Effects of (-)-cubebin (Piper cubeba) on cytotoxicity, mutagenicity and expression of p38 MAP kinase and GSTa2 in a hepatoma cell line

Original

Research

Article

Effects

of

( )-cubebin

(

Piper

cubeba

)

on

cytotoxicity,

mutagenicity

and

expression

of

p38

MAP

kinase

and

GSTa2

in

a

hepatoma

cell

line

Andressa

Megumi

Niwa

a,

*

,

Juliana

Cristina

Marcarini

b

,

Daniele

Sartori

a

,

Edson

Luis

Maistro

c

,

Ma´rio

Se´rgio

Mantovani

a

a_{DepartamentodeBiologiaGeral,UniversidadeEstadualdeLondrina(UEL),Londrina(PR),Brazil} b_{InstitutodeBiocieˆncias,UniversidadeEstadualPaulista–UNESP,RioClaro,Sa˜oPaulo,Brazil}

c_{DepartamentodeFonoaudiologia,UniversidadeEstadualPaulistaJuliodeMesquitaFilho(UNESP),Marı´lia,Sa˜oPaulo,Brazil}

1. Introduction

ThepepperPiper cubeba, known asthecubeb pepper,tailed pepperorJavapepper,is apopularspiceconsumedthroughout Europeaswellasinmanyothercountries,includingArabia,India, IndonesiaandMorocco(Junqueiraetal.,2007).Thispepperhas beenusedsincetheMiddleAgesasbothaspiceandinthepractice oftraditionalmedicineforthetreatmentofvariousdiseases.The genusPiperincludesmorethan1000speciesofplants,whichgrow asweeds,brushor,lessfrequently,asstandingorsustainedtrees (Junqueiraetal.,2007),andtheyareprimarilyfoundinAsiaand theNewWorld(Nunesetal.,2007).

Recentstudieshaveshownthatthelignan dibenzylbutyrolac-tone( )-cubebincanbeextractedfromtheseedsofP.cubeba.This lignan has several beneficial properties, including trypanocidal (Bastos et al., 1999; de Souza et al., 2005), analgesic, anti-inflammatory(Bastosetal.,2001),leishmanicidal(Bodiwalaetal., 2007)andanti-proliferative(Yametal.,2008)activities.Lignans

(fromtheLatinlignum,meaningwood)arephytoestrogensfound inawidevarietyofplants(Krajcˇova´ etal.,2009).Theyconsistof dimers formedvia theoxidative couplingof cinnamyl alcohols witheachotherorwithcinnamicacids(BarbosaFilho,2004).

Despite the substantial consumption of P. cubeba in various countries,fewstudieshaveevaluatedtherisksassociatedwiththe consumption of this species. Given the promising therapeutic potentialofthislignan,theobjectiveofthisstudywastoassessthe cytotoxicity,mutagenicityandcellproliferationkineticsinHTCcells (aRattusnorvegicushepatomacellline)treatedwith( )-cubebin. Wealsoexaminedcellsexposedto( )-cubebintodeterminethe expressionofp38MAPkinase,whichisinvolvedinseveralcellular functions,suchasinflammation,cellgrowth,differentiation,cell cycleregulation andcelldeath (Zarubin andHan,2005),and of glutathioneS-transferase(GST),whichisinvolvedinthemetabolism of xenobiotics.Collectively,these experimentsprovide datathat contributetothesafetherapeuticusageof( )-cubebin.

2. Materialsandmethods

2.1. ( )-Cubebinextractionandpreparation

( )-Cubebinwasobtained frompowderedseedsofP.cubeba imported from India via maceration with 96% ethanol. Details

ARTICLE INFO

Articlehistory:

Received22February2012

Receivedinrevisedform7December2012 Accepted20December2012

Keywords:

( )-Cubebin

Pipercubeba

Cytotoxicity Mutagenicity Cellproliferation HTCcells Foodsafety Foodanalysis Foodcomposition

ABSTRACT

( )-CubebinisalignanextractedfromtheseedsofthepepperPipercubeba,acommonlyeatenspicewith beneficial properties, including trypanocidal, anti-inflammatory, analgesic, anti-proliferative and leishmanicidal activities. Because of its therapeutic potential, we investigated theeffects of ( )-cubebinonthecytotoxicity,cellproliferationkinetics,mutagenicityandexpressionofp38MAPkinase andglutathioneS-transferasea2(GSTa2)usingreal-timeRT-PCRinRattusnorvegicushepatomacells.We foundthat280mM( )-cubebinwascytotoxicafter24,48and72hofexposure,butnotmutagenicat

0.28mM,2.8mMand28mMafter26h.Similarly,exposureto0.28mM,2.8mMand28mM( )-cubebin

for24,48,72and96hdidnotalterthecellproliferationkinetics.Cellsexposedto28mM( )-cubebinfor

24hdidnotexhibitchangesinp38MAPkinaseandGSTa2expression,indicatingthatcellularchanges werenot induced byextracellular stimuli andthat ( )-cubebinis likely notmetabolized viathis pathway.Ourresultssuggestthathighlevelsof( )-cubebinshouldbeconsumedwithcautionduetothe cytotoxiceffectobservedatthehighestconcentration.However,atlowerconcentrations,nocytotoxic, mutagenicorproliferativeeffectswereobserved,providingfurtherevidenceofthesafetyofconsuming ( )-cubebin.

ß2013ElsevierInc.

*Correspondingauthorat:DepartamentodeBiologiaGeral–CCB,Universidade EstadualdeLondrina–CampusUniversita´rio,RodoviaCelsoGarciaCid,Pr445Km 380,CampusUniversita´rio,P.O.Box6001,Londrina,Parana´,CEP:86051-980,Brazil. Tel.:+554333714977.

E-mailaddress:andressamn@yahoo.com.br(A.M.Niwa).

ContentslistsavailableatSciVerseScienceDirect

Journal

of

Food

Composition

and

Analysis

j our na l h ome p a ge : w ww . e l se v i e r. co m/ l oc a te / j f ca

0889-1575ß2013ElsevierInc.

http://dx.doi.org/10.1016/j.jfca.2012.12.003

Open access under the Elsevier OA license.

regardingtheisolationof( )-cubebinwereprovidedbyMaistro etal. (2011). The ( )-cubebin, which was estimatedto have a purityof99%byHPLCandspectraldataanalyses,withayieldof 2.24mg/g dry seed. The ( )-cubebin was diluted in dimethyl sulfoxide(DMSO)(MallinckrodtChemicals,USA).The concentra-tionofDMSOdidnotexceed0.25%oftheculturemedium.

2.2. InductionofDNAdamage

Doxorubicin (18.4

mM;

Adriblastina1_{, Pharmacia, Italy) was} dissolvedinculturemediumandusedtoinduceDNAdamagefor the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) cytotoxicity and cell proliferation kinetics assays. For the cytokinesis-block micronucleus assay, the mutagen benzo[a]pyrene (Fluka, Switzerland), which indirectly induces DNAdamage,wasusedataconcentrationof79

mM

anddissolved in0.8%DMSO.

Theuseofdoxorubicinwasbasedonpreliminarystudiesfrom ourgroupjusttoselectwhichthecytotoxicconcentration.Theuse ofbenzo[a]pyreneaspositivecontrolinmicronucleusassaywas duetotheuseofHTCcells (fromrathepatoma) toverifytheir functionalmetabolicactivity.Benzo[a]pyreneisanindirect-acting mutagen,i.e.,mustbemetabolizedbycellstocauseDNAdamage (Vermaetal.,2012),providingevidencethatHTCcellsduringthe micronucleusassayweremetabolicallyactive.

2.3. HTCcells

AR.norvegicushepatomacellline(HTCcells)wasacquiredfrom theRiodeJaneiroCellBank(RJBC/UFRJ).HTCcellsweregrownat 37₈C in culture flasks (25cm2_{) containing a 1:1 mixture of} Dulbecco’smodifiedEagle’smedium:nutrientmixtureF-12(Ham) (Gibco, USA) and supplemented with an antibiotic–antimycotic solution(Gibco,USA)and10%fetalbovineserum(FBS)(Gibco,USA).

2.4. MTTcytotoxicityassay

The MTT assay (Invitrogen, Life Technologies, USA) was performed according to the protocol described by Mosmann (1983).Seeded96-wellcultureplates(2.5104_{cells/well)were} incubated at 37₈C for 24h. After this period, the following treatmentswereadministeredfor24,48and72h:control(0.25% DMSO); cytotoxicity inducer (18.4

mM

doxorubicin); and ( )-cubebinat0.028

mM,

0.28

mM,

2.8

mM,

28

mM

and280

mM.

The selectionofconcentrationsusedinMTTassaywasbasedoninvivo studyofMaistroetal.(2011).DMSOataconcentrationof0.25% wasalsoadministeredtowellscontainingculturemediumalone (no cells) for use as a ‘‘blank’’ control. Three independent experiments were performed, and each experiment included twelvereplicates.

2.5. Cellproliferationkinetics

Changes in cell proliferation were quantified in HTC cells exposed to ( )-cubebin for 24, 48, 72 and 96h by manually countingcells.Approximately105cellsperculturetube(10cm2) wereincubatedunderthefollowingtreatmentconditions:control (0.025%DMSO);DNAdamageinducer(18.4

mM

doxorubicin);and ( )-cubebin(0.28

mM,

2.8

mM

and28

mM).

Thesametreatment conditions were tested in the remaining experiments. Three independentrepetitionswereperformedineachtreatment.

2.6. Cellviabilityassay

Cellviabilitywasmeasuredbymanuallycountingcellsstained with0.2%trypanbluereagent(Gibco,USA)inaNeubauerchamber,

accordingtoMauroetal.(2011).Every24h,100cellsfromeach treatmentwerecountedoveratotalperiodof96h.Thenumberof viableversusnonviablecellswasexpressedasapercentageofthe total cell viability. Three independent experiments were per-formedforeachtreatmentcondition.

2.7. Cytokinesis-blockmicronucleusassayanddeterminationofthe nucleardivisionindex

HTCcells(106_{)weregrownincultureflasks(25cm}2_)for24h priortotheadministrationofthedifferenttreatments.Binucleated cellswereobtainedviaincubation with3

mg/mL

cytochalasin-b (Sigma–Aldrich,Germany)for27h.Afterthisperiod,thecellswere harvestedaccordingtoapreviouslypublishedprotocol(Martinsde Oliveiraetal.,2002).Theslideswereanalyzedaccordingtocriteria determined by Fenech (2000). Three independent experiments wereperformed.

The nucleardivision index(NDI) was alsodetermined from theseslidesandverifiedaccordingtoEastmondandTucker(1989). Threeindependentexperimentswereperformed.

2.8. Real-timeRT-PCR

Toevaluatep38MAPkinaseandGSTa2geneexpression,106_HTC cells were grown in culture flasks (25cm2_{) for 24h and then} exposed to 28

mM

( )-cubebin for a period of 12h. After this period,thecells weretrypsinized,and totalRNAwasextracted usingTRIzolLS(Invitrogen,LifeTechnologies,USA).Theextracted RNA was treated with 1U of DNase I Amplification Grade (Invitrogen, Life Technologies, USA). The purity of the sample wasconfirmed using a spectrophotometer(Eppendorf Biophot-ometer,Germany),andsampleintegritywasverifiedvia electro-phoresisona0.8%agarosegel.

cDNA wassynthesizedfrom1

mg

of totalRNAin a reaction containing0.25mM dNTPs(Invitrogen, LifeTechnologies,USA), 10pM oligo-dT (Invitrogen, Life Technologies, USA), 4U of RNaseOUTTM_{RecombinantRibonucleaseInhibitor(Invitrogen,Life} Technologies, USA) and 200U of M-MLV reverse transcriptase (Invitrogen,LifeTechnologies,USA)inatotalreactionvolumeof 20

mL.

cDNAsynthesiswascarriedoutinathermocyclerat37₈C for50min,followedby70₈Cfor15min.

Real-timePCRwasperformedinaPTC200DNAEngineCycler thermocyclerusingtheChromo4detectionsystem(MJResearch BIO-RAD).PCRamplificationofeachsamplewasperformedina totalreactionvolumeof20

mL,

whichincluded2

mL

ofcDNAand 0.25

mM

of eachforwardandreverseprimer,toamplifypartial regionsofthep38MAPkinaseand GSTa2genes(Table1).cDNA amplificationwasmeasuredaccordingtothefluorescenceemitted bytheSYBRGreenfluorophore(10

mL;

containedinthePlatinum SYBRGreenqPCRSupermix-UDGkit,Invitrogen,LifeTechnologies, USA). The PCR cycling conditions were as follows: an initial denaturationstepat508Cfor2minfollowedby958Cfor3min

Table1

Oligonucleotideprimersusedforreal-timeRT-PCR.

Gene Primersequence Reference

GAPDH F:50_{ACAAGATGGTGAAGGTCGGTGTCA3}0 _{Tsoetal.(1985)}

R:50_{AGCTTCCCATTCTCAGCCTTGACT3}0

p38 F:50_{AAGTCATTAGCTTTGTGCCACCGC3}0 _Inthisstudya

R:50_{AGTGGGATGGACAGAACAGAAGCA3}0

GSTa2 F:50_{AGCCATGGCCAAGACTACCTTGTA3}0 _{Pickettetal.(1986)}

R:50_{AGAGGTCAGAAGGCTGGCATCAAA3}0

and39cyclesof958Cfor20s,608Cfor30sand728Cfor20s,with afinalstepat958Cfor10sfollowedby408Cfor1min.Melting curve analysis was performed at the end of the reaction by increasingthetemperaturefrom508Cto958Cinstepsof0.58C every5s.EachPCRamplificationwasperformedintwobiological experimentsandwiththreemechanicalreplicates.Thedatawere normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)gene.

2.9. Statisticalanalysis

ThedataobtainedfromtheMTT,cellproliferationkineticsand micronucleus assays were assessed using analysis of variance (ANOVA)followedbyDunnett’stest.Comparisonswereperformed betweenthe( )-cubebintreatmentsandthecontrolgroupusing GraphPad Instat, where p< 0.05 was considered statistically significant.

The expression levels of p38 MAP Kinase and GSTa2 were determinedusingamethodpreviouslypublishedbyPfaffl(2001). ThestatisticalanalyseswereperformedusingREST-384software (Pfaffletal.,2002).

3. Results

TheabsorbancereadingsforHTCcellsexposedto0.028,0.28, 2.8 and 28

mM

( )-cubebin for 24, 48 and 72h were not significantly different from the control group, suggesting that ( )-cubebin is not cytotoxic when administered at these

concentrations. However, exposure to 280

mM

( )-cubebin resulted in a significant difference from the control group, suggestingthatthissubstanceiscytotoxicathighconcentrations (Fig.1).Weomittedthe280

mM

( )-cubebintreatmentcondition fromfurthertestingbecausecelldeathmayskewtheresultsofthe followingexperiments.Next,weevaluatedwhetherthe prolifera-tionofHTCcellsexposedto0.28,2.8and28

mM

( )-cubebinwas altered after 24, 48, 72 and 96h of exposure. At these three concentrationsof( )-cubebin,thecellproliferationkineticsofthe HTCcellswerenotalteredatanyofthetimepointsexamined,and there wereno significantdifferences detectedcompared tothe controlgroup(Fig.2).Weconfirmedthattherewasnocytotoxicity attheseconcentrationsusinga cellviabilityassay,inwhichno significantdifference wasobservedinthecells exposedto( )-cubebincomparedtothecontrolgroupatanyofthetimepoints examined(Fig.3).

To determine whether ( )-cubebin is able to induce DNA damage,acytokinesis-blockmicronucleusassaywasperformed. Nosignificantdifferencewasfoundinthenumberofmicronuclei detectedinthegroupsexposedto( )-cubebinfor26hcompared to the control group, demonstrating that ( )-cubebin is not mutagenicatconcentrationsof0.28,2.8and28

mM

(Fig.4).There was also no significant difference in theNDI compared tothe controlgroupundertheseconditions(Fig.4).

Inthereal-timePCRreactionsweconsideredtheaveragevalues ofefficiency80%.Next,weassessedwhetherHTCcellsexposedto 28

mM

( )-cubebinfor24hexhibitedalteredlevelsofp38MAP kinaseandGSTa2geneexpression.p38MAPkinaseandGSTa2are

Fig.1.AverageabsorbanceobservedinMTTcytotoxicityassayafter24,48and72hofincubationwith( )-cubebin.Control,culturemedium0.5%DMSO;DXR,18.4mM

doxorubicin(DNAdamageinducer).*Significantdifferencecomparedwiththecontrol(p<0.05).

Fig.2.GrowthcurveofHTCcellsexposedto( )-cubebinfor24,48,72and96h.

Control,culturemediumwith0.025%DMSO; DXR,18.4mMdoxorubicin (DNA

damageinducer).

involved in cell signaling and the metabolism of xenobiotics, respectively.Theanalysisoftheirexpressioncouldprovideinsight into the cellular alterations resulting from exposure to ( )-cubebin.However, wedidnot observesignificantdifferencesin p38MAPkinaseandGSTa2expressionincellsexposedto28

mM

( )-cubebincomparedtocontrolcells(Fig.5).

4. Discussion

The useof medicinalplantsbythegeneral population dates backquitealongtimeand continuestobeacommon practice, highlightingtheimportanceofperformingstudiestoassesstheir genotoxicity(Teixeiraetal.,2003).( )-Cubebinisalignanofgreat interestdue toitstrypanocidal(Bastosetal.,1999;Esperandim etal.,2013;deSouzaetal.,2005),analgesic,anti-inflammatory (Bastoset al.,2001), leishmanicidal(Bodiwalaet al.,2007)and anti-proliferativeactivities(Yametal.,2008).Duetothepromising therapeuticeffectsof( )-cubebinandconsideringthesubstantial consumption of P. cubeba in some countries, we examined a numberoffactorsrelatedtoitssafeconsumption.TheR.norvegicus hepatoma cells (HTC cells) usedin this study are proficientat metabolizingxenobiotics.Thus,HTCcellsrepresentanidealmodel systemforuseintoxicitystudiesbecausetheymimictheroleof theliverinmetabolizingxenobiotics,primarilyfromfoodsources inhumans.

Published reports on the mutagenicity of ( )-cubebin are scarce.Maistroetal.(2011)observedaclastogeniceffectinmurine bonemarrowcellstreatedwith500mg/kgand2000mg/kg( )-cubebin.Inthesamestudy,nogenotoxiceffectwasobservedinthe peripheralbloodcellsofmicetreatedwith250mg/kg( )-cubebin

for24handatconcentrationsof500mg/kgand2000mg/kgfor 4h.

Themutagenicityandrecombinogenicityeffectsof( )-cubebin alone orin combination withdoxorubicin wereinvestigatedin DrosophilamelanogasterbydeRezendeetal.(2011).Theseauthors found that treatment with 0.25, 0.5 or 1.0mM ( )-cubebin reduced the DNA damage induced by 0.2mM doxorubicin. However, administration of doxorubicin in combination with 2mM( )-cubebinsignificantlyincreasedDNAdamage, suggest-ing that this lignan can positively or negatively influence doxorubicin-inducedmutagenicity,dependingonthe concentra-tionapplied(deRezendeetal.,2011).

In addition to the work performed examining ( )-cubebin directly,someresearchershavestudiedplantextractscontaining ( )-cubebin(Junqueiraetal.,2007;Medolaetal.,2007;Resende etal.,2010).InastudybyJunqueiraetal.(2007),micetreatedwith seedextractsfromP.cubebaatconcentrationsof1.0g/kgand1.5g/ kgfor48and72hwereexaminedforclastogenicity.Thisanalysis demonstratedasignificantincreaseinmicronucleated polychro-matic erythrocytesintheblood whencomparedwitha control group.Similarly,treatmentwiththeseconcentrationsoftheseed extractfor24hsignificantlyincreasedthenumberofmicronuclei inmousebonemarrowcells.Ourdatasuggestthat( )-cubebinis notthecomponentofP.cubebaseedsthatisresponsibleforthe genotoxicityobservedinvivo.Theconcentrationofthecompound usedandtheexposuretimecouldalsocontributetothevariance observedbetweenthesestudies.

Inadditiontoperformingmorphologicalanalyses,weevaluated whethertreatmentwith28

mM

( )-cubebinalterstheexpression of genes involved in cell signaling (p38 MAP kinase) and the metabolismofxenobiotics(GSTa2).MAPkinasesarekeymembers ofasignalingpathwaythatplaysaroleinresponsetoavarietyof extracellular stimuli(Zarubinand Han, 2005). TheMAP kinase familyhasbeendividedintothefollowingfourdistinctsubgroups: (1) ERKs, which are kinases that areregulated byextracellular stimuli;(2)JNK/SAPKA-c-JunN-terminalkinase,whichisactivated bystress;(3)BMK1-ERK5/largeMAPkinase1;and(4)theprotein kinasep38group(OnoandHan,2000).Thep38kinaseshavebeen showntobeinvolvedininflammation,cellgrowth,differentiation, cell cycle regulation and cell death (New and Han, 1998). The glutathioneS-transferases(GSTs)aremembersofamultigenefamily involvedinthedetoxificationand,insomecases,activationofa widevarietyofchemicals(EatonandBammler,1999).GSTproteins arethemajordetoxifyingenzymesinvolvedinphaseII detoxifica-tionandareprimarilyfoundinthecytosol(Sheehanetal.,2001). WefoundthattheexposureofHTCcellsto28

mM

( )-cubebinfor 24hdidnotresultinanalterationoftheexpressionofp38MAP kinase,suggestingthat( )-cubebindoesnotinduceanysignificant cellularchanges.Thisfindingconfirmstheresultsobtainedinthe cytotoxicityandproliferationassays,inwhich28

mM

( )-cubebin was not found to be cytotoxicand did not affect cell growth. Additionally,HTCcellsexposedto28

mM

( )-cubebinfor24hdid notshowalteredexpressionofGSTa2,suggestingthat( )-cubebin is not metabolizedvia this pathway. The geneexpression data furtherimplythat( )-cubebinissafeanddoesnotalterimportant cellularevents.

5. Conclusion

Ourdatasuggestthat( )-cubebin,whichisobtainedfromP. cubeba, should be consumed with caution, as the highest concentration tested (280

mM)

was found tobe cytotoxic. The other three concentrations used here (0.28

mM,

2.8

mM

and 28

mM)

werenotmutagenic,andtheresultsofthesetreatments provideinsightintothesafeuseofthislignan.Consistentwiththis finding and in support of ( )-cubebin’s therapeutic potential,

Fig.4.Meanandstandarddeviationofthenumberofmicronuclei(MN)andNDIin HTCcellsexposedto( )-cubebinfor26h.Control,culturemediumwith0.04% DMSO;B[a]p,79mMbenzo[a]pyrene(DNAdamageinducer);MN,micronucleus;

NDI, nucleardivision index.*Significantdifferencecomparedwiththecontrol (p<0.05).

Fig.5.Evaluationoftherelativeexpressionlevelsofp38MAPkinaseandGSTa2by real-timeRT-PCRinHTCcellsexposedto28mM( )-cubebinfor12h.Thedata

( )-cubebin does not affecttheproliferation of ratliver tumor cells.Finally,HTCcellsexposedto28

mM

( )-cubebinfor24hdid notshowalteredexpressionofp38MAPkinaseandGSTa2,again indicatingthat( )-cubebincanbeconsumedsafely,asitdoesnot alterimportantcellularpathways.

Acknowledgements

Thisresearchhad thefinancial supportof CNPq,CAPES and Fundac¸a˜oArauca´ria.

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