50-Hz electromagnetic fields enhance cell proliferation and(3)

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/InBaratioasanin …… 此处隐藏：6415字，全部文档内容请下载后查看。喜欢就下载吧 ……