswere washed with Tris-buffered saline con-taining 0.1% Tween 20 and incubated withan alkaline phosphatase-conjugated goat anti-mouse secondary antibody. Immunoreactivenitrotyrosine-containing protein (3-NT) wasdetected using a chemiluminescent assay kit(Immun-Star; Bio-Rad, USA), and the intensityof the bands was estimated by densitomet-ric analysis with a ChemImager 5500 system(Alpha Innotech). The protein concentration inthe lysates was measured using the Bradfordreagent.

Nicotine and Cotinine SerumConcentrationsThe serum concentrations of nicotine and

cotinine were determined by gas chromatog-raphy (Agilent Technologies 6890N) with anitrogen/phosphorus detector (Man et al.2006). In brief, 150 μl serum was diluted in150 μl of 0.1 M phosphate buffer (pH 6).Prolintan was used as an internal standard. Thesamples were subjected to solid-phase microex-traction with a 130-mg Bond Elut Certify SPEcartridge (Varian, USA) that was previously con-ditioned with 2 ml methanol and 2 ml 0.1 Mphosphate buffer (pH 6). The cartridge waswashed twice with 3 ml milli-Q water and3 ml of 0.1 M HCl and maintained undera vacuum (20 mbar) for 7 min until com-pletely dry. The cartridge was then washedwith 9 ml methanol. Nicotine and cotininewere eluted from the cartridge with a solutionof dichloromethane/isopropanol/ammoniumhydroxide (at a ratio of 12:3:0.3) that wasfreshly prepared, evaporated under a streamof nitrogen, and resuspended with 50 μlmethanol for the injection of 2 μl intothe gas chromatograph (GC). The chromato-graph conditions were set as follows: injec-tor temperature, 250◦C; injection mode, split-less; column, HP-5MS 30 × 0.25 × 0.1(Agilent, Palo Alto, CA); helium as the car-rier gas (1.2 ml/min); oven program: initialtemperature, 100◦C, ramp 10◦C/min, finaltemperature, 250◦C, detector temperature,300◦C.

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Statistical AnalysisGPx, GR, GST and SOD activities and the

levels of MDA and 3-NT were analyzed usingStudent’s t-test for independent samples. Dataare presented as means ± SD or SEM whereindicated. Differences with a probability of 95%(p < .05) were considered significant.

RESULTS

The exposure parameters, such as CO lev-els, whole-blood COHb, and plasma nicotineand cotinine levels, are shown in Table 1.

The activities of GPx, GR, GST, and SODand levels of MDA levels in Group I areillustrated in Figure 2. Immediately after anacute exposure, there was a significant rise inGST and GR activities and in MDA levels inthe hippocampus in ETS-exposed mice. TheETS-exposed mice also showed a significantincrease in GPx and SOD activities in prefrontal

cortex and a significant elevation in GST activityand MDA levels in cerebellum and striatum.Three hours after an acute exposure (GroupII), there was a significant rise in SOD activ-ity in the hippocampus. However, the activitiesof GPx, GR, GST, and SOD were all markedlydecreased in prefrontal cortex, whereas MDAlevels were increased (Figure 3).

Animals exposed to ETS for 14 d andeuthanized immediately after the last exposure(Group III) displayed a significant decrease inGPx activity and significant elevation in GRand GST activity in the hippocampus. Thesemice also had significantly enhanced GPx andGST activity in the prefrontal cortex, increasedGPx activity in cerebellum, and elevated GSTactivity in striatum (Figure 4).

Figure 5 shows that mice exposed to ETSfor 14 d and euthanized 3 h after the last expo-sure (Group IV) showed significantly increasedGR and GST activity in the prefrontal cor-tex and elevated GST activity in striatum. No

TABLE 1. Levels of CO in the Chamber, COHb in Whole Blood, and Nicotine and Cotinine inthe Plasma in Mice Exposed to ETS

CO (ppm) COHb (%)Nicotine(ng/ml)

Cotinine(ng/ml)

Control Not detectable 1.4 ± 0.2 Not detectable Not detectableETS 500.9 ± 158.6 16.6 ± 1.1 183.3 ± 25.2 145.6 ± 17.3

Note. The data are presented as means ± SD.

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FIGURE 4. GPx, GR, GST and SOD activity and MDA levels in Group III (0 h after repeated exposures) in the indicated brain structures.(a) GPx activity; (b) GR activity; (c) GST activity; (d) SOD activity; (e) MDA levels. HC, hippocampus, FC, prefrontal cortex, CE, cerebellum,and ST, striatum. The data are expressed as percent of control ± SEM. Asterisk indicates significant difference at p < .05.

significant differences were detected in 3-NTlevels between the ETS-exposed and controlanimals. Animals that were repeatedly exposedto ETS for 14 d gained less weight compared tocontrols (Figure 6).

DISCUSSION

The developing central nervous system(CNS) is more vulnerable to toxic agents thanthe mature CNS (Rice and Barone 2000).To the best of our knowledge, this is the firststudy showing a differential response to acute

and repeated ETS exposure in distinct brainregions in infant mice. Most studies examinedthe influence of ETS on adult brain, withthe exception of one study by Stangherlinet al. (2009), which was performed with ratpups. Our study differs from the aforemen-tioned study in that distinct brain regions (ratherthan the whole brain) were examined. Datademonstrated alterations in the CNS antioxi-dant defenses in mice exposed to ETS, whichwere brain-region specific and dependent onexposure duration. Our primary finding wasthat acute ETS exposure increased MDA levels,

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FIGURE 5. GPx, GR, GST and SOD activity and MDA levels in Group IV (3 h after repeated exposures) in the indicated brain structures.(a) GPx activity; (b) GR activity; (c) GST activity; (d) SOD activity; (e) MDA levels. HC, hippocampus, FC, prefrontal cortex, CE, cerebellum,and ST, striatum. The data are expressed as percent of control ± SEM. Asterisk indicates significant difference at p < .05.

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whereas repeated exposures to ETS exerted nomarked effect on lipid peroxidation, suggest-ing a type of tissue adaptation and an efficientantioxidant response during repeated insult inthe infant mouse brain.

The brain is particularly sensitive to oxida-tive damage, but compared to other organs,such as the liver and kidney, the brain has lowerlevels of the antioxidant enzymes SOD, GPx,and catalase. Further, neurons contain low lev-els of GSH, a primary antioxidant that is respon-sible for the elimination of cytosolic perox-ides (Floyd 1999; Mamelak 2007; Markesberry1999; Slivka et al. 1987). Relatively few studies

evaluated oxidative stress in the CNS afterexposure to tobacco smoke. Similar to a studyby Stangherlin et al. (2009), Anbarasi et al.(2006b) using total homogenate of adult ratbrains showed a decrease in GSH levels, a fallin the activity of the antioxidant enzymes SOD,GPx, GR, and CAT, and marked apoptosis inrats that were exposed to ETS twice daily for12 wk. Our results are consistent with those ofManna et al. (2006), who observed an increasein ROS and lipid peroxidation in the frontalcortex, cerebellum, hippocampus, and striatumof adult A/J mice that were exposed to ETS7 h/d for 6 mo. On the other hand, Delibaset al. (2003) did not find any marked changein lipid peroxidation or antioxidant enzymesin the hippocampus of ETS-exposed adult rats.Baskaran et al. (1999) noted increased lipidperoxidation in liver, lungs, and kidneys of ani-mals that were exposed to cigarette smoke butfound no change in the brain or heart. Withrespect to antioxidant enzymes, Baskaran et al.(1999) found a rise in the activity of GST butnot catalase, SOD, or GPx in whole brains ofadult rats. Our findings reaffirmed that ETS alsoproduced adverse effects on developing mouseCNS via disturbances in the antioxidant system,which is a more vulnerable animal model.

The rapid reaction of O2•- and nitric oxide

(NO•) leads to the formation of the potentoxidizing agent peroxynitrite anion (ONOO-),

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which is involved in the oxidation and nitrationof lipids, DNA strand leakage, nitration of pro-teins resulting in formation of 3-nitrotyrosine(3-NT), and disruption of structural proteinssuch as actin and neurofilament L (Bao and Liu2002; Beckman et al. 1994). Although changesin 3-NT formation were not noted, one cannotpresume that ONOO- was not formed. It is pos-sible that an efficient antioxidant system may beresponsible for the lack of 3-NT formation.

The prefrontal cortex displayed a distinctlydifferent response compared to other CNSregions in terms of nearly all of the antioxidantenzymes and MDA levels after an acute ETSexposure. The lack of an increase in MDA lev-els in prefrontal cortex may be associated withincreased GPx and SOD activity at 0 h. Whenthe evaluation occurred at 3 h post ETS expo-sure, SOD activity decreased, as did the activityof all of the other antioxidant enzymes, therebyleading to higher MDA levels. Data thus suggesta disruption in the antioxidant system and a lossof protection against lipid peroxidation. The fallin SOD activity maintained O2

•- levels, which,together with NO•, formed ONOO- and ledto OH• formation. Because the decrease inGR activity was only present in the prefrontalcortex, it is postulated that this adverse effectmay also be due to GSH consumption and adeficiency in GSH regeneration.

The hippocampus, cerebellum, and stria-tum showed qualitatively similar response pat-terns. All three brain regions showed higherMDA levels and higher GST activity at 0 h afteran acute ETS exposure. Only the hippocampusdisplayed elevated GR activity. These findingssuggest that GST may be responsible for theimmediate response following an exposure toETS. Three hours after acute ETS exposure,MDA levels returned to control levels, suggest-ing an adaptive response.

In summary, the present study suggests abrain region- and time-dependent response ofthe enzymatic antioxidant system in the mouseCNS. The prefrontal cortex showed a responsepattern that was distinct from other regions.Furthermore, although antioxidant defenses didnot prevent the adverse effects of ETS follow-ing acute exposure, antioxidant enzymes were

able to diminish these effects after repeatedexposures to ETS, suggesting that the brainmight display an adaptive capacity.

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