Assessing the mechanism of DNA damage induced by lead through direct and indirect interactions

HaoZhanga,KaiWeia,MengyuZhanga,RutaoLiua,⇑,YadongChenbabSchoolofEnvironmentalScienceandEngineering,ShandongUniversity,27ShandaNanlu,Jinan250100,PRChinaLaboratoryofMolecularDesignandDrugDiscovery,SchoolofBasicScience,ChinaPharmaceuticalUniversity,24Tongjiaxiang,Nanjing210009,PRChinaarticleinfoabstract

Leadstillpossessesgreatthreatstohumanhealthowingtoitswidespreaddistributionintheenviron-mentcausedbyhumanactivities,althoughvariousactionshavebeentakentocutdowntheuseanddis-tributionoflead.Inthiswork,mechanismsofDNAdamagecausedbyleadthroughindirectanddirectinteractionswereinvestigated.Resultsfromcometassayshowedleadat1–10lMinducedDNAstrandbreaksinmicelivercellsaccordingtoolivetailmomentanalysis.SignalsofDNA–proteincrosslinks(DPC)werenotsignificantlydetecteduntilexposedat100lMPb2+.Furthermore,directinteractionsbetweenPb2+andDNAwereexploredtodeterminethebindingmodebetweenthemusingspectraanal-ysis,isothermaltitrationcalorimetrystudiesandmoleculardockinginvestigations,whichindicatedthatPb2+couldbindtoDNAwithfourbindingsitestoformPb2+–DNAcomplexbyminorgroovebindingeffectsandelectrostaticforces,resultingindamagetothestructureofDNAdoublehelix.Combinedstud-iesofleadgenotoxicityinindirect(cometassayandDPCassay)anddirect(bindingmodeinvestigations)interactionscanbeappliedtostudythepotentialdamagestoDNAinducedbyheavymetalpollutants.Ó2014ElsevierB.V.Allrightsreserved.Articlehistory:Received8March2014Receivedinrevisedform22April2014Accepted24April2014Availableonline5May2014Keywords:LeadgenotoxicityCometassayDNA–proteincrosslinksIsothermaltitrationcalorimetryBindingmode1.IntroductionLeadhasbeenwidelyusedinanthropogenicactivities[1],suchasleadedgasoline,leadpaints,leadedplumbingcomponents,carrepair,andbatterymanufacturing,formanyyearsbecauseofitshigherdensity,softness,ductility,malleabilityandpoorerelectri-calconductivitycomparedtoothermetals[2].Theuseofleadinhumanproductshasresultedinlargeamountsofleadspreadingintotheenvironment,whichmakesthebodymoresusceptibletoexposetolead[3–5],andcausesadversehealthproblemsincludingneurologicaldiseases[6],hematologicaldamage[7],cognitivedef-icits[8],renaldisorders[9]andimmunologicalpathologies[10].Leadisalsoregardedasacarcinogen(GroupB2,possiblehumancarcinogen)bytheInternationalAgencyforResearchonCancer(IARC)[11],whichcanbesupportedbystudiesfromepidemiolog-icalandexperimentalevidencesthatinvestigatedthepossiblelinkbetweenleadexposureandcancer[12–14].Theknowledgeofmechanismsbywhichleadcausecancerisstillrathercomplexandnotfullyunderstood,butseveralpossiblemechanismshavebeenproposedtoexplainthecarcinogenicprop-ertiesofleadatthecellularandmolecularlevelthatleadmay⇑Correspondingauthor.Tel./fax:+8653188365489.E-mailaddress:rutaoliu@sdu.edu.cn(R.Liu).http://dx.doi.org/10.1016/j.jphotobiol.2014.04.0201011-1344/Ó2014ElsevierB.V.Allrightsreserved.promoteorenhancetheprocessofcarcinogenesisbyincreasingthepossibilityonDNAdamagethroughindirectanddirectinterac-tions.TheindirectmechanismsofDNAdamagecausedbyleadhavebeenstudiedthroughseveralaspects:leadcaninducetheproductionofreactiveoxygenspecies(ROS)andthenresultinDNAstrandbreaks[15];leadcaninterferewiththeprocessofbaseexcisionrepair(BER)andnucleotideexcisionrepair(NER)toinhi-bitDNArepairmachinery[16],andreplacezincinDNAbindingproteins[17].ThedirectevidenceofinteractionsbetweenleadandkidneyDNAfromsilvercruciancarpwasstudiedbyHongetal.[18]thatleadcouldchangethesecondarystructureofDNAbycovalentlybindingwiththeoxygenatomofnucleicacidornitrogenatomofbasepairsofDNA.Tajmir–Riahietal.investigatedtheeffectsofPb2+onconformationalchangesofcalfthymusDNAbyFTIRspectroscopy[19]andlaserRamanspectroscopy[20],whichindicatedleadmainlyboundtothephosphategroupsofDNAbackbone,anddidnotinteractwithnucleicbases,butboundtoN-7atomoftheguaninebases.However,thedirectbindingmodebetweenleadandDNAremainsunclear.ThereisalackofstudyfocusingonbothdirectandindirectinteractionsbetweenDNAandlead.Directinteractionsbetweenmacromolecules(protein,DNA)andsmallmoleculesatthemolec-ularlevelhavebeenthoroughlystudiedbyourgroup[21–23],whichcandeterminebindingmodesofligandsonfunctionalbio-moleculesandprovideevidenceforpotentialtoxicmechanismsH.Zhangetal./JournalofPhotochemistryandPhotobiologyB:Biology136(2014)46–5347ofpoisoncompoundsinvivo.ThepresentstudyaimstoevaluatetheDNAdamagelevelcausedbyleadbycometassayandDNA–proteincrosslinking(DPC)assay(indirectinteractions),anddirectbindingsbetweenleadandDNAusingmultiplespectroscopictech-nologies,includingfluorescencespectroscopy,UV–visabsorptionspectroscopyandcirculardichroismspectroscopy,isothermaltitrationcalorimetry(ITC)measurement,andmoleculardockingstudies.TheresultsobtainedfromcometandDPCassayindicatedleadcaninducesignificantDNAdamageinlowexposureconcen-trations.Thespectroscopicresultsshowedthatboththeelectro-staticeffectsplayedkeyrolesinPb2+–DNAbindings,andITCandmoleculardockingstudiesofferedthespecificbindingsitesofDNAtoPb2+.Thisworksuggestsbothdirectandindirectmecha-nismsofDNAdamageinducedbyxenobioticsshouldbeconsidered.2.Materialsandmethods2.1.ChemicalsReagentsusedincometassayandDNA–proteincrosslinkingassaywerelistedasfollows:0.75%ofnormalmeltingagarose(NMA,purchasedfromBiowest,Spain)and1%lowmeltingagarose(LMA,purchasedfromBiowest,Spain)weredispensedbyPBSbuf-fer(0.1mol/L,pH=7.4),respectively.TritonX-100,DMSO,propidi-umiodide,proteinaseKandHoechst33258wereallgotfromBiodee,China.CalfthymusDNA(ctDNA,Sigma–Aldrich,St.Louis,MO,USA)wasdissolvedinwaterasthestocksolution(100lg/mL),andtheconcentration(2.35Â10À4mol/L)wasdeterminedaccordingtotheDNAabsorbanceat260nmusingeDNA(p)=6600LmolÀ1cmÀ1afterdeterminingtheabsorbanceratioA260/A280intherangeof1.80–1.90.Astocksolutionofleadchloride(purchasedfromTianjinChemicalReagent,China)waspreparedbydissolved0.2781gofPbCl2in100mLwaterandfilteredby0.22lmsyringefilters.MethylenebluewasboughtfromAmrescoanddissolveddirectlyinwateratafinalconcentrationof1Â10À3mol/Lasthestocksolution.Adenine,cytosineandthymine(Sigma–Aldrich,St.Louis,MO,USA)weredissolvedinwatertomakethestocksolutionof1Â10À4mol/L,andguaninewassuspendedinNaOHsolution(0.01mol/L).Tris–HClbuffer(0.02mol/L,pH=7.4)wasusedtocontrolacidity.2.2.AnimalsThree-month-oldmalemice(C57BL6-J)wereobtainedfromSchoolofLifeScience,ShandongUniversity,China.Theanimalroomwasmaintainedat23±2°Candthehumidityof30–70%undera12/12-hlight/darkcycle.Allfeedweresterilizedbyultra-violetsterilization.Waterandbeddingweresterilizedbyautoclav-ing.Micewerekilledbycervicaldislocationafter24hstarvationperiodtocollectlivers,whichwerethenwashedandmincedinCa2+andMg2+freeD-Hank’sbalancedsaltsolutiontoharvestlivercellsuspensions,andthenexposedtoPb2+ofvaryingconcentra-tions(0,1,5,10,100lM)andmaintainedinDulbecco’sModifiedEaglesMedium(DMEM)with10%fetalbovineserumand1%pen-icillin–streptomycinsolutionsunderstandardcellculturecondi-tions(37°C,5%CO2)for24h.2.3.CometassayAmodifiedmeasurementofthecometassayinalkalinecondi-tions(pH>13)originallydescribedbyOliveandBanath[24]wasexploredtodetermineDNAdamagelevelinleadexposedlivercells.Thecometslidesweremadebytwolayersofagarosegel,andcellsofcontrolledandtreatedgroupssuspendedin1%LMAwereplacedonaslidepre-coatedwith0.75%NMA.Aftergelling,theslideswereimmersedintoalysissolution(2.5MNaCl,0.1MNa2EDTA,10mMTris,1%TritonX-100,10%DMSO,pH=10)at4°Cfor2h.Afterwashedwithultrapurewater,theslidesweresubjectedtoDNAdenaturationincoldelectrophoresisbufferat4°Cfor20min.Electrophoresiswasthenconductedat25Vand300mAforanother20minat4°C.Afterelectrophoresis,theslideswerewashedthreetimeswith0.4MTris–HCl(pH=7.4),andthenstainedwithpropidiumiodide(5mg/L)for15minandanalyzedusingfluorescencemicroscope(EclipseTi,Nikon,Japan)coupledwithchargecoupleddevice(CCD)camera.100cellswererandomlyselectedfromeachgroupandanalyzedbyCASPsoftware,andolivetailmoment(OTM)ofeachcometwascalculatedtoprobethelevelofDNAdamage.Makesurethatallexperimentalstepswerecon-ductedindarktopreventsecondaryDNAdamage.2.4.DNA–proteincrosslinks(DPC)determinationTheprocedureofDPCwasbasedonQuievrynetal.withsomemodifications[25].Firstly,thestandardcurveforthedetermina-tionofDNAcontentswasdeveloped.AseriesofconcentrationsofctDNA(0,100,200,300,400,500,750,1000,2000,3000,5000ng/mL)weresuccessivelyprobedbyHoechst33258(400ng/mL)andthefluorescenceintensitiesofeachgroup(kex=350nm,kem=450nm)wererecordedafter30minincuba-tionindark.Thetreatmentcellswerecentrifugedat6000rpmfor3minandthenharvestedbyPBS(0.5mL,pH=7.5).Theresus-pendedcellswerelysedin2%SDSat65°Cfor10min,thecellularlysateswerepassedthroughby1mLpipettetipstoshearedDNAfragments,andDPCwereprecipitatedbytheadditionof1MKCl(dissolvedin20mMTris–HCl,pH=7.5).Thesupernatantscon-tainingfreeDNAwerecollectedusing5mLeppendorftubes,andDPCwerepresentedintheprecipitations.Thisprocessshouldberepeated3timestocollectfreeDNAandDPCcompletely.DNApresentedinDPCwasdissociatedfromDPC–SDS–KClcomplexesbypre-treatmentofsampleswith0.2g/LproteinaseKat50°Cfor3h.ThenconcentrationsoffreeDNA(C1)andDNAinDPC(C2)canbemeasuredusingfluorescencespectrophotometerafterdyedbyHoechst33258.TheDPCcoefficient(Q)wascalculatedusingthefollowingequation,Q¼C2C1þC2whereC1andC2canbecalculatedaccordingtothestandardcurve.2.5.FluorescencemeasurementsThefluorescencespectrawererecordedonanF-4600fluorime-ter(Hitachi,Japan).Theexcitationandemissionslitwidthsweresetat5.0nm.Thescanspeedwas1200nm/min.PMT(photomul-tipliertube)voltagewas850V.Theexperimentswerecarriedoutasfollows:toaseriesof10mLtesttubes,1.0mLof0.2MTris–HClbuffer(pH=7.4),1.0mLof40lMMBsolutionand1.0mLof2.35Â10À4MctDNAweresuccessivelyadded;then,differentamountsofPb2+wereaddedanddilutedto10.0mLwithultrapurewater.2.6.UV–visabsorptionmeasurementsTheabsorptionspectraofMB–DNA,DNAinfluencedbydifferentconcentrationsofPb2+,andeffectsofPb2+onDNAbases(A,T,G,C)wereallmeasuredonaUV-2450spectrophotometer(Shimadzu,Kyoto,Japan).48H.Zhangetal./JournalofPhotochemistryandPhotobiologyB:Biology136(2014)46–532.7.Circulardichroism(CD)spectrameasurementCDspectraweremeasuredusingaJ-810circulardichroismspectrometer(JASCO).TheCDspectrawerecollectedfrom200–300nm,andthreescansweremadeandaveragedforeachCDspectrum.2.8.IsothermaltitrationcalorimetryexperimentsIsothermaltitrationcalorimetry(ITC)experimentswereper-formedonaMicrocalITC200microcalorimeter(MicrocalInc.,Northampton,MA)at298Kbytitrating200lLofctDNA(0.0235mM)withapproximately40lLofPb2+(1mM)usingstir-ringspeedat1000rpm.BothofDNAandPb2+weredissolvedin0.02MTris–HClbuffer(pH7.4),andthespacingtimebetweeneachinjectionwassetto120stoachievethermodynamicequilibration.2.9.MoleculardockinginvestigationTheinteractionsbetweenPb2+andDNAwereconfirmedbymoleculardockingstudiesusingmolecularoperatingenvironmentsoftware(MOE)(Version2009,ChemicalComputingGroupInc,Canada).ThecrystalstructureofDNA(PDBcode1BNA),withthesequenceofduplex50-d(CGCGAATTCGCG)2-30,wasdownloadedfromtheProteinDataBank(http://www.pdb.org/).The3Dstruc-tureofleadchloridewasgeneratedandenergyminimizedusingtheMOEBuildermoduleandtheEnergyMinimizemodule,respectively.3.Resultsanddiscussion3.1.LeadinducedindirectDNAdamagedetectedbySCGEandDPCThemeasurementofindirectDNAdamageinmicelivercellsinducedbyleadwasexploreduponthesinglecellgelelectropho-resis(SCGE)underalkalineconditions.Fig.1showstypicalcometimagesofhepatocytesexposedtodifferentconcentrationsofPb2+(0,1,5,10lM).Controlgrouppresentsintactcellnucleiwithnotails,andexperimentalgroupsshowlongercomettailswiththeincreaseofPb2+.AsignificantincreaseinthevaluesofOTM,%tailDNAandtaillengthshowninTable1wereobservedinPb2+ABCDFig.1.TypicalcometimagesofleadinducedDNAdamageinmicelivercells.(A)untreatedcontrol,(B)1lMPb2+,(C)5lMPb2+and(D)10lMPb2+.Table1

AssessmentofPb2+inducedDNAdamageinmicelivercellsanalyzedusingdifferentparametersofcometassay.LeadOlivetailmomentTailDNA(%)Taillengthconcentrations(Arbitraryunit)(lm)(lM)00.009454±0.018230.06791±1.16953.01±0.0995013.710±0.7665***12.03±2.605***32.69±7.813***56.861±1.018***19.62±2.902***50.88±9.746***109.382±1.699***21.20±3.622***68.06±10.41******P<0.001.Fig.2.OlivetailmomentlevelofDNAfromlivercellsofmicetreatedwithleadattheconcentrationsof0,1,5,and10lM.Table2

DeterminationofDPCcoefficientsinlivercellsofmiceexposedtolead.C(Pb2+)/lMDPC(ng/mL)FreeDNA(ng/mL)DPCcoefficient(%)00898.540101068.54059.561198.140.791021.77954.142.2100126.31872.4412.6exposuregroups.StudieshaveinvestigatedthatOTMcanbethemostrelevantparameterstoprobeDNAdamagelevelcomparedtoothercometassaymeasurements,suchastaillength,%tailDNAandanothermeasuresoftailmoment[26].ResultsofleadgenotoxicityreflectedbyOTMvaluesareshowninFig.2.TheOTMvaluesofthetreatmentgroupsweresignificantlyhigherthanthecontrolgroup(P<0.0001),whichindicatedthatPb2+cancauseadose-dependentincreaseinthefrequencyofDNAstrandbreaksinmicelivercells[27].DPCsinvivoarecreatedwhenDNAfragmentsbecomecova-lentlyboundtoproteinsnearby,whichplaysakeyroleincausingcarcinogenicandmutagenicprocessofmammaliancells[28,29].Studieshavedemonstratedthatseveralmetalcompounds,suchasnickel[30],arsenic[31]andchromium[32],caninvolveDPCsbyoxidativemechanisms.AstandardcurvetodetermineDNAcon-centrationsisshowninSIFig.1(supportinginformation),andthecontentsoffreeDNAandDNAinDPCindifferentexposuregroupsareanalyzedinTable2.Noormicro-amountofDPCsweredetectedintheincubatedrangesof0–10lMPb2+usedincometassay,andtheDPCcoefficientreachedupto12.6%inPb2+exposureat100lM.H.Zhangetal./JournalofPhotochemistryandPhotobiologyB:Biology136(2014)46–5349UnderlowPb2+(<10lM)exposure,wefoundsignificantincreaseinOlivetailmomentofDNAcomparedwiththecontrol,butfewchangesinamountofDPCs,whichcanbeinferredthatgenerationofDPCsneedaggravatedDNAdamageandconsiderablefragmentsofDNA,soDPCcanberegardedasanindicatorofDNAdamageinseverelevels.Thecytobiologicalandmolecularmecha-nismsontheformationofDNAfragmentsinducedbyleadhavebeenstudiesthroughseveralaspects.ThestrongcorrelationswerefoundbetweenDNAdamagelevelandcellularredoxstate[33,34].Fracassoetal.[35]reportedleadinducedapositiverelevanceonDNAstrandbreakswiththeproductionofROS,andanegativecor-relationwithGSHlevels.Liuetal.[36,37]foundthatquercetin,aflavonoid,couldrelieveDNAdamageinratkidneyandlivercellsinducedbyleadbyquenchingROSproductionandrenewingtheactivitiesofantioxidantenzymes.OtherstudiesfocusedontheabilityofleadonalkylatedDNAbases.WozniakandBlasiak[38]observedsignificantincreaseinthecomettaillengthaftertreat-mentwithPb2+andAlkA(3-methyladenine-DNAglycosylaseII),andtheresultissupportedbyBrowne[39]thatPb2+isoneofthemostreactiveionsintermsofcatalyzingDNAalkylationandfragmentation.However,thedirectinteractionsandthebindingmodesbetweenPb2+andDNAremainunclear,whichmaybeanotherpossiblemechanismofDNAdamageinducedbylead.3.2.DirectbindinginteractionsofleadwithDNATherearethreenon-covalentbindingmodesofsmallmoleculestoDNA:intercalationamongthestackedbasepairsofDNA,electrostaticeffectswiththenegativelychargednucleicacidsugar-phosphatestructure,andbindinginteractionswithmajororminorgrooveoftheDNAdoublehelix[40].ThespecificbindinginteractionsbetweenPb2+andctDNAwerestudiedusingfluores-cenceemission,UV–visabsorption,andcirculardichroismspectra,andthespecificbindingsitesofDNAtoPb2+wereexploredbyiso-thermaltitrationcalorimetryandmoleculardockingtools.3.2.1.CharacterizationofbindinginteractionsbetweenleadandDNAbyspectrainvestigationsMethyleneblue(MB)isafluorescenceprobetostudyinterac-tionsofmetalionswithDNA[41].MBcanintercalateintoDNAbasepairscausingthedecreasedfluorescenceintensities.Thefluo-rescenceemissionspectraofctDNAinthepresenceofMBwith12001050Aytais1000ne1000etnI 950ebcnec900syertoiu850lsFn800ea800tn0246810I [Pb] 10-5molL-1ec600necsero400ulF2000660680700720740wavelength (nm)Fig.3A.EffectsofPb2+onthefluorescenceintensitiesofMB–DNAsystem.Conditions:ctDNA:2.35Â10À5mol/L;methyleneblue:4Â10À5mol/L;Pb2+/(10À5mol/L)(a–e):0,1,3,6,9;pH=7.4;T=293K.Inset:thecomparisonsonthefluorescencepeaksofMB(a)andMB–DNA(b)changedbyPb2+.ConditionsarethesamewithFig.3A.differentPb2+concentrationsareshowninFig.3A.Thefluores-cencepeakofMB–DNAsystemincreasedefficientlywitharedshift(from698nmto701nm)withsuccessiveadditionofPb2+,indicat-ingthatPb2+interactedwithDNAinonecertainbindingmodeofthefollowingthreehypotheses:(1)Pb2+directlyinfluencedthefluorescencepropertiesofMBbyformingPb–MBcomplex.(2)Pb2+competitivelyenteredintothebindingsiteofMB–DNAcomplex,andfreeMBwasreleased.Inotherwords,Pb2+bindstoDNAthroughintercalation.(3)ThestructureofDNAdoublehelixwaschangedwiththeadditionofPb2+,whichhinderedthecombinationofDNAandMB.Inordertotesthypothesis(1),effectsofPb2+ionsonthefluo-rescenceintensityoffreeMBhadbeenexplored(Fig.3Ainset)thattheMBemissionspectraexhibitsbarelychangesafterleadincuba-tion,whichdemonstratesthatPb2+hadweakeffectsonthestruc-tureofMB.Sohypothesis(1)isinvalid[42].ThenUV–visabsorptionspectraofctDNAintheabsenceandpresenceofPb2+(Fig.3B)thatDNAhastwoabsorptionpeaksat200nmcausedbyp–p*andn–r*transitionsofDNA’ssugar-phosphatestructureand260nmresultedfromp–p*transitionofthenitrogenousbases[18,43,44].Hypochromismeffectswereobservedbothat200nmand260nm,andaslightlyredshift(Dk=3nm)wasalsooccurredat260nm.Thecharacteristicabsorptionsignalsofintercalationareaccompaniedwithlargechangesintheabsorbance(hypochro-mismratioP35%)andanappreciableshiftatbandposition(DkP15nm)probablyduetop-electronstackingbetweentheintercalated-ligandsandDNAbasepairs,andtheelectrostaticandgroovebindingsshowminorchangesonintensityorpositioninwavelength[45].Therefore,hypothesis(2)isincorrect.Circulardichroism(CD)measurementisasimpleandeffectivemethodforanassessmentofDNAconformationalchanges[46].ThetypeofPb2+–DNAinteractionwasconfirmedbyCDspectrashowninFig.3C.AsaclassicalB-formDNA,ctDNAdisplaystwoconservativeCDbandsintheUVregion:anegativebandat245nmowingtothehelicityandapositiveoneat275nmduetobasestacking[47].AsthedifferentadditionsofPb2+(0,10and40lmol/L)intoDNA,theintensityofthenegativebanddecreased(shiftingtozerolevels)withaslightblueshiftandthepositivebanddecreasedwithoutanysignificantshiftinpeakposition,whichdemonstratedthatPb2+inducedthestructureofDNAhelix2.0aB1.5dsbA1.00.50.0200240280320Wavelength (nm)Fig.3B.Uv–visabsorptionspectraofDNAinthepresenceofPb2+.Conditions:ctDNA:2.35Â10À5mol/L;methyleneblue:4Â10À5mol/L;Pb2+/(10À5mol/L)(a–d):0,1,2,3;pH=7.4;T=293K.50H.Zhangetal./JournalofPhotochemistryandPhotobiologyB:Biology136(2014)46–531.51.0C0.5a)0.0egdm-0.5(c DC-1.0c-1.5-2.0a-2.5230240250260270280290Wavelength (nm)Fig.3C.EffectsofPb2+oncirculardichroismspectraofDNA.Conditions:ctDNA:2.35Â10À5mol/L;Pb2+/(10À5mol/L)(a–d):0,1,4;pH=7.4;T=293K.uncoilingandthebindingsiteofPb2+onDNAlocatedatthehelicalstructureformedbysugar-phosphatebackbone,notinbasestack-ingareas[48].Newpeaksarenotpresentedinthespectraindicat-ingthatthereisnoconformationaltransition(Bformremaining)duringtheinteraction[49].AccordingtotheresultsillustratedfromFig.3(A–C),thebindingmodebetweenPb2+andDNAisnon-intercalationcombination,butwhethergroovebindingorelectrostaticbindingisresponsibleforthePb2+–DNAinteractionremainsunknown.Theanionicphos-phategroupsofDNAofferthebindingpositionforcations(Pb2+)withelectrostaticforces,sodifferentconcentrationsofNaClsolu-tionswereaddedintoPb2+–DNAcomplextodeterminewhethertheelectrostaticbindingoccurredbetweenPb2+andDNA.TheresultsshowninFig.4(curvea)revealedthatthefluorescenceintensityofaconstantconcentrationofthePb2+–MB–DNAcom-plexpresentedanobviousdecreasewiththeincrementaladditionofNaCl.WeinferthatNa+canweakenbindingforcebetweenPb2+andDNAbyneutralizingthenegativechargesofbackbonephos-phategroups,indicatingthatelectrostaticforceplaysaroleintheinteractionofPb2+andDNA.CurvebinFig.4showslessinfluenceofNa+concentrationonthefluorescenceintensityofDNA–MBsystem(justaslightincrease),andthefluorescenceintensitiesMB–DNAcomplexwereweakerthanthatofPb2+–MB–DNAsystemincubatedinthesameNa+concentration,1000950aytis900netnI 850ecnecs800eroulF750700b0.000.020.040.060.080.10c(NaCl)/mol·L-1Fig.4.EffectsofionicstrengthonfluorescencepeaksofPb–MB–DNA(a)andMB–DNA(b)systems.Conditions:ctDNA:2.35Â10À5mol/L;methyleneblue:4Â10À5mol/L;Pb2+:1Â10À4mol/L;Na+:0,0.02,0.04,0.06,0.08,0.1mol/L.whichcanbesupposedthattheelectrostaticeffectisthemainbindingforcebetweenPb2+andDNA,andgroovebindingmayalsoexistinPb2+–DNAinteractions.3.2.2.IdentificationofthespecificbindingsitesofDNAtoPb2+byITCandmoleculardockingstudiesIsothermaltitrationcalorimetry(ITC)canbeusedtoinvestigatethebindingaffinityconstant(k),numberofbindingsites(n),enthalpy(DH)andentropy(DS)effectsbetweenligandsandDNA[50,51]andfree-energychange(DG)canbecalculatedbythethermodynamicequation:DG=ÀRTlnKa=DH–TDS(RistheuniversalgasconstantandTistheabsolutetemperature).TheITCresultsillustratedinFig.5werecorrectedfordilutionheatbysubstractingthebaselinedatameasuredinidenticalseriesofinjectionsintoTris–HClbuffer,andthemostmatchednumbersofbindingsitesarefourbythecalculationusing‘‘sequentialbind-ingsites’’model.ThetopgraphinFig.5showsheatflowofeachtitration(lcal/s)intimeofminutes(min),andthebottompartillustratesintegratedheatintermsofkcal/molofinjectantplottedagainstmolarratioofPb2+/ctDNA.Thermodynamicparametersofeachbindingsiteincludingka,DH,DSandDGweresummarizedinTable3.NegativeDGvaluesoffourbindingsitesrevealedinterac-tionsbetweenPb2+andDNAoccurredspontaneously.Morespecif-ically,allthebindingconstantsareintherangeof102–103magnitudes,whicharerathersmallerthantheintercalatedbind-ingsofligandsonDNA,suchasethidiumbromide-DNA(Ka=2Â106)[52]andMB–DNA(Ka=1.65Â105)[53]combina-tions.PositiveDHandDSarecalculatedinn1andn3bindingsites,andnegativeDHandDSarefiguredoutinn2andn4areas,whichcanbeinferredthattherearetwoactionmodesinPb2+–ctDNAinteractions.ITCresultsshowedthatbothofelectrostaticeffectsandgroovebindingsmayexistinPb2+–DNAcomplex.Time (min)0102030400.00-5.00ces/laµ-10.00-15.004.00tnat2.00cejni fo 1-0.00lom lack-2.00-4.0001234567Molar RatioFig.5.ITCprofilesforthebindingofPb2+toctDNAat298K.ThetoppanelshowstherawdataforheatflowofeachtitrationofPb2+intoctDNA,andthebottomgraphrepresentsintegratedheatdataaftercorrectionsofdilutionheatagainstmolarratioofPb2+/ctDNAfittedtothe‘‘sequentialbindingsites’’.H.Zhangetal./JournalofPhotochemistryandPhotobiologyB:Biology136(2014)46–53Table3ThermodynamicparametersforinteractionofPb2+withDNAbyITC.ThermodynamicparametersnKa(LmolÀ1)DH(JmolÀ1)DS(JmolÀ1KÀ1)DG(kJmolÀ1)Pb2+–DNAn1(2.72±0.5)Â103(2.26±0.69)Â104141.7À19.65n2(5.31±1.1)Â103À(2.25±0.41)Â104À58.6À5.03n3(2.07±0.83)Â103(3.80±0.66)Â104143À4.64n4(8.7±1.5)Â102À(8.14±0.76)Â104À260À3.8851Fig.6.MoleculardockingresultsofPb–DNAsystembyMOE.TheleftfigureshowsfourbindingsitesofDNA,andthedetailedcombinationsareillustratedineachsite.PbCl2,markedingreen,bindswithOatoms(redballs)ofphosphatesonDC_21andDG_22insite1,N_7(blueball)ofguanineinsite2,OatomofphosphatesonDG_12,andOatomofdeoxyriboseonDG_16,imidazoleringofadenineonDA_17insite4.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)ThemoleculardockingmethodhasoftenbeenperformedtostudythedetailedinformationonbindingmodesbetweensmallmoleculesandDNAorproteins[54,55].Inourstudy,thereasonablecombinationsbetweenPb2+andDNAwereinvestigatedthroughmolecularoperatingenvironment(MOE).ThewholeDNAmoleculewasselectedasthepotentialbindingsites,andallpossibledockingresultsshowninFig.6clearlydemonstratethattherearefourbind-ingsitesonDNAforPb2+,whichisconsistentwiththeITCresults:site1andsite3obtainedthepositionsforelectrostaticbindingsofPb2+withoxygenatomsofphosphatecombinedwithDC_21,DG_22andDG_12,respectively;Pb2+enteredintoDNAminorgrooves,bindingtoN-7ofDG_4insite2,andoxygenatomindeoxyriboseofDG_16,imidazoleringinadenineofDA_17insite4.AtomsinvolvedintheinteractionofPb2+withDNAandbindingdistancesbetweenthemareshowninTable4.DockingresultscouldbesupportedbyinteractionsofPb2+withDNAbases(A,T,G,C),presentedinsupportinginformation.Thetwomainabsorptionpeaksofbasesat208nmand260nmincreasedgradu-allywiththeadditionofPb2+,indicatingthatPb2+couldaffecttheconformationofpurinesandpyrimidinesbybindingtothemwithelectrostaticeffectsorcoordinationbonds.4.ConclusionsInthisarticle,themechanismsofDNAdamagecausedbyleadaredevelopedonthebasisofindirectinteractionsbycometassayandDPCassayanddirectbindinginteractionsbymultiplespectraanalysis,ITCmeasurementandmoleculardockingstudies.ResultsaccordingtocometandDPCassaysindicatedthatleadcouldinduceadose-dependentincreaseinthefrequencyofDNAstrandbreaksinlowexposureconcentrations,andresultinformationofDPCsin100lMoflead.Furthermore,studiesfromdirectinterac-tionsbetweenPb2+andDNArevealedleadcanbindtophosphatebackboneofDNAthroughelectrostaticforces,andenterintominorgroovesofDNAbycombinationswithpurinesandpyrimidines.Thecombinationstudiesofleadgenotoxicityincellular(cometandDPCassays)andmolecular(bindingmodeinvestigations)lev-elscanbeappliedtostudythepotentialDNAdamagesinducedbyheavymetalpollutants.AcknowledgmentsThisworkissupportedbyNSFC(21277081),theCultivationFundoftheKeyScientificandTechnicalInnovationProject,Table4

AtomsinvolvedinbindingsitesofDNAwithPb2+andthedistancesbetweenthem,analyzedfromthemoleculardockingresults.BindingsitesSite1Site2Site3Site4ResiduesDC_21DG_22DG_4DG_12DG_16DA_17AtomsorgroupsOONOOImidazoleringDistances(Å)2.082.072.772.152.57–52H.Zhangetal./JournalofPhotochemistryandPhotobiologyB:Biology136(2014)46–53ResearchFundfortheDoctoralProgramofHigherEducation,Min-istryofEducationofChina(708058,20130131110016),Indepen-dentinnovationprogramofJinan(201202083)andIndependentinnovationfoundationofShandongUniversitynaturalsciencepro-jects(2012DX002)arealsoacknowledged.AppendixA.SupplementarymaterialDNAstandardcurve;Uv–visabsorptionspectraofMB–DNAinthepresenceofPb2+;effectsofPb2+onUV–visabsorptionspectraofDNAbases(A,T,G,C).ThismaterialisavailablefreeofchargeviatheInternetathttp://www.sciencedirect.com.Supplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,athttp://dx.doi.org/10.1016/j.jphotobiol.2014.04.020.References[1]K.Koller,T.Brown,A.Spurgeon,L.Levy,Recentdevelopmentsinlow-levelleadexposureandintellectualimpairmentinchildren,Environ.HealthPerspect.112(2004)987–994.[2]G.H.El-Sokkary,G.H.Abdel-Rahman,E.S.Kamel,Melatoninprotectsagainstlead-inducedhepaticandrenaltoxicityinmalerats,Toxicology213(2005)25–33.[3]W.T.Wu,Y.J.Lin,S.H.Liou,C.Y.Yang,K.F.Cheng,P.J.Tsai,T.N.Wu,Braincancerassociatedwithenvironmentalleadexposure:evidencefromimplementationofaNationalPetrol-LeadPhase-OutProgram(PLPOP)inTaiwanbetween1979and2007,Environ.Int.40(2012)97–101.[4]H.Hu,R.Shih,S.Rothenberg,B.S.Schwartz,Theepidemiologyofleadtoxicityinadults:measuringdoseandconsiderationofothermethodologicissues,Environ.HealthPerspect.115(2007)455–462.[5]M.Ahamed,M.K.Siddiqui,Environmentalleadtoxicityandnutritionalfactors,Clin.Nutr.26(2007)400–408.[6]C.D.Toscano,T.R.Guilarte,Leadneurotoxicity:fromexposuretomoleculareffects,BrainRes.BrainRes.Rev.49(2005)529–554.[7]L.R.Feksa,E.Oliveira,T.Trombini,M.Luchese,S.Bisi,R.Linden,D.B.Berlese,D.B.Rojas,R.B.Andrade,P.F.Schuck,L.M.Lacerda,M.Wajner,C.M.Wannmacher,T.Emanuelli,Pyruvatekinaseactivityanddelta-aminolevulinicaciddehydrataseactivityasbiomarkersoftoxicityinworkersexposedtolead,Arch.Environ.Contam.Toxicol.63(2012)453–460.[8]R.A.Shih,H.Hu,M.G.Weisskopf,B.S.Schwartz,Cumulativeleaddoseandcognitivefunctioninadults:areviewofstudiesthatmeasuredbothbloodleadandbonelead,Environ.HealthPerspect.115(2007)483–492.[9]P.Muntner,J.He,S.Vupputuri,J.Coresh,V.Batuman,BloodleadandchronickidneydiseaseinthegeneralUnitedStatespopulation:resultsfromNHANESIII,KidneyInt.63(2003)1044–1050.[10]R.R.Dietert,M.S.Piepenbrink,Leadandimmunefunction,Crit.Rev.Toxicol.36(2006)359–385.[11]I.W.G.o.t.E.o.C.R.t.Humans,Inorganicandorganicleadcompounds,IARCMonogr.Eval.Carcinog.RisksHum.87(2006)1–471.[12]M.C.Rousseau,M.E.Parent,L.Nadon,B.Latreille,J.Siemiatycki,Occupationalexposuretoleadcompoundsandriskofcanceramongmen:apopulation-basedcase-controlstudy,Am.J.Epidemiol.166(2007)1005–1014.[13]E.K.Silbergeld,M.Waalkes,J.M.Rice,Leadasacarcinogen:experimentalevidenceandmechanismsofaction,Am.J.Ind.Med.38(2000)316–323.[14]E.K.Silbergeld,Facilitativemechanismsofleadasacarcinogen,Mutat.Res.533(2003)121–133.[15]A.Kumar,M.N.Prasad,V.MohanMuraliAchary,B.B.Panda,Elucidationoflead-inducedoxidativestressinTalinumtriangularerootsbyanalysisofantioxidantresponsesandDNAdamageatcellularlevel,Environ.Sci.Pollut.Res.Int.20(2013)4551–4561.[16]J.Garcia-Leston,J.Roma-Torres,M.Vilares,R.Pinto,J.Prista,J.P.Teixeira,O.Mayan,J.Conde,M.Pingarilho,J.F.Gaspar,E.Pasaro,J.Mendez,B.Laffon,GenotoxiceffectsofoccupationalexposuretoleadandinfluenceofpolymorphismsingenesinvolvedinleadtoxicokineticsandinDNArepair,Environ.Int.43(2012)29–36.[17]N.H.Zawia,T.Crumpton,M.Brydie,G.R.Reddy,M.Razmiafshari,Disruptionofthezincfingerdomain:acommontargetthatunderliesmanyoftheeffectsoflead,Neurotoxicology21(2000)1069–1080.[18]F.Hong,C.Wu,C.Liu,L.Wang,F.Gao,F.Yang,J.Xu,T.Liu,Y.Xie,X.Li,DirectevidenceforinteractionbetweenleadionsandkidneyDNAfromsilvercruciancarp,Chemosphere68(2007)1442–1446.[19]H.A.Tajmir-Riahi,M.Naoui,R.Ahmad,TheeffectsofCu2+andPb2+onthesolutionstructureofcalfthymusDNA:DNAcondensationanddenaturationstudiedbyFouriertransformirdifferencespectroscopy,Biopolymers33(1993)1819–1827.[20]H.A.Tajmir-Riahi,M.Langlais,R.Savoie,AlaserRamanspectroscopicstudyoftheinteractionofcalf-thymusDNAwithCu(II)andPb(II)ions:metalionbindingandDNAconformationalchanges,NucleicAcidsRes.16(1988)751–762.[21]X.C.Zhao,R.T.Liu,Recentprogressandperspectivesonthetoxicityofcarbonnanotubesatorganism,organ,cell,andbiomacromoleculelevels,Environ.Int.40(2012)244–255.[22]Z.X.Chi,R.T.Liu,H.Zhang,Noncovalentinteractionofoxytetracyclinewiththeenzymetrypsin,Biomacromolecules11(2010)2454–2459.[23]Z.X.Chi,R.T.Liu,Y.J.Sun,M.J.Wang,P.J.Zhang,C.Z.Gao,InvestigationonthetoxicinteractionoftoluidinebluewithcalfthymusDNA,J.Hazard.Mater.175(2010)274–278.[24]P.L.Olive,J.P.Banath,Thecometassay:amethodtomeasureDNAdamageinindividualcells,Nat.Protoc.1(2006)23–29.[25]G.Quievryn,A.Zhitkovich,LossofDNA–proteincrosslinksfromformaldehyde-exposedcellsoccursthroughspontaneoushydrolysisandanactiverepairprocesslinkedtoproteosomefunction,Carcinogenesis21(2000)1573–1580.[26]L.Ahmad,S.Jalali,S.A.Shami,Z.Akram,S.Batool,O.Kalsoom,EffectsofcryopreservationonspermDNAintegrityinnormospermicandfourcategoriesofinfertilemales,Syst.Biol.Reprod.Med.56(2010)74–83.[27]S.Dwivedi,Q.Saquib,A.A.Al-Khedhairy,J.Musarrat,Butachlorinduceddissipationofmitochondrialmembranepotential,oxidativeDNAdamageandnecrosisinhumanperipheralbloodmononuclearcells,Toxicology302(2012)77–87.[28]I.G.Minko,I.D.Kozekov,A.Kozekova,T.M.Harris,C.J.Rizzo,R.S.Lloyd,MutagenicpotentialofDNA-peptidecrosslinksmediatedbyacrolein-derivedDNAadducts,Mutat.Res.637(2008)161–172.[29]J.Shaham,Y.Bomstein,A.Meltzer,Z.Kaufman,E.Palma,J.Ribak,DNA–proteincrosslinks,abiomarkerofexposuretoformaldehyde-invitroandinvivostudies,Carcinogenesis17(1996)121–125.[30]K.S.Cameron,V.Buchner,P.B.Tchounwou,Exploringthemolecularmechanismsofnickel-inducedgenotoxicityandcarcinogenicity:aliteraturereview,Rev.Environ.Health26(2011)81–92.[31]P.Ramirez,L.M.DelRazo,M.C.Gutierrez-Ruiz,M.E.Gonsebatt,ArseniteinducesDNA–proteincrosslinksandcytokeratinexpressionintheWRL-68humanhepaticcellline,Carcinogenesis21(2000)701–706.[32]A.Macfie,E.Hagan,A.Zhitkovich,MechanismofDNA–proteincross-linkingbychromium,Chem.Res.Toxicol.23(2010)341–347.[33]K.Jomova,M.Valko,Advancesinmetal-inducedoxidativestressandhumandisease,Toxicology283(2011)65–87.[34]B.Pourrut,S.Jean,J.Silvestre,E.Pinelli,Lead-inducedDNAdamageinViciafabarootcells:potentialinvolvementofoxidativestress,Mutat.Res.726(2011)123–128.[35]M.E.Fracasso,L.Perbellini,S.Solda,G.Talamini,P.Franceschetti,LeadinducedDNAstrandbreaksinlymphocytesofexposedworkers:roleofreactiveoxygenspeciesandproteinkinaseC,Mutat.Res.515(2002)159–169.[36]C.M.Liu,J.Q.Ma,Y.Z.Sun,Quercetinprotectstheratkidneyagainstoxidativestress-mediatedDNAdamageandapoptosisinducedbylead,Environ.Toxicol.Pharmacol.30(2010)264–271.[37]C.M.Liu,J.Q.Ma,Y.Z.Sun,Puerarinprotectstheratliveragainstoxidativestress-mediatedDNAdamageandapoptosisinducedbylead,Exp.Toxicol.Pathol.64(2012)575–582.[38]K.Wozniak,J.Blasiak,Invitrogenotoxicityofleadacetate:inductionofsingleanddoubleDNAstrandbreaksandDNA–proteincross-links,Mutat.Res.535(2003)127–139.[39]K.A.Browne,Metalion-catalyzednucleicacidalkylationandfragmentation,J.Am.Chem.Soc.124(2002)7950–7962.[40]Y.Shi,C.Guo,Y.Sun,Z.Liu,F.Xu,Y.Zhang,Z.Wen,Z.Li,InteractionbetweenDNAandmicrocystin-LRstudiedbyspectraanalysisandatomicforcemicroscopy,Biomacromolecules12(2011)797–803.[41]C.Tong,Z.Hu,MethyleneblueasaDNAprobeforacomparativestudyofCd2+,Pb2+andCr3+ionsbindingtocalfthymusDNA,J.Lumin.131(2011)2133–2139.[42]C.Tong,G.Xiang,Y.Bai,InteractionofparaquatwithcalfthymusDNA:aterbium(III)luminescentprobeandmultispectralstudy,J.Agr.FoodChem.58(2010)5257–5262.[43]D.Voet,W.B.Gratzer,R.A.Cox,P.Doty,Absorptionspectraofnucleotides,polynucleotides,andnucleicacidsinthefarultraviolet,Biopolymers1(1963)193–208.[44]Y.Ma,J.Pan,G.Zhang,Y.Zhang,BindingpropertiesofbutylatedhydroxytoluenewithcalfthymusDNAinvitro,J.Photochem.Photobiol.B126(2013)112–118.[45]Y.J.Guo,J.B.Chao,J.H.Pan,Studyontheinteractionof5-pyridine-10,15,20-tris-(p-chlorophenyl)porphyrinwithcyclodextrinsandDNAbyspectroscopy,Spectrochim.ActaA:Mol.Biomol.Spectrosc.68(2007)231–236.[46]N.C.Garbett,P.A.Ragazzon,J.B.Chaires,Circulardichroismtodeterminebindingmodeandaffinityofligand-DNAinteractions,Nat.Protoc.2(2007)3166–3172.[47]L.D.Mello,R.M.Pereira,A.C.Sawaya,M.N.Eberlin,L.T.Kubota,ElectrochemicalandspectroscopiccharacterizationoftheinteractionbetweenDNAandCu(II)-naringincomplex,J.Pharm.Biomed.Anal.45(2007)706–713.[48]J.Chai,J.Wang,Q.Xu,F.Hao,R.Liu,Multi-spectroscopicmethodscombinedwithmolecularmodelingdissecttheinteractionmechanismsofractopamineandcalfthymusDNA,Mol.Biosyst.8(2012)1902–1907.[49]P.UmaMaheswari,M.Palaniandavar,DNAbindingandcleavagepropertiesofcertaintetrammineruthenium(II)complexesofmodified1,10-phenanthrolines-effectofhydrogen-bondingonDNA-bindingaffinity,J.Inorg.Biochem.98(2004)219–230.H.Zhangetal./JournalofPhotochemistryandPhotobiologyB:Biology136(2014)46–53[50]R.Trotta,S.DeTito,I.Lauri,V.LaPietra,L.Marinelli,S.Cosconati,L.Martino,M.R.Conte,L.Mayol,E.Novellino,A.Randazzo,Amoredetailedpictureoftheinteractionsbetweenvirtualscreening-derivedhitsandtheDNAG-quadruplex:NMR,molecularmodellingandITCstudies,Biochimie93(2011)1280–1287.[51]L.Y.Li,H.N.Jia,H.J.Yu,K.J.Du,Q.T.Lin,K.Q.Qiu,H.Chao,L.N.Ji,Synthesis,characterizationandDNA-bindingstudiesofrutheniumcomplexes[Ru(tpy)(ptn)]2+andRu(dmtpy)(ptn)]2+,J.Inorg.Biochem.113(2012)31–39.[52]S.Bi,H.Zhang,C.Qiao,Y.Sun,C.Liu,StudiesofinteractionofemodinandDNAinthepresenceofethidiumbromidebyspectroscopicmethod,Spectrochim.ActaA:Mol.Biomol.Spectrosc.69(2008)123–129.53[53]M.Hossain,P.Giri,G.S.Kumar,DNAintercalationbyquinacrineandmethyleneblue:acomparativebindingandthermodynamiccharacterizationstudy,DNACellBiol.27(2008)81–90.[54]S.Charak,M.Shandilya,G.Tyagi,R.Mehrotra,SpectroscopicandmoleculardockingstudiesonchlorambucilinteractionwithDNA,Int.J.Biol.Macromol.51(2012)406–411.[55]Z.X.Chi,R.T.Liu,Phenotypiccharacterizationofthebindingoftetracyclinetohumanserumalbumin,Biomacromolecules12(2011)203–209.