50-Hz electromagnetic fields enhance cell proliferation and(2)

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.

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