50-Hz electromagnetic fields enhance cell proliferation and

BiochimicaetBiophysicaActa1743(2005)120–

129

/locate/bba

50-HzextremelylowfrequencyelectromagneticfieldsenhancecellproliferationandDNAdamage:Possibleinvolvement

ofaredoxmechanism

FedericaI.Wolfa,*,AngelaTorselloa,BeatriceTedescoa,SilviaFasanellaa,AlmaBoninsegnaa,

MarcelloD’Ascenzob,ClaudioGrassib,GianBattistaAzzenab,AchilleCittadinia

b

InstituteofGeneralPathologyandGiovanniXXIIICancerResearchCenter,L.goF.Vito,1-00168Rome,Italy

InstituteofHumanPhysiology,CatholicUniversityofSacredHeart,FacultyofMedicine,L.goF.Vito,1-00168Rome,Italy

Received11March2004;receivedinrevisedform2September2004;accepted8September2004

Availableonline19September2004

a

Abstract

HL-60leukemiacells,Rat-1fibroblastsandWI-38diploidfibroblastswereexposedfor24–72hto0.5–1.0-mT50-Hzextremelylowfrequencyelectromagneticfield(ELF-EMF).Thistreatmentinducedadose-dependentincreaseintheproliferationrateofallcelltypes,namelyabout30%increaseofcellproliferationafter72-hexposureto1.0mT.ThiswasaccompaniedbyincreasedpercentageofcellsintheS-phaseafter12-and48-hexposure.TheabilityofELF-EMFtoinduceDNAdamagewasalsoinvestigatedbymeasuringDNAstrandbreaks.Adose-dependentincreaseinDNAdamagewasobservedinallcelllines,withtwopeaksoccurringat24and72h.AsimilarpatternofDNAdamagewasobservedbymeasuringformationof8-OHdGadducts.TheeffectsofELF-EMFoncellproliferationandDNAdamagewerepreventedbypretreatmentofcellswithanantioxidantlikea-tocopherol,suggestingthatredoxreactionswereinvolved.Accordingly,Rat-1fibroblaststhathadbeenexposedtoELF-EMFfor3or24hexhibitedasignificantincreaseindichlorofluorescein-detectablereactiveoxygenspecies,whichwasbluntedbya-tocopherolpretreatment.CellsexposedtoELF-EMFandexaminedasearlyas6haftertreatmentinitiationalsoexhibitedmodificationsofNFnB-relatedproteins(p65-p50andInBa),whichweresuggestiveofincreasedformationofp65-p50orp65-p65activeforms,aprocessusuallyattributedtoredoxreactions.TheseresultssuggestthatELF-EMFinfluenceproliferationandDNAdamageinbothnormalandtumorcellsthroughtheactionoffreeradicalspecies.ThisinformationmaybeofvalueforappraisingthepathophysiologicconsequencesofanexposuretoELF-EMF.D2004ElsevierB.V.Allrightsreserved.

Keywords:8-OHdG;Singlestrandbreak;Cellcycle;DCF;NFnB;InB;a-Tocopherol

1.Introduction

Environmentalexposuretoextremelylowfrequency(ELF)electromagneticfields(EMF)isstronglyincreasedindevelopedcountriesasaconsequenceofthedistributionanduseofelectricity.

SinceWertheimerandLeeper[1]arguedthatthefrequencyofchildhoodcancercorrelatedtotheelectrical

*Correspondingauthor.FedericaI.WolfInstituteofGeneralPathology,CatholicUniversityofSacredHeart,FacultyofMedicine,L.goF.Vito,1-00168Rome,Italy.Tel.:+39063016619;fax:+39063012753.

E-mailaddress:fwolf@rm.unicatt.it(F.I.Wolf).0167-4889/$-seefrontmatterD2004ElsevierB.V.Allrightsreserved.doi:10.1016/j.bbamcr.2004.09.005

wiringconfigurationrunningnearbytheirhouses,thecorrelationbetweenELF-EMFexposureandcancerriskhasbecomeamatterofpublicconcern.Asaconsequence,thepossibleeffectsofELF-EMFonbiologicalsystemswereextensivelyinvestigated.Someepidemiologicalstud-iesofferedpositiveevidenceforacorrelationbetweenexposuretoELF-EMFandincreasedincidenceofbrain,breastandhematologicalmalignancies,buttheseresultshavenotbeenconfirmedinotherstudies[2–5].Studieswithlaboratoryanimalssimilarlyproducedinconclusiveorcontradictoryresults;thesmallnumberofanimalsexam-inedandthelackofproperlystandardisedexposureparametersmayhavecontributedtosuchnegativeor

F.I.Wolfetal./BiochimicaetBiophysicaActa1743(2005)120–129121

inconsistentresults[6,7].InvitrostudieshaveexploredthepotentialeffectsofELF-EMFoncellproliferation[8],apoptosis[9,10],differentiation[11],genotoxicity[12]andproto-oncogenemodulation[13–15];again,theresultsremainedinconclusiveoropentodebate.ItisunclearhowELF-EMFwouldinfluencecellularbehavior,butaplausiblehypothesisisthatELF-EMFaffectmembranestructureandpermeabilitytosmallmolecules.Inarecentstudy,wehaveshownthatinneuroendocrinecellsELF-EMFexposureincreasedCa2+currentsduetooverexpres-sionofvoltage-gatedCa2+channels,anoccurrencethatmaywellbecorrelatedwithproliferativeevents[16].AnotherinterestinghypothesisisthatELF-EMFinterferewithchemicalreactionsinvolvingfreeradicalproduction(seeRef.[17]forreview).

Itiswellestablishedthatfreeradicalshavepleiotropiceffectswhichmayvaryfromcytotoxictomitogenicresponsesdependingonthedoseintensity,thedurationofexposure,andthetypeofcellortissue[18].Freeradicals,e.g.,hydroxylradicals,caninteractwithDNAandformprimarily8-OHdGadducts,resultinginsinglestrandbreaks[19].Adductsorstrandbreaksarerecognisedandusuallyremovedquiteefficientlybyspecificrepairmechanisms.Nevertheless,DNAdamagecouldbecomeasiteofmutationandakeysteptocarcinogenesisifthedamagewereextensiveenoughtoovercometherepaircapacityofthecell[20,21].Ontheotherhand,ithasbeendemonstratedthatlowlevelsofreactiveoxygenspeciestriggerintracellularsignalsthatinvolvethetran-scriptionofgenesandleadtoresponsesincludingproliferation[22,23].Oneofthebestcharacterizedredox-modulatablesignalsinvolvesNFnB,whichcantriggerproliferationorapoptosis[24,25].NFnB/Relproteins,primarilycomposedofheterodimersofp50/p65,arepresentinthecytoplasmintheirinactiveformassociatedwithIkBs.Activationoccursafterphosphor-ylationofInBaandtranslocationofp50/p65intothenucleuswhere,afterfurtherphosphorylationofthep65subunit,itbindstoDNAandfunctionsasatranscriptionfactor.Inseveralstudies,overexpressionandnucleartranslocationofp65wereshowntocorrelatewiththetranscriptionactivityofNFkB[26].ActivationofNFnBthroughphosphorylationofInBhasalsobeeninvestigatedindetailandshowntobeassociatedwithreceptor-mediatedsignalslikeCTLA4[27].Otheractivationmechanisms,mediatedbyredoxreactions,havealsobeendescribed.

Inthisstudyweevaluatedtheeffectsof50-HzELF-EMFoncellproliferation,cellcycledistributionandDNAdamageinnormalcells(embryonichumanlungfibroblasts,WI-38),neoplasticcells(humanpromyelocyticleukemiacells,HL-60),andimmortalizedcells(ratfibroblasts,Rat-1).Weshowthat24–72-hexposureto0.5–1-mTELF-EMFincreasedcellproliferationandDNAdamageinallcelltypes,andthattheseeffectsmightbemediatedbyformationofreactiveoxygenspecies.

2.Materialsandmethods2.1.Cellculture

HumanpromyelocyticHL-60cellsweregrownat378Cin5%CO2/airatmosphereinRPMI1640(SigmaItalia,Milano)supplementedwith10%heat-inactivatedfetalbovineserum(FBS)(LifeTechnologiesItalia,Milano).Rat-1fibroblastswereculturedat378Cin5%CO2/airatmosphereinEMEMmedium(Sigma)supplementedwith10%fetalbovineserum(FBS)(LifeTechnologies).WI-38diploidfibroblastsderivedfromembryonichumanlungprimaryculturewerepurchasedfromIstitutoZooprofilatticodell’Emilia-Romagna(Brescia,Italy).Cellswerereceivedfromtheproviderat11populationdoublings(PDs)andweregrownat378Cunder5%CO2/airatmosphereinEaglebasalmedium(BME)(SigmaItalia)supplementedwith10%fetalbovineserum(FBS)(LifeTechnologiesItalia)and1mMsodiumpyruvate.Cellswereusedforexperimentswithin21PDs.

Experimentswereroutinelycarriedoutontriplicatecultures.AtspecifiedtimescellswereharvestedandduplicatehemocytometercountswithCoulterZ1wereperformed.Thetrypanblueexclusiontestwasusedtoevaluatethepercentageofviablecells.Inallcases,cellswereusedforexperimentsonlywhenviabilitywasN95%.2.2.Anti-oxidanttreatment

Whereindicated,cellswereexposedtotheantioxidantdl-a-tocopherol(FlukaChemika-bioChemika,Buchs,Swit-zerland).After24hthemediumwasreplacedandcellswereexposedtoELF-EMF.dl-a-Tocopherolwasdeliveredtothecellsusingtetrahydrofuran(THF)or96%ethanol(Fluka)asasolvent.WhenTHFwasused0.25g/LBHTwasaddedtoavoidtheformationofperoxides.Stocksolutionsofa-tocopherolwerepreparedimmediatelybeforeeachexperi-mentandjudged~98%purebyHPLC.Aliquotsofstocksolutionswererapidlyaddedtotheculturemediumtoachieveafinalconcentrationof10AM.Controlexperimentsshowedthat10AMwasthelowestconcentrationatwhicha-tocopherolinterferedwithELF-EMFwhilenotelicitinganydirecteffectoncellproliferation(datanotshown).TheamountofTHFaddedtothecellsneverexceededN0.5%(v/v).ControlexperimentsshowednodifferencebetweenuntreatedculturesandthosetreatedwithTHForethanolaloneintermsofcellnumber,viability,andoxidativeevents(ROSproductionand8-OHdGformation).Whereindicated,bcontrolcellsQthereforereferstountreatedcells.2.3.Electromagneticfieldexposure

Asolenoidfieldgeneratorproducingsinusoidalwave-formwithamplitudeof0.05–1.0mTandfrequencyof1–100Hzwasplacedinatissuecultureincubator.Fortechnicaldetails,seeRef.[16].Controlandexposedcellswereplacedinthesameincubatoroutsideorinsidethe

122F.I.Wolfetal./BiochimicaetBiophysicaActa1743(2005)120–129

Fig.1.EffectofELF-EMFexposureto1.0mT/50Hzfor72honproliferationofHL-60cells,Rat-1andWI-38fibroblasts.Afterplatingcellsweregrownfor72hundercontinuousELF-EMFexposure(closedsymbols)orincontrolcondition(opensymbols).Every24hcellswereevaluatedbyanautomaticCoultercounter.Insertsreport%netgrowthincreaseofexposedcomparedtocontrolcells.DataaremeanFS.D.offiveseparateexperiments;*Pb0.05vs.controlcellsbyStudent’st

test.

solenoid,respectively.Cellswereexposedtoasinusoidal50-Hzelectromagneticfieldat0.50,0.75and1.0mTfor3–72hinculturedishesinsertedintoplexiglascylinderplacedinsidethesolenoid.AthermometricprobeplacedincellculturedishesinsideandoutsidetheELF-EMFgeneratorrevealednosignificanttemperaturedifferencebetweenculturemediaofexposedorunexposedcells.Aftertreat-ment,cellswerewashedandrapidlyusedfortheassays.2.4.Assaysfor8-OHdGandDNAstrandbreaks

Cytospinsampleswerepreparedaccordingtothefollowingprocedure.Cellsweredilutedinsucrosebuffer(0.25Msucrose,1.8mMCaCl2,25mMKCl,50mMTris,pH7.5)atadensityofabout2Â106cells/ml.Next,50Alwasaddedtocarbowax-ethanolbuffer(carbowaxstock:77-mlPEG-1000in50-mlwater,1-mlstockin74-ml70%ethanol)(SigmaItalia)andmixed.Aliquotsof150Alwereplacedintocytospinfunnelsandcentrifugedat300rpmfor5min.Slideswerecoatedwithaminopropyl-triethoxysilane(Kindler,Freiburg,Germany).Sampleswereair-driedfor10–30min,fixedin95%coldethanol(À208C)for10min,andstoredatÀ208C.

Detectionof8-OHdGbyimmunocytochemistrycoupledwithDAB(Vector,Burlingham,USA)wascarriedoutessentiallyasdescribedbyYabouroughetal.[28].Themonoclonalantibodyfor8-OHdG1F7waskindlyprovidedbyDr.R.M.Santella,ColumbiaSchoolofPublicHealth,NY.Semi-quantitativeevaluationofthestainingwascarriedoutbyanopticalmicroscope(ECLIPSEE600,Nikon,at400Â)connectedtoanImage-ProplusVersion4.1(MediaCybernetics,USA).Nuclearstainingwasevaluatedinapproximately100cellsofrandomlychosenimagesbyoperatorswhowereblindtothestatusofcelltreatment,asrecommendedinRef.[28].Negativeandpositivecontrols(untreatedand0.5mMH2O2-treatedcells,respectively)wereincludedwithineachbatchofslides.Dataarereportedasunitsofopticaldensity(OD)Â1000.

DetectionofSSBbysinglecellmicrogelelectrophoresiswasperformedbythemethodofSingh[29],withminormodifications[30].Dataarereportedastailmoment[31],evaluatedbyImage-ProplusVersion4.1.Atleast50randomlyselectedrepresentativecometswerecalculatedforeachblindsample.

2.5.CytofluorimetricanalysisofcellcycledistributionTrypsinizedcellswerecollectedandwashedtwicewithPBS.About1Â106cellsweresuspendedin1-mlPBS,fixedin5mlof70%ethanolandstoredat48C.Atthetimeofanalysis,cellswerecollectedbycentrifugationandthepelletswereresuspendedin0.2mg/mlpropidiumiodideinPBScontaining0.6%NonidetP-40andRNAase(1mg/ml);suspensionswereincubatedinthedarkatroomtemperaturefor30min.ThecellsuspensionswerethenfilteredandanalyzedforDNAcontentonCoulterEPICS753

flow

Fig.2.EffectofELF-EMFexposurefrom0.5to1.0mT/50Hzfor48honproliferationofHL-60cells,Rat-1andWI-38fibroblasts.CellswereexposedtoELF-EMFof0.5,0.75,and1.0mT/50Hzfor48handtheirgrowthratecomparedtomatchedcontrolcultures.Data(meanFS.D.offiveexperiments)areexpressedas%growthincrease.

F.I.Wolfetal./BiochimicaetBiophysicaActa1743(2005)120–129

Table1

EffectofELF-EMF(1mT–50Hz)oncellcycledistributioninRat-1cells

Cellcycledistribution(%)12hG0/G1

ControlELF-EMF

50.626.3

S

31.5

38.8*(+23%)

24hG0/G167.763.5

S

20.1

23.5(+17%)

48hG0/G155.452.2

S

27.5

34.3*(+25%)

72hG0/G157.467.3

S

123

24.0

17.0(À30%)

Dataaremeansofthreeexperiments,S.D.beingb15%.*Pb0.05byStudent’st

test.

cytometer.ThepercentageofcellsinthedifferentphasesofthecellcyclewasdeterminedusingtheMulticyclesoftwareversion2.53.

2.6.Westernblotanalysesofproteinexpression

Cells(10Â106)wereharvestedandpelletsweresus-pendedinlysisbuffer(150mMNaCl,50mMTris–HCl,pH8.0,0.05%NaN3,1%Tritonand1mMPMSF)for30minat48C.Afterincubationsampleswerecentrifugedat14,000rpmfor15minat48C.ThesupernatantswereassayedforproteincontentbytheBioradproteinassaymethod(BioradlaboratoriesGmbH,Munchen,Germany)andstoredatÀ808C.Westernblotanalysiswasperformedwithequalamountsofproteinsfromeachsample(generallyb100Ag),separatedbySDS-PAGE(12%)andtransferredtoimmobilon-Pmembranes(Millipore,Bedford,MA)at100Vfor1h.Immunodetectionwasperformedusingtheenhancedchem-iluminescencekitforwesternblottingdetection(AmershamPharmaciaBiotech,Freiburg,Germany).Thepolyclonalantibodiestop65,p50andInBa(1:1000dilution)werefromSantaCruzBiotechnology(SantaCruz,CA).Thepolyclonalantibodytoh-actin(1:200)(SantaCruzBiotechnology)wasusedasinternalcontrol.2.7.IntracellularROSevaluation

IntracellularROSweredetectedindichlorodihydrofluor-escein-diacetate(H2DCF-DA)-loadedcells(MolecularProbe,Leiden,Netherlands),usingaCytofluor2300/2350(Millipore,Billerica,MA).Samplesof2Â106cellsplacedon

Corning6-wellsplateswerepreincubatedwith5AMH2DCF-DAfor1hat378C.Plateswerecentrifugedat1200rpmfor10minandthefluorescenceofcontrolandtreatedcellswasreadintheCytofluor(excitationat504nm,emissionat526nm).Alpha-tocopherolattheconcentrationof10AMdidnotalterthebasalfluorescenceofDCF(BackgroundofDCFwasusually60–70relativefluorescentunits/F.U.).2.8.Statisticalanalysis

DataaregivenasmeansFS.D.ofatleastthreeseparateexperiments.Inthefigures,S.D.areindicatedbyverticalbars;valueswithoutverticalbarshaveS.D.withinthesymbols.StatisticalanalyseswereperformedbyunpairedStudent’sttest,anddifferenceswereconsideredsignificantwhenPb0.05.

Multifactorialtwo-wayanalysisofvariance(ANOVA)wasadoptedtoassessdifferencesamongmultiplesetsofdataobtainedwithuntreatedortreatedcellsatdifferenttimesofcultureandexposure.Whensignificantvalueswerefound(Pb0.05),posthoccomparisonsofmeansweremadeusingtheTukey’sHonestlySignificantDifferencestest.Otherdetailsaregiveninthelegendstofiguresandtables.

3.Results

3.1.EffectofELF-EMFonproliferation

Weinvestigatedtheeffectsof50HzELF-EMFexposuresofdifferentintensities(from0.5to1.0mT)forupto72hon

Fig.3.EffectofELF-EMFexposureat0.5–1.0mT/50Hzfor72honDNAdamageevaluatedasstrandbreaksinHL-60cells,Rat-1andWI-38fibroblasts.CellswereexposedfordifferentextentoftimetoELF-EMFfrom0.5to1.0mT/50HzandsinglestrandbreakswereevaluatedbyCometassay.Data(meanFS.D.ofthreeexperiments)areexpressedastailmoment.Valuesofbasaldamageweresubtractedfromallpoints.SeeMaterialsandmethodsfortechnicaldetails.

124F.I.Wolfetal./BiochimicaetBiophysicaActa1743(2005)120–129

Table2

Effectofa-tocopherolpretreatmentofELF-EMF-inducedproliferation

Increaseofcellgrowth(%)HL-60

Exposure(h)244872

a

Rat-1

+a-Ta

1.4(À15.6)**24.029.0

ELF-EMF14.020.024.3

+a-T6.5(À7.5)*12.2(À7.8)*24.0

WI-38ELF-EMF17.223.031.0

+a-T2.0(À15.2)**8.7(À14.3)**2.2(À28.8)**

ELF-EMF17.024.029.1

Cellswerepreincubatedwith10AMa-tocopherolfor24hpriortoexposuretoELF-EMF.Notethat10AMa-tocopheroldidnotinfluenceproliferationrateinnon-exposedcontrolcellsduring24–72-hincubations.*Pb0.05byStudent’sttest.**Pb0.005byStudent’st

test.

theproliferationrateofHL-60leukemiacells,Rat-1immortalizedfibroblasts,andWI-38diploidfibroblasts.Fig.1showsthat1-mTELF-EMFincreasedtheproliferationofthethreecelltypesinatime-dependentmanner,reachingastatisticalsignificancevs.unexposedcellsafter48-hexposure(Pb0.05).Theincreasesinproliferationratewereproportionaltotheexposuretime,asshownintheinsetsinwhichdatawereexpressedaspercentrelativetounexposedcells.At72h,thegrowthrateincreased20–30%inallcells.Fig.2showsthatinallcelltypesELF-EMFenhancedcellproliferationinadose-dependentmanner.After48-hexposureto0.75mTtheaveragecellgrowthincreasedbyapproximately15–20%.SincetheeffectofELF-EMFwasproportionaltothedoseintensity,allsubsequentexperi-mentswereperformedat1.0mT,soastomaximizetheeffectsinducedbyELF-EMF.

Toconfirmdataobtainedbycellcounts,wemeasuredtime-relatedchangesincellcycledistributionduringthecourseof72-hexposuretoELF-EMF(1mT/50Hz)paredtocontrols,ELF-EMF-exposurecausedasignificantincreaseofthepercentageofcellsinSphaseat12handat48h.At72hthecellsinSphasedecreasedby30%suggestingthat,undertheseconditions,theexposedcellsreachedconfluenceearlierthancontrols.SimilarresultswereobtainedwithHL-60cellsandWI-38fibroblasts(datanotshown).

3.2.EffectofELF-EMFonDNAdamage

Fig.3reportsDNAdamage,measuredasDNAstrandbreaksbythecometassay,inthethreecelllinesafter72-hexposureto0.5–1.0-mTELF-EMF.Attime0,thebasallevelsofstrandbreaksweresubstantiallyhigherinHL-60cellsthaninRat-1andWI-38fibroblasts,asonewouldexpecttofindinahyperdiploidneoplasticpopulation[32].IrrespectiveofthebasallevelsofDNAdamage,however,24-hexposuretoELF-EMFincreasedstrandbreaksinallcelllinesinadose-dependentmanner.Thiseffectwasmuchevidentat1.0-mTELF-EMF,especiallyinnon-neoplasticcellslikeRat-1(eightfoldincrease)andWI-38fibroblasts(16-foldincrease).Inthefollowing24hofexposure,strandbreaksreturnedtobasallevelsbutincreasedagainat72h.ItisworthnotingthatDNAdamageappeared12hafterthepeakofSphase(cf.Table1).TherepairofDNAdamageafter72-hexposuretoELF-EMFwasalsoinvestigated.After24hofpost-exposurerecoveryRat-1cellsshowedlevelsofDNAdamagethatwerereducedbyabout92%comparedtothelevelsdeterminedattheendof72-hexposuretoELF-EMF(nettailmoments,30vs.399).UndercomparableconditionsHL-60cellsshowedlevelsofDNAstrandbreaksthatdecreasedonly44%(nettailmoments,234vs.414).Thislatterfindingdidnotcomeunexpected,aswe

Fig.4.EffectofantioxidanttreatmentonDNAdamageinducedbyELF-EMFat1.0mT/50Hzupto72hinHL-60cells,Rat-1andWI-38fibroblasts.Cellswerepretreatedwith10AMa-tocopherolfor24h.Afterremovalofexcessantioxidant,treatedcellswereexposedtoELF-EMFfrom24to72h.DataexpressedastailmomentaremeanFS.D.ofthreedifferentexperiments.Valuesofbasaldamageweresubtractedfromallpoints.

F.I.Wolfetal./BiochimicaetBiophysicaActa1743(2005)120–129125

Fig.5.EffectofantioxidanttreatmentonoxidativeDNAdamageevaluatedas8-OHdG,inducedbyELF-EMFat1.0mT/50Hzupto72hinHL-60cells,Rat-1andWI-38fibroblasts.Cellswerepretreatedwith10AMa-tocopherolfor24hpriortoexposuretoELF-EMFfrom24to72h.8-OHdGadductsidentifiedbythemonoclonalantibody1F7coupledwithDABwerequantifiedevaluatingopticaldensity(OD)(seeMaterialsandmethodsforfurtherdetails).Basalstainingwassubtractedfromexperimentaldata.DataaremeansFS.D.ofthreedifferent

experiments.

havepreviouslyshownthatneoplasticcellsarecharac-terizedbyinsufficientrepairmechanisms[32].

3.3.EffectofantioxidantonELF-EMFproliferationandDNAdamage

CellsweretreatedwithanantioxidantpriortotheirexposuretoELF-EMF.a-Tocopherolat10AM,addedasdescribedunderMaterialsandMethods[33],preventedstimulationofcellproliferationinallcellpopulationsexaminedafter24-hexposuretoELF-EMF(Table2).After48-hexposurethesameeffectwaspresentinRat-1andWI-38cells,butnotinHL-60cells;after72-hexposure,a-tocopherolinhibitedproliferationonlyinWI-38cells(seealsoTable2).Similarresultswereobtainedwhentheeffectsofa-tocopherolonDNAdamagewereevaluated.Fig.4showsthata-tocopherolsignificantlyreducedDNAstrandbreaksinallcellstrains,andthiseffectwasmorepersistentinRat-1andWI-38cellsthaninHL60.

TobettercharacterizewhetherDNAwasdamagedbyoxygen-centeredfreeradicals,wemeasuredthelevelsof8-OHdGadducts,whichindicateoxidativedamagebyhydroxylradicalsorhydroxylradical-typespecies[20].Fig.5showsthatinallcelltypes8-OHdGlevelspeakedat24and72hafterexposuretoELF-EMF,similartothatdeterminedbythecometassay(seeFig.4).Alpha-tocopherolprevented8-OHdGformationby~50%through-outELF-EMFtreatmentinallcelltypes.

3.4.InvolvementofROSinELF-EMFinducedDNAdamageandproliferation

HavingshownthattheeffectsofELF-EMFoncellproliferationandDNAdamagewereinhibitedbyantiox-idants,weperformedexperimentstoobtaindirectevidencethatELF-EMFcausedtheformationoffreeradicalspecies.

WethereforemeasuredintracellularROSinDCF-loadedRat-1fibroblasts.Fig.6(panelA)showsthatthecellularlevelsofROSincreased~18%asearlyas3hafterexposuretoELF-EMFandthatsuchanincreasepersistedafter24-hexposure.Undercomparableconditions,a

-tocopherol

Fig.6.EffectsofELF-EMFexposureat1.0mT/50HzonROSproductioninRat-1fibroblastswithorwithouta-tocopherolpretreatment.DCF-detectableROSweremeasuredincontrolorELF-EMF-exposedcells(3-and24-hexposures).Valueswereexpressedasrelativefluorescenceunits.PanelAshowsthattreatment/timeinteractionwassignificant(Pb0.05).Valuesnotsharingthesamesuperscriptweresignificantlydifferent(candd:Pb0.05vs.aandb,respectively).PanelBshowstheeffectof24-hpretreatmentwith10AMa-tocopherolonELF-EMF-inducedROSproduction.DataarereportedaspercentofDCFfluorescenceincreasevs.relativecontrolsat3and24hofexposuretoELF-EMF(b:Pb0.05vs.a;c:Pb0.001vs.a)(Tukey’stest).

126F.I.Wolfetal./BiochimicaetBiophysicaActa1743(2005)120–129

almostcompletelypreventedROSincreaseat3-hexposure,andreducedROSincreaseby~50%at24-hexposure(Fig.6,panelB).

3.5.EffectofELF-EMFonredox-mediatedsignalsandproliferation

Wedeterminedpossiblechangesintheexpressionlevelsofproteinsthatareinvolvedinredox-mediatedsignals.Fig.7AshowstheexpressionofNFnBp65andp50inRat-1cells.ItcanbeobservedthatbothNFnBp65andp50expressionwasincreasedat12hofexposureto1mT/paredtocontrols,p65expressionincreased120%at12handremainedhighupto24h.Theincreaseofp50expressionwasrelativelysmallerbutstillevidentandpersistentatboth12and24h.Undercomparableconditions,pretreatmentwith10AMa-tocopheroldidnotmodifyp65expressionincontrolandexposedcells(Fig.7B).TheeffectsofELF-EMFonthelevelsofthetotalinhibitorysubunitInBawerealsoinvestigated.WhereasincontrolcellstotalInBatendedtoincreasefrom6to24h,incellsexposedtoELF-EMFInBaunderwentasignificantdecrease;ofnote,pretreat-mentofcellswitha-tocopherolincreasedtotalInBaboth

intheabsenceandpresenceofELF-EMF(Fig.8A).Tobetterevaluatethefunctionalimplicationsofthemodu-lationoftotalInBa,wedeterminedthep65/InBaratioasanindexofNFkBactivity.Fig.8Bshowsthat(i)cellsexposedtoELF-EMFdisplayedasignificantincreaseoftheactiveformofNFnB,whichpeakedat6handremainedhigherthanincontrolcellsat12and24hexposure;(ii)pretreatmentofcellswitha-tocopherolalwaysabolishedtheincreasedratioofp65toInBainducedbyELF-EMF.Thesedataconfirmedthatredox-mediatedsignalswereinvolvedinthecontrolofcellproliferationinducedbyELF-EMF.

4.Discussion

Inthisstudyweutilizedhumanlymphoblasticleukemia(HL-60cells),immortalizedbutnottransformedfibroblasts(Rat-1),andhumandiploidfibroblasts(WI-38cells)withtheaimofinvestigatingtheeffectofELF-EMFonnormalandtumorcells.OurresultsshowedthatELF-EMFdose-dependentlyincreasecellproliferation,leadingtoa30%increaseofcellnumberafter72hexposure(cf.Figs1and2).Increasedproliferationwasparalleledbya

significant

Fig.7.Expressionlevelsofp65andp50NFkB-relatedproteinsinRat-1cellsexposedtoELF-EMF1mT/50Hzfordifferentextentoftime.Effectofantioxidantpretreatment.ProteinsfromcontrolandELF-EMF-exposedcellsfrom3to36hwererunonagelelectrophoresisandstainedwithmonoclonalantibodiesforp65-Rel1,p50anda-actinashousekeepingprotein.PanelAshowstheexpressionofp65andp50from3-to36-hincubationincontrolandexposedcells(70-Agproteinwasloadedintothegel).Ontheleft,densitometricevaluationsshowthepercentageincreaseofproteinexpressioncomparedtovaluesat3hforp65andat6hforp50,respectively.PanelBreportstheeffectof24-hpretreatmentwith10AMa-tocopherolontheexpressionlevelofp65from6-to24-hincubations(40-Agproteinloadedintothegel).Resultsconfirmedbytwoseparateexperiments.

F.I.Wolfetal./BiochimicaetBiophysicaActa1743(2005)120–129127

Fig.8.ExpressionlevelsofInBa,theNFkB-inhibitorysubunit,inRat-1cellsexposedtoELF-EMF1mT/50Hzwithorwithoutantioxidant.PanelA:TheexpressionlevelsofInBa,normalizedtoa-actindensitometricquantification,areexpressedaspercentageofthecontrolcellsat6-hculture.Whenindicated,cellswerepretreatedwith10AMa-tocopherolfor24hpriortoincubationwithorwithoutELF-EMFexposure.PanelB:Ratiooftheexpressionlevelsofp65toInBa(p65/InBa),anindexofNFkBactiveform.Resultsconfirmedbytwoseparateexperiments.

increaseofthepercentageofcellsintheSphase,whichreachedamaximumof25%inthecaseofRat-1cellsafter12-hexposure.Interestingly,theenhancedrecruitmentofcellsintheSphasedisplayedtwopeaksafter12-and48-hexposure.ParallelevaluationofDNAdamageshowedthatELF-EMFsignificantlyincreasedbothDNAstrandbreaksand8-OHdGlevels,whichreachedtheirmaximaaftertheSphasepeaks(cf.Figs3and4andTable1).ThisobservationsuggeststhattheexposuretoELF-EMFcausedatransientmitogeniceffectat12h,followedbyaDNAdamagingeffectat24h.

Fromapathogeneticviewpoint,itcanbehypothesizedthatshort-termexposurestoELF-EMF,andconsequentformationofROS,inducegrowthstimulationandincreasethenumberofDNAsynthesizingcells.Long-termexpo-surestoELF-EMFandcontinuousgenerationofROSeventuallycauseaccumulationofDNAdamage,whichslowsdowntheprogressionofthecellcycle.Collectively,thepercentageofcellsintheSphasewasinverselyrelatedtoDNAdamageanddirectlyrelatedtoDNArepair(cf.Table1andFig.3).TheseresultsareconsistentwiththerecentevidenceforarelationshipbetweencellcycledistributionandsusceptibilitytooxidativeDNAdamage[34].AccordingtothisinterpretationcellcyclearrestinG1protectsagainstoxidativeDNAdamage,demonstratinganinverserelationshipbetweencellproliferationandoxidativeDNAdamage.ReactiveoxygenspecieshavebeententativelyproposedtomediatetheeffectsofELF-EMF[35].WehaveextendedandvalidatedthisconceptbydemonstratingthatasizableincreaseofROSoccurredshortlyafterexposuretoELF-EMFandprecededactivationofmoleculareventsabletoinducecellproliferation(cf.Figs.6–8).Inaddition,wehaveshownthatanantioxidantlikea-tocopherolbluntedtheincreaseofROSandconsistentlypreventedproliferationandDNAdamageinducedbyELF-EMF(cf.Table2andFigs.4–7).Itisworthnotingthattheeffectofa-tocopherolwasmorepersistentinWI-38cellsthaninHL-60cells,andexhibitedanintermediatedurationinRat-1cells(cf.Table2andFig.4).Thismightbeattributedtocell-specificratesofincorporationandmetabolismofa-tocopherol,asshowninpreviousstudiesofotherantioxidants[36].

WealsoprovidenovelinformationaboutthemechanismwherebyROScanmediatetheeffectsofELF-EMFoncellproliferation.Infact,ourdatademonstratethatELF-EMFincreasethelevelsofNFnB-relatedproteins,mostnotablyp65,whilealsodecreasingthelevelsofInBa(cf.Figs7and8).Theincreasedlevelsofp65,inconjunctionwiththereducedlevelsoftotalInBa,haveafunctionalroleinregulatingtranscriptionprocesses,asp65-p65[37]homo-dimersorp50-p65heterodimers[26]areknowntoactivatetranscription.Antioxidantpretreatmentwitha-tocopheroldoesnotinterferewiththeeffectsofELF-EMFonp65orp50butincreasesthelevelsofInBainsuchamanner

that

128F.I.Wolfetal./BiochimicaetBiophysicaActa1743(2005)120–129

theratioofp65toInBareturnstoessentiallythesamelevelsofuntreatedcells.Bydoingso,a-tocopherolprecludesnucleartranslocationofp65-p50activecom-plexes[38].

TheprecisemechanismsthroughwhichELF-EMFincreasecellularROSproductionremainunknown.Like-wise,wecannotspeculatewhichparticularreactivespeciesmediatedtheproliferativeandDNA-damagingeffectsandhowsuchreactivespeciescouldbescavengedbya-tocopherol.DichlorofluoresceiniswidelyusedfordetectingcellularROS,andreportedlyexhibitsapeculiarspecificityforhydrogenperoxide[39];however,chemicalstudiesshowthatDCFactuallydetectsamuchbroaderarrayofreactiveintermediates[40].Alpha-tocopherolisoneofthemosteffectivelipid-solubleantioxidantsand,assuch,wouldbeexpectedtoscavengelipidcenteredradicalsratherthanhydrogenperoxideorthehydroxylradicalsthateventuallyform8-OHdGadductssuchasthosedetectedinourstudy.Nonetheless,previousstudiesshowedthata-tocopherolinhibitedtheformationofDCF-detectableROSincells[41],andwasabletodiminishDNAstrandbreaksinducedbyhydrogenperoxide[42].Ourfindingsthata-tocopheroldecreasestheyieldofDCF-detectableROSincellsexposedtoELF-EMF,whilealsodiminishingproliferationandDNAoxidativedamage,mustthereforebeappreciatedwithinthecontextofanoxidizingtoneinwhichmorethanoneoxidantwasinvolvedanda-tocopherolprobablyactedonmorethanonefreeradicalspecies.

ThebiologicaleffectsofELF-EMFremainamatterofdebate,asindicatedbyrecentpaperssupporting[8,43–46]ordisproving[47–49]aneffectofELF-EMFoncellgrowthandDNAdamage.Thesediscrepanciesmightatleastinpartbeattributedtoexperimentalfactorslikeintensityanddurationofexposureorthecelltypesusedinthedifferentstudies.HerewehaveshownthatELF-EMFfirstincreaseproliferationandtheninducereversibleDNAdamageinnormalandleukemiccellculturescharacterizedbydifferentgrowthrates.Thus,ourmodelshowsthatbothcellgrowthandDNAdamagecanbeobservedincellpopulationsthatareexposedtoELF-EMFforasufficientlylongtimeandthenareallowedtorecoveraftertreatment.Theconsequen-cesofthesedualandtime-dependenteffectswoulddependontheDNArepaircapacityofcells,ortheaccumulationofpotentiallycarcinogenicmutations,orthecouplingofDNAdamagewithapoptosis.HavingoneconsequenceortheotherwillalsodependonenvironmentalornutritionalfactorsthatmodulatetheimpactofELF-EMFonthecell.Theseresultsmaythereforebeofvaluetodevelopresearchandnewinvestigationalmodelsonthepotentiallydelete-riouseffectsofELF-EMF.

Inconclusion,thedatareportedinthispaperconfirmthatELF-EMFinfluencecellproliferationandsusceptibilitytoDNAdamage,anduncovermechanismsthroughwhichROSmaybeimportantinthesesettings.Sucheffectsareseeninnormalortransformedcellswithverydifferentproliferationrates,andthushighlighttheimportanceof

characterizingtheroleofELF-EMFinthecomplexprocessofcarcinogenesisortumorprogression.

Acknowledgements

ThisworkwassupportedbyCOFINnos.2001064293and2003067599toF.I.Wolfand2002064819toC.G.byISPESL(IstitutoSuperioreperlaPrevenzioneelaSicurezzadelLavoro)andUCSC.

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