0731-8898/10/$35.00 © 2010 by Begell House, Inc. 185

Journal of Environmental Pathology, Toxicology, and Oncology, 29(3):185-197 (2010)

Protective Effects of a By-Product of the Pecan Nut Industry (Carya illinoensis) on the Toxicity Induced by Cyclophosphamide in Rats Carya illinoensis Protects Against Cyclophosphamide-

Induced Toxicity

D.M. Benvegnú,a R.C.S. Barcelos,a N. Boufleur,b P. Reckziegel,b C.S. Pase,b L.G. Müller,b

N.M.B. Martins,c C. Vareli,d *M.E. Bürgera

aProgramadepósGraduaçãoemFarmacologia,UniversidadeFederaldeSantaMaria,RS,Brazil;bDepartamentodeFisiologiaeFarmacologia,UniversidadeFederaldeSantaMaria,RS,Brazil;cDepartamentodePatologia,UniversidadeFederaldeSantaMaria,RS,Brazil;

dDepartamentodeQuímica,UniversidadeFederaldeSantaMaria,RS,Brazil

Correspondenceauthor.*Email:mariliseeb@yahoo.com.br

This study investigated theantioxidanteffectsof pecannut (Carya illinoensis) shell aqueousextract(AE)ontoxicityinducedbycyclophosphamide(CP)intheheart,kidney,liver,blad-der,plasmaanderythrocytesof rats.RatsweretreatedwithwaterorpecanshellAE(5%)ad libitum,replacingdrinkingwaterfor37daysuptotheendof theexperiment.Onday30,half of eachgroupreceivedasingleadministrationof vehicleorCP200mg/kg-ip.After7days,theorganswereremoved.RatstreatedwithCPshowedanincreaseinlipidperoxidation(LP)anddecreaseinreducedglutathione(GSH)levelsinallstructures.Catalase(CAT)activitywasincreasedintheheartanddecreasedinliverandkidney.Besides,CPtreatmentdecreasedplas-maticvitaminC(VITC)levelsandinducedbladdermacroscopicalandmicroscopicaldam-ages.Incontrast,co-treatmentwithpecanshellAEpreventedtheLPdevelopmentandtheGSHdepletioninallstructures,exceptintheheartandplasma,respectively.CATactivityintheheartandliveraswellastheplasmaticVITClevelsremainedunchanged.Finally,AEpre-ventedCP-inducedbladderinjury.Thesefindingsrevealedtheprotectiveroleof pecanshellAEinCP-inducedmultipleorgantoxicity.

KEYWORDS: cyclophosphamide, Carya illinoensis, pecan nut shells, oxidative stress,antioxidant

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Introduction

Carya illinoensis (Wangenh.)K.Koch (Juglandaceae),popularly known as pecan, is a tree native to thesouthernUnitedStatesandnorthernMexico,whichreachestoSouthAmerica.1Pecannutsareasourceof monounsaturated fatty acids, which stimulatestheir cultivation and consumption.2 Pecan process-ing results in a great amount (40-50%) of shells(by-product).Thisby-productof thepecanindustryrepresents a promising source of antioxidant com-pounds.3 Recently, Villarreal-Lozoya et al. (2007)4demonstratedthatpecanshellshavehigherlevelsof phenolic compounds and condensed tannin, whichshowed a greater antioxidant capacity than theker-nels. In fact, pecan shells are popularly used in teaformtotreatdiseasesrelatedtotobacconicotineaswellastopreventdifferentpathologies,mainlythoserelated to xenobiotic toxicity.5 Food and its deriva-tiveshavebeenwidelyusedtomaintainthefunctionalintegrityof thebodyandareanimportantsourceof newproductswithlowercostsandlowincidenceof undesirable side effects, often associatedwith drugtreatments.6

Cyclophosphamide(CP)isanantineoplasticdrugwidelyusedinthetreatmentof cancerandimmuno-suppressioninductionbeforeorgantransplantation.7Thetherapeuticeffectsof CPareassociatedwiththephosphoramidemustard,whiletheacroleinislinkedwithitstoxicsideeffects.8Thesemetabolitesaregen-eratedby thehepaticmicrosomalcytochromeP450mixedfunctionaloxidasesystem.9Themajorlimita-tionof CPtreatmentisthedamagetonormaltissues,leading tomultiple organ toxicity.10,11Besides lethalcardiotoxicity,12theCPtreatmentisrelatedtohepato-toxicity,13,14nephrotoxicity,15andurotoxicity.16,17ThebladderisespeciallyaffectedbyCPtreatment,whosedeleterious effects include mucosal edema, hemor-rhage,ulceration,fibrosis,necrosis,contracture,andvesico-ureteral reflux.18 In fact, most of the side-effects of CP, including the urotoxicity, are relatedtoreactiveoxygenspecies(ROS)generation,19whichhavebeenimplicatedintheactionof manycytostaticdrugs,20 such as CP. CP is closely related to oxida-tivestress(OS)andtissuedamageresultingfromtheincrease of lipid peroxidation (LP) and depletionof antioxidant agents,21 such as glutathione (GSH),catalase(CAT),andsuperoxidedismutase(SOD).22-24Inthissense,biologicalcompoundswithantioxidantpropertiesmaycontributetotheprotectionof tissues

againstdeleteriouseffectsof ROS.Multiple clinical studies have suggested that

theuseof antioxidant supplements in combinationwith chemotherapy can prolong the survival timeof patientscomparedwithexpectedoutcomewith-outsupplementation.25-27Thus,naturalantioxidants,especially from plants, foods or nutritional supple-ments,havebecomeanimportantresearchissueataworldwidelevel.

Consideringthatthepecannutshellisanaturalproduct widely used in folkloric medicine to treatxenobiotictoxiceffectsandthatitschemicalcompo-sition is rich inpolyphenols suchasflavonoidsandcondensedtannins,thepresentstudywasperformedtoevaluate theprotectiveeffectsof pecanshellex-tractonCP-inducedtoxicityinvitaltissuesof rats.

Materials and Methods

Drugs and Chemicals

Cyclophosphamide(Genuxal®,AstaMédica,Brazil)was donated from HUSM (Santa Maria UniversityHospital)andwasdissolvedindistilledwaterjustbe-foreuse.

Vegetal Material

Therawmaterialwaskindlydonatedbyapecanpro-cessingcompany,whichalsoprocessesthenutshellsforthepreparationof tea.Thisproductiscurrentlysoldinsupermarkets,withpermissionof theMinis-tryof Agriculture,Brazil.

Aqueous Extract Preparation

Shellsof C. illinoensis wereleftovernightat40°Cinahotairovenandfinelypowdered.Theaqueousex-tract(AE)of theshellswasfreshlypreparedbyinfu-sion(5%,90°C),filteredusingfilterpaper,andcooledtoroomtemperature.Duringthisprocedure,theex-tract was protected from light. Since experimentaldatawerenot found in the literature, thechoiceof the extract concentrationwasbasedonpreliminarystudiesperformed inour laboratory,which showedthehighpotentialantioxidantof thepecanshellex-tract.

Characterization of Aqueous Extract

Furtherqualitativeandquantitativeanalysesof C. il-

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linoensis shellAEwereperformedusingHPLC(con-dition:column XTerraRP-18(4.6x250mm,5µM);eluent:methanol:water(80:20),0.4%aceticacid;flowrate:1mL/min;detection:UVat290nm).Themajorconstituentof theAEdetectedwasgallicacid(GA),correspondingto1.8g/100gof extract(Fig.1Aand1B).

Animals

Wistaradultmalerats(330±40g)wereused.Groupsof seven animalswere kept in Plexiglas cageswithfree access to food andwater in a roomwith con-trolled temperature (22–23°C)andona12h-light/darkcyclewith lightsonat7:00a.m.Animalsweremaintainedandusedinaccordancewiththerulesof the Committee on Care and Use of ExperimentalAnimalof theFederalUniversityof SantaMaria,RS,Brazil.

Experimental Protocol

After2weeksof acclimatization,28ratswereran-domly allocated into four experimental groups(n=7),designatedascontrol(C),cyclophosphamide-treated(CP),pecannutshellaqueousextract(AE),andextractplusCP-treated(AE+CP).Tapwater(CandCPgroups)orthefreshpecanshellAE(AEandAE+CPgroups), similar to tea,which ispopularlyused,wasdailyoffered to animalsad libitum.After30daysof treatmentwithpecanshellAEorvehicle,thegroupsdesignatedasCPandAE+CPreceivedasingledoseof CP(200mg/kgbodyweight-i.p.),whileCandAEgroupswere injectedwithvehicle(distilledwater)andmaintainedwiththeoraltreat-ment (pecan shell AE or vehicle) for another 7days.Onday8,theanimalswereanesthetizedwiththiopental (50 mg/kg/mL, i.p.) and sacrificed byexsanguination (thebloodwascollectedbycardiac

FIGURE 1.Highperformanceliquidchromatogramof gallicacid(GA)authenticstandard(A)andpecanshellAE(B).tR=3.68minand1.8gGA/100gpecanshellAE(monitoredat290nm).

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puncture).Thebloodwascentrifugedforseparationintoplasmaanderythrocytesusedintheevaluationsdescribedbelow.Theheart, liverandkidneyswereremoved,homogenizedin10volumes(10mL/gtis-sue) of 10mMTris-HCl buffer (pH-7.4), and cen-trifuged (5000 rpm/20min) for biochemical evalu-ations. After removal, the bladder was observedmacroscopically and fixed in 10% buffered form-aldehyde for subsequent histological analysis. Thisprocedurewasespeciallydonewiththebladderbe-causetheurotoxicityisthemostserioussideeffectrelatedtoCPtreatment.

Thiobarbituric Acid Reactive Species (TBARS) Levels

TBARSassaymeasuresLPwhichoccursbyexcessiveROSgeneration.LPwasestimatedthroughthepinkchromogen produced by the reaction of thiobarbi-turic acid (TBA) with malondialdehyde (MDA) at100ºC,measuredspectrophotometricallyat535nm.

Inplasmaanderythrocytes,TBARSwasestimatedbythemethoddescribedbyLapennaetal.(2001),28aftermodifications.29Inthetissues,TBARSlevelswerede-terminedinaccordancewithOhkawaetal.(1979).30Resultswere expressed as nmolMDA/mLplasma,nmolMDA/mLerythrocytesandnmolMDA/gtis-sue,respectively.

Estimation of Antioxidants

Reduced Gluthatione (GSH) Levels

GSH tissue content was determined after reactionwith5,5’-dithiobis-(2-nitrobenzoicacid).Theyellowcolorformedwasreadat412nm,inaccordancewithBoyneandEllman(1972),31aftermodifications.29Astandard curveusing cysteinewasused to calculatethecontentof GSHintissuesamples,expressedasmmolGSH/gtissue.

Red blood cell pellets (RBC) obtained aftercentrifugation of whole blood were hemolyzed

FIGURE 2.Effectsof pecanshellAEonTBARS(A)andGSH(B)levelsandCATactivity(C)inheartof ratstreatedwithCP.*IndicatesasignificantdifferencefromCgroup(P<0.05);+IndicatesasignificantdifferencefromAEplusCPgroup(P<0.05).

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with 10% triton solution and the protein fractionwasprecipitatedwith20%trichloroaceticacid fol-lowedbycentrifugation.Thecolorimetricassaywascarried out as described above. A standard curveusing GSH was constructed in order to calculatethecontentof GSH,expressedasnmolGSH/mLerythrocytes.

GSH was assayed in plasma using the samemethodof BoyneandEllman(1972).31AstandardcurveusingGSHwasalsoutilized.Resultswereex-pressedasnmolGSH/mLplasma.

Vitamin C Levels

PlasmavitaminC(VITC)wasestimatedasdescribedbyGalley et al. (1996)32with somemodifications.29Thismethodproducesanorangechromogenbythereaction with dinitrophenylhydrazine at 37ºC,mea-suredspectrophotometricallyat520nm.Astandardcurve using ascorbic acidwas used to calculate thecontentof VITCandisexpressedasmgVITC/mLplasma.

CAT Enzyme Activity

CAT activity was spectrophotometrically quantifiedintissuesbythemethodof Aebi(1984)33whichin-volvesmonitoringthedisappearanceof H2O2inthepresenceof cellhomogenate(pH7at25°C)at240nm. The enzymatic activity was expressed in µmolH2O2/min/gtissue.

Macroscopical Analysis

Bladdertissueswereanalyzedbythreeobserversandscoredaccordingtoadamagescaleof 0(normal)to4(severechanges).Thedamagewasscoredaccordingtothetissuecolorintensity.

Histological Evaluation

Afterfixation (minimum time18-24h), thebladdertissuesweretrimmedinto2-4mmthicksectionsforprocessing and sectioning.The tissueswere embed-dedinparaffinandatleastfourcrosssections4-5mmthickweretakenfromeachbladderandstainedwithhematoxylin-eosin(H&E).Histopathologicalexami-nationwasperformedbyapathologistandmicropho-tographsweretakenusingthesoftwareWinTV2000.

Statistical Analysis

Levene’s testwas applied toverify thevarianceho-mogeneity. Parametric data were analyzed by two-way ANOVA followed byDuncan’smultiple rangetest when appropriate. Values were expressed asmean±S.E.M.NonparametricdatawereanalyzedbyKruskal-Wallisanalysisof variancefollowedbytwo-tailedMann-WhitneyU testwhen appropriate, andexpressed as median±quartiles. Statistica softwarepackageforWindowsversion8.0wasusedandvaluesof P<0.05wereconsideredstatisticallysignificantforallcomparisonsmade.Valuesof TBARSof alltissueswereexpressedasmedian±quartile.Valuesof GSH,VITClevelsandCATactivityshowednormaldistri-butionandthereforewereexpressedasmean±S.E.M.

Results

Treatmentswithcyclophosphamide(CP),pecanshellAE(AE),orthecombinationof cyclophosphamidepluspecanshellAE(CP+AE)ontheoxidativestressparameters of the heart, liver, kidneys, plasma anderythrocytesareshowninFig.2-6,respectively.

Heart:TheCPtreatmentincreasedTBARSlevelsintheheartinrelationtoCgroup,andthiseffectwasnotpreventedbytheextract.Infact, theco-treatedgroup showed similar TBARS levels to CP group,whiletheAEtreatedgroupshowedsimilarvaluestoCgroup(Fig.2A).CPdecreasedtheGSHlevels intheheartwhencomparedtocontrol,andthiseffectwaspreventedbytheextractof pecanshells.Theex-tractaloneincreasedtheGSHlevelswhencomparedtoCgroup(Fig.2B).RatstreatedwithCPpresenteda significant increase in CAT activity in relation tocontrol. Alone, pecan shell extract did not changeCATactivity,butpreventedtheeffectof CPintheheart tissue when administered concomitantly (Fig.2C).

Liver: The CP treatment increased the hepaticTBARSlevelswhencomparedtocontrol,andthisef-fectwaspartiallypreventedbytheco-treatmentwiththeextract.Theextract treatedgroup(AE)showedsimilar values to control (Fig. 3A). CP significantlydecreased the hepatic GSH levels in relation to Cgroup,andthiseffectwaspartiallypreventedbytheco-treatment with pecan shell extract. The extractalonedidnotchangetheliverGSHlevels,whichweresimilartoCgroup(Fig.3B).RatstreatedwithCPpre-sentedasignificantdecreaseinliverCATactivityinrelationtoCgroup,andthiseffectwaspartiallypre-ventedbytheextract.TheAEgroupshowedhigher

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TABLE 1.Effectsof pecanshellAEonmacroscopicalchangesintheurinarybladderof ratstreatedwithCP(valuesareexpressedasmedian±quartile,n=7)

Groups Score

C 0(0-0)AE 0(0-0)CP 4(4-4)

AE+CP 0(0-1)0(normal)and4(severechanges).*IndicatesasignificantdifferencefromCgroup(P<0.05);+IndicatesasignificantdifferencefromAEplusCPgroup(P<0.05)

FIGURE 3.Effectsof pecanshellAEonTBARS(A)andGSH(B)levelsandCATactivity(C)inliverof ratstreatedwithCP.*IndicatesasignificantdifferencefromCgroup(P<0.05);+IndicatesasignificantdifferencefromAEplusCPgroup(P<0.05).

FIGURE 4.Effectsof pecanshellAEonTBARS(A)andGSH(B)levelsandCATactivity(C)inkidneyof ratstreatedwithCP.*IndicatesasignificantdifferencefromCgroup(P<0.05);+IndicatesasignificantdifferencefromAEplusCPgroup(P<0.05)

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CATactivitythancontrol(Fig.3C).Kidney: The CP treatment increased renal

TBARSlevelsinrelationtoCgroup,andthiseffectwas partially prevented by the extract. In fact, theco-treatedgroupshowedhigherrenalTBARSlevelsthantheCgroup,whiletheAEgroupshowedsimilarvalues to control (Fig. 4A).CPdecreased theGSHlevelsinkidneywhencomparedtocontrol,andthiseffect was partially prevented by the co-treatmentwith pecan shell extract. The extract alone did notchange theGSH levels when compared to control(Fig. 4B). TheCP treatment significantly decreasedthe renalCATactivitywhencompared toCgroup.Alone, theextract increased theCATactivity in re-lationtocontrol,butdidnotprevent theCPeffectwhenadministeredtogether(Fig.4C).

Plasma: The CP treatment increased TBARSlevelsinplasma,andtheco-treatmentpreventedthiseffect.TheextractalonedidnotchangetheTBARSlevels(Fig.5A).CPreducedtheGSHlevelsinplasmawhencompared tocontrol, and thepecanshell ex-tractdidnotpreventthiseffect.GSHlevelswerenotchangedbytheextract treatment(Fig.5B).TheCPtreatmentreducedtheplasmaticVITClevelswhencomparedtocontrol,andtheextractpreventedthiseffect.PecanshellextractshowedVITClevelssimi-lartocontrol(Fig.5C).

Erythrocytes: The CP treatment increased theTBARS levels in relation to control and this effect

was partially prevented by the co-treatment withpecan shell extract. In fact, the co-treated groupshowedhigherTBARSlevelsinerythrocytesthantheCgroup.TheextractdecreasedtheTBARSlevelsinerythrocytes, when compared to control (Fig. 6A).ThetreatmentwithCPdecreasedtheGSHlevelsinerythrocytesinrelationtocontrol.Theco-treatmentwithpecanshellextractpartiallypreventedthiseffectof CPandtheextractalonedidnotaltertheerythro-cytes’GSHlevels(Fig.6B).

Treatmentswithcyclophosphamide(CP),pecanshellAE(AE)orthecombinationof cyclophospha-midepluspecanshellAE(CP+AE)on themacro-scopicalevaluationof thebladderareshowninTable1,andthehistopathologicalevaluationsareshowninTable2andFig.7.

CP treatment induced damages in the bladder,which were prevented by pecan shell extract. Theco-treated group showed damage scores similar tocontrol.Theextractalonedidnotmodifythescoresof thebladder(Table1).

Urinary bladders from control group presentnormal cytology, showing transitional epithelial lin-ing, narrow lamina propria with epithelial lining infoldsandnormalmusclelayer(Table2;Fig.7Aand7B). Rats treated with CP showed widened laminapropriawithseveredegreeof hemorrhageandedemaaswellasmoderateleukocyteinfiltrationandvascularproliferation.We can also observe a thickening of

FIGURE 5.Effectsof pecanshellAEonTBARS(A),GSH(B)andVITC(C)levelsinplasmaof ratstreatedwithCP.*IndicatesasignificantdifferencefromCgroup(P<0.05);+IndicatesasignificantdifferencefromAEplusCPgroup(P<0.05).

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FIGURE 6.Effectsof pecanshellAEonTBARS(A)andGSH(B)levelsinerythrocytesof ratstreatedwithCP.*IndicatesasignificantdifferencefromCgroup(P<0.05);+IndicatesasignificantdifferencefromAEplusCPgroup(P<0.05).

FIGURE 7.Effectsof pecanshellAEonhistologicalchangesintheurinarybladderof ratstreatedwithCP.Theleftcolumnrepresentslowmagnification(40X)andtherightcolumnrepresentshighmagnification(100X).AandB–controlgroup;CandD–pecanshellAEgroup;EandF–cyclophosphamidegroup;GandH–pecanshellAEpluscyclophos-phamidegroup.

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enceof hydroxylgroups,mainlyattheparaposition,is especially efficient for the antioxidant activity of GA.36

LPmediatedby excessiveROSproductionnotneutralized is shown here through an increase of 60.2%of heartTBARSlevels.Otherauthorsalsoob-servedLPdevelopmentinhearttissueof ratstreatedwithCP.37,38Inthissense,cardiacmuscleisparticular-lysusceptibletoFRinjury,mainlybecauseitcontainslowlevelsof enzymaticantioxidantsandGSH.39,40Inourfindings,theCPtreatmentreducedaround37.6%of GSH levels and increased 73%of CAT activityinthehearttissue.Recentlyitwasreportedthatthealkylant agents are potent inactivators of glutathi-one reductase.41 In fact, thealkylantmetabolitesof CPcanreactwithsulfhydrylgroupsof GR,and inturnreducetheregenerationof GSHfromoxidizedglutathione.38 The same authors reported that CPdecreasestheheartCATactivity,whileweobservedanincreaseof itsactivity.Infact,theseexperimentalparadigmswereperformedusingdifferentdosesof CPandthereforecannotbeconsideredcontradicto-ry.Thus,theheartenzymaticchangesdemonstratedherewerepreventedbypecanshellextract,showingits antioxidant potential. The cardiotoxicity of CPtreatment is a serious side effect of CP treatment,mainly by lethal cardiotoxicity described after hightherapeuticdosesof thischemotherapy,12emphasiz-ingtheneedfornovelcompounds,suchasplantsorfoods,whichwouldprotect thenormal tissue fromchemotherapy-inducedtoxicity.

Liver disorderswere observedwhen the thera-peuticdoseof CPneedstobeincreased.13,14,42Herewe could show the liver toxicity by CP treatment,evidenced by an increase of 116.7% in LP and adecreaseof antioxidantdefensesof 84.9%forGSHlevels and 58.12% for CAT activity.Other authorsalso published similar findings,43,44 confirming the

muscle layer in these tissues (Fig. 7E and 7F).Theco-treatmentwithpecanshellextractshowedanim-provementinbladdertissuesbecausetheypresentedmildthickeningof laminapropriawithoutedemaandvascularproliferationaswellaslowdegreeof hemor-rhageandleukocyteinfiltration.Onlythemusclelayerdidnotshowanychangesinthisgroup(Fig.7Gand7H).Pecanshellextractdidnotalterthehistologyof bladdertissueinrelationtocontrol(Fig.7Cand7D)

Discussion

Themost commoneffectof the chemotherapeuticagents is the cytotoxicity in different tissues,whichisrelatedtoanincreaseinthefreeradicals(FR).34Inourstudy,TBARSlevelswereincreasedintheheart,liver,kidneys,plasma,andredbloodcellsduetotheLPinducedbyCP.Theseeffectsmightbeduetotheincreasedproductionof FRand/ordecreasedanti-oxidantdefensesystem.Animalsco-treatedwithpe-canshellsAEplusCPshowedlowerTBARSvaluesinliver,kidney,plasma,andredbloodcells,indicatingreduced levelsof LP in these tissues. In this sense,theCP-inducedOSwaspreventedorattenuatedbypecanshellextract.Thishypothesisisinaccordancewithpreviousexperimentsperformedinourlabora-tory,whenthepecanshellsshowedhighin vitroandex vivoantioxidantpotential(submittedresults).

Theeffectsof pecanshellAEontheoxidativedamages induced by CP may be explained by thepresence of phenolic compounds and condensedtannins inpecanshells.4,35Of particular importanceto our findings, GA (3,4,5-trihydroxybenzoic acid)wasthemostabundantpolyphenolobserved inpe-can shells. Recently, Lu et al. (2006)36 attributed ascavenger activity to this compound on superoxideanion, hydroxyl radicals, singlet oxygen or peroxylradical, showinghealthypotential. In fact, thepres-

TABLE 2.Effectsof pecanshellAEonhistologicalchangesintheurinarybladderof ratstreatedwithCP.

Groups Hemorrhage EdemaLeukocyte infiltration

Vascular proliferation

Thickening of muscle layer

Thickening of lamina propria

C ND ND ND ND ND NDAE ND ND ND ND ND NDCP +++ +++ ++ ++ ++ +++

AE+CP + ND + ND ++ ++++Severe,++Moderate,+Mild,ND=notdemonstrated.

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leukocyteinfiltrationinthebladder.Theco-treatmentwithpecanshellextractshowedonceagain itshighantioxidantpotential observed through the absenceof these deleterious effects of CP on the bladder.Theuseof antioxidantcompoundssoundspromis-ingasalternativestopreventCP-inducedurotoxicity.In fact,Mesna (2-mercaptoethanesulfonic acid) is athiol clinically used as a uroprotective compound.53However this medicine decreases the incidence of cystitisinonly5%.19Thismodestbenefitof Mesnaon theCP-inducedurotoxicity points to the searchformorepotentcompoundsthatmaypreventthesedamages.

Clinical studies have shown that patients whoreceiveantioxidantswiththestandardchemotherapytoleratethetreatmentbetterandhaveprolongedsur-vivaltimecomparedwithexpectedoutcomewithoutthe antioxidant supplements.26,54Moreover, chemo-therapyandantioxidantsmayenhancetheeffective-nessof thetreatment.55,56Thus,naturalantioxidantsmayoffercomparativelysaferalternativestosyntheticantioxidants,whichmay cause seriousor unaccept-able adverse side effects.57 The natural antioxidantstudiedhererevealedapotenteffecttominimizethechemotherapydamagecausedinhealthytissues.

Weknowthatinnovativesubstancescanbefrac-tionatedandisolatedleadingtoanewdrugandanewindustrialtherapeuticarsenal.However,whenanewchemicalsubstanceisisolatedfromthecrudeextractof plants it becomes a new drugwith side effects.On theotherhand, thepopularknowledgemaybeextremelyuseful tominimize side effects related toconventionalpharmacotherapyaswellastocontrib-ute to the therapeuticeffects. In this sense, theuseof natural compounds such as foods or beveragesintheir integralform,asdemonstratedhere,canbeespeciallybeneficialwhenassociatedtomedicines.

Pecan shell is a by-product of the pecan nutindustry with high antioxidant potential which waseffectiveinreducingtheCPdamageonvitaltissues.Thisextractmaybeusefultopreventdeleteriousef-fectsrelatedtothischemotherapy.Fromtheseobser-vations,itispossibletoconcludethatCPtreatmentresults in pronounced damage in the heart, liver,kidneys,bloodandbladdermediatedbyOSduetoitstoxicmetabolites.Pecanshellextractshowedprotec-tiveeffectsagainstthegeneraltoxicityof CP,anditschemicalconstituentsdeservefurtherstudies.

Acknowledgements

toxicityof CPintheliver.Pecanshellextractshowedbeneficial effects through the prevention of theseoutcomesintheliver.

CPtreatment isalsorelatedtonephrotoxicity,45which has been attributed to acrolein metabolite.46Recently, Sugumar andAbraham (2007)47 observedan increaseof 47%of renalLPand adecreaseof 77%of GSHlevels.Themechanismof CP-inducedrenal damage is limited, and oxidative stress isthoughttoplayacentralroleintheseevents.21Thisway,wefoundsimilarresultsobservedbyanincreaseof TBARS (266.8%) aswell as low levels of GSH(74.4%) and CAT activity (32.9%) in renal tissue,demonstrating the wide renal damage mediated byCP.Animportantfindinginthepresentstudyisthatthehighantioxidantpropertyof pecanshellextract35wasabletopreventtheLPandrestoretheGSHlev-elsthatCPinducedinthisvitaltissue.

ThebloodcanalsobeaffectedbyCPtreatment,whichmightbeobservedinplasmaanderythrocytesservingasmarkersof bodydamage.48Here,CPtreat-mentincreased42.4%of plasmaTBARSlevelsandthelevelsof GSHandVITCwerereducedin56.9%and26.3%,respectively.Lowlevelsof plasmaticVITCduringCPtreatmentmayincreasethesusceptibilityof tissuestoROSdamage.49Ourfindingsshowedpe-canshellextractpreservedtheplasmaticVITClevelsandprevented theLP, strengthening its antioxidantpotential.

Redbloodcellmembrane is susceptible tooxi-dativestressdue to itshighcontentof polyunsatu-rated fatty acids, which are vulnerable to oxidativedamage.50 Recently, a study showed an increase of TBARSand adecreaseof GSH in erythrocytesof ratstreatedwithCP,whichwerereversedbythean-tioxidantpropertiesof thesqualene.38Theseresultsare inaccordancewithours,whichshowedthatthepecanshellextractpreservedtheLPandGSHlevelsmodifiedbyCPinerythrocytes.

Oneof themajorsideeffectsof CPadministra-tionisurotoxicity.51Sincethebladderistheprimarystorageorganforurine,thecontentof CPmetabo-lites ishigher than theother areasof urinary tract,increasingthesensitivityof thebladdertooxidativedamage.52 These deleterious effects of CP includeurothelialdamage,edema,necrosis,ulceration,hem-orrhage, neovascularization, and leukocyte infiltra-tion.51 Inourfindings, theCP induced accentuateddamage to theendothelial layer, edemaandhemor-rhage,aswellasmoderatevascularproliferationand

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TheauthorswishtothankPecantea®forprovidingpecanshellsandDolesReagentes®whichprovidedthecommercialkits.FinancialsupportwasprovidedbyCAPES,CNPq.

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