Polyhydroxy Fullerenes: Beneficial Effects on Growth and Lifespan in Diverse Biological Models
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Title: Polyhydroxy Fullerenes: Beneficial Effects on Growth and Lifespan in Diverse Biological Models
Series Title: PLoS One. 2011;6(5):e19976
Physical Description: Journal Article
Creator: Folta, Kevin
Gao, Jie
Wang, Yihai
Krishna, Vijay
Bai, Wei
Indeglia, Paul
Georgieva, Angelina
Nakamura, Hideya
Koopman, Ben
Moudgi, Brij
Publisher: Public Library of Science
Place of Publication: San Francisco, CA
Publication Date: May 27, 2011
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Abstract: Recent toxicological studies on carbon nanomaterials, including fullerenes, have led to concerns about their safety. Functionalized fullerenes, such as polyhydroxy fullerenes (PHF, fullerols, or fullerenols), have attracted particular attention due to their water solubility and toxicity. Here, we report surprisingly beneficial and/or specific effects of PHF on model organisms representing four kingdoms, including the green algae Pseudokirchneriella subcapitata, the plant Arabidopsis thaliana, the fungus Aspergillus niger, and the invertebrate Ceriodaphnia dubia. The results showed that PHF had no acute or chronic negative effects on the freshwater organisms. Conversely, PHF could surprisingly increase the algal culture density over controls at higher concentrations (i.e., 72% increase by 1 and 5 mg/L of PHF) and extend the lifespan and stimulate the reproduction of Daphnia (e.g. about 38% by 20 mg/L of PHF). We also show that at certain PHF concentrations fungal growth can be enhanced and Arabidopsis thaliana seedlings exhibit longer hypocotyls, while other complex physiological processes remain unaffected. These findings may open new research fields in the potential applications of PHF, e.g., in biofuel production and aquaculture. These results will form the basis of further research into the mechanisms of growth stimulation and life extension by PHF.
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PolyhydroxyFullerenes(FullerolsorFullerenols): BeneficialEffectsonGrowthandLifespaninDiverse BiologicalModelsJieGao1,2* ,YihaiWang3,KevinM.Folta3,VijayKrishna1,WeiBai2,PaulIndeglia2,AngelinaGeorgieva1, HideyaNakamura1,BenKoopman1,2,BrijMoudgil1,4*1 ParticleEngineeringResearchCenter,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica, 2 DepartmentofEnvironmentalEngineeringSciences, UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica, 3 PlantMolecularandCellularBiologyProgram,HorticulturalSciencesDepartment,Universityof Florida,Gainesville,Florida,UnitedStatesofAmerica, 4 DepartmentofMaterialsScienceandEngineering,UniversityofFlorida,Gainesville,Florida,UnitedStatesof AmericaAbstractRecenttoxicologicalstudiesoncarbonnanomaterials,includingfullerenes,haveledtoconcernsabouttheirsafety. Functionalizedfullerenes,suchaspolyhydroxyfullerenes(PHF,fullerols,orfullerenols),haveattractedparticularattention duetotheirwatersolubilityandtoxicity.Here,wereportsurprisinglybeneficialand/orspecificeffectsofPHFonmodel organismsrepresentingfourkingdoms,includingthegreenalgae Pseudokirchneriellasubcapitata ,theplant Arabidopsis thaliana ,thefungus Aspergillusniger ,andtheinvertebrate Ceriodaphniadubia .TheresultsshowedthatPHFhadnoacuteor chronicnegativeeffectsonthefreshwaterorganisms.Conversely,PHFcouldsurprisinglyincreasethealgalculturedensity overcontrolsathigherconcentrations(i.e.,72%increaseby1and5mg/LofPHF)andextendthelifespanandstimulatethe reproductionof Daphnia (e.g.about38%by20mg/LofPHF).WealsoshowthatatcertainPHFconcentrationsfungal growthcanbeenhancedand Arabidopsisthaliana seedlingsexhibitlongerhypocotyls,whileothercomplexphysiological processesremainunaffected.ThesefindingsmayopennewresearchfieldsinthepotentialapplicationsofPHF,e.g.,in biofuelproductionandaquaculture.Theseresultswillformthebasisoffurtherresearchintothemechanismsofgrowth stimulationandlifeextensionbyPHF.Citation: GaoJ,WangY,FoltaKM,KrishnaV,BaiW,etal.(2011)PolyhydroxyFullerenes(FullerolsorFullerenols):BeneficialEffectsonGrowthandLifespani n DiverseBiologicalModels.PLoSONE6(5):e19976.doi:10.1371/journal.pone.0019976 Editor: RichardC.Willson,UniversityofHouston,UnitedStatesofAmerica Received December5,2010; Accepted April21,2011; Published May27,2011 Copyright: 2011Gaoetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisresearchwassupportedbyaresearchgrantfromParticleEngineeringResearchCenter(PERC)(perc.ufl.edu)andCenterforNano-BioSensors (CNBS,cnbs.centers.ufl.edu).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanu script. PublicationofthisarticlewasfundedinpartbytheUniversityofFloridaOpen-AccessPublishingFund. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:dencyl@ufl.edu(JG);bmoudgil@perc.ufl.edu(BM)IntroductionSincetheirdiscoveryin1985,C60fullereneshavebeenamong themostwidelystudiedandusedcarbon-basednanomaterials duetotheiruniquestructuralandelectronicpropertiesthat enablenumerousindustrial,electricalandmedicalapplications [1,2,3].Comparedwiththepristineform,functionalized fullereneshavemorepotentialapplicationsduetotheirenhanced watersolubility.Functionalizedfullereneshavebeenreportedto reduceoxidativestressbyscavengingreactiveoxygenspecies [4,5]andhavebeenexaminedfortheirantioxidantproperties. Conversely,C60fullereneanditswater-solublederivativeshave beenthesubjectofconcernbecauseoftheirrapidlygrowing productionandpotentialenvironmentalandhealthimplications [6].Oneheavilystudiedfunctionalizedfullereneisthepolyhydroxyfullerene(PHF,alsonamedfullerolorfullerenol,Figure1), whereinthefullerenesaredecoratedwith12–42hydroxylgroups permoleculeleadingtoenhancedsolubility.PHFhasbeen shown,forexample,toinducetheproductionofreactiveoxygen speciesandcausemembranedamageinratlivermicrosomes[7], andexhibitcytotoxicityandphototoxicitytohumanepithelial cells[8,9]. Astheglobalproductionoffullerenesandtheirderivativesis growingrapidly,fullerene-containingproductswillinevitablyenter variousenvironmentaltheatresdur ingtheirproduction,deployment anddisposal.Amongtheseenvironments,aquaticandsoilsystemsare likelytheultimatesinksforcarbon-basednanomaterials[6,10,11], thusexposingaquaticandsoilorganismstofullerenes,especiallythe watersolublederivatives.Therefore,itiscriticaltoidentifythe potentialimpactsofthisnanomaterialinecologicalsystems. TheobjectiveofthisstudywastoinvestigatetheeffectsofPHFon growth,development,andreproductioninrepresentativebiological systems.Thestudyexploitstheadaptivecapacities,sensoryspectra and/orreproductiveabilitiesofthesebiologicalsystemsthat ultimatelyserveaslivingsensorsofbio-activecompoundsintheir environments.Theirbehaviorsprovidevaluableinformationthat describeimpactsongrowthanddevelopment,andmayevendetail themechanismofPHFingressthatshapesbiologicalprocesses.The short-termorchronicgrowthand/orreproductiveeffectsofPHF areinvestigatedforamodelfreshwaterphytoplanktonicorganism, thegreenalgae Pseudokirchneriellasubcapitata ,amodelplant Arabidopsis thaliana ,amodelfungus Aspergillusniger ,andamodelzooplankter, theinvertebrate Ceriodaphniadubia PLoSONE|www.plosone.org1May2011|Volume6|Issue5|e19976

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ResultsThefirsttestswereperformedon Pseudokirchneriellasubcapitata ,a standardalgalspeciescommonlyusedtoassesstoxicityofatest substanceinanaqueoussystem.AllPHFconcentrationsexceptfor 0.001mg/Land0.1mg/Lshowedsignificanteffects(p 0.05).After fourdaysofgrowth,thenumberof P.subcapitata cellswas72%higher inthepresenceof1mg/Land5mg/LPHFrelativetothenoPHF controltreatment(Fig.2A).PHFconcentrationsof0.001mg/Land 0.1mg/Ldidnothavesignificanteffectsat a =0.05,eventhough meanvalueswereslightlyelevatedrelativetocontrols. Lighthasaprofoundeffectontheregulationofplantgrowth.The effectsaremostsalientinthedevelopingseedling.Discreteportionsof theelectromagneticspectrum,nutrients,toxinsandotherenvironmentalfactorsoftentimeshavepotenteffectsonhypocotyl(stem) growth.Thedevelopingseedlingisthereforeanexcellentbiosensorto describehowagivencompoundmaycontributetogrowthresponses. Duetoitsshortlifecycle,welldefinedphysiologyandsubstantial geneticresources, Arabidopsisthaliana isoneofthepopularplant modelsforplantbiologyandgeneticresearch.Inthisstudy,theroot andhypocotyllengthsofArabidopsisseedlingsweremeasuredafter fourdaysofgrowthonvariousPHFconcentrations.Theresults Figure1.Chemicalstructureofapolydroxyfullerene(PHF) molecule(x=12to42). doi:10.1371/journal.pone.0019976.g001 Figure2.Effectsofpolyhydroxyfullerens(PHF)ongrowthand/orlifespan. (A)Normalizedalgalgrowthcomparedtountreatedcontrols. Theculturesweregrowninthepresenceofthetreatmentfor4days(n=9).(B)Normalized Arabidopsis hypocotylgrowthagainstuntreatedcontrols. Datarepresentmeanofthreetrials,whereineachtrialcomprisesatleast25seedlingmeasurements.(C)Normalized Daphnia reproductionas measuredbythenumberofneonatesproducedoverthelifetimeof5maturedaphnids(n=4).(D)Lifespanoftesteddaphnidsatconcentrationsfrom 0.001to20mg/L(n=20).Errorbarsindicatestandarderrorofthemean.*:p 0.05;**:p 0.01. doi:10.1371/journal.pone.0019976.g002 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org2May2011|Volume6|Issue5|e19976

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indicatethatseedlingsgerminatedinthepresenceof100and 200mg/LPHFhavesignificantly(p 0.01)longerhypocotyls comparedtolowerconcentrationsorcontrols(Fig.2B).Noeffects wereobservedinrootelongation(Fig.3)oronphototropicgrowth (notshown).Thedifferenceinhypocotylelongationwasnotobserved indarknessormonochromaticlightconditions(Fig.4).Theresults indicatethattheeffectsonseedlinggrowtharespecific,dependon integrationofsignalsmultiplephotosensorypathways,andaffectonly discreteplantorgans. TheeffectsofPHFwereassessedonthe Aspergillusniger ,the causativeorganismofblackmold.Weobservedthatconcentrationsabove10mg/Lstimulatedgrowth.Treatmentof A.niger with PHFfor120hours(media:amendedRPMI1640)causedmore sporestoformcomparedtocontrols(Fig.5),indicatingthatPHF athigherconcentrationcanstimulatereproduction. ThegrowthandreproductiveeffectsofPHFwerealsotested on Ceriodaphniadubia ,anestablishedEPA-recommended freshwaterbiologicalinvertebratesystem.PHFtreatmentover thelifetimeofthedaphnidsincreasedthenumberofneonatesat 20mg/LPHFby38%(Figure2C).Thedaphnidswere monitoredforPHFeffectonlifespan.At20mg/LofPHFtheir lifespanincreased38%overcomparablenon-PHFtreated controls(p 0.05)(Figure2D).LowerPHFconcentrationshad nosignificanteffect.Figures6a-cshowsamplemicrographstaken forthedaphnidsinthecontrol,0.001mg/Land20mg/Lof PHFtreatmentsafter2-dayexposure.AccumulationofPHFwas clearlyobservedinthe Daphnia GItractexposedto20mg/Lof PHF(Figure6c;arrow).Duringthefirst8days,thebodylength ofeachtesteddaphnidwasmeasureddailyandthelengthwas determinedfromthebaseofthetailspinetothebaseofthehead [12](whitelinesinFigure6a–c).AsshowninFigure6d,there werenoobservableeffectsonthe Daphnia growthatconcentrationsof5mg/Lorlower.At20mg/LofPHF,thegrowthrate wasaffectedwithinthefirsthoursoftreatment,butwasmostly unaffectedthereafter,resultinginasmallerdaphnidsizeoverthe testperiodof8days( a =0.05).DiscussionAlthoughitisunclearhowPHFsaffectcellulargrowthor expandlifespan,theunexpectedobservationsmaybeattributed tothefactthatPHFisoneofthemostpotentROSscavengers andcouldreduceoxidativechallengeexperiencedbyorganisms duringagingandgrowth[13,14].Itwasfoundthatfullerene cagecanabsorbupto6electronsanddispersethemthroughthe 20benzeneringsoveritssurface[15].InastudybyQuicketal. [16],anothertypeoffunctionalizedfullerene,carboxyfullerene, whichisaknownantioxidant,hasbeenshowntoextendthe lifespanofmiceby5%,andtoreverseage-relatedcognitive impairment.However,noclearcorrelationhasbeenobserved betweenantioxidantsandlifespanintheliterature,sofar,and othermechanismsmayberesponsibleforthelifespaneffect [17]. Recentdiscoveriesthatcertainspeciesoffungicandecompose PHFandincorporatethemintobiomass[18]andthatPHFcan bemineralizedtootherinorganiccarbonbydirectphotolysis[19] shedlightonotherpossibilitiesthatalgal,fungalorplantcells maybeabletoutilizePHFmoleculesasanutritionsource.In addition,thefactthatstemgrowthisaffectedunderlight conditions,butrootgrowth,phototropiccurvatureanddarkstem growtharenotaffected(Figures2B,3and4),indicatesthatPHF effectsaremechanisticallydiscreteandnotsimplypleiotropic effectsofagenerallytoxiccompound.Theseresultsindicatethat PHFeffectsarespecificandareaffectingseedlinggrowthdueto interactionwithgrowthregulatoryprocesses,particularlythose associatedwithstemelongationinlight.Theprincipleeffectthat isseenisalongersteminPHFtreatedseedlings.Theeffectisnot light-qualityspecific,sotheeffectislikelyinresponsetolight, ratherthansensingoflight.Examplesofsuchresponsepathways Figure3.NormalizedrootlengthsofArabidopsisseedlingsinvariousPHFconcentrationsunderwhitelightcondition. No observabledifferenceonrootelongationwasdetectedinthePHFtreatedseedlingscomparedwithcontrolseedlings(p 0.05).Rootlengthswereall normalizedtocontrolwithineachexperimentalreplicate.Atleast25seedlingspertreatmentweremeasuredineachreplicate.Errorbarsrepresent s thestandarddeviationofthethreeindependenttrials. doi:10.1371/journal.pone.0019976.g003 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org3May2011|Volume6|Issue5|e19976

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mayincludethegibberellicacidsensing/responsemechanism,a cellelongationsystemthatisinhibitedbylight.PHFmakes hypocotylslonger,suggestingthatthecompoundispossibly interactingwiththemechanismsthatadjuststemgrowthinthe lightenvironment,suchasahypersensitivityto,orproductionof, planthormones. Theslightreductionof Daphnia bodylengthinthe20mg/LPHF (Fig.6)treatmentindicatesapossibledelayinagingcauseddirectly [16]orindirectly(e.g.,dietaryrestrictionatthisconcentration, whichhasbeenknowntoextendlifespaninavarietyoforganisms [20,21,22]byahighdoseofPHF.Inaddition,theincreaseand improvementofalgalbiomassmayaddfavorablenutrientstothe Daphnia foodandthusincreasetheirlifespan. Frombacteriatomammaliancellstomammaliansystems, polyhydroxyfullereneshavegenerallybeendescribedastoxic [9,23,24,25].TheresultsobtainedfromthisstudyareunexpectedbecausePHFfunctionedoftenasagrowthstimulant,or atleastagrowthregulator.Ourfindingsalsosuggestthat applicationofPHFhasdiscreteb iologicalconsequencesthatare speciesandtissuespecific.Thus,itisimportanttonotethat PHF’seffectscanbegenerallyre gardedasbeneficial,withno evidenceofdeleteriouseffectsd escribedforothernanomaterials.Thesefindingsaresignif icantbecausetheyindicatethat PHFhasthepotentialtoserveasagrowthregulatoror modulatorofbiologicalprocesse sthatspanseveralkingdoms, openingthedoortonewapplicationsthatmaybenefitindustry andagriculture.Suchapplicati onsmayincludeinstanceswhere growthstimulationisdesired,f orinstance,inalgalproduction forbiofuelapplication,aquacu lture,orinmodulatingplant statureordevelopment.MaterialsandMethods 1.PreparationofPHFsolutionPHFusedinthealgaeand Daphnia studywerepurchasedfrom Nano-C,Inc.(Westwood,MA),andfromBuckyUSA(Houston, TX)forthefungalstudy,bothsampleswithassignedpossible compositionC60(OH)24–26.PHFwerealsosynthesizedthrough analkaliroute[26]andusedintheplantstudy.Briefly,inadry 250mlroundbottomflask160mgC60wasmixedwith55ml toluene(Aldrich)andstirredfor30mincoveredwithrubber septumandequippedwithNitrogenfilledballoon.Tothisdark purplesolutionwasadded0.3ml40%aqueoustetrabutyl ammoniumhydroxide(FisherSci)followedbyadditionof5ml Figure4.NormalizedrootorhypocotyllengthsofArabidopsisseedlingsunderbluelight(BL)orredlight(RL)conditions. PHF concentrationwas100mg/L.Nodifferenceonrootandhypocotylelongationwasobservedunderthosetwomonochromaticlightconditions (p 0.05).Rootandhypocotyllengthswereallnormalizedtocontrolthatgrownunder0.2mmolm2 2s2 1BLorRLwithineachexperimentalreplicate. Atleast25seedlingspertreatmentweremeasuredineachreplicate.Errorbarsrepresentthestandarddeviationofthethreeindependenttrials. doi:10.1371/journal.pone.0019976.g004 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org4May2011|Volume6|Issue5|e19976

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50%aqueousNaOH(Acros).Reactionmixturewasstirredat roomtemperaturefor5daysuntilthepurplecolordisappeared. Thenflaskwasplacedinafreezer( 2 10 u C)andthecleartoluene layerwasdecantedoutofthefrozenmixture.Tothemixturewas added40mlnanopurewaterandmixturestirredatroom temperaturefor2days.Thedarkbrownmixturewasfreeze-dried toremovethewaterandtheremainingpowderwaswashedwith methanol5X20ml.Itwascentrifugedandmethanolwas decantedeachtime.Theresidualpowderdriedinvacuumoven atroomtemperature(340mg)andpurifiedonneutralSephadex sizeexclusioncolumnpreparedwithpurewaterand20g SephadexG-25(Sigma-Aldrich).Collectedwere25mlfractions, darkfractions1–3werecombinedandfreeze-driedtogive 200mgdarkbrownpowder,completelysolubleinwater.Priorto useinthisstudy,thePHFsolutionswerecharacterizedas describedelsewhere[27]. Astocksolutioncontaining1000mg/LofPHFwasprepared bydissolving10mgofPHFin10mLofNanopure H waterand theresultantsolutiondisplayedacharacteristicdarkbrownhue.2.96-hourPseudokirchneriellasubcapitataGrowthAssayMaintenanceofthealgalculturewasdescribedinGriffittetal. [28].Briefly,theculturemediumwaspreparedfromstock solutionsaccordingtoEPAstandardmethodforpreliminaryalgal assayprocedure[29].ThepHoftheculturemediumwasadjusted to7.5 6 0.1with0.1NNaOHor0.1NHClandthenfiltered througha0.45mmmembraneandsterilizedbyautoclaving.A purecultureof P.subcapitata wasobtainedfromHydrosphere Research(Alachua,FL)andgrowninPreliminaryAlgalAssay Procedure(PAAP)mediumwithEDTAat25 6 1 u C.Lightsource (86 6 8.6mEm2 2s2 1)andcontinuousaerationwereprovided 24hoursperday.Newcultureswerepreparedeveryweekunder sterileconditionsbytransferringapproximately20–30mLofthe matureculturesto1–2Loffreshsterilemedia. Thegrowthassaywasperformedinautoclaved125mL ErlenmeyerflasksaccordingtotheEPAprotocol[29].Allsample dilutions(i.e.culturemediaspikedwithincreasingconcentrationsof PHFfrom0.001to20mg/L)andnegativecontrolswererunin triplicatesandinoculatedwith1mLofa4to7-dayoldalgal cultures.Allflaskswereplacedunderthefluorescentlightsinthe samegrowthconditions.Thealgalgrowthafter96-hwas determinedbycellnumbercountbetween3.4and8mm[30]using aCoulterMultisizerIII(BeckmanCoulter,Inc.Brea,CA,USA).3. Arabidopsis GrowthExperimentsArabdopsisthaliana seeds(ecotypeColumbia-0)wereplacedontoa minimalmedium(1mMKCl,1mMCaCl2;solidifiedwith1% phytoagar)containing0–200mg/LPHFinsquarePetridishesand stratifiedat4 u Cfor48h.Theseedswerethentreatedwithwhite light(20mmolm2 2s2 1)for15minutestosynchronizegermination. Thentheplatesweremovedtoexperimentalconditionsinavertical positionsuchthatseedlingswouldgrowuprightontheagarsurface. Theexperimentalconditionsweredarkness,whitelight(coolwhite fluorescent;20mmolm2 2s2 1),blueorredLEDlight(various fluenceratestestedforblueandredlightat0.2,2or20mmolm2 2s2 1).Seedlingsweregrownfor96hinexperimentalconditions, imagedonaflatbedscanner,andthenmeasuredusingImageTool 3.0againstknownstandards.Atleast25seedlingsweremeasured pertreatmentinatleastthreeindependentexperimentalreplicates.4.FungiGrowthExperimentsFungigrowthexperimentswereconductedina96-wellplateby followingstandardmicro-dilutionprotocolwithtwodifferentgrowth media:RPMI1640with2%glucoseandRPMI1640withpotato dextrosebroth.TheprotocolinvolvespreparingaseriesofPHF dilutionsina96wellplate,inoculatingthewellswith A.niger ,andusing theabsorbanceat600nmasanindic atorofabiomassconcentration. Visiblelightabsorbancecorrelatestobiomasstoindicatefungal growthorinhibitionrelativetotheabsorbancemeasuredfroma controlwithoutPHFastaughtinSchwalbeetal.[31]. Pre-preparedRPMI-1640wasobtainedfromMediatechInc. (Manassas,VA).Potatodextrosebrothwaspreparedinthe laboratory.PHFstocksolutionswerepreparedintheculture mediaatconcentrationof2000mg/L,followedby10folddilution Figure5.Aphotographicreproductionofa96-wellplatetestafter120-hourincubation. Thecolumnoftestcellshaving A.niger exposed to1,000mg/LPHFshowssignificantlyhighergrowththancontrolorotherPHFquantities. doi:10.1371/journal.pone.0019976.g005 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org5May2011|Volume6|Issue5|e19976

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withtherespectivemediatogiveconcentrationsof200,20and 2mg/L.AserialdilutionofthePHFstocksolutionwascarriedout in96wellplates.Eachplatecomprised12columnsand8rows. Eachcellwasidentifiedbyitsrowletter(A–H)andcolumn number(1–12).Thus,thetop,leftmostwellwouldbecellA1,and thebottom,rightmostcellwouldbecellH12.Afterdilution, inoculationof A.niger wascarriedoutfollowingthestandard protocoldescribedinSchwalbeetal.[31].Theplateswere coveredwithaluminumfoil,keptinanorbitalshakerwith temperatureat37 u Cand75RPM. Figure6.Microscopicimages(10Xmagnification)ofdaphnidsexposedtocontrolandpHtreatmentsafter2days. (A)Control,(B) treatmentwith0.001mg/LofPHF,and(C)treatmentwith20mg/LofPHF.(D)Meanbodylengthofdaphnidsoveradurationof8daysinresponse tovariousconcentrationsofPHF(n=2). doi:10.1371/journal.pone.0019976.g006 BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org6May2011|Volume6|Issue5|e19976

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5.SurvivalandReproductionAssayUsing Ceriodaphnia dubiaMaintenanceofthe Daphnia culturewasdescribedinGaoetal. [10]andGriffittetal.[28].Briefly,moderatelyhardwater(MHW) preparedfollowingtheEPAstandardmethod[29]wasusedas culturemediainthistest.Purecultureof C.dubia wasobtained fromHydrosphereResearch(Alachua,FL)andkeptin1L beakerscontaining500mLofMHWinaPervicalTMmodel # E30BXenvironmentalchamberat25 u Cwithconstantaeration. Thephotoperiodwas16-hrlight/8-hrdark. C.dubia wasfedwith concentrated P.subcapitata cellsandYCT(madefromyeast,cereal leavesandtroutchow).Thedaphnidswerefedeveryotherday with6.67mLYCTand6.67mLalgaesolution/Lculture. Neonatesoflessthan24hourswereseparatedfromadultsdaily andusedfortesting. Thesurvivalandreproductionassayswereperformedaccording toEPA’sprotocol[29].MHWservedasnegativecontrolandas thediluenttopreparemediawithincreasingconcentrationsof PHF.Neonateslessthan24hourswereseparatedfromadultsand fed2hourspriortoteststart.Foreachtest,groupsof5 Daphnia neonatesweretransferredinto30mlplasticcupscontaining20ml ofMHW(controls)orMHWplusPHFatvariousconcentrations (i.e.0.0001,0.001,0.01,0.1,1,5and20mg/L)for7daystoassess theeffectsofPHFongrowth,survivalandreproduction.The daphnidswereculturedat25 6 1 u Cwiththesamephotoperiod andfedwith40mLofYCTand P.subcapitata dailyperdaphnid. Theculturemediumwasrenewedthreetimesaweek.Everyday thesurvivalandnumberofnewbornswererecordedandthe offspringsproducedwereseparatedfromthetestcontainers.All sampledilutionsandnegativecontrolswereruninfourreplicates andeachtreatmenthadatotalof20daphnids.6.GrowtheffectsofPHFon CeriodaphniadubiaAteachconcentration,twoneonateswerekeptinseparatecups (i.e.,onepercup)atsameconditionsasothertestdaphnidsand observedbyopticalmicroscopy1hourafterfeedingeveryday. Themicrographstakenwereanalyzedforsize(i.e.corebody length)ofexposeddaphnidsanddistributionofPHFinthebody.7.EffectofPHFonthelifespanof CeriodaphniadubiaThesamedaphnidsusedinthesurvivalandreproductionassay werekeptandtestedfortheirlifespanwithandwithoutthe exposuretoPHF.Thedaphnidswereculturedandfedinthesame manneraspreviouslydescribed.Theculturemediumwasrenewed threetimesaweek.Everydaythesurvivalandnumberof newbornswererecordedandtheoffspringsproducedwere separateddailyuntilthedeathofalldaphnids.8.DataAnalysisAlltheexperimentswererunintriplicateorgreater,anddata wereillustratedasmean 6 standarderror(SE).Statisticalanalyses wereperformedusingone-wayanalysisofvariance(ANOVA) followedbyDunnett’stestwithanNCSS2004(Kaysville,Utah). A p valueoflessthan0.05wasconsideredtobestatistically significant.AuthorContributionsConceivedanddesignedtheexperiments:JGKMFBM.Performedthe experiments:JGYWWB.Analyzedthedata:JGKMFBK.Contributed reagents/materials/analysistools:VKAG.Wrotethepaper:JGYWKMF WBPIAGHNBKBM.References1.ChaudhuriP,ParaskarA,SoniS,MashelkarRA,SenguptaS(2009)Fullerenolcytotoxicconjugatesforcancerchemotherapy.AcsNano3:2505–2514. 2.BakryR,VallantRM,Najam-Ul-HaqM,RainerM,SzaboZ,etal.(2007) Medicinalapplicationsoffullerenes.IntJNanomed2:639–649. 3.WangHB,DeSousaR,GasaJ,TasakiK,StuckyG,etal.(2007)Fabricationof newfullerenecompositemembranesandtheirapplicationinprotonexchange membranefuelcells.JMembrSci289:277–283. 4.ChenYW,HwangKC,YenCC,LaiYL(2004)Fullerenederivativesprotect againstoxidativestressinRAW264.7cellsandischemia-reperfusedlungs. 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30.HallJA,GoldingLA(1998)Freshwateralgae( Selenastrumcapricornutum ):chronic toxicitytestprotocol.Wellington,NewZealand:NIWAreportfortheMinistry fortheEnvironment.MFE80205Appendix4MFE80205Appendix4. 31.SchwalbeR,Steele-MooreL,GoodwinAC(2007)Antimicrobialsusceptibility testingprotocols.BocaRatonFL:CRCPress.BeneficialEffectsofPolyhydroxyFullerenes PLoSONE|www.plosone.org8May2011|Volume6|Issue5|e19976


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