Nanoparticle toxicity and molecular mechanisms in fish: A

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Nanoparticle toxicity and molecular mechanismsin fish: A case study with silver nanoparticlesJiejun GaoPurdue University

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Is approved by the final examining committee:

To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material.

Approved by Major Professor(s):

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Jiejun Gao

Nanoparticle Toxicity and Molecular Mechanisms in Fish: A Case Study with Silver Nanoparticles

Doctor of Philosophy

Dr. Maria S Sepúlveda Dr. Qing DengChair

Dr. Alexander Wei

Dr. Cecon T. Mahapatra

Dr. Henry C. Chang

Maria S Sepúlveda

Robert G Wagner 11/14/2016

NANOPARTICLETOXICITYANDMOLECULARMECHANISMSINFISH:ACASESTUDYWITHSILVER

NANOPARTICLES

ADissertation

SubmittedtotheFaculty

of

PurdueUniversity

by

JiejunGao

InPartialFulfillmentofthe

RequirementsfortheDegree

of

DoctorofPhilosophy

December2016

PurdueUniversity

WestLafayette,Indiana

ii

ACKNOWLEDGMENTS

Thisthesisbecamearealitywithgreatsupportandhelpfrommanycollaborators.Theauthor

wouldliketoacknowledgethecontributionsofanumberofpeoplewithoutwhom,thiswork

couldnothavebeencompleted:

AllmembersfromDr.MariaS.Sepúlveda’slabgroup,includingChrisKlinkhamerforintroducing

meintothefieldofnanotoxicology;Dr.CeconMahapatra,SamGuffeyandDr.MatthewCharles

forhelpwithexperimentaldesign;Dr.DavidCoulterforhelpwithstatisticalanalysis;andDr.

EugeneGaoforteachingmelotsofmoleculartechniques.AhmedAbdel-moneim,GaryHoover,

DaraghDeeganandTimMalinichhelpedinthecollectionofsmallmouthbassplasma.

AllmembersfromDr.AlexanderWei’slabgroup,especiallyDr.OscarMorales,Dr.NaveenR.

Kadasala,MariaKhmebnikovaandLuLinforhelpingwithnanoparticlecharacterization.Dr.Alex

Weiforhisinputonexperimentaldesignandmanuscriptcritiques,whichhelpedmebecomea

betterscientificcommunicatorandacademicwriter.

AllmembersfromDr.JenniferFreeman’sandDr.DonnaM.Fekete’slabgroups.Specialthanks

toDr.JenniferFreemanforprovidingtransgeniczebrafishembryosandtoDr.Ten-TsaoandDr.

DonnaM.Feketeforsupportwithwildtypezebrafish.

Manythankstothecampus-wideMassSpectrometryCenterandResearchInstrumentation

CenterintheChemistryDepartment,PurdueUniversityforguidance,trainingonsample

processing,useoffluorescentmicroscopes,andICP-MSanalyses.ThankstoDr.ChrisGilpinfrom

theLifeScienceMicroscopyFacilityforhelpwithTEManalyses.

VickiHedrickfromPurdue’sProteomicsFacility,withoutherguidanceandtrainingontheLC-

MS/MS,Icouldn’thavefinishedmyresearchontime.

Tomycommitteemembers,Dr.HenryChangandDr.DengQingfortheiradviceand

contributionsforimprovingmywork.

iii

MostimportantlytomyadvisorDr.MariaSepúlvedafornotonlygivingmetheopportunityto

workcloselywithherforfourandahalfyears,butalsoforhergreatmentorshipandhelpto

makemebecomeabetterscientistandabetterpersonthroughoutmyPh.D.studyprocess.

IgratefullyacknowledgethefinancialsupportoftheChinaScholarshipCouncilandthe

DepartmentofForestryandNaturalResources,PurdueUniversity.

Finally,Iwouldliketoexpressmygratitudetomyfamilyandmyfriendsforinspiringand

supportingmeduringmystudieshere,especiallytomyyoungersister,thanksforsupporting

andbelievinginmethroughoutmytimeatPurdue.

iv

TABLEOFCONTENTS

Page

LISTOFTABLES.................................................................................................................vii

LISTOFFIGURES..............................................................................................................viii

ABSTRACT........................................................................................................................xiii

CHAPTER1INTRODUCTION...............................................................................................1

1.0Summary...............................................................................................................................1

1.1Nanoparticlesintheenvironment........................................................................................1

1.2Vasculardevelopment...........................................................................................................2

1.3CriticalissuesinassessingthetoxicityofNPs.......................................................................4

1.4VasculartoxicityofNPsinvitro.............................................................................................5

1.5VasculartoxicityofNPsinvivo............................................................................................11

1.6Conclusions..........................................................................................................................15

CHAPTER2PROTEINCORONAFROMSILVERNANOPARTICLESEXPOSEDTOFISHPLASMA

..........................................................................................................................................16

2.0Summary.............................................................................................................................16

2.1Introduction.........................................................................................................................17

2.2Materialsandmethods.......................................................................................................19

2.2.1Smallmouthbassplasmacollection.............................................................................19

2.2.2WesternblottingofVTGandVEGF..............................................................................19

2.2.3CharacterizationofPVP-AgNPsbeforeandafterincubationwithfishplasma............20

2.2.4PC-PVP-AgNPpelletpreparationandsilverstainofSDS-PAGE....................................20

2.2.5Liquidchromatography-massspectrometry/MSanalysisofproteincorona...............21

2.2.6Statisticalanalysis.........................................................................................................24

2.3Results.................................................................................................................................24

2.3.1CharacterizationofPVP-AgNPproteincorona.............................................................24

v

Page

2.3.2Silverstain....................................................................................................................25

2.3.3LC-MS/MSproteomicanalysis......................................................................................26

2.4Discussion............................................................................................................................26

2.5Conclusions..........................................................................................................................32

2.6APPENDIX............................................................................................................................38

CHAPTER3VASCULARTOXICITYOFSILVERNANOPARTICLESTODEVELOPINGZEBRAFISH

(DANIORERIO)..................................................................................................................47

3.0Summary.............................................................................................................................47

3.1Introduction.........................................................................................................................48


3.2.1CharacterizationofPVP-AgNPs....................................................................................50

3.2.2Toxicitytests.................................................................................................................50

3.2.3SilverionreleasefromPVP-AgNPs...............................................................................51

3.2.4Effectsonthecardiovascularsystem...........................................................................52

3.2.5Dataanalysis.................................................................................................................54

3.3Results.................................................................................................................................54

3.3.1CharacterizationandstabilityofPVP-AgNPs................................................................54

3.3.2Vasculardevelopmentalmorphology..........................................................................56

3.3.3Functionaltests............................................................................................................57

3.3.4Molecularmechanisms:geneandproteinexpression.................................................58

3.4Discussion............................................................................................................................59

3.5Conclusions..........................................................................................................................62

3.6APPENDIX............................................................................................................................64

CHAPTER4NANOSILVERCOATEDSOCKSANDTHEIRTOXICITYTOZEBRAFISH(DANIO

RERIO)EMBRYOS..............................................................................................................68

4.0Summary.............................................................................................................................68

4.1Introduction.........................................................................................................................69


4.2.1Preparationofexposuresolutions...............................................................................70

4.2.2Embryoexposureandtoxicitytesting..........................................................................70

vi

Page

4.2.3QuantificationoftotalsilverandcharacterizationofAgNPs.......................................71

4.2.4Geneexpressionanalysis.............................................................................................71

4.2.6Dataanalysis.................................................................................................................74

4.3Resultsanddiscussion.........................................................................................................74

4.3.1Quantificationandcharacterizationofsilvernanoparticles........................................74

4.3.2Toxicitytests.................................................................................................................76

4.3.3Oxidativestressgeneexpression.................................................................................76

4.4Conclusions..........................................................................................................................77

CHAPTER5RESEARCHSUMMARY,GLOBALCONCLUSIONS&FUTUREDIRECTIONS.......78

5.1ProteincoronaofAgNPsinfishplasma..............................................................................78

5.1.1Impacttofield..............................................................................................................78

5.1.2Futureresearchneeds..................................................................................................78

5.2VasculartoxicityofAgNPs...................................................................................................79

5.2.1Impacttofield..............................................................................................................79


5.3ToxicityofcommercialproductscontainingAgNPs............................................................79

5.3.1Impacttofield..............................................................................................................79


LISTOFREFERENCES.........................................................................................................81

VITA..................................................................................................................................91

vii

LISTOFTABLES

Table Page

Table1.1Toxicityofnanoparticlestoendothelialcells..................................................................7

Table1.2Toxicityofnanoparticlestothevascularsystemofvertebrates...................................11

Table2.1Proteinsisolatedfromtheproteincorona(PC)ofPVP-AgNPsafterincubationfor1or

24hwithplasmafromeitheradultfemale(F)ormale(M)smallmouthbass.CaMKII:

Calcium/calmodulin-dependentproteinkinase;Ig:Immunoglobulin;VEGF:Vascular

endothelialgrowthfactor;VTG:Vitellogenin;ZP:Zonapellucida........................................32

Table3.1Zetapotentialvaluesinfishembryomedium...............................................................54

Table4.1Primersequencesforsodandactingenes....................................................................71

Table4.272h-LC50andEC50responsesinzebrafishembryosexposedtosock-AgNP,spun-AgNP

andAgNO3.Concentrationsareinmg/L...............................................................................75

viii

LISTOFFIGURES

Figure Page

Figure1.1Effectsofnanoparticles(NPs)onendothelialcells(ECs).Endothelialnitricoxide

synthase(eNOS);Glutathioneperoxidase(GSH-Px);Interleukin1beta(IL-1b);Lactate

dehydrogenase(LDH);Malondialdehyde(MDA);Nitricoxide(NO);Phosphoinositide3-

kinase(PI3K);ProteinkinaseB(PKB,alsoknownasAkt);Proto-oncogenetyrosine-protein

kinaseSrc(Srckinase);Reactiveoxygenspecies(ROS);Vascularendothelialgrowthfactor

(VEGF)......................................................................................................................................5

Figure1.2Intersegmentalvessels(ISVs,whitearrows)oftransgeniczebrafish(TG:fli1a:EGFP)at

24hourspostfertilizationincontrolsand10mg/LPVP-AgNPtreatedembryos.AgNPs

decreasedgrowthofISVsandinhibitedfilopodiaformationonthetipsofISVs..................14

Figure2.1Westernblotofplasmafromadultfemaleandmalesmallmouthbasssampledinthe

peakofspawningseasonfornorthernIndiana(March).Asexpected,vitellogenin(VTG)was

onlyfoundinfemaleplasmawhereasvascularendothelialgrowthfactor(VEGF)was

constitutivelyexpressedinallfishandthususedasareferenceprotein.............................19

Figure2.2UV-VisofnakedPVP-AgNPsandofPVP-AgNPsafterincubationwithplasmafrom

adultfemaleormalesmallmouthbassfor1hor24h.AllPVP-AgNPsUV-Visabsorptions

shiftedtohigherwavelengthsafterincubationwithfishplasmacomparedwithnakedPVP-

AgNPs....................................................................................................................................21

Figure2.3Nanotrackinganalysis(NTA)showingchangesinthesizeofPVP-AgNPs(mode±

standarderror)andZpotentialafterincubationwithplasmafromadultfemale(F)ormale

(M)smallmouthbassfor1hor24h.*p<0.05comparedwith1hPVP-AgNPszeta

potential;#:p<0.05comparedwith24hPVP-AgNPszetapotential..................................22

Figure2.4Nanotrackinganalysis(NTA)ofPVP-AgNPsafterincubationwithsmallmouthbass

plasmafromadultfemale(F)ormale(M)for1h(A)or24h(B).ShownarealsoNTA

analysesfromfishplasma(noPVP-AgNPs)andnakedPVP-AgNPs......................................23

ix

Figure Page

Figure2.5SDS-PAGEgelsilverstainofproteincorona(PC)isolatedfromPVP-AgNPsafter

incubationwithplasma(P)fromadultfemale(F)ormale(M)smallmouthbassforeither1

hor24h.BothlengthofincubationandgenderaffectedPC.Smallersizedproteins(<24

kDa)wereonlyfoundinPCfromM(blackarrows).Adecreaseintheabundanceofproteins

50-80kDawasdetectedafter24hfromFplasma.PlasmawithoutPVP-AgNPsarealso

shownforcomparison...........................................................................................................24

Figure2.6Venndiagramofproteinsidentifiedinproteincorona(PC)ofPVP-AgNPsincubated

withfishplasmafromeitherfemale(F)ormale(M)smallmouthbassfor1hor24h........25

Figure2.7Percentdistributionofdifferentsizeproteinsobtainedfromproteincorona(PC)from

PVP-AgNPsincubatedwithplasmafromadultfemale(F)ormale(M)smallmouthbassfor1

hor24h.PlasmawithoutPVP-AgNPsarealsoshownforcomparison...............................27

Figure2.8Percentdistributionofdifferentclassesofproteinsobtainedfromproteincorona(PC)

fromPVP-AgNPsincubatedwithplasmafromadultfemale(F)ormale(M)smallmouthbass

for1hor24h.PlasmawithoutPVP-AgNPsarealsoshownforcomparison.Eachgraph

showsauniquesetofproteins.CaMKII:Calcium/calmodulin-dependentproteinkinase;

VTG:Vitellogenin;ZP:Zonapellucida.A.Includesthemostfrequentproteinsdetected;B.

Includeslessfrequentlyobservedproteins...........................................................................29

Figure2.9Proposedmechanismsofsilvernanoparticles(AgNPs)distributioninsideafish.When

AgNPsenterintofishbloodplasma,proteinscouldimmediatelybindtothesurfaceof

particles,inducingreceptor-mediatedphagocytosis,bloodclotting,andmoveintovascular

endothelialcells.LipoproteinsandapolipoproteinscouldalsobindtoAgNPs,whichcould

facilitatethebiodistributionofparticlestoalmostavarietyoftissuesandorgans.

VitellogeninbindingtothesurfaceofNPscoulddriveparticlestransporttodeveloping

follicleswithintheovarieswhichmightresultinmaternaltransporttoembryos...............31

Figure3.1A.Representativetrunkregionoftransgenic(TG)(fli1a)zebrafishembryofroma24-

hourpostfertilization(hpf)control.BoxedareacontainsfiveISVsusedtomeasurethese

parameters;arrow“a”pointstolengthofISV(dashedline);arrow“b”pointstoangle

betweenISVandDA;arrow“c”pointstointervalspacingbetweenISVs.B.Embryostreated

with0.1mg/LPVP-AgNPsexhibitdisorderedISVs;C,D.Embryostreatedwith1.0and10

mg/LPVP-AgNPsexhibitatrophiedISVs,relativetoA..........................................................51

x

Figure Page

Figure3.2Changesinintersegmentalvessel(ISV)morphologyacrossdevelopmentincontrol

andPVP-AgNPtreatedembryos.A.ISVlengthincreasedwithdevelopment.Areductionof

ISVsproutgrowthwasobservedat24hpfinembryosexposedto1and10mg/L(n=20),

andat48hpfinalltreatedembryos(n�10).B.AnglebetweenISVsandDAdecreasedwith

development.Anglewassignificantlylargerat72hpf(n=10)and96hpf(n=5)inembryos

exposedto10mg/LPVP-AgNP,relativetocontrol.C.IntervaldistancebetweenISVs

increasedwithdevelopmentstage.At72hpfand96hpf,embryostreatedwith10mg/L

PVP-AgNPhadasmallerintervaldistancerelativetocontrol.*p<0.05;#p<0.1.Lines

overtopsofbarsdenotesignificantdifferences...................................................................55

Figure3.3EffectsofPVP-AgNPsoncommoncardinalvein(CCV)regression;boxedareaindicates

regionofinterest.TheCCV(whitearrow)inthecontrolspecimenisatubeofvascular

endothelialcellsalongtheanteriormarginoftheyolkat72hpf;however,noregressionis

observedinembryostreatedwith10mg/LPVP-AgNP.Bar=200μm.................................56

Figure3.4Heartbeats(mean±SE)ofzebrafishembryosat48and72hpf.Significantreductionin

heartbeatratewasobservedat48hpfforembryostreatedwith10mg/LPVP-AgNP;a

decreaseinheartbeatratewasalsoobservedat72hpfforembryostreatedwith1mg/L

PVP-AgNP(*p<0.05).C=control........................................................................................57

Figure3.5Changesinoxygenlevel(mean±SE)afterexposingembyrostoPVP-AgNPs,upto96

hpf.A.ContinuousdecreaseinoxygenlevelwasobservedforzebrafishembryosatallAgNP

doses,butwithsignificantlylessconsumptionversuscontrolat84hpf(a–c:0.1,1.0and10

mg/Lgroups,p<0.05);at96hpf,onlythetwohighestAgNPdoses(1.0and10mg/L)

differedfromcontrol(b,c:p<0.05).B.Expansionofboxedarea........................................58

Figure3.6Bodylength(mean±SE)ofzebrafishlarvaeafterexposuretoPVP-AgNPsfor96h;

larvaeexposedto1and10mg/LAgNPsweresmaller(*p<0.05).C=control...................59

Figure3.7RelativeincreasesinmRNAexpression(mean±SE)forvegf,vegfr,plc,pkc,pi3k,and

bcl2,atdifferenttimepointsofexposuretoPVP-AgNPs(*p<0.05).C=control...............60

Figure3.8RelativeincreaseofmRNAexpressionforhypoxia-inducedfactor(hif),inducedby10

mg/LPVP-AgNPsat12,24,48,and72hpf(*p<0.05).........................................................61

xi

Figure Page

Figure3.9VEGFproteinexpressionbywholezebrafishembryos,asafunctionofPVP-AgNP

exposure(inmg/L).A.RepresentativeWesternblotsofVEGFandβ-actinproducedby

embryosat12and24hpf,showingnegligiblechangesinVEGFproduction.B.Changesin

VEGFproteinexpression(mean±SE)at12and24hpf;again,nosignificantdifferences

wereobservedbetweengroups(p>0.05).C=control........................................................62

Figure3.10MechanisticmodelaccountingfortheobservedeffectsofPVP-AgNPsonthe

cardiovasculardevelopmentinzebrafishembryos.A.PVP-AgNPscoatthesurfaceofeggs,

inducinghypoxiaandsubsequentexpressionofgenesintheVEGFsignalingpathway,with

peakexpressionat12hpfandreductiontobackgroundlevelsby72hpf.Inductionofthese

genes,however,doesnotleadtoincreasedproteinproduction.At48hpf,delayedvascular

developmentandbradycardiabecomeevident;at85hpf,oxygenconsumptionis

significantlydecreased.Uponhatching(>96hpf),larvaeexposedtoPVP-AgNPsare

releasedintonormoxiabutarealsosmallerrelativetocontrolspecimens.B.Proposed

molecularmechanismofvasculartoxicitybyPVP-AgNPs.Attheembryonicstage,the

chorioniscoveredbyPVP-AgNPs,blockingoxygenintake.Hypoxia-inducedexpressionof

VEGFpathwaygenesincreasesrapidly;atthesametime,intracellularPVP-AgNPsenterthe

endoplasmicreticulum(ER)andblockproteinsynthesis.Thelattereffectnotonlyblocks

hypoxia-inducedangiogenesis,butcanalsoincreasethemortalityofzebrafishembryosata

laterdevelopmentstage.......................................................................................................63

Figure4.1A.Measuredconcentrationsoftotalsilverinsocks-AgNPandspun-AgNPsolutions

usinginductivelycoupledplasmamassspectrometry(ICP-MS)fromfourindependent

experiments.B.Particlesizedistributionofsilvernanoparticlesinsocks-AgNPandspun-

AgNPsolutionsfromthreeindependentexperiments.Valuesweremeasuredatthestartof

theexperiment......................................................................................................................72

Figure4.2Transmissionelectronmicrograph(TEM)andEnergy-dispersiveX-rayspectroscopy

(EDX)ofsilvernanoparticles:(A&B)Silvernanoparticles(<20nm)foundinthesock-AgNP

solution.AgglomerationofAgNPsisseenonthefabricsurfaceofAgNP-coatedsocks

(arrows).(C)EDXanalysisconfirmsthepresenceofsilver.(D)Fiberresiduefoundinspun-

AgNPsolution.NoAgNPswerefoundinthespun-AgNPsolution.(E)EDXanalysisshows

thatnosilverpeakswerepresentinthefiber......................................................................73

xii

Figure Page

Figure4.3Exampleofthemajortypesofabnormalitiesobservedinsilvernanoparticlesexposed

zebrafishembryosafter72hrs.(A)Normallarvae(B&C)deformedlarvaea.Pericardial

edema.b.Yolksacedema.c.Spinalcurvature.....................................................................74

Figure4.4Relativesuperoxidedismutase(sod)mRNAlevelsinzebrafishembryosexposedto

differentconcentrationsofsock-AgNP,spun-AgNPandAgNO3solutionsquantifiedbyqRT

PCR(n=3pertreatment).....................................................................................................76

xiii

ABSTRACT

Jiejun,Gao,Ph.D.,PurdueUniversity,December2016.NanoparticleToxicityandMolecularMechanismsinFish:ACaseStudywithSilverNanoparticles.MajorProfessor:Dr.MariaS.Sepúlveda.Nanoparticles(NPs)arewidelyusedinamyriadofcommercialandindustrialproductsmaking

theirentrytotheenvironmentalikelyevent.NPshaveuniquephysical-chemicalpropertiesthat

resultfromtheirsmallsizeandhighsurfaceareatovolumeratio,makingthemhighlyreactive

andpotentiallytoxic.InChapter1,wesummarizetheeffectsandmechanismsofmetal-based

NPsonthevascularsystem.InvitrostudieshaveshownthatNPsareanti-angiogenicbecause

theycauseinflammation,oxidativestress,andapoptosisofendothelialcellsresultingin

increasedpermeabilityanddecreasedproliferationandmigration.Wholeanimalstudies

examiningtheeffectsofNPsonthevascularsystemarescarceandalthoughthefewavailable

studiesdonotdisprooftheanti-angiogenicpropertiesofNPs,themechanismsandlong-term

effectsofthesechangesarepoorlyunderstood.Consideringthekeyphysiologicalroleofthe

vascularsystem,thereisaneedformorestudiesinthisarea.InChapter2,weshowthatblood

proteinscanquicklybindtothesurfaceofNPs,whichmightgreatlychangetheirbiological

identity.Wepresentnoveldataontheformationofa“proteincorona”(PC)afterincubationof

polyvinylpyrrolidone-coatedAgNPs(PVP-AgNPs,50nm)infish(smallmouthbass)plasma.Both

genderandlengthofincubationaffectedthetypesofproteinsidentifiedfromPC.Themost

commonproteinswererelatedtoimmunefunction,redbloodcellfunctionandclotting,and

lipidandcholesteroltransport.Vitellogeninandzonapellucidaeggproteinswereonlydetected

inPCsincubatedwithfemaleplasma.Weproposethatfishplasmaservesasagoodmediafor

thecharacterizationofPCinfuturestudieswithNPsandthatbindingofeggproteinstothe

surfaceofPVP-AgNPscouldresultinenhancedmovementofparticlestodevelopingoocytes

andmaternaltransfertodevelopingembryos.InChapter3,vasculareffectsofPVP-AgNPs(60

nm)wereevaluatedontransgeniczebrafish(TGfli1a:EGFP)embryos.Exposureto1and10

xiv

mg/LPVP-AgNPsduringtheperiodofvasculardevelopmentcausedadelayinvascular

development;however,expressionofgeneswithinthevascularendotheliagrowthfactor(VEGF)

signalingpathwaywasenhanced.Thisapparentcontradictionwasexplainedbytheinductionof

hypoxicconditionsintheembryoviaagglomerationofNPsonthesurfaceofeggs.Hypoxiaisa

potentstimulantoftheVEGFsignalingpathway,butbloodvesseldevelopmentisnotenhanced

becausenoincreasedproductioninVEGFproteinwasobserved,likelybecauseofimpaired

translationduetoAgNPtoxicitytotheendoplasmicreticulum.InChapter4,weevaluatedthe

toxicityofacommercialproduct(socks)containingAgNPs.Importantly,wedemonstratethat

thetoxicityofthisleachatetozebrafishembryoswasnotduetoAgNPs,butinsteadtothe

presenceofunknownchemical(s).Atthetimethisstudywaspublished,therewasnodataon

thetoxicityofAgNPsreleasedfromanycommercialproducts.Overall,thissetofstudies

advancesourcurrentunderstandingonthemechanismsoftoxicityofAgNPsbyprovidingnovel

molecularandwholeanimaldatathatcanbeusedforfutureriskassessmentforthisemerging

classofcontaminants.

1

CHAPTER1 INTRODUCTION

1.0Summary

Nanoparticles(NPs)arewidelyusedinamyriadofcommercialandindustrialproductsmaking

theirentrytotheenvironmentalikelyevent.OrganismscanbeexposedtoNPsviainhalation,

orallyordermally.Thevascularsystemplayscriticalrolesinnutrienttransport,gasexchange,

andregulatinghomeostasisandisoneofthefirstfunctionalorgansystemsthatdevelopin

embryos.Thismini-reviewfocusesontheeffectsofmetalormetaloxideengineeredNPsonthe

vertebratevascularsystem.Westartbybrieflyreviewingtheoverallmechanismsinvolvedinthe

developmentofthevascularsysteminvertebrates.Next,wediscusssomeuniquechallengesin

nanotoxicologyresearch.WethenseparatevascularNPtoxicityfindingsbetweeninvitroandin

vivostudiesanddiscussthedifferentmolecularmechanismsreported.Weendwithsomemajor

conclusionsandneedsforfutureresearchinthisarea.

1.1Nanoparticlesintheenvironment

Becauseoftheirsmallsize(1-100nm)nanoparticles(NPs)arehighlyreactiveandcatalyticand

thereforehavefoundtheirwayinamyriadofapplicationsincludingcosmetics,textilesandfood

packaging.Governmentandindustryaroundtheworldaredevelopingnanotechnologiesin

variousfields,andasaresult,themarketfortheseproductswasestimatedatover$3trillionUS

dollarsin2015(FlynnandWei,2005).Themostcommonlyusedcorematerialforengineered1-

dimensionalNPsaresilicadioxide(SiO2),titaniumdioxide(TiO2)andsilver(Ag).Next-generation

2-and3-dimensionalNPsarealsobeingproducedinlargeamountsandappliedtoallsortsof

products(Huangetal.,2014;Kerativitayananetal.,2015).Althoughthefateandtransportof

NPsintheenvironmentislargelyunknown,modelsestimatethattheenvironmental

concentrationsofNPsinsurfacewater,wastewaterandsedimentsrangefrom0.003partsper

trillion(fullerenesinsurfacewater)upto89partsperbillion(TiO2NPsinsludge-treatedsoil)

(MuellerandNowack,2008;Gottschalketal.,2013).Therefore,whetheritisthroughtheuseof

2

cosmetics,householdproducts,medicalapplications,ordrinkingofcontaminatedwater,

exposuretoNPsislikely.

NPscanenterorganismsviadermal,nasal,ocular,respiratoryandgastrointestinalroutesand

distributethroughthecirculatoryandlymphaticsystemstomajororgans(Buzeaetal.,2007;

Asharanietal.,2008).AlthoughrecentreviewshaveaddressedtoxicityofNPstomajororgans

andsystems(Cardetal.,2008;Croseraetal.,2009;SimkóandMattsson,2010;Lettieroetal.,

2012),specificeffectsofmetal-basedNPstothevascularsystemhasbeenlargelyignored.This

isincontrasttocarbon-basedNPswhereonereviewpublishedin2009,summarizedthe

literatureontheireffectsonthecardiovascularsystemmostlyinducedviainflammatory

pathwaysthatleadtofibrosisandlossoffunction(SimeonovaandErdely,2009).

Inthecurrentreview,wesummarizetheeffectsofmetalormetaloxideengineeredNPsonthe

vertebratevascularsystem.NaturallyoccurringNPs(suchasthoseproducedfrom

photochemicalreactions,volcaniceruptions,forestfires,orsimpleerosion),carbonairpollution

particles,andorganicNPsarenotincludedinthisreview.Wedonotfocusoneffectsonthe

heartperse,sincewecouldnotidentifynanotoxicitystudieswiththeNPsofinterestthathave

focusedspecificallyoncardiaceffects.Inaddition,studiesthatreportverygeneral

cardiovasculartoxicityeffectssuchaspericardialedemaorchangesinheartratesafterNP

exposure,arenotreportedhere.Westartbybrieflyreviewingtheoverallmechanismsinvolved

inthedevelopmentofthevascularsysteminvertebrates.Next,wediscusssomeunique

challengesinnanotoxicologyresearch.WethenseparatevascularNPtoxicityfindingsbetween

invitroandinvivostudiesanddiscussmolecularmechanismsreportedfromdifferenttypesof

endothelialcells(ECs)aswellasvascularresponsesinwholeanimals.BecausetoxicityofNPsis

notonlyrelatedtodose,butalsotosize,shape,coating,andsurfacecharge(Ehrhartetal.,

2015)wealsopresentthisinformationwheneverpossible.Weendwithsomeconclusionsand

needsforfutureresearchinthisarea.

1.2Vasculardevelopment

Theheartandvascularsystemarethefirsttofunctionallydevelopinembryos.Vascular

formationoccursintwophases:vasculogenesis,thedenovovascularformationviaassemblyof

endothelialprecursorscalledangioblasts;andangiogenesis,thesproutingofpre-existingvessels

bymigrationandproliferationofECs(BeckandD'Amore,1997;Ellertsdóttiretal.,2010).

3

NormalvasculardevelopmentdependsonECdifferentiation,migrationandproliferation;

extracellularmatrixremodeling;andcell-cellcommunication(HerbertandStainier,2011;Talet

al.,2014).ECshavetheremarkablecapacityofgivingrisetovesselswithdiversefunctional,

morphologicalandmolecularsignatures(HerbertandStainier,2011).Proangiogenicsignals

inducefundamentalchangesinECbehaviorincludinglooseningofcell-celljunctionsleadingto

degradationofthesurroundingbasementmembraneandinitiatingnewbloodvesselsprouting

(HerbertandStainier,2011).AsmallproportionofECs(TipCells,TCs)resultinnewlysprouting

vessels(Gerhardtetal.,2003;DeSmetetal.,2009).ECsthattrailTCs,orStalkCells(SCs),are

lessmotilebutsupporttheextensionofsproutingvessels,generatingthetrunkofnew

capillariesandmaintainingconnectivitywithintheparentalvessel(Kameietal.,2006;Adams

andAlitalo,2007;Iruela-ArispeandDavis,2009).AfterTCscontactotherECs,tightjunctionsare

generatedandfusewithrecipientvesselstoformalumen(HerbertandStainier,2011).

Decadesofresearchhaveuncoveredkeysignalingpathwaysinvolvedinvasculardevelopment.

Thevascularendothelialgrowthfactor(VEGF)andNotchsignalingpathwaysaremajor

controllersofECbehaviorduringbloodvesselsprouting.VEGFsignalingpromotesECs

proliferation,filopodiaextension,degradationoftheextracellularmatrix,andchemotaxis

(HerbertandStainier,2011)viaactivationofmultipledownstreamintermediariessuchas

mitogen-activatedproteinkinases(MAPKs),phosphoinoside3-kinases(PI3Ks),proteinkinaseB

(PKB,alsoknownasAKT),phospholipaseCγandsmallGTPases(HerbertandStainier,2011).

Over-expressionofvegfleadstoseveralabnormalitiesinheartdevelopmentandembryonic

lethalityinmice(vegfa,vegf-mousegenomeinformatics)(Miqueroletal.,2000);formationof

abnormalendothelialprecursorcellsanddisorganizedvascularstructuresinXenopusembryos

(vegf)(Cleaveretal.,1997);andectopicvasculaturedevelopmentinzebrafishembryos(vegfa,

bothisoforms,includingvegf165andvegf121)(Liangetal.,2001).Knock-downofvegfain

zebrafishresultsinbloodvesseldeficiencieswithnoorreducednumbersofcirculatingred

bloodcellsandenlargementofthepericardium(Naseviciusetal.,2000).Mouseembryonicstem

cellsfromembryoswithdysfunctionalVEGFreceptor2(VEGFR2)areunabletomigratetothe

correctlocationandformbloodislands(Shalabyetal.,1997)anddiewithoutdevelopingECs

andyolk-sacbloodislands(Shalabyetal.,1995).

4

1.3CriticalissuesinassessingthetoxicityofNPs

Thefieldof‘nanotoxicology’isrelativelynew,withthefirstmanuscriptspublishedinlate2004

(asearchusing“WebofScience”(InformationScienceInstitute,www.isi.edu)between2004-

2016identifiedatotalof1,518manuscriptsusing“nanotox*”asthekeyword).Thisbodyof

literaturehascharacterizedawiderangeofNPtoxicitiesaffectingallmajororgansystems

(Bormetal.,2006;DeJongandBorm,2008;Aillonetal.,2009).Challengesoftestingthetoxicity

ofNPsarerelatedtotheiruniquephysical-chemicalproperties,largely,theirlargesurfacearea

tovolumeratio,whichmakesthemhighlybiologicallyandchemically-active(Oberdörsteretal.,

2005;Buzeaetal.,2007).Inaddition,NPselicitquantumeffects,creatingoptical,electricaland

magneticbehaviorsthataresometimesvastlydifferentfrombulkmaterials(Buzeaetal.,2007).

Studieshavedemonstratedthatsmaller-sizeNPsaregenerallymoretoxiccomparedtolarger

ones.ThisislikelybecausesmallerNPscangeneratemoreradicaloxygenspecies(ROS)and

inflammationcomparedtolarger-sizeparticles(ChoiandHu,2008;Karlssonetal.,2009;Scown

etal.,2010).Forexample,thecytotoxicityofgoldnanoparticles(AuNPs)indicatethat15nmare

nontoxic,but~10timeslowersize(1.4nm)increasesthecytotoxicity60to100-fold.

Interestingly,thetypeofcellularresponsewasalsosize-dependent:1.4nmAuNPscausedcell

deathvianecrosiswhereas1.2nmAuNPsinducedprogrammedcelldeathorapoptosisinstead

(Panetal.,2007).AgNPcelltoxicityhasalsobeenshowntobesize-dependentinmacrophages,

with15nmcausingmoreoxidativestressandinflammationcomparedto30and50nm(Carlson

etal.,2008).

AnotheruniquefeatureofNPtoxicityistheformationofabiomolecularlayerontheirsurface,

referredtoas“ProteinCorona”(PC).ViavanderWaals,electrostatic,hydrogenbondingand

hydrophilic/hydrophobicinteractions,biomoleculessuchaslipids,sugarsandespecially

proteins,willbindtothesurfaceofNPsonceincontactwithbiologicalfluids(Xiaetal.,2010;

Monopolietal.,2012).ThecompositionofthisPCisimportantbecauseresearchhasshownit

canaffectagglomeration,toxicokinetics,signaling,andultimately,toxicity(Tenzeretal.,2013).

Forinstance,negativelychargedNPswillattractpositivelychargedbiomolecules,whichinturn

willincreaseinteractionswiththenegativelychargedcellmembrane(Setyawatietal.,2015).

Thisalsohasimplicationsforinterpretingtoxicityresultsacrossdifferentanimalmodelsand

exposurescenarios(Shannahanetal.,2016).

5

1.4VasculartoxicityofNPsinvitro

ResearchershaveuseddifferentEClinestoinvestigatethevasculartoxicmechanismsofNPsin

vitro.Ingeneral,effectsofNPsonECsarecorematerial-andconcentration-dependent.For

instance,zincoxide(ZnON),magnesiumoxide(MgON)andcopperoxide(CuO)NPsincreasedEC

permeabilityandoxidativestressstartingfrom1ppm,whileAuNPsandironoxide(Fe3O4)NPs

weresignificantlylesstoxicatthresholdconcentrationsof25and400ppm,respectively(Table

1.1.)(Sunetal.,2011).

SizeofNPshasalsobeenidentifiedasakeyfactorinfluencingECuptakeandvasculartoxicity.

NPs(50-80nm)enterECsviacaveolin-mediatedendocytosis,whilelargerNPs(100-200nm)

Figure1.1Effectsofnanoparticles(NPs)onendothelialcells(ECs).Endothelialnitricoxidesynthase(eNOS);Glutathioneperoxidase(GSH-Px);Interleukin1beta(IL-1b);Lactatedehydrogenase(LDH);Malondialdehyde(MDA);Nitricoxide(NO);Phosphoinositide3-kinase(PI3K);ProteinkinaseB(PKB,alsoknownasAkt);Proto-oncogenetyrosine-proteinkinaseSrc(Srckinase);Reactiveoxygenspecies(ROS);Vascularendothelialgrowthfactor(VEGF).

6

interactwithsurfacereceptorstriggeringtheformationofclathrincoatedpitswhichtransport

NPsintocellsviaclathrin-mediatedendocytosis(Fröhlich,2012;Setyawatietal.,2015).Smaller

AuNPs(15nm)inhibitedVEGF-inducedECproliferationandmigrationatalowerobservedeffect

concentration(24ppm),comparedto59ppmfor50nmAuNPs(Karthikeyanetal.,2010;Panet

al.,2014a).Asalreadydiscussed,surfacechargecanalsoinfluencevasculartoxicity.The

kallikrein-kininsystem(KKS)isinvolvedinregulatingvascularpermeability.Onlynegatively

chargedAgNPsactivatetheplasmaKKSpathwaybytriggeringHagemanfactorauto-activation

(Longetal.,2016).

Regardlessofdifferencesonphysical-chemicalproperties,allNPstestedareanti-angiogenicas

theyinhibittheVEGFsignalingpathwayaffectingECpermeability,proliferationandmigration.

Theydothisviainductionofinflammation,oxidativestress,andapoptosis(Fig.1.1)(Gurunathan

etal.,2009;Kalishwaralaletal.,2009;Rosas-Hernándezetal.,2009;Sheikpranbabuetal.,2009;

Sheikpranbabuetal.,2010;Changetal.,2015).InflammationbyNPsincreasesendothelium

permeabilityresultinginenhancedNPcellularuptake,andinhibitionofAkt,Srckinase,Pi3kand

VEGFsignalingpathwaysbyNPsresultsinECdeath.InHumanUmbilicalVeinEndothelialCells

(HUVECs),AuNPsbindtothevascularpermeabilityfactor/VEGFandbasicfibroblastgrowth

factorinhibitingendothelialandfibroblastcellproliferationduetoadecreaseinVEGFR-2

phosphorylationandintracellularcalciumrelease(Mukherjeeetal.,2005).NPsinduceoxidative

stress,interferingwithlysosomalhydrolases(LH),cathepsinsB(CTSB)andcathepsinsD(CTSD),

whichnegativelyimpactmitochondriamembranepotential(Gojovaetal.,2007;Apopaetal.,

2009;LiuandSun,2010;Zhuetal.,2010;HalamodaKenzaouietal.,2012;Suetal.,2012;Duan

etal.,2013a;Duanetal.,2013b).ECproliferationcanalsobeinhibitedbyNPsviatriggering

productionofnitricoxide(NO)andendothelialnitricoxide(eNOS),whicharecriticalfor

maintainingvascularhomeostasisandvesseldilation,protectingfromplateletaggregation,

leukocyteadhesionandcontrollingproliferationandmigrationofvascularsmoothmusclecells

(Rosas-Hernándezetal.,2009;Zhuetal.,2010;Suetal.,2012).

7

Table1.1Toxicityofnanoparticlestoendothelialcells.

Nanoparticle Size(nm) Cellline Response Ref(s).

Copperoxide

(CuO)

40 HUVEC 1-100mg/L↓cellsurvival (Changetal.,

2015)

180 HCMEC 1-100mg/L↑permeability,

inflammatoryresponse,

expressionvcam-1,icam-1,mcp-

2,IL-8,LDH

(Sunetal.,

2011)

Gold(Au) 5 HUVEC From67nM↓VEGFR2

phosphorylation,intracellular

calciumrelease,RhoAactivation

leadingto↓

endothelial/fibroblastcell

proliferation,permeabilityand

angiogenesis

(Mukherjeeet

al.,2005)

15 24mg/L↓VEGFmigrationand

tubeformationbyaffectingcell

surfaceultrastructure,

cytoskeletonandmightaffect

angiogenesisviatheAkt

pathway

(Panetal.,

2014a)

50

BRPEC

59mg/L↓VEGF-,IL-1β-

inducedproliferationand

migrationbysuppressionofSrc

kinasesignalingpathway

(Karthikeyanet

al.,2010)

8

Ironoxide

(IO)

8(uncoated

andoleic

acid-coated)

HCEC >30µgNPs/cm2↑autophagy

andROSinterferingwithLH,

CTSDandCTSB

(Halamoda

Kenzaouietal.,

2012)

Fe2O3 22 HUEC 2-100mg/L↑NO,oxidative

stress,apoptosis,↓

mitochondriamembrane

potential

(Zhuetal.,

2010)

47 HAEC 50mg/Lnoinflammation (Gojovaetal.,

2007)

298 HMVEC

50mg/L↑permeability,ROS

oxidativestress-modulated

microtubuleremodeling,

Akt/GSK-3β-signalingpathways

(Apopaetal.,

2009)

Fe3O4 15-

20

HUVEC 400mg/L↑NO,Enosactivity,

↓CAV1

(Suetal.,2012)

43 HUEC 2-100mg/L↑oxidativestress,

NO,apoptosis

(Zhuetal.,

2010)

Magnesium

oxide(MgO)


inflammatoryresponse,


2,il-8,LDH

(Sunetal.,

2011)

Silica(Si) 20,62

HUVEC

≥25mg/L↓SOD,GSH-Px,

mitochondrialmembrane

potential≥50mg/L,JNK,C-Jun,

P53,NF-Kbsignalingpathways,

↑oxidativestress,apoptosis

(LiuandSun,

2010;Duanet

al.,2013a;

Duanetal.,

2013b)

9

25,50 HCEC >30µgNPs/cm2↑autophagy


CTSDandCTSB

(Halamoda

Kenzaouietal.,

2012)

600

(mesoporous

coated)

RBC 20-100mg/Lmembrane

deformation,internalizationof

NPsinsideofRBCs,hemolysis

(Zhaoetal.,

2011)

Silver(Ag) 40-50 PREC 5mg/L↓VEGF-,IL-1β-induced

endothelialpermeability

throughinactivationofSrc

kinasepathway

(Sheikpranbabu

etal.,2009;

Sheikpranbabu

etal.,2010)

BREC 54mg/L↓VEGF-inducedcell

proliferation,migration,

capillary-liketubeformation,

angiogenesisviainhibitionof

PI3K/Aktsignalingpathways,

↑apoptosisviaCASP3activity

(Gurunathanet

al.,2009;

Kalishwaralalet

al.,2009)

42 HREC TriggeringHagemanfactor

autoactivationplasmaKKS,

bradykinin,B2receptors,VE-

cadherin,paracellular

permeability

(Longetal.,

2016)

45 CEC ≤10mg/L↓cell

proliferation,↑

vasoconstriction

byinhibiting

Ach-induced

NO-mediated

relaxation

50&100

mg/L↑NO-

dependent

proliferation

viaactivation

ofeNOsand

vasodilation

(Rosas-

Hernándezet

al.,2009)

10

Titanium

dioxide

(TiO2)

21 HCEC >15µgNPs/cm2↑autophagy


CTSDandCTSB

(Halamoda

Kenzaouietal.,

2012)

Yttrium

oxide(Y2O3)

20-60 HAEC ≥10mg/L↑inflammatory

response,expressionicam-1,il-

8,mcp-1

(Gojovaetal.,

2007)

Zincoxide

(ZnO)



2,il-8,LDH

(Sunetal.,

2011)

100-200 HAEC ≥10mg/L↑inflammatory

response,expressionmcp-1

(Gojovaetal.,

2007)

Acetylcholine(Ach);BovineRetinalEndothelialCells(BRECs);BovineRetinalPigmentEpithelialCells(BRPECs);Caspase-3(CASP3);CathepsinsB(CTSB);CathepsinsD(CTSD);Caveolin-1(CAV1);c-JunN-terminalkinase(JNK);CoronaryEndothelialCells(CECs);HumanAorticEndothelialCells(HAECs);HumanCardiacMicrovascularEndothelialCells(HCMECs);HumanCerebralEndothelialCells(HCECs);HumanMicrovascularEndothelialCells(HMVECs);HumanRetinalEndothelialCells(HRECs);HumanUmbilicalVeinEndothelialCells(HUVECs);Endothelialnitricoxidesynthase(Enos);Glutathioneperoxidase(GSH-Px);Glycogensynthasekinase3beta(GSK-3b);Intercellularadhesionmolecule1(icam-1);Interleukin1beta(IL-1b);Interleukin8(IL-8);Lactatedehydrogenase(LDH);Lysosomalhydrolases(LH);Monocytechemoattractantprotein2(mcp-2);Nitricoxide(NO);Nuclearfactorkappa-light-chain-enhancerofactivatedBcells(NF-Kb);Phosphoprotein53(P53);PorcineRetinalEndothelialCells(PRECs);ProteinkinaseB(PKB,alsoknownasAkt);Proto-oncogenetyrosine-proteinkinaseSrc(Srckinase);Rashomologgenefamily,membraneA(RhoA);Reactiveoxygenspecies(ROS);Redbloodcells(RBCs);Superoxidedismutase(SOD);Vascularcelladhesionprotein1(vcam-1);Vascularendothelialgrowthfactor(VEGF).

11

1.5VasculartoxicityofNPsinvivo

InvivostudiesexaminingthevasculotoxicityofNPsarescarce.NPshavebeenidentifiedas

responsiblefortheformationofbloodclots,leadingtovesselobstructionandslowingofblood

flow.Andsimilarlytowhathasbeenreportedinvitro,NPshavebeenshowntohaveanti-

angiogenicpropertiesinvivo(Table1.2.).

Table1.2Toxicityofnanoparticlestothevascularsystemofvertebrates.

Nanoparticle Size(nm) AnimalModel/Age Response Ref.

Copperoxide

(CuO)

40 ZebrafishTG

(nacre/fli1:EGFP)/

embryos

100mg/Lfor5d↓

numbersubintestinal

vessels,↓expression

vegfAa,vegfr1

(Changet

al.,2015)

Gold(Au) 5 Nudemice/6-8weeks

old

Twoinjectionsof10

µL,670nmol/LAuNPs

intomiceearon2nd

and4thday

↓VEGF121-induced

permeabilityand

angiogenesis

(Mukherjee

etal.,2005)

13.5 MaleICRmice >550µg/kg↓RBCs,

bodyweightand

hematocrit

(Zhanget

al.,2010)

Iron

oxide

(IO)

Fe2O3 6.8 BALB/Cjmice/7-10

weeks

10mg/Lfor12-24h↓

arterialblood

pressure,contractility

ofsmallmesenteric

arteries

(Iversenet

al.,2013)

12

Fe3O4 15-20 BALB/cmice/15-20g

bodyweight

20mg/kgfor72h

damageto

endotheliuminthe

aorticroot,↑serum

NO,eNOS,↓

expressionCAV-1

(Suetal.,

2012)

Silica

(Si)

62 Zebrafish/

embryos

100mg/Lfor96h↓

expressionp-VEGFR2,

p-ERK1/2à↓

angiogenesis,

abnormalheart

development

(Duanetal.,

2013a)

ICRmice/8weeksand

20-22ginbodyweight

178mg/Lfor14d

autophagicactivityin

ECSandpericytesà

↓angiogenesis

(Duanetal.,

2014)

Silicadioxide

(SiO2)

170 CD-1mice 450mg/kgfor10d®

thrombosisof

endocardium,heart

andlung,anemia

600mg/kg↓

hemoglobin,↑

alanine

aminotransferase

(Yuetal.,

2012)

Silver

(Ag)

10(coated

with

mecaptoun-

deonicacid)

C57BL/6mice/10

weeksoldmaleand22

ginbodyweight

500mg/Lfor1h↑

retinalvascular

permeability,KKS

signalingpathway

(Longetal.,

2016)

13

50(coated

withPVP)

Zebrafish

TG(fli1a:EGFP)/embryos

10mg/Lfor96h

↓growth

intersegmental

vessels,↑VEGF

signalingpathway

(Gaoetal.,

2016)

Titanium

dioxide

(TiO2)

20 Malematuresyrian

mice/2weeksoldand

30ginbodyweight

10,100mg/Lfor14

days↑lymphocytes,

↓neutrophils

(Mahdiehet

al.,2015)

21 Sprague-Dawleyrats/

250-275gFemale;300-

325gMale

Theparentratswere

inhalationexposedto

TiO2NPsfor7days

withdailydeposition

of43.8µg.Pupswere

deliveredandgrew

intoadulthoodwith

endothelium-

dependentdilation

andactive

mechanotransduction

incoronaryand

uterinearterioles

impaired,combining

withreductionin

mitochondrial

respirationinleft

ventricleanduterus.

(Stapleton

etal.,2015)

Caveolin-1(CAV-1);Endothelialcells(ECs);Endothelialgrowthfactor(EGF);endothelialnitricoxidesynthase(eNOS);Kallikrein-kininsystem(KKS);Mitogen-activatedproteinkinase(MAPK,alsoknownasERK);NitricOxide(NO);Polyvinylpyrrolidone(PVP);Redbloodcells(RBCs);Transgenic(TG);Vascularendothelialgrowthfactor(VEGF);Vascularendothelialgrowthfactorreceptor1(VEGFR1);Vascularendothelialgrowthfactorreceptor2(VEGFR2).

14

NPscauseplateletaggregationcausingabnormalbloodflow.Forinstance,femaleCD-1mice

exposedtononporousnanospheresSiO2(170nminDIwater,450mg/kg)causethrombosisof

endocardiumandlungsleadingtoheartcongestionandfailure(Yuetal.,2012).Besidesaltering

platelets,NPshavealsobeenassociatedwithdecreasedredbloodcells(anemia)(Zhangetal.,

2010;Mahdiehetal.,2015).

TwostudieswereidentifiedwhereNPexposurewasrelatedwithalteredcontractilityofblood

vessels.Inonestudy,aninjectionofpolyacrylicacid(PAA)coatedΥ-Fe2O3NPs(6.8nm,10

mg/kg)toBALB/Cjmicecausedanacutedecreaseinmeanarterialbloodpressureafter12-24

hoursassociatedwithadecreasedcontractionofsmallmesentericarteries(Iversenetal.,2013).

Inanotherstudy,inhalationofTiO2NPs(21nm)bySprague-dawleyfemaleratsresultedin

microvascularimpairmentsinoffspring.Inthisstudy,multigenerationaleffectswerealso

observed.PupsbornfrommothersexposedtoTiO2NPsdevelopedimpairedendothelium-

dependentdilationincoronaryanduterinearteriolesandhadreducedmaximalmitochondrial

respirationintheseheartanduterusasadults(Stapletonetal.,2015).Theseresults

demonstrateprenatalNPexposurecanpotentiallyleadtolater-in-lifeadverseeffectstothe

vascularsystem.

EffectsonbloodvesselformationisacommonfindinginanimalsexposedtoNPs.Indeed,NPs

changevascularpermeability,inhibitvasculardevelopment,andinsomeinstances,leadto

abnormalheartdevelopment(Yuetal.,2012;Duanetal.,2013a;Duanetal.,2014;Changetal.,

2015;Gaoetal.,2016;Longetal.,2016).ENPsshowedautophagicactivityimpairingthe

endotheliumoftheaorticrootandnegativelyimpactedangiogenesisinvolvingCAV-1,ICAM-1,

Figure1.2Intersegmentalvessels(ISVs,whitearrows)oftransgeniczebrafish(TG:fli1a:EGFP)at24hourspostfertilizationincontrolsand10mg/LPVP-AgNPtreatedembryos.AgNPsdecreasedgrowthofISVsandinhibitedfilopodiaformationonthetipsofISVs.

15

VCAM-1,andLC3(Suetal.,2012;Duanetal.,2014).Inaddition,disruptionofangiogenesisby

NPshasbeenassociatedwithboththeVEGFR2-mediatedautophagyandtheVEGFsignaling

pathwaysinzebrafishembryosandnudemouse(Mukherjeeetal.,2005;Duanetal.,2013a;

Duanetal.,2014;Changetal.,2015;Gaoetal.,2016).

Ourresearchgrouprecentlyinvestigatedthevasculareffectsofpolyvinylpyrrolidone-coated

AgNPs(PVP-AgNPs,60nm)ontransgeniczebrafish(TGfli1a:EGFP)embryos.Ourresultsshow

thatexposureto1and10mg/LPVP-AgNPsduringtheperiodofvasculardevelopmentcausesa

delayinthegrowthofintersegmentalvessels(Fig.1.2).Similarresultswerereportedinanother

studywithTGzebrafishexposedtoCuONPs(Changetal.,2015).Inourstudy,however,

expressionofgeneswithintheVEGFsignalingpathwaywasenhanced.Thisapparent

contradictionwasexplainedbytheinductionofhypoxicconditionsintheembryovia

agglomerationofNPsonthesurfaceofeggs.HypoxiaisapotentstimulantoftheVEGFsignaling

pathway,butbloodvesseldevelopmentisnotenhancedbecausenoincreasedproductionin

VEGFproteinwasobserved,likelybecauseofimpairedtranslationduetoAgNPstoxicitytothe

endoplasmicreticulum(Gaoetal.,2016).

1.6Conclusions

NPsarecurrentlybeingusedinavarietyofcommercialproducts,therefore,humanexposureis

likely.Inthisreview,wesummarizetheeffectsandmechanismsofmetal-basedNPsonthe

vascularsystem.InvitrostudiesshowthatNPsareanti-angiogenicbecausetheycause

inflammation,oxidativestress,andapoptosisofECsresultinginincreasedpermeabilityand

decreasedproliferationandmigration.MechanisticstudiesshowthatNPsinduceNF-Kb,P53,IL-

8andeNOSactivity,whileinhibitingtheVEGFsignalingpathway.Wholeanimalstudies

examiningtheeffectsofNPsonthevascularsystemarescarce.Althoughthefewavailable

studiesdonotdisprooftheanti-angiogenicpropertiesofNPs,themechanismsandlong-term

effectsofthesechangesarepoorlyunderstood.Consideringthekeyphysiologicalroleofthe

vascularsystem,thereisaneedformorestudiesinthisarea,particularlyassessingtheimpact

ofdifferentNPproperties(size,shape,surfacecoatings,etc.)onthedevelopmentofthe

vascularsystem.

16

CHAPTER2 PROTEINCORONAFROMSILVERNANOPARTICLESEXPOSEDTOFISHPLASMA

2.0Summary

Immediatelyafterexposure,nanoparticles(NPs)comeincontactwithbiologicalfluids,resulting

inphysicalandchemicalchangestotheNPsthemselveswhichcouldimpacttheirbiodistribution

andtoxicity.Oneofthesechangesinvolvestheformationofaproteincorona(PC)onthe

surfaceofNPs.Studieswithmammalianserum/plasmaandcellshaveshownthatPCformation

changesintracellularuptakeandtoxicityofNPs.Inthisstudy,wereportforthefirsttime,the

formationofPCsafterincubationofpolyvinylpyrrolidone-AgNPs(PVP-AgNPs,50nm)infish

(smallmouthbass,Micropterusdolomieu)plasma.WehypothesizedthataPCwouldform

similarlytowhathasbeenreportedinmammals,andthatbothlengthofincubation(1or24h)

andgenderwouldinfluencetheproteinprofileofPCs.Particlecharacterizationanalyses

indicatedincreasedinstabilityandagglomerationofNPsafterbeingincubatedwithplasma.Size

ofNPsincreasedwithlengthofincubation(overallincreaseof3.95nmand10nmafter1and24

hincubation,respectively).Sizechangeswerealsogender-dependentwithparticlesincubated

withmaleplasmaresultinginoverallsmallerparticlesizes(averageincreaseof8.6nm

comparedtoanincreaseof11.4nmforfemaleplasma).Usinglabel-freemassspectrometry-

basedproteomicapproaches,wedeterminedthatbothgenderandlengthofincubation

affectedthetypesofproteinsidentified.Over300differentproteinswereidentified,andas

withpreviousstudies,themostcommonproteinsrelatedtoimmunefunction(complement,

immunoglobulins,ceruloplasmin,andtransferrin),redbloodcellfunctionandclotting

(hemoglobin,plasminogen,andfibrinogen/fibronectin),andlipidandcholesteroltransport

(lipoproteinsandapolipoproteins).Causesforthemuchlargernumberofuniqueproteins

identifiedfromPCsformedwithmaleplasmadeservesfurtherinvestigation.Asexpected,

vitellogeninandzonapellucidaeggproteinswereonlydetectedinPCsincubatedwithfemale

plasma.WeproposethatfishplasmaservesasagoodmediaforthecharacterizationofPCin

17

futurestudieswithNPsandthatbindingofeggproteinstothesurfaceofPVP-AgNPscould

resultinenhancedmovementofparticlestoovariesanddevelopingeggs.

2.1Introduction

Themanipulationofmaterialsatthenanoscale(1-100nm),hasbecomeamulti-billiondollar

industryresultinginthedevelopmentofamyriadofproductscontainingdifferenttypesof

nanoparticles(NPs)manyofwhichareusedincommonhouseholdandothercommercial

products.Therefore,exposuretoNPsbyhumansandotherorganismsisalikelyevent

(Eigenheeretal.,2014).Immediatelyafterexposure,NPscomeincontactwithbiologicalfluids,

resultinginphysicalandchemicalchangestotheNPsthemselveswhichcouldimpacttheir

biodistributionandtoxicity.Oneofthesechangesinvolvestheformationofaproteincorona

(PC)onthesurfaceofNPs(TreuelandNienhaus,2012).StudieshaveshownthatPCformation

onthesurfaceofNPschangestheirintracellularuptakeasbindingwithmembranesurface

ligandscouldallowparticlestointeractwithcomplementarymoleculesorreceptorsonthecell

membrane,resultinginreceptor-mediatedendocytosis(DecuzziandFerrari,2007;Neletal.,

2009).

ThebindingofproteinstoNPsisgovernedbytheaffinityofproteinstoNPsaswellasprotein-

proteinbindingaffinity.Proteinlayerswithhighbindingaffinityformwhatisknownas“hard

corona”,whileproteinlayersinvolvinglowbindingaffinityarereferredtoas“softcorona”

(Milanietal.,2012).SeveralfactorsinfluencethecompositionofPCsincludingsize,shape,

surfacematerial,surfacechargeandcorematerialofNPs;compositionofbiologicalfluids;and

exposureduration(Lundqvistetal.,2008;Tenzeretal.,2013;Pozzietal.,2015).Forinstance,

profilesofhumanPCformedonsilica(Si)andpolystyrene(Ps)NPsofvaryingsizes(30to150

nm)andsurfacefunctionalizationfoundchangesintheamountofboundproteinsasafunction

oftime(from0.5to120min),withcompositionstayingaboutthesame(Tenzeretal.,2013).

Therefore,theinteractionofproteinswithNPsisadynamicprocess,withabundantproteins

withhighassociationrateconstantsinitiallyoccupyingthesurfaceofNPs,andlaterbeing

replacedbylessabundantproteinswithslowerexchangeratesandhigheraffinity.

SilverNPs(AgNPs)areoneofthebeststudiedinrelationtoPCformation.Regardlessofrouteof

exposure(dermal,oral,inhalation)AgNPstranslocateintothecirculatorysystemleadingtothe

formationofPCs.Moststudiessofarhaveusedmammalianbiologicalfluids(serumorplasma)

18

orcellsforstudyingPCs(Treueletal.,2010;Dingetal.,2013;Shannahanetal.,2013;Wenetal.,

2013;Eigenheeretal.,2014;Walkeyetal.,2014).Arecentstudyusinghumanplasma

investigatedtheimpactofgenderintheformationofPCincitrate-AgNPs(20nm)andpolyvinyl

pyrrolidone-AgNPs(PVP-AgNPs,20nm)andreported>70%oftheproteinswereshared

betweenfemalesandmalesforbothtypesofNPs(Huangetal.,2016).Nootherstudieswere

identifiedthathaveexaminedtheimpactofgenderonPCcomposition.

TheformationofaPCcanchangecellularuptakeandtoxicityofNPs.Astudywithcitrate-coated

gold(Au)NPs(15,40,and80nm)reportedincreasedinternalizationandcytotoxicityforthe

smallestNPs,butonlywhenincubatedwithoneofthetwomediumtested(RoswellPark

MemorialInstituteMedium,RPMI)(Maioranoetal.,2010).Inanotherstudy,silicaNPs

(AmSiNPs,30nm)pre-coatedwithhumanplasmafor<30minwerelesstoxictohumanvascular

endothelialcells(HUVECs)andmicrovascularendothelialcelllines(ISO-HAS1),whilePCsformed

afterprolongedexposuretoplasma(>30min)didn’tcounteracttoxicityfurther,indicatingthe

biologicalrelevanceofshortexposuretime(Tenzeretal.,2013).Inaddition,internalizationof

SiNPs(50nm)intoA549cellsdecreasedsignificantlyresultinginlessaccumulationinthe

cytosol,comparedtonakedNPsincubatedunderserum-freeconditions(Lesniaketal.,2012).

Inthisstudy,wereportforthefirsttime,theformationofPCsafterincubationofPVP-AgNPs

(50nm)infish(smallmouthbass,Micropterusdolomieu)plasma.WehypothesizedthataPC

wouldformsimilarlytowhathasbeenreportedinmammals,andthatbothlengthofincubation

(1or24h)andgenderwouldinfluencetheproteinprofileofPCs.Femalefish(andallegg-laying

animals)producehepaticproteins(vitellogenin,VTG;zonapellucida,ZP)thatareneededforthe

properdevelopmentofeggs.Therefore,wecollectedthefishatthepeakoftheirspawning

season(lateMarchinnorthernIndiana)andhypothesizedtheseeggproteinswouldbe

identifiedonlyfromPCsincubatedwithfemaleplasma.Weidentifiedover300different

proteinsandPCsdifferedintheircompositionbasedonlengthofincubationandgender,with

VTGandZPpresentonlyinPCsincubatedwithfemaleplasma.Wediscussthesefindingsin

relationtopreviousresearchwithmammalianbiologicalfluidsandproposethatbindingofVTG

andZPtothesurfaceofPVP-AgNPscouldresultinenhancedmovementofparticlestoovaries

anddevelopingembryos.

19

2.2Materialsandmethods

2.2.1Smallmouthbassplasmacollection

Adultbass(6femalesand4males)werecollectedfromtheSt.JosephRiver,Elkhart,northern

Indiana,USA,duringthepeakoftheirspawningseason(lateMarch).Fishwerecapturedusing

electroshockingandtransportedaliveinanaeratedlivewell,andwithin<3hofcapture,bled

fromthecaudalvein.Bloodsamples(~1mL)weresavedinlithiumheparinizedvials,keptonice

untilcentrifugedat1,000gfor20min,andplasmastoredat-80°Cuntilprocessed.After

bleeding,fishwereeuthanizedwith200mg/Ltricainemethanesulfonate(MS-222)and

dissectedforfinalgenderdetermination.

2.2.2WesternblottingofVTGandVEGF

WesternblotwasusedtoconfirmthepresenceorabsenceofVTGinplasmafromfemalesand

males,respectively.Totalplasmaproteinconcentrationwasdeterminedusingbicinchoninicacid

150

VEGF

250

100

75

50

37

25

20

15

10

Female

MalesVTG

kDa Marker

Figure2.1WesternblotofplasmafromadultfemaleandmalesmallmouthbasssampledinthepeakofspawningseasonfornorthernIndiana(March).Asexpected,vitellogenin(VTG)wasonlyfoundinfemaleplasmawhereasvascularendothelialgrowthfactor(VEGF)wasconstitutivelyexpressedinallfishandthususedasareferenceprotein.

20

(BCAAssayKit,Thermoscientific,Rockford,IL,USA).Atotalof20µgproteinwasloadedontoa

sodiumdodecylsulfate(SDS)-12%polyacrylamidegel(PAGE)andproteinstransferredtoa

polyvinylidenedifluoride(PVDF)membrane.Theprimaryantibodyusedwasapolyclonalanti-

VTGfromBiosense(Bergen,Norway)andthesecondaryantibodywasereIR-DYE700(Li-Cor,

Lincoln,NE,USA).Inaddition,vascularendothelialgrowthfactor(VEGF)wasusedasareference

proteinsinceitwasstablyexpressedinallfishplasma(Fig.2.1).VEGFwasdetectedusinga

polyclonalanti-primaryantibodyfromAnaspec(Fremont,CA,USA)andasecondaryantibody

(IR-DYE800)fromLi-Cor.WesternblotimageanalysiswasdoneusinganOdysseyinfrared

imager.SinceonlyfemaleplasmahadVTGwithnonebeingpresentinmales,plasmasamples

werepooledbygenderandstoredat-80°CforallPVP-AgNPincubationexperiments.

2.2.3CharacterizationofPVP-AgNPsbeforeandafterincubationwithfishplasma

PVP-AgNPs(NanocomposixInc,SanDiego,CA,USA)wereincubatedwitheitherfemaleormale

bassplasma(ratioofNPstoproteinswas1:500µg/µg)for1hor24h.AggregationofNPswas

evaluatedwithultraviolet–visiblespectroscopy(UV-Vis)testsperformedusingaVarianCary50

UV-Visspectrophotometer(Varian,Inc.PaloAlto,CA,USA).Nanoparticletrackinganalysis(NTA)

(NanosightLM-10,MalvernInstruments,Worcestershire,UK)wasusedforquantifyingparticle

sizedistributionat25°C.Allsampleswerediluted10,000timesusingparticle-freewater.

StabilityofNPsolutionswasevaluatedbymeasuringzetapotentialusingaMalvernZetasizer

NanoZS(Malvern,UK,λ=633nm)alsoat25°C.Sampleswereequilibratedinsidetheinstrument

for30spriortoanalysisandmeasurementswerebasedonthemeanofthreerunsperformed

withratesof10kcps.PVP-AgNPsnotincubatedwithfishplasma(“naked”particles)aswellas

fishplasmawithnoPVP-AgNPswerealsoanalyzed.

2.2.4PC-PVP-AgNPpelletpreparationandsilverstainofSDS-PAGE

PVP-AgNPsandplasmaproteins(1:1µg/µg)wereincubatedfor1hor24hat30°C.After

incubation,sampleswerecentrifugedat15,300gat4°Cfor20mininordertoseparatetheNP-

proteincomplexesfromplasma(Docteretal.,2014).Thesupernatantwasdiscardedandthe

pelletwashedwithaphosphate-bufferedsolutiontwiceandpreservedat-80°Cuntilprocessed.

AnSDS-PAGEsilverstainedgelwasusedtoimagethesizedistributionofPCsinall4incubation

groupsaswellasfromplasmanotincubatedwithNPs.Atotalof50µLlysisbuffer(8Murea,2%

21

CHAPS,storedat-20°C)wasaddedtothePC-PVP-AgNPpelletstoeluteboundproteinsfromthe

surfaceofNPsaftera5minincubationat95°C.Sampleswerethencentrifugedat15,300gat

roomtemperaturefor15minandthesupernatanttransferredtoafreshtubeandloadedontoa

12%SDS-PAGEandstainedwithasilverstainkit(ThermoFisherScientificInc.Rockford,IL,USA)

(Docteretal.,2014).

2.2.5Liquidchromatography-massspectrometry/MSanalysisofproteincorona

Proteincoronapelletsweredigestedin10µLof10mMofdithiothreitol(DTT)and25Mmof

ammoniumbicarbonate(ABC)andincubatedfor1hat37°C.Next,10µLofafreshlymade

alkylationreagentmixture(97.5%acetonitrile,ACN;0.5%triethylphosphine,TEP;and2%

iodoethanol,IEtOH)wasaddedtoeachsampleandincubatedat37°Cfor1h.Sampleswere

driedfor30minunderavacuumcentrifugeafterincubatedwith80µLofanLys-C/trypsin

mixture(dissolvedin25mMABCtoafinalconcentration0.05µg/µL).Samplesweretransferred

tobarocyclermicrotubes,sealedandplacedinabarocycler(PressureBiosciences,SouthEaston,

MA,USA)setat50°C;50sat20kpsi;and10satatmosphericpressureforatotal120cycles(2

h).Afterdigestion,reversephaseC18columns(silicabasedC18ultramicrospincolumnkit,The

400 600 800 10000.00

0.02

0.04

0.06

0.08

0.10

Wavelength (nm)

Abs

orba

nce

24 h AgNP F 24 h AgNP M1 h AgNP F 1 h AgNP M

AgNPs

Figure2.2UV-VisofnakedPVP-AgNPsandofPVP-AgNPsafterincubationwithplasmafromadultfemaleormalesmallmouthbassfor1hor24h.AllPVP-AgNPsUV-VisabsorptionsshiftedtohigherwavelengthsafterincubationwithfishplasmacomparedwithnakedPVP-AgNPs.

22

NestGroup,Inc.Southborough,MA,USA)wereusedforsamplepurification.Columnswere

conditionedbyadding100µL100%ACNandcentrifugedfor1minat110g,followedwith

washeswithpurifiedwater(100µL)andcentrifugedeachtimeat110gfor1min.Next,

peptideswereloadedontothecolumnandcentrifugedat110gfor1min.Atotalof100µL

0.1%formicacid(FA)(v/v)wasthenloadedintothecolumnandcentrifugedfor1minat110g

twicetoremoveorganicbuffers.Peptideswereelutedusing50µL80%CAN/0.1%FA(v/v)and

centrifugedat110gfor1minthreetimes.Sampleswerevacuumed-driedandre-suspendedin

97%purifiedwater/3%CAN/0.1%FA(v/v)forLC-MS/MSanalysis(Hedricketal.,2015).

ProteinsampleswererunonaDionexUltiMate3000RSLCNanoSystemcoupledtoaQ

ExactiveTMHFHybridQuadrupole-OrbitrapMS(ThermoScientific,Waltham,MA,USA).Peptides

wereloadedintoatrapcolumn(20µmx350mm)andwashedwith98%purifiedwater/2%

ACN/0.01%FAusingaflowrateof5µL/min.Thetrapcolumnwasthenswitchedin-linewiththe

analyticalcolumnafter5min.Peptideswereseparatedusingareversephaseacclaimpepmap

1h-PVP-AgNPs

1h-PVP-AgNPs-

F

1h-PVP-AgNPs-

M

24h-PVP-AgNPs

24h-PVP-AgNPs-

F

24h-PVP-AgNPs-

M

ZetaPotential(mV) -30.23 -20.73 -16.63 -34.67 -20.13 -17.17

Size(nm) 52.6 57.3 56.1 49.4 60.8 58

-40

-20

0

20

40

60

Size(nm)

Zeta Potential(mV)

* * # #

Figure2.3Nanotrackinganalysis(NTA)showingchangesinthesizeofPVP-AgNPs(mode±standarderror)andZpotentialafterincubationwithplasmafromadultfemale(F)ormale(M)smallmouthbassfor1hor24h.*p<0.05comparedwith1hPVP-AgNPszetapotential;#:p<0.05comparedwith24hPVP-AgNPszetapotential.

23

RSLCC18(75µmx15cm)columnfor120minataflowrateof300nL/min.MobilephaseA

consistedof0.01%FAinwaterandmobilephaseBconsistedof0.01%FAin80%ACN.Thelinear

gradientstartedwith5%Bandreached30%Bin80min,45%Bin91min,and100%Bin93min.

Thecolumnwasheldat100%Bfor5minbeforebeingbroughtbackto5%Bandheldfor20

min.SampleswereinjectedintotheQEHFusingaNanosprayFlexTMIonsourcefittedwithan

emissiontip.Dataacquisitionwasperformedbymonitoringthetop20precursorsat120,000

resolutionswithaninjectiontimeof100milliseconds.LC-MS/MSdatabase(NCBInr-Chordata)

searchwasperformedusingMascotMS/MSIonServer(www.matrixscience.com).Search

0 100 200 300 400 5000

1.0´107

2.0´107

3.0´107

4.0´107

NTA 24 h

Particle size (nm)

Parti

cles

/mL

AgNPs F

AgNPs MAgNPs

Plasma FPlasma M

0 100 200 300 400 5000

1.0´107

2.0´107

3.0´107

4.0´107

NTA 1 h

Particle size (nm)

Parti

cles

/mL

AgNPs F

AgNPs MAgNPs

Plasma FPlasma M

Figure2.4Nanotrackinganalysis(NTA)ofPVP-AgNPsafterincubationwithsmallmouthbassplasmafromadultfemale(F)ormale(M)for1h(A)or24h(B).ShownarealsoNTAanalysesfromfishplasma(noPVP-AgNPs)andnakedPVP-AgNPs.

24

parameterswereasfollows:peptidetolerance±0.05Da;MS/MStolerance±0.1Da;and

peptidechargeof2+,3+and4+.Decoyselectionwascheckedandfinalresultswereexported

withafalsediscoveryrate(FDR)setat<5%.

2.2.6Statisticalanalysis

AllstatisticalanalyseswereconductedusingSPSS22.0.One-wayanalysisofvariance(ANOVA)

followedbypost-hocTukey’smultiplecomparisontestswereusedtocomparemeansacross

treatments.

2.3Results

2.3.1CharacterizationofPVP-AgNPproteincorona

ResultsfromUV-VisanalysesindicatedthatpurePVP-AgNPshadauniqueabsorptionat430nm,

whereastheabsorbanceofPVP-AgNP-PCmixturespeakedat~440nmindicatinga“redshift”

PC-M-24h PC-M-1h M-P F-P PC-F-1h PC-F-24h Marker

150 100

75 50

37

25

20

15

10

250 kDa

Figure2.5SDS-PAGEgelsilverstainofproteincorona(PC)isolatedfromPVP-AgNPsafterincubationwithplasma(P)fromadultfemale(F)ormale(M)smallmouthbassforeither1hor24h.BothlengthofincubationandgenderaffectedPC.Smallersizedproteins(<24kDa)wereonlyfoundinPCfromM(blackarrows).Adecreaseintheabundanceofproteins50-80kDawasdetectedafter24hfromFplasma.PlasmawithoutPVP-AgNPsarealsoshownforcomparison.

25

(Fig.2.2).After1hincubationwithbassplasmathesizeofPVP-AgNPsincreasedfroman

averageof52.6nmtoanaverageof57.3nminfemaleplasmaandtoanaverageof56.1nmin

maleplasma(Figs.2.3and2.4A).After24h,thesizeofpurePVP-AgNPsdecreasedto49.4nm,

butcontinuedtoincreasetoanaverageof60.8nminfemaleplasmaand58.0nminmale

plasma(Figs.2.3and2.4B).Increaseinsizewasnotstatisticallysignificant,butasdiscussed

below,anincreasein>5nmisconsideredbiologicallysignificant.Zetapotentialdataindicated

thatthesurfacechargeofPVP-AgNPschangedsignificantlyafterincubationwithfishplasma

regardlessoflengthofincubationandfishgender(Fig.2.3).

2.3.2Silverstain

ArepresentativeimageofanSDS-PAGEsilverstaingelisshowninFig.2.5.Thesizeandamount

ofproteinsbindingtothesurfaceofparticleswasgender-specificandtime-dependent.

Comparedtoproteinsfromfemaleplasma,theproteinprofilefrommaleplasmacontained

Figure2.6Venndiagramofproteinsidentifiedinproteincorona(PC)ofPVP-AgNPsincubatedwithfishplasmafromeitherfemale(F)ormale(M)smallmouthbassfor1hor24h.

26

smallerproteins(~20-23kDa)(seearrowsinFig.2.5).Regardlessofgender,therewerealso

moreproteinsboundtotheparticlesaftera1hincubationcomparedto24h.

2.3.3LC-MS/MSproteomicanalysis

AVenndiagramsummarizingthedifferencesandsimilaritiesoftheproteinsidentifiedfromPC

populationsacrossthefourtypesofincubationconditionsisshowninFig.2.6.Overall,atotalof

135and147proteinswereidentifiedfromthePCoffemaleplasmaincubatedfor1hand24h,

respectively.Inmales,thenumberofproteinswere194and193forthesameincubation

periods.Sixtyproteinsweresharedacrossallconditions.Withinfemales,89proteinswere

sharedacrossconditions,and19and26proteinswereuniquetothe1hand24hincubation

periods,respectively.Inmales,109proteinsweresharedacrossallconditions,and67and24

wereonlydetectedaftera1hor24hofincubation,respectively(Fig.2.6).

AsalreadypresentedinFig.2.5,thesizeofproteinsidentifiedrangedfrom<20kDato>125

kDa(Fig.2.7).Regardlessofincubationconditionsandcomparedtopurefishplasma,most

proteinsfoundboundtothesurfaceofPVP-AgNPswere<70kDa(%fractionrangingfrom89-

92%).Inparticular,therewasahigherpercentageofsmallerproteins(<20kDa)inPCsof

femaleswhichincreasedwithlengthofincubation.Medium-sizedproteins(50-70kDa)were

morecommoninPCsincubatedwithmaleplasma(seealsoFig.2.5).

Asummaryshowingthepercentcontributionofthedifferentproteinsidentifiedbasedontheir

functionsfromallconditionstestedispresentedinFigs.2.8andA.2.1.Adetailedlistwiththe

namesofallproteinsidentifiedisshowninTable2.1(TableA.2.1alsoincludesdetailed

informationonaccessionnumbersandnumberofsignificantpeptidesequencesidentified).The

majorcomponentsofallPCsconsistedofdifferentformsofimmunoglobins(Ig),hemoglobin

(Hb),fibronectin/fibrinogen,apolipoproteins,andcomplementcomponents.Female-specific

proteinsVTGandZPweredetectedonlyinfemaleplasmaregardlessoflengthofincubation

(Figs.2.8andA.2.1).AngiotensinogenwasonlyfoundinPCofmalesafter24h(Figs.2.8and

A.2.1).

2.4Discussion

Inthisstudy,weinvestigatedtheeffectsoffishgenderandlengthofincubationonthe

formationofPConthesurfaceofnegativelychargedPVP-AgNPs(50nm).Sincethisisthefirst

27

studythatusesfishplasmatocharacterizePCsfromNPs,wefirstvalidatedmethodsdeveloped

inmammalsforcharacterizationofPCformationandlaterisolationofproteinsfor

identification.WeshowthataPCdevelopedonthesurfaceofPVP-AgNPsandthatbothgender

andlengthofincubationaffectedthesizeofthePCformedandthetypesofproteinsisolated.

WeproposethatfishplasmaservesasagoodmediaforthecharacterizationofPCinfuture

studieswithNPsasourfindingsmoreorlessreplicatewhatothershavereportedusinghuman

androdentplasma/serum.

IncubationofPVP-AgNPswithfishplasmaresultedinincreasedagglomeration(Fig.2.2)and

increasedinstability(Fig.2.3)oftestsolutionsandthesechangesweremostevidentinthecase

ofmaleplasmaincubatedfor24h.Thezetapotentialforsamplesincubatedwithfemaleplasma

increasedby31%and42%after1hand24hofincubation,respectively.Thesesamevaluesfor

samplesincubatedwithmaleplasmawere45%and49%.Similarresultshavebeenreported

withnegativelychargedNPs(Casalsetal.,2010;Tenzeretal.,2013;Huangetal.,2016).

WealsoobservedchangesinthesizeofPVP-AgNPsafterbeingincubatedwithfishplasma(Figs.

2.3and2.4)withincreasedlengthofincubationresultinginincreasedNPsize.After1h

F-P

PC-F-1h

PC-F-24h

M-P

PC-M-1h

PC-M-24h

0

50

100

ProteinFractio

n(%

)

<20kDa20-25kDa25-50kDa50-70kDa70-100kDa100-125kDa>125kDa

Figure2.7Percentdistributionofdifferentsizeproteinsobtainedfromproteincorona(PC)fromPVP-AgNPsincubatedwithplasmafromadultfemale(F)ormale(M)smallmouthbassfor1hor24h.PlasmawithoutPVP-AgNPsarealsoshownforcomparison.

28

incubationwithfishplasma,thesizeofPVP-AgNPsincreasedanaverageof3.95nm(anoverall

8%increase).Thisincreaseinsizewasmoredramaticafter24h,withanaverageincreaseof10

nm(20%increase).Sizechangewasalsogender-dependentwithparticlesincubatedwithmale

plasmaresultinginoverallsmallerparticlesizes,butthiswasnoticeableonlyafter24hof

incubation.Indeed,particlesincreasedanaverageof8.6nmwhenincubatedwithmaleplasma,

comparedtoanincreaseof11.4nmforfemaleplasma.

OveralltheseresultssuggestthatPCswereaffectedbybothincubationlengthsandgenderof

fish.Similarresultshavebeenreportedwithcitrate-coatedAgNPs(10nmsize;red-shiftof~5

nminwavelength)incubatedwithhumanubiquitinfor24h(Manginietal.,2014),silica

nanoparticles(30nm;140nm,from0.5minupto120min)incubatedwithhumanplasma

(Tenzeretal.,2013),andmetallicAuNPs(4-40nm,from0hto48h)afterincubationwith10%

fetalbovineserum(Casalsetal.,2010).Theonlystudyidentifiedevaluatinggendereffectson

PCsfromcitrateandPVP-AgNPs(20nm)incubatedwithhumanplasmafoundminimal

differencesbetweengenders(Huangetal.,2016).

Atotalof337proteinswereidentifiedfromallPCs(Fig.2.6).However,>82%(n=278)ofthese

proteinswereuniquetomales.Further,therewasalargenumberofproteinsuniquetothe1h

(n=67)and24h(n=64)maleincubationconditions.Incontrast,thenumberofuniqueproteins

isolatedfromfemaleplasmaincubatedwithNPswasmuchlower(n=59),with19and26being

uniquetothe1hand24htreatments,respectively.Thisisaninterestingfindinganddeserves

furtherinvestigation.

Ingeneral,PCsmirroredthesizedistributionofproteinsidentifiedintheplasmaofthefish

studied(Fig.2.7).Interestingly,smallerproteins(<25kDa)weremorecommoninPCsformedin

femaleplasma,increasinginpercentagewithlengthofincubation.Thisshiftresultedinaslight

decreaseinthepercentofmedium-sizedproteins(50–70kDa).Proteins>70kDawere

uncommoninthefishplasmausedforthesestudieswhichwasreflectedintheiroveralllow

representationinthePCsevaluated.Previousstudieshavealsoreportedsmallmolecularweight

plasmaproteins(70%ofPCswere<60kDa)fromthesurfaceofcitrate-andPVP-coatedAgNPs

(20nm)afterincubatedwithhumanplasma(Huangetal.,2016).Inanotherstudy,sizeof

proteinswascore-materialdependentwithPCsfrompolystyrenenanoparticles(PsNPs)

29

containingmostlyproteins~60-70kDa,comparedtoPCsfromSiNPswhichcontainedlargersize

proteins(150-200kDa)(Tenzeretal.,2013).

Themostcommonclassofproteinsidentifiedrelatedtoimmunefunction(complement,Ig’s,

andacute-phaseproteins,includingamyloidA,antitrypsin,kallikrein,kininogen,andvitaminK-

dependentprotein),redbloodcellfunctionandclotting(Hb,plasminogen,and

F-P

PC-F-1h

PC-F-24h

M-P

PC-M-1h

PC-M-24h

0

50

100ProteinMassF

raction(%)

ImmunoglobinHemoglobinFibrinogen/FibronectinApolipoproteinLipoproteinComplementParvalbuminVTG/ZP

F-P

PC-F-1h

PC-F-24h M-

P

PC-M-1h

PC-M-24h

0

5

10

ProteinMassFraction(%)

TransferrinMacroglobulinAngiotensinogenCeruloplasminCaMKIIPlasminogenAcute-phaseproteins

A.

B.

Figure2.8Percentdistributionofdifferentclassesofproteinsobtainedfromproteincorona(PC)fromPVP-AgNPsincubatedwithplasmafromadultfemale(F)ormale(M)smallmouthbassfor1hor24h.PlasmawithoutPVP-AgNPsarealsoshownforcomparison.Eachgraphshowsauniquesetofproteins.CaMKII:Calcium/calmodulin-dependentproteinkinase;VTG:Vitellogenin;ZP:Zonapellucida.A.Includesthemostfrequentproteinsdetected;B.Includeslessfrequentlyobservedproteins.

30

fibrinogen/fibronectin),andlipidandcholesteroltransport(lipoproteinandapolipoprotein)

(Figs.2.8andA.2.1).Proteinsassociatedwithcoagulation,platelet,complementactivationand

immuneresponseswerealsofoundtobindtothesurfaceofPVP-AgNPs(20nm),citrated-AgNPs

(20nm),SiNPs(35,120and140nm),andPsNPs(35,120and140nm)inpreviousstudies

(Lundqvistetal.,2008;Tenzeretal.,2013;Huangetal.,2016).Proteinsinvolvedinthecontrol

ofbloodpressure(angiotensinogen)aswellasintheproductionofeggs(VTGandZP)were

identifiedinthepresentstudy.Forthemostpart,thesameproteinsdetectedinfreeplasma

sampleswerealsofoundinthedifferentPCsexamined(Fig.2.8),withsomeinteresting

exceptions.AngiotensinogenwaspresentonlyinthePCfrommaleplasmasamplesincubated

for24h,despitetheirpresenceinfreeplasmafrombothgenders.Similarly,parvalbuminwas

almostabsentfromPCs,butwasdetectedinplasmafrombothgenders.Withtheexceptionof

complementproteins,thefrequencydistributionofproteinsdifferedbetweenfreeplasma

samplesandPCs.Forinstance,Ig’s,ceruloplasminandplasminogenweremorecommonlyfound

associatedwithPCs.Incontrast,macroglobulin,lipoproteinsandapolipoproteinswereless

common.TheonlyproteinsthatwerefemalespecificweretheeggproteinsVTGandZPand

bothwerealsodetectedinfemalePCs.Thiswasexpectedsincefishwerecollectedinthepeak

ofthebreedingseasonwhenVTGandZParehighest.

ApoliporoteinsbindingtothesurfaceofNPscouldaffectfateandtransportintocellsandorgans

(Fig.2.9).Highdensitylipoproteins(HDL,ApoA)wereassociatedwithPVP-AgNPsregardlessof

genderorlengthofincubation.ApoB-100andApoEwerefoundinallPCs,indicatingthatPVP-

AgNPswereassociatedwithverylowdensitylipoproteins(VLDL)andlowdensitylipoproteins

(LDL),similarlytowhatwasreportedbyLundqvistetal.(Lundqvistetal.,2008).Bindingof

CaMKIIcoulddriveNPsreuptakeincardiomyocytes.Inaddition,AgNPsbindingtoangiotensin

mightalsoaffectvasoconstrictionandbloodpressurecontrol.

ThisisthefirststudyreportingfemalespecificeggproteinsinPCs.Thisisofimportance,

becauseitcouldleadtoincreasedmovementofNPstotheovarysincetheseproteinsaretaken

upbydevelopingfolliclesviareceptor-mediatedendocytosis(Panetal.,1969;Wallace,1985).

ThiscouldatleastpartiallyexplainmovementofdifferenttypesofNPstofishovaries(Liuetal.,

2016;Zhangetal.,2016)leadingtoabnormalfolliculardevelopmentandultimatelynegatively

impactingfecundityandreproductivesuccess(ChatterjeeandBhattacharjee,2016).Inaddition,

31

thiscouldresultinexposureofearly-lifestages(embryosandlarvae)toNPsresultingin

decreasedsurvival(Austinetal.,2012;Leeetal.,2012;Tabatabaeietal.,2015;Morishitaetal.,

2016)(Fig.2.9).However,thishypothesisneedstobeconfirmedwithadditionalstudies.

Figure2.9Proposedmechanismsofsilvernanoparticles(AgNPs)distributioninsideafish.WhenAgNPsenterintofishbloodplasma,proteinscouldimmediatelybindtothesurfaceofparticles,inducingreceptor-mediatedphagocytosis,bloodclotting,andmoveintovascularendothelialcells.LipoproteinsandapolipoproteinscouldalsobindtoAgNPs,whichcouldfacilitatethebiodistributionofparticlestoalmostavarietyoftissuesandorgans.VitellogeninbindingtothesurfaceofNPscoulddriveparticlestransporttodevelopingfollicleswithintheovarieswhichmightresultinmaternaltransporttoembryos.

32

2.5Conclusions

Inthischapter,wereportforthefirsttime,theformationandcharacterizationofPCsinfish

plasma.BothgenderandlengthofincubationaffectedthechangesinNPsizeobservedaswell

asthetypesofproteinsidentified,with>300differentproteinsidentifiedoverall.Aswith

previousstudies,themostcommonproteinsidentifiedrelatedtoimmuneandredbloodcell

function.CausesforthemuchlargernumberofuniqueproteinsidentifiedfromPCsformedwith

maleplasmadeservesfurtherinvestigation.ThelipoproteinandApobindingtothesurfaceof

NPscoulddriveparticletoavarietyoforgansandtissues.Finally,eggproteinswereonly

detectedinPCsincubatedwithfemaleplasma.WeproposethatbindingofVTGandZPtothe

surfaceofPVP-AgNPscouldresultinenhancedmovementofparticlestoovariesanddeveloping

embryos(Fig.2.9).

Table2.1Proteinsisolatedfromtheproteincorona(PC)ofPVP-AgNPsafterincubationfor1or24hwithplasmafromeitheradultfemale(F)ormale(M)smallmouthbass.CaMKII:Calcium/calmodulin-dependentproteinkinase;Ig:Immunoglobulin;VEGF:Vascularendothelialgrowthfactor;VTG:Vitellogenin;ZP:Zonapellucida.

Protein PC-F-1 PC-F-24 PC-M-1 PC-M-24

Angiotensinogen

angiotensinogen Ö

angiotensinogenprecursor Ö

Apolipoprotein

A-I Ö Ö Ö Ö

A-Iprecursor Ö Ö Ö Ö

A-IV Ö Ö Ö

B Ö Ö Ö Ö

B-100 Ö Ö Ö Ö

C-II Ö

33

D Ö

E Ö Ö Ö Ö

beta-2-glycoprotein1(apolipoproteinH) Ö Ö

CaMKII

subunitalphaisoform1 Ö Ö Ö

subunitdelta Ö Ö Ö Ö

Ceruplasmin

ceruloplasmin Ö Ö Ö Ö

ceruloplasminisoformX1 Ö Ö Ö Ö

RedBloodCellFunction

BloodCoagulation

alpha-1-antitrypsin Ö Ö Ö

alpha-1-antitrypsinhomolog Ö

antithrombin-III Ö

beta-fibrinogen Ö

fibrinogenalphachain Ö

fibrinogenbetachain Ö Ö Ö Ö

fibrinogenbetachainprecursor Ö Ö Ö

fibrinogengammachain Ö Ö Ö Ö

fibrinogengammaprotein Ö Ö Ö

gammafibrinogen Ö Ö Ö

kallikrein Ö Ö Ö

34

kininogen Ö

kininogen-1isoformX1 Ö

plasminogen Ö Ö Ö Ö

plasminogenprecursor Ö Ö Ö

serumamyloidA Ö Ö Ö Ö

serumamyloidAprotein Ö Ö Ö Ö

vitaminK-dependentproteinC Ö Ö Ö Ö

Hemoglobin

betachain Ö Ö

betaembryonic-2 Ö

beta-2subunit Ö Ö

embryonicsubunitalpha Ö Ö

subunitalpha-1 Ö Ö Ö Ö

subunitalpha-2 Ö Ö Ö Ö

subunitalpha-A Ö Ö Ö Ö

subunitbeta Ö Ö

subunitbeta-1 Ö Ö Ö Ö

Transferrin

serotransferrin Ö Ö Ö

transferrin Ö Ö Ö Ö

ImmuneFunction

Complement

35

C1qprotein4 Ö Ö Ö

C1qB Ö

C1qC Ö

C1r/s-Aisotype Ö Ö Ö

C1ssubcomponent Ö

C3 Ö Ö Ö Ö

C3-2 Ö

C3-2precursor Ö Ö

C3isoformX1 Ö Ö Ö

C4 Ö Ö Ö Ö

C4-B Ö

C5 Ö Ö Ö Ö

C6 Ö Ö Ö Ö

C7 Ö Ö Ö Ö

C8alpha Ö Ö Ö Ö

C8beta Ö Ö Ö Ö

C8gammachainisoformX1 Ö Ö Ö

C9 Ö Ö Ö Ö

factorB Ö

factorH Ö Ö

factorI Ö

pro-C3-1 Ö Ö Ö Ö

36

Immunoglobulins

deltaheavychain Ö Ö Ö

domain-containingreceptor1 Ö Ö Ö

gammaheavychainvariableregion Ö

heavychain Ö Ö Ö

heavychainBrE-3-mouse(fragment) Ö

heavychainprecursor Ö

heavychainvariableregion Ö Ö Ö Ö

kappachainVregionMem5 Ö Ö

kappachainV-IIregionRPMI6410precursorprotein Ö Ö

kappachainV-IVregionJIprecursor Ö

kappalightchain Ö

lambdalightchainVLJregion Ö

lambdapolypeptide1precursor Ö

lightchain Ö Ö Ö Ö

lightchainisotype1 Ö Ö

lightchainisotypeL1 Ö Ö

lightchainprecursor Ö

lightchainprecursorL Ö Ö Ö Ö

lightchaintype1 Ö Ö Ö

Mheavychain Ö Ö Ö Ö

muheavychain Ö Ö Ö Ö

37

muheavychainsecretoryform Ö

muheavychainvariableregion Ö

mu/tauheavychain Ö Ö Ö

tauheavychain Ö Ö Ö Ö

Macroglobulins

alpha-2-macroglobulin Ö Ö Ö Ö

alpha-2-macroglobulin-1 Ö

alpha-2-macroglobulin-P Ö Ö

VEGF

chainA Ö Ö Ö Ö

Female-specificEggProteins

VTG Ö Ö

VTGA Ö

VTGAa Ö Ö

VTGAb Ö Ö

VTGC Ö Ö

VTG-1 Ö Ö

ZPglycoprotein2.3precursor Ö Ö

38

2.6Appendix

TableA.2.1Majorproteinsidentifiedfromthedifferentproteincoronas(PC)withtheir

accessionnumberfromMascotDaemonandthenumberofsignificantpeptidesequences

identifiedforeachprotein.A:PC-F-1h;B:PC-F-24h;C:PC-M-1h;D:PC-M-24h.

Protein

AccessionNumber

Num.ofsig.seq.

A B C D

Angiotensinogen

angiotensinogen gi|343459201 0 0 0 2

angiotensinogen

precursor

gi|213511078 0 1 0 0

Apolipoproteins

A-I gi|736298162 1 1 1 1

A-Iprecursor gi|118344628 1 1 1 1

A-IV gi|551495886gi|734628151gi|498985280

gi|736294062

3 0 2 2

B gi|740529756gi|226731853 3 3 1 3

B-100 gi|657567867gi|734622166gi|499036286

gi|734622170gi|736191546gi|617302261

gi|542201992gi|657567865gi|432945483

21 7 11 9

C-II gi|193795860 1 0 0 0

D gi|657534183 0 0 0 1

E gi|193795858 5 5 5 5

39

beta-2-glycoprotein

1(apolipoprotein

H)

gi|617447201gi|323650112 0 0 1 2

CaMKII

subunitalpha

isoform1

gi|395504846 0 1 1 1

subunitdelta gi|657737225 2 2 3 2

Ceruplasmin

ceruloplasmin gi|657597079gi|736207854gi|348543049

gi|734642589

15 15 11 18

isoformX1 gi|551507474gi|657739145 8 10 9 6

RedBloodCellFunction

BloodCoagulation

alpha-1-antitrypsin gi|397776428gi|209981964 0 1 1 2

alpha-1-antitrypsin

homolog

gi|657564670gi|498942077 0 0 0 2

antithrombin-III gi|734648335 0 0 1 0

beta-fibrinogen gi|683842253 0 0 3 0

fibrinogenalpha

chain

gi|617449639 0 0 1 0

fibrinogenbeta

chain

gi|551518621gi|723586559gi|373906010

gi|499043505

3 3 14 9

fibrinogenbeta

chainprecursor

gi|146447341gi|147900556 3 3 0 8

40

fibrinogengamma

chain

gi|617493935gi|542187118gi|432847401

gi|530453430gi|336111758gi|46811245

4 8 11 11

kallikrein gi|551520679 0 1 1 1

kininogen gi|46576222 0 1 0 0

kininogen-1isoform

X1

gi|499022758 1 0 0 0

plasminogen gi|551509957gi|311223464gi|742250469

gi|395537460gi|410916093gi|542250797

gi|657550476gi|617464853gi|507642775

13 12 26 16

plasminogen

precursor

gi|157278425gi|41393105 0 2 5 3

serumamyloidA gi|401721571gi|401721573gi|530653512

gi|465990098

4 4 2 3

vitaminK-

dependentprotein

C

gi|657580165gi|657811911gi|734645142 1 1 3 2

Hemoglobin

betachain gi|157284022 3 0 0 2

betaembryonic-2 gi|47086345 0 0 2 0

beta-2subunit gi|116488092 0 2 3 0

embryonicsubunit

alpha

gi|523704559 4 0 4 0

subunitalpha-1 gi|432868060 4 4 4 3

subunitalpha-2 gi|269969355 2 2 2 2

41

subunitalpha-A gi|734603612 2 2 2 2

subunitbeta gi|115502222gi|122603gi|734603610

gi|524948366gi|225716372

0 3 9 0

subunitbeta-1 gi|551527417gi|225706810 20 21 20 1

Transferrin

serotransferrin gi|6136039 1 0 2 1

transferrin gi|374431112gi|10567299gi|7339632 4 3 7 6

ImmuneFunction

Complement

C1qB gi|429509793 0 0 1 0

C1qC gi|429509799 0 0 0 1

C1qprotein4 gi|397511022 1 1 0 1

C1r/s-Aisotype gi|67772028 0 1 1 1

C1ssubcomponent gi|734635084 0 0 0 1

C3 gi|498928208gi|657760797gi|498929264

gi|736214174gi|734648978gi|657587421

gi|551507245gi|499047883gi|542233247

gi|657592798gi|617471088gi|410917688

gi|617491246gi|657592798gi|303305915

gi|315570434

29 32 24 41

C3isoformX1 gi|348525110gi|734619056 0 3 5 3

C3-2 gi|339269297 7

C3-2precursor gi|157311657 0 3 4 0

C4 gi|684939571gi|636571980 1 1 3 1

42

C4-B gi|498938159 0 0 1 0

C5 gi|499042231gi|657589422gi|734649196

gi|736187868

7 7 13 8

C6 gi|684939384gi|551522012gi|348505142

gi|465986824

1 1 4 3

C7 gi|429508173gi|410922874gi|6682831

gi|542188839gi|657814026

3 5 4 4

C8alpha gi|429508163gi|734635553 2 2 1 1

C8beta gi|429508165gi|20138051gi|328677213

gi|410924816gi|734635551

13 15 19 14

C8gammachain

isoformX1

gi|617385810 1 0 1 1

C9 gi|352962746gi|408689299gi|410903672

gi|736216070gi|323650010

8 7 10 9

pro-C3-1 gi|573026038 9 9 9 12

factorB gi|657542545gi|736297094 0 0 0 2

factorH gi|736308413 0 0 1 1

factorI gi|542244580 0 0 0 1

Immunoglobulins

deltaheavychain gi|226860390gi|559775728 2 2 3 0

domain-containing

receptor1

gi|677385174 0 1 1 1

gammaheavychain

variableregion

gi|700652874 0 0 1 0

43

heavychain gi|11890707gi|11890645gi|2852433 0 1 3 1

heavychainBrE-3-

mouse(fragment)

gi|2137439 0 0 1 0

heavychain

precursor

gi|5006471 0 0 2 0

heavychain

variableregion

gi|326632403gi|28394637gi|326632423

gi|28394669gi|732549929gi|81302953

gi|732549192gi|732550005

3 8 13 13

kappachainV

regionMem5

gi|229367590 0 0 7 5

kappachainV-II

regionRPMI6410

precursorprotein

gi|528762398 0 0 2 2

kappachainV-IV

regionJIprecursor

gi|209738284 0 0 2 0

kappalightchain gi|225625776 0 0 1 0

lambdalightchain

VLJregion

gi|21669497 0 0 1 0

lambdapolypeptide

1precursor

gi|229366314 0 0 2 0

lightchain gi|33340577gi|33317504gi|34577135

gi|291165397gi|158602744gi|11323075

gi|33340577gi|197700160gi|349299

23 26 32 22

lightchainisotype1 gi|119067935 0 0 3 2

lightchainisotype

L1

gi|111278861 0 0 3 2

44

lightchain

precursor

gi|14289265 0 0 0 2

lightchain

precursorL

gi|37779042 5 5 6 4

lightchaintype1 gi|30692167 0 2 2 2

Mheavychain gi|532579074 15 15 22 23

muheavychain gi|149394324gi|322423472gi|111228036

gi|566036242gi|566036234gi|334362362

11 9 18 10

muheavychain

secretoryform

gi|629632982 3 0 0 0

muheavychain

variableregion

gi|372467865 0 0 3 0

mu/tauheavychain gi|566036203gi|566036207 0 3 3 5

tauheavychain gi|566036209 1 1 1 1

Macroglobulins

alpha2 gi|734650793gi|697870411gi|734650085

gi|742195782gi|736193097gi|42415863

7 3 1 8

alpha2-1 gi|323650130 0 0 0 1

alpha-2-P gi|734650797 1 0 0 2

VEGF

chainA gi|157836259 1 1 1 1

Female-specificEggProteins

VTG gi|5852935gi|260159577gi|703999526

gi|687670861gi|391353202

12 10 0 0

45

VTGA gi|291465276 2 0 0 0

VTGAa gi|374923101 4 4 0 0

VTGAb gi|326375569 4 4 0 0

VTGC gi|71011912gi|374923105 3 2 0 0

VTG-1 gi|410921648gi|657576314gi|736193115

gi|657576314

4 3 0 0

ZPglycoprotein2.3

precursor

gi|185132234 2 2 0 0

46

FigureA.2.1Heatmapofidentifiedproteinfractions(%)fromproteincorona(PC)isolatedfrom

PVP-AgNPsincubatedwithplasmafromadultfemale(F)ormale(M)smallmouthbassfor1hor

24h.ProteinfractionsarecomparedtoplasmanottreatedwithPVP-AgNPs(firsttwocolumns).

F.P

M.P

PC.M

.1h

PC.M

.24h

PC.F.1h

PC.F.24h

Immunoglobin

VTG/ZP

Acute−phase proteins

Transferrin

Macroglobulin

Ceruloplasmin

Plasminogen

Angiotensinogen

CAMKII

Complement

Hemoglobin

Fibrinogen/Fibronectin

Lipoprotein

Apolipoprotein

Parvalbumin

5

10

15

20

25

30

35

47

CHAPTER3 VASCULARTOXICITYOFSILVERNANOPARTICLESTODEVELOPINGZEBRAFISH(DANIORERIO)

Reproducedfrom:Gao,J.,Mahapatra,C.T.,Mapes,C.D.,Khlebnikova,M.,Wei,A.,&Sepúlveda,M.S.(2016).Vasculartoxicityofsilvernanoparticlestodevelopingzebrafish(Daniorerio).Nanotoxicology,1-10(Gaoetal.,2016).

3.0Summary

Nanoparticles(NPs,1–100nm)canentertheenvironmentandresultinexposuretohumans

andotherorganismsleadingtopotentialadversehealtheffects.Theaimofthepresentstudyis

toevaluatetheeffectsofearlylifeexposuretopolyvinylpyrrolidone-coatedsilvernanoparticles

(PVP-AgNPs,50nm),particularlywithrespecttovasculartoxicityonzebrafishembryosand

larvae(Daniorerio).PreviouslypublisheddatahassuggestedthatPVP-AgNPexposurecan

inhibittheexpressionofgeneswithinthevascularendothelialgrowthfactor(VEGF)signaling

pathway,leadingtodelayedandabnormalvasculardevelopment.Hereweshowthatearly

acuteexposure(0–12hourpost-fertilization,hpf)ofembryostoPVP-AgNPsat1mg/Lorhigher

resultsinatransient,dose-dependentinductioninVEGF-relatedgeneexpressionthatreturnsto

baselinelevelsathatching(72hpf).Hatchingresultsinnormoxia,negatingtheeffectsofAgNPs

onvasculardevelopment.Interestingly,increasedgenetranscriptionwasnotfollowedbythe

productionofassociatedproteinswithintheVEGFpathway,whichweattributetoNP-induced

stressintheendoplasmicreticulum(ER).Theimpairedtranslationmayberesponsibleforthe

observeddelaysinvasculardevelopmentatlaterstages,andforsmallerlarvaesizeathatching.

Silverion(Ag+)concentrationswere<0.001mg/Latalltimes,withnosignificanteffectsonthe

VEGFpathway.WeproposethatPVP-AgNPstemporarilydelayembryonicvasculardevelopment

byinterferingwithoxygendiffusionintotheegg,leadingtohypoxicconditionsandERstress.

48

3.1Introduction

Thevascularsystemisoneofthefirstorganstodevelopinembryonicvertebrates.Vascular

developmentoccursintwophases:vasculogenesis,denovoformationofvessels,and

angiogenesis,whichinvolvesthesproutingofnewvesselsfrompre-existingones(Shirinifardet

al.,2013).Thevascularendothelialgrowthfactor(VEGF)signalingpathwayregulates

vasculogenesisandangiogenesisviaendothelialcellproliferation,promotingcellmigrationand

inhibitionofapoptosis(HerbertandStainier,2011).Inzebrafish(Daniorerio),VEGFaisthemain

driverofvasculardevelopment;vegfaistransferredmaternallyandexpressedataveryearly

stage(1-4cellstage),decliningtoverylowlevelsat8.5hourspostfertilization(hpf)or80%

epiboly(Liangetal.,2001;Covassinetal.,2009).Expressionofembryonicvegfabeginsat12hpf

reachingpeaklevelsat24-30hpf(Liangetal.,2001).Lossofendothelium-specificreceptor

tyrosinekinase,VEGFR-2,andVEGFfunctioninhibitvasculardevelopment(Goreetal.,2012).In

contrast,overexpressionofvegfaresultsinectopicvasculaturedevelopmentaswellas

pericardialedemainlaterstagesofzebrafishembryos(Liangetal.,2001).Similarresultshave

beenreportedinmicewithoverexpressionofVEGFresultinginsevereabnormalitiesinheart

developmentandembryoniclethality(Miqueroletal.,2000).

Hypoxiaisamajorregulatorofbloodvesselformation,andstimulatesangiogenesisinorderto

increaseoxygendeliverytohypoxicregions.Hypoxiaincreasesexpressionofseveralgenes

withintheVEGFsignalingpathway(Liuetal.,1995;Stoneetal.,1995;Neufeldetal.,1999).A

verysensitivemolecularmarkerofhypoxiaisthehypoxia-induciblefactor1(HIF1),which

triggerstheVEGFsignalingpathwayinvascularendothelialcells(Liuetal.,1995).

Zebrafishisapopularvertebratemodelforinvestigatingvasculartoxicity,asitsgenepatternof

vasculogenesisandangiogenesisissimilartohumans.Embryoniczebrafishdevelopexuteroand

arealsotransparent,allowingforthevisualizationofvascularmorphologicalchangesinreal

time(Stainieretal.,1993;Serbedzijaetal.,1999;LiuandStainier,2012;Quaifeetal.,2012;

Delovetal.,2014).Forinstance,vasculardevelopmentinzebrafishlarvaecanbetrackedinreal

timebyvisualizingthewholecirculatorysystemusingtransgeniclineswithfluorescentmarkers

(RaldúaandPiña,2014).Inaddition,zebrafishembryosarenotcompletelydependentona

functionalcardiovascularsystemduringearlyembryodevelopment,allowingforthestudyof

severecardiovasculardefects(Bakkers,2011).

49

Silvernanoparticles(AgNPs)arecommonlyusedasbactericidesinmanycommercialproducts.

Nearly400commercialproductsareknowntocontainAgNPs,culminatingintheglobal

productionofapproximately320tonsofAgNPsperyear(Vanceetal.,2015).Ahandfulofin

vitrostudieshavereportedthatAgNPsarevasculotoxic.Inendothelialcells,AgNPs(40–50nm:5

mg/L)inhibitvasculardevelopmentviatheSrckinaseandPI3K/Aktpathways,bothdownstream

oftheVEGFsignalingpathway(Gurunathanetal.,2009;Kalishwaralaletal.,2009;

Sheikpranbabuetal.,2009).Otherinorganicnanoparticles(copperoxide:100mg/L;15-and40-

nmgoldparticles:25mg/L)canalsoinhibitvasculogenesisviatheVEGFsignalingpathway(Pan

etal.,2014b;Changetal.,2015).Inhumanumbilicalveinendothelialcells,goldNPsinhibitcell

migrationandtubeformationviaAktphosphorylationdownstreamofVEGFR2/PI3Kaffecting

cell-surfaceultrastructureandcytoskeletonformation(Panetal.,2014b).Finally,bothmicron-

sizedparticles(PM2.5)andultrafineparticles(carbonblack,<100nm)alterthedevelopmentof

endothelialcells.Inhepaticvessels,theycauseplateletaccumulation,whichhasbeen

associatedwithprothromboticchangesandelevatedriskofmyocardialinfarction(Petersetal.,

2001;Khandogaetal.,2004).

Evidenceforthevasculartoxicityofnanoparticlesonwholeorganismsismostlyanecdotal,with

themajorityofpublishedstudiesnotdesignedtospecificallytestfortheseeffects.Zebrafish

embryosexposedtoAgNPs(5–35nm:>50mg/L)respondedwithdecreasedbloodflow,

pericardialedemaandcardiacarrhythmia(Asharanietal.,2008).Theseare,however,non-

specificresponsesthatcanbeinducedbyamyriadofcauses.Inafollow-upstudy,thesame

AgNPswereshowntocauseabnormalcardiacdevelopmentandcirculatorydefects(Asharaniet

al.,2011).Further,increasedmortalityanddeformityrateswerepartlyattributedtodepressed

heartratesresultingindiminishedbloodflowtothebrainandthespinalcord(Asharanietal.,

2008;Massarskyetal.,2013).

Theobjectiveofthepresentstudyistoevaluatethevasculartoxicityofpolyvinylpyrrolidone-

coatedsilvernanoparticles(PVP-AgNPs)onembryoniczebrafish,followingexposureduring

vascular(2–96hpf)andheart(48–96hpf)development.Basedonpreviouslypublisheddata,we

reasonedthatexposureofembryoniczebrafishtoPVP-AgNPswouldinhibittheVEGFsignaling

pathway,leadingtodelayedandabnormalvasculardevelopment.Thisisthefirststudyto

systematicallyinvestigatethemechanismsofvasculartoxicitybyPVP-AgNPsinvivo.

50


3.2.1CharacterizationofPVP-AgNPs

PVP-AgNPs(50nm)werepurchasedfromNanocomposixInc.(SanDiego,CA,USA).Theoriginal

concentrationofPVP-AgNPswas4,730mg/Landwasstoredat4°Cunderdarkconditions.PVP-

AgNPsolutionswerediluted10,000timesinembryomedia(Replenish®,SeachemLaboratories

Inc.,Madison,GA,USA:13–14%calcium,1%magnesium,0.12%potassium,and0.6–0.7%

sodium)andparticle-freewaterseparatelybeforeanalysis.Particlesizedistributionswere

obtainedusingnanoparticletrackingAnalysis(NTA)(NanosightLM-10,MalvernInstruments,

Worcestershire,UK).Zetapotentialsofparticlesinfishembryomedia(12μLReplenishin100

mLtestsolution)weremeasuredusingaMalvernZetasizerNanoZS(Malvern,UK;λ=633nm)at

25°C(Table1)(EPA,2002).Sampleswereallowedtoequilibratefor30sinsidetheinstrument

priortoanalysis;measurementswerebasedonthemeanof12–16runsperformedatratesof

30–70kcps.

3.2.2Toxicitytests

Transgenicadultzebrafish(TG(fli1a:EGFP))expressingenhancedgreenfluorescentprotein

(EGFP)undercontrolofthefli1promotorwereobtainedfromZFIN(Eugene,OR,USA).

TransgenicandABwild-type(WT)zebrafishweremaintainedseparatelyattheAquaticEcology

Laboratory(PurdueUniversity)atatemperatureof28±1°Candaphotoperiodof14L:10D.Fish

werefedtwicedailywithacombinationofhatchedArtemianaupliiinthemorningand

commercialfood(Tetramin)intheafternoon.Genderswerehousedseparatelyuntiltheday

beforebreeding,thenplacedintanksata2:1male:femaleratio.Fishwereleftundisturbed

overnightandfertilizedembryoswerecollected1hafterthelightwasturnedonthenext

morning.Embryoswithin1hpfwererandomlyplacedinplasticpetridishescontaining25mL

exposuresolutions.Halfoftheexposuresolutionwasreneweddaily.Eachtestconsistedof

threereplicatesandeachexperimentwasrepeatedatleastthreetimesfromembryoscollected

fromdifferentmatingevents.Ultrapurewater(Millipore;R>18MΩ·cm)wasusedfordiluting

PVP-AgNPsintoaseriesofconcentrations(0.1,1.0,and10mg/L)asexposuresolutions.Every

100mLofexposuresolutioncontained12μLembryomedium.Mortalitywasrecordedat24,48,

72and96h.

51

3.2.3SilverionreleasefromPVP-AgNPs

Ag+releasefromPVP-AgNPswasquantifiedfromexposuresolutionsat0,24,48,72and96h

usinganion-selectiveelectrode(ISE,Model9616BNWP,ThermoFisherScientific,USA).The

systemwascalibratedwithaseriesofsilvernitrate(AgNO3)concentrations(1–20,000µg/L).

Concentrationswerequantifiedimmediatelypriortoexposuretests.

200µm

Control24hpf

b

a

cYolk

A

B DC

100μm

Figure3.1A.Representativetrunkregionoftransgenic(TG)(fli1a)zebrafishembryofroma24-hourpostfertilization(hpf)control.BoxedareacontainsfiveISVsusedtomeasuretheseparameters;arrow“a”pointstolengthofISV(dashedline);arrow“b”pointstoanglebetweenISVandDA;arrow“c”pointstointervalspacingbetweenISVs.B.Embryostreatedwith0.1mg/LPVP-AgNPsexhibitdisorderedISVs;C,D.Embryostreatedwith1.0and10mg/LPVP-AgNPsexhibitatrophiedISVs,relativetoA.

52

3.2.4Effectsonthecardiovascularsystem

3.2.4.1VasculardevelopmentandsizeoflarvaeathatchingDevelopmentofintersegmentalvessels(ISV)inzebrafishfollowsawell-definedpatternwith

pairsofISVssproutingfromthedorsalaorta(DA)atregularspatialandtemporalintervals

followingananteriortoposteriorsequence(Childsetal.,2002;Isogaietal.,2003).Thecommon

cardinalvein(CCV)growsacrosstheyolkofzebrafishembryosandisextensivelyremodeledand

regressesastheheartmigratesdorsallywithinthepericardium.

ISVlength,intervalbetweenISVs,anglesbetweenISVsandtheDA,andregressionofCCVwere

recordedfromTG(fli1a:EGFP)embryosusinganOlympusBX-51opticalmicroscopefittedwith

aDP71camera(OlympusAmerica,Jupiter,FL,USA).Measurementsofeggswerecollectedafter

enzymaticdechorionationusingpronase(2mg/mLinembryomediafor1min)andafter

anesthesiawithtricanemethanesulfonate(MS222,0.2g/kg).Measurementsweretakenfrom

digitalimagesfromatleast15embryosperdoseanddevelopmentalstages(24,48,72and96

hpf)fromthefirst5ISVslocatedanteriortotheyolksacextension(seeinsertinFig.3.1A,red

rectangleindicatesthepositionofISVs).Additionalendpoints,whichcouldbequantifiedin

transparentWTembryos,includedmeasurementofstandardlarvaelengthusingaNikonSMZ

1500(NikonUSA,Melville,NY,USA)andNIS-ElementsD3.2software,andquantificationof

heartbeatratesusinganOlympusCX-41withaDP70camera.

3.2.4.2OxygenconsumptionOxygenintakebyWTzebrafishlarvaewasquantifiedusingamulti-frequencyphasefluorometer

(MFPF)(TauThetaLLC,USA).Oxygensensorswerepreparedbymixinghydrophobic

platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin(PtTFPP)(Frontier

Scientific,Logan,UT)andTiO2microparticles(Sigma-Aldrich,St.Louis,MO,USA)ina

polystyrenematrix.Theseweredepositedontothebottomsof1.5-mLclearglassvialsusing

200-μLmicropipettetips,whichwerecuredinthedarkovernight(Stensbergetal.,2014b).

Larvae(48-hpf)wereaddedintoeachvialwithtestsolution,whichwereimmediatelysealed

withTeflon-linedcaps.Changesinoxygenlevelswererecordedevery6huntil96hpfusingTau

thetasoftware(TauThetaLLC).NegativecontrolswithPVP-AgNPsdispersedin“fishtank”water

withoutlarvaeweretestedinparallel.Eachtreatmenthadaminimum8biologicalreplicates.

53

3.2.4.3GeneexpressionGeneexpressionwithintheVEGFpathway(vegf,vegfr,pi3k,pkc,plc,bcl2)wasquantifiedat

severaltimepoints(2,4,6,12,24and72hpf)usingquantitativeRT-PCR(qRT-PCR).Expression

ofhif1inthe10mg/LPVP-AgNPgroupandcontrolswasquantifiedat12,24,48,and72hpf.For

moreinformationonprimersused,seeTableA.2.1.Aminimumofthreebiologicalreplicates

consistingofgroupsofsix-pooledzebrafishembryos/larvaewasusedperconditionandwere

ranintriplicate.TotalRNAwasextractedusingTriSure(Bioline)followingre-suspensionofRNA

inwatertreatedwithdiethylpyrocarbonate.TotalRNAwasquantifiedusingaNanodrop2000

spectrophotometer(ThermoFisherScientific,USA).DNaseI(ThermoScientific,Lithuania)was

addedat37°Cfor30mintoremovegenomicDNAcontamination,andcDNAwassynthesized

usinghigh-capacityreverse-transcriptionkits(AppliedBiosystems,USA)followingthe

manufacturer’sinstruction.Elongationfactor1alpha1(ef1a)wasusedasthehousekeeping

gene,asitisstablyexpressedthroughoutzebrafishdevelopmentandisunaffectedbyAgNP

exposure(datanotshown).PrimerswerepurchasedfromIntegratedDNATechnologies

(Coralville,IA,USA)andgeneexpressionquantifiedusingaBio-RadCFX96system(Bio-Rad

Laboratories,Philadelphia,PA,USA).Eachreaction(20μLtotalvolume)contained10μLof2X

iQTMSYBRGreenSupermix(Bio-RadLaboratories170-8880),10μMgenespecificprimers

(forwardandreverse),60ngofcDNAtemplate,andnuclease-freewater.AllquantitativePCR

reactionswereperformedinduplicate.Geneexpressionlevelsweredeterminedbythe

comparativeCqmethod(LivakandSchmittgen,2001).

3.2.4.4ProteinexpressionWesternblottingwasusedtocharacterizetheeffectofPVP-AgNPsonVEGFproteinexpression

at12and24hpf.Twentyembryoswereplacedinatubeandhomogenizedusingalysisbuffer

(50mMTris,pH7.8,150mMNaCl,1%NonidetP-40).Totalproteinconcentrationwas

determinedusingbicinchoninicacid(BCAAssayKit,ThermoScientific,Rockford,IL,USA).

Sampleswereresolvedusinga12%SDS–polyacrylamidegel,thentransferredtopolyvinylidene

difluoride(PVDF)membranesforWesternblotanalysisusinganOdysseyinfraredimager(Li-Cor,

Lincoln,NE,USA).Primaryantibodiesusedincludepolyclonalanti-VEGF(Anaspec,Fremont,CA,

USA)andpolyclonalantiβ-actin(GenScript,Piscataway,NJ,USA),whichshowedstabilityat

differentdevelopmentalstagesofzebrafish,includingPVP-AgNPtreatedgroups.Secondary

54

antibodieswerelabeledwithIR-DYE800andIR-DYE700(Li-Cor).IntensityoftheVEGF

immunoreactivitywasnormalizedtothelevelofβ-actininthesample.

Table3.1Zetapotentialvaluesinfishembryomedium.

PVP-AgNPstestsolution 0.1mg/L 1mg/L 10mg/L

Temperature(°C) 25

Meanvalue(mV) -7.52 -9.27 -13.10

Standarddeviation(mV) 7.20 7.80 15.6

3.2.5Dataanalysis

AllstatisticalanalyseswereconductedusingSPSS22.0.Unlessotherwisenoted,resultsare

expressedasmeans±SE(standarderror)ofthreeormorebiologicalreplicates.Datawasfirst

assessedfornormality.One-wayanalysisofvariance(ANOVA)followedbypost-hocTukey’s

multiplecomparisontestsandLSDtests(forWesternblotproteinexpressiontest)wereusedto

comparemeansacrosstreatments.

3.3Results

3.3.1CharacterizationandstabilityofPVP-AgNPs

ThehydrodynamicdiameterofPVP-AgNPsinfreshlypreparedfishembryomediumwas

determinedbyNTAtobe51.1nmwitharelativestandarddeviationof4%,comparabletothe

sizeofPVP-AgNPsdispersedinpurewater(53.3;Fig.A.3.1).Allzetapotentialvalueswere

negative,rangingfrom–7.52to–13.10mV(Table2.1).ForAg+concentrations,anISEstandard

curvewasdevelopedfortherangebetween1and20,000µg/Landappliedtowardtheanalysis

ofAgNPsolutionsinfishmedia,whichweremonitoredforupto96h.Inallcases,Ag+

concentrationswerebelowdetectionlevels(1µg/L;datanotshown).

55

50

100

150

200

250

300

24 48 72 96IS

V Le

ngth

(μm

)Hour post fertilization (hpf)

Control 0.1mg/L 1mg/L 10mg/LA

* ** * *

65

70

75

80

85

24 48 72 96

Angl

e (o )

Hour post fertilization (hpf)

B

##

50

75

100

125

150

24 48 72 96

Inte

rval

(μm

)

Hour post fertilization (hpf)

* *

C

Figure3.2Changesinintersegmentalvessel(ISV)morphologyacrossdevelopmentincontrolandPVP-AgNPtreatedembryos.A.ISVlengthincreasedwithdevelopment.AreductionofISVsproutgrowthwasobservedat24hpfinembryosexposedto1and10mg/L(n=20),andat48hpfinalltreatedembryos(n�10).B.AnglebetweenISVsandDAdecreasedwithdevelopment.Anglewassignificantlylargerat72hpf(n=10)and96hpf(n=5)inembryosexposedto10mg/LPVP-AgNP,relativetocontrol.C.IntervaldistancebetweenISVsincreasedwithdevelopmentstage.At72hpfand96hpf,embryostreatedwith10mg/LPVP-AgNPhadasmallerintervaldistancerelativetocontrol.*p<0.05;#p<0.1.Linesovertopsofbarsdenotesignificantdifferences

56

3.3.2Vasculardevelopmentalmorphology

EarlyISVsproutingandsproutextensiondevelopmentoccursbetween20-25hpf.Disruptionof

somiteshapeorontheVEGFsignalingpathwaycanperturbtheorientationofISVsprouting

relativetotheDA.Asexpected,effectsonvasculardevelopmentwerehighlydependentonthe

embryonicstage.Comparedwith

the24-hpfcontrolgroup,embryos

exposedto1.0and10mg/LPVP-

AgNPsexperiencedadelayin

vasculogenesis,madeevidentbya

decreaseinISVslengthanda

completelackofISVdevelopment

(Figs.2.1B-Dand2.2A).Thisdelay

wasalsoevidentinthe0.1mg/L

groupat48hpf.Nodifferencesin

ISVlengthwereobservedat72hpf

betweentreatedandcontrol

specimens,howeverthehighest

dose(10mg/L)causedadecrease

inintervaldistancebetweenISVs

(Fig.3.2C).Incontrast,theanglebetweenISVandDAdecreasedbyroughly20%during

developmentbetween24to96hpf.Thereductioninsprout-to-DAangleoccurredatadelayed

rate,asnochangeswereobservedbetween48and96hpf(Fig.3.2B).CCVistheonlyvessel

passingovertheyolkforthefirst60hofzebrafishdevelopment,providingallvenousbloodand

returntotheheartduringthefirstweek(Belloetal.,2004).Weobservedacleardelayin

regressionoftheCCVinthePVP-AgNPtreatedgroup.Incontrolspecimensandatthelowest

PVP-AgNPdose,theCCVwasremodeledintoatubeby72hpf(Fig.3.3),however,regression

hadnotyetoccurredinlarvaetreatedwith1.0or10mg/LPVP-AgNP.But,by96hpf,therewas

nolongeranydifferenceinthedegreeofCCVregressionbetweengroups(datanotshown).

100μm

Control 0.1mg/L

1mg/L 10mg/L

Figure3.3EffectsofPVP-AgNPsoncommoncardinalvein(CCV)regression;boxedareaindicatesregionofinterest.TheCCV(whitearrow)inthecontrolspecimenisatubeofvascularendothelialcellsalongtheanteriormarginoftheyolkat72hpf;however,noregressionisobservedinembryostreatedwith10mg/LPVP-AgNP.Bar=200μm.

57

3.3.3Functionaltests

PVP-AgNPeffectsonheartrate(48and72hpf),oxygenconsumption(48to96hpf),and

standardbodylength(96hpf)werequantifiedinordertoexaminewhole-organismresponses.

PVP-AgNPsinduceddose-dependentbradycardiainlarvaeat48and72hpfexposure,startingat

1mg/LPVP-AgNP(Fig.3.4).Further,asignificantdecreaseinoxygenconsumptionwasobserved

inallconcentrationsstartingat84hpf(Fig.3.5A).From91to96hpf,groupstreatedwith1.0

and10mg/LPVP-AgNPrespondedwithasignificantdecreaseinoxygenconsumptioncompared

withcontrols(Fig.3.5B).Oxygenwasalsomeasuredovertimeincontrolvialsfilledwithfish

embryomediawithoutlarvae,withnoapparentchanges(datanotshown).Thisconfirmedthat

oxygenconsumptionwasdrivenbylarvaeratherthanmicrobialsources(e.g.,bacteria).Finally,

asignificantreductionintotalstandardlengthwasobservedforlarvaeexposedtoPVP-AgNPsat

1.0and10mg/L,andhighmortalitywascausedat10mg/Lgroupat96hpf(Figs.3.6andA.3.2).

0

10

20

30

40

50

60

C 0.1 1 10

Heartbeats/20second

s

PVP-AgNPsConcentration(mg/L)

48hpf 72hpf

** *

0

10

20

30

40

50

60

C 0.1 1 10

Heartbeats/20second

s

PVP-AgNPsConcentration(mg/L)

48hpf 72hpf

** *

Figure3.4Heartbeats(mean±SE)ofzebrafishembryosat48and72hpf.Significantreductioninheartbeatratewasobservedat48hpfforembryostreatedwith10mg/LPVP-AgNP;adecreaseinheartbeatratewasalsoobservedat72hpfforembryostreatedwith1mg/LPVP-AgNP(*p<0.05).C=control.

58

3.3.4Molecularmechanisms:geneandproteinexpression

TheVEGFsignalingpathwayplaysacriticalroleoncardiovasculardevelopmentandisup-

regulatedunderhypoxia.HIF1isatranscriptionfactorhighlyinduciblebycellularhypoxic

conditions.ToinvestigatethemolecularmechanismsofPVP-AgNPs’effectonvascular

developmentinembryos,relativeexpressionofgeneswithintheVEGFpathway(vegf,vegfr,

pi3k,pkc,plcandbcl2)wasquantifiedatsixtimepoints(2,4,6,12,24and72hpf)(Figs.3.7and

A.3.3A).Exceptforbcl2,theexpressionofallothergeneschangedinadose-andtime-

dependentmanner.ThehighestconcentrationofPVP-AgNPs(10mg/L)significantlyinduced

bc

B

0

2

4

6

8

82 84 86 88 90 92 94 96 98

OxygenPe

rcen

tage

HoursPostFertilization(hpf)

abc

bc

0

5

10

15

20

25

45 55 65 75 85 95OxygenPe

rcen

tage

HoursPostFertilization(hpf)

Control 0.1mg/L 1mg/L 10mg/L

abc

A

bc bc

Figure3.5Changesinoxygenlevel(mean±SE)afterexposingembyrostoPVP-AgNPs,upto96hpf.A.ContinuousdecreaseinoxygenlevelwasobservedforzebrafishembryosatallAgNPdoses,butwithsignificantlylessconsumptionversuscontrolat84hpf(a–c:0.1,1.0and10mg/Lgroups,p<0.05);at96hpf,onlythetwohighestAgNPdoses(1.0and10mg/L)differedfromcontrol(b,c:p<0.05).B.Expansionofboxedarea.

B

59

vegf,vegfr,plc,pkc,andpi3kfromearlylifestageembryos(4and6hpf).Apeakinexpressionof

pi3kwasobservedat4hpf,whereasexpressionoftheothergenespeakedat12hpfand

decreasedthereafter.By24hpf,gene

expressioninspecimenstreatedwith

PVP-AgNPwasmuchreducedalthough

stillhigherthanthecontrols,andwas

completelybacktobackgroundlevels

aroundthetimeofhatching(72hpf).

Expressionofthesamesixgeneswas

alsomeasuredinembryosexposedto

silverion(AgNO3,0.01,0.1and1.0

mg/L)at2,4,6,12and24hpf(Fig.

A.3.3B);withtheexceptionofbcl2,

geneexpressiondidnotchange

significantlyduringthisperiod.Expressionofhif1wasalsoquantifiedat4timepoints(12,24,48

and72hpf)incontrolsandgroupstreatedwith10mg/LPVP-AgNP(Fig.3.8).PVP-AgNPstreated

group,hif1peakedinexpressionat12hpfanddecreasedgraduallythereafteruntilreaching

baselinelevelsat72hpf.Todeterminewhetherchangesingeneexpressioncouldbeinducedby

othernanoparticles,weperformedacontrolstudywith50-nmPVP-AuNPsat10mg/L.However,

itsimpactonmRNAexpressionofVEGF-relatedgeneswasmodestatbest,relativetothat

observedforPVP-AgNPs(Fig.A.3.3C).Finally,todetermineiftheabovechangesinmRNAwere

translatedtotheproteinlevel,changesintheexpressionofVEGFafterPVP-AgNPexposurewas

performedin12and24hpfzebrafishembryos(Fig.3.9).However,VEGFexpressiondidnot

differacrosstreatmentgroups.

3.4Discussion

Weevaluatedthevasculartoxicityof50-nmPVP-AgNPsonzebrafishembryosandlarvaeforup

to96hpf.NTAandzetapotentialvaluesindicatedtheAgNPswerestableinsolutionsoverthis

period.Veryimportantly,ourresultsindicatethatthevasculotoxiceffectsareinducedbythe

AgNPsthemselves,astheAg+concentrationswereconsistently<1μg/L.Weshowthatanacute

(0–12hpf)exposureofembryostoPVP-AgNPsresultsinareversibledose-dependentinduction

inVEGF-relatedgeneexpression,whichrevertstobaselinelevelsjustaroundhatching.

C

0.1 mg/L

1 mg/L

10 mg/L

2500 3000 3500 4000

BodyLength(µm)

**

Figure3.6Bodylength(mean±SE)ofzebrafishlarvaeafterexposuretoPVP-AgNPsfor96h;larvaeexposedto1and10mg/LAgNPsweresmaller(*p<0.05).C=control.

60

Increasedgenetranscription,however,wasnotfollowedbythetranslationandproductionof

VEGF.CorrelatingwiththelackofVEGFexpression,vasculardevelopmentofPVP-AgNPstreated

embryoswasdelayedratherthanenhanced.Thethresholdfordelayedvasculardevelopment

andgrowthbyPVP-AgNPswasdeterminedtobe1mg/L.

WeproposethatzebrafisheggsexposedtoPVP-AgNPsat10mg/Lexperienceaphysical

interferencethatlimitsthediffusionofoxygen,leadingtoembryonichypoxiaandsubsequent

alterationsingenetranscription(Fig.3.10A).Inthisscenario,theoverexpressionofgeneswithin

theVEGFpathwayintheearlylifestagesofembryosmaybeinducedfromthelackofoxygen.

Thisargumentissupportedbythehighlevelsofexpressionofhif1at12hpf,withreversionto

0

2

4

6

2hpf 4hpf 6hpf 12hpf 24hpf 72hpf

Foldchange

vegfC 0.1mg/L1mg/L 10mg/L


vegfrC 0.1mg/L1mg/L 10mg/L

* * **

*

0

2

4

6


Foldchange

plc

**

*


pkc

** *

*

*

0

2

4

6


Foldchange

pi3k

**

*


bcl2

*


pkc

** *

*

*

*

* ** *

Figure3.7RelativeincreasesinmRNAexpression(mean±SE)forvegf,vegfr,plc,pkc,pi3k,andbcl2,atdifferenttimepointsofexposuretoPVP-AgNPs(*p<0.05).C=control.

61

baselinelevelsby24-72hpf(Fig.3.8).ExposuretoPVP-AgNPs,however,didnotresultin

increasedVEGFproduction,possiblybecauseofNPaggregationontheendoplasmicreticulum

(seebelow)(Fig.3.10B).Hatchingresultsinnormoxia,effectivelyremovingthehypoxia-driven

effectsofNPsongeneexpression.

EffectsoncardiovasculardevelopmentafterexposuretoPVP-AgNPswereconsistentwith

previousstudieswithzebrafish

embryosreportingbradycardia

anddecreasedperipheral

circulation(Asharanietal.,2008;

Massarskyetal.,2013).Abnormal

ordelayeddevelopmentofISVs

anddorsalmigrationoftheCCV

causedbyPVP-AgNPscould

explainsmallergrowthratesof

larvaeathatching.

NPsareknowntoaggregateon

thechorionsurfaceoffisheggs(Labanetal.,2010),causingthedepletionofoxygenexchange

andhypoxia(Zhuetal.,2012).InJapanesemedaka(Oryziaslatipes),35-nmAgNPs(62.5–1,000

μg/L)havebeenreportedtodisruptthenetworkoffinefilamentsfromeggs,resultingin

increasedanaerobicrespirationbyembryos(WuandZhou,2012).IndividualAgNPs(upto46

nm)areknowntobetransportedintoembryosthroughchorionporecanals(CPCs)(Leeetal.,

2007);AgNPscanalsoaggregateonthechorionexteriorofzebrafishembryoswithinthefirst24

hours(Osborneetal.,2013).Theseearlierobservationssupportourhypothesisthattheearly

induction(12hpf)ofmRNAexpressionofhif1andothergeneswithintheVEGFsignaling

pathwayiscausedbythephysicalblockageofoxygendiffusionbyAgNPs.

TheabsenceofVEGFproduction,despiteathreetofive-foldupregulationofvegf,isinaccord

withrecentfindingsindicatingtheinhibitionofproteintranslationbyNPaggregationonthe

endoplasmicreticulum(ER).InternalizationofNPsintotheERhasbeenreportedforcitrate-

AgNPsandPVP-AgNPs(75–100nm),aswellaszincoxideNPs(42nm)andhydroxyapatiteNPs

(60nm)(Zuckeretal.,2013;Chenetal.,2014;Hanetal.,2014).TheaccumulationofNPscan

0

5

10

15

12hpf 24hpf 48hpf 72hpf

Foldchange

Control PVP-AgNPs10mg/L

*

Figure3.8RelativeincreaseofmRNAexpressionforhypoxia-inducedfactor(hif),inducedby10mg/LPVP-AgNPsat12,24,48,and72hpf(*p<0.05).

62

induceERstress,aself-protectionmechanismagainstERdamageordysfunction(Linetal.,

2008;Gargetal.,2012).ERstresshasbeenstudiedinhumanChanglivercellsandChinese

hamsterlungfibroblastsexposedtoAgNPs<100nm,andisthoughttobecausedbythe

phosphorylationofRNA-dependentproteinkinase-likeERkinase(PERK)anditsdownstream

eukaryoticinitiationfactor2α,

phosphorylationofinositol-

requiringprotein1(IRE1)

(Zhangetal.,2012).Inzebrafish

embryosandhepatocytes,120-

nmAgNPsatloadingsaslowas

0.1mg/LinducedERstressas

measuredbyincreased

transcriptionofthebiomarkers

biPandsynv(Christenetal.,

2013).TheinductionofER

stressinzebrafishembryosby

PVP-AgNPsmayexplainwhy

theinducedexpressionof

VEGF-relatedgenesisnot

accompaniedbyprotein

translation.

3.5Conclusions

PVP-AgNPscausesdose-dependenthypoxiainzebrafishembryosstartingatloadingsof1mg/L,

resultingindelayedvasculardevelopmentanddecreasedlarvaegrowth.PVP-AgNPsalsocaused

bradycardiaandimpairedthetoleranceoflarvaetohypoxia.Theadverseeffectonvascular

developmentappearstobespecifictoAgNPs,asaparallelstudywithPVP-AuNPsdidnot

produceasignificantresponse(Fig.A.3.3C).Thisisthefirstinvivostudytofocusspecificallyon

thevasculartoxicityofPVP-AgNPs;theconsequencesoflowerlarvaesizeathatchingonlong-

termviabilityremainunknown,andmayrequireadditionalstudiesforamorecomprehensive

correlationwithNP-inducedchangesinvasculardevelopment.Wenotethat1mg/LofAgNPisa

VEGF(24h)β-ACTIN(24h)

VEGF(12h)β-ACTIN

(12h)

A.

0

50

100

150

200

C 0.1 1 10VEG

F/ActinRatio%

PVP-AgNPsConcentrationmg/L

12hpf 24hpfB.

Figure3.9VEGFproteinexpressionbywholezebrafishembryos,asafunctionofPVP-AgNPexposure(inmg/L).A.RepresentativeWesternblotsofVEGFandβ-actinproducedbyembryosat12and24hpf,showingnegligiblechangesinVEGFproduction.B.ChangesinVEGFproteinexpression(mean±SE)at12and24hpf;again,nosignificantdifferenceswereobservedbetweengroups(p>0.05).C=control.

63

relativelyhighconcentrationthatisunlikelytobeenvironmentallyrelevant(Gottschalketal.,

2009;Gottschalketal.,2013).Lastly,theapparentdisconnectbetweenAgNP-inducedmRNA

expressionandproteintranslationmaybeattributabletothesimultaneousinductionofER

stressbythesameNPs.

Figure3.10MechanisticmodelaccountingfortheobservedeffectsofPVP-AgNPsonthecardiovasculardevelopmentinzebrafishembryos.A.PVP-AgNPscoatthesurfaceofeggs,inducinghypoxiaandsubsequentexpressionofgenesintheVEGFsignalingpathway,withpeakexpressionat12hpfandreductiontobackgroundlevelsby72hpf.Inductionofthesegenes,however,doesnotleadtoincreasedproteinproduction.At48hpf,delayedvasculardevelopmentandbradycardiabecomeevident;at85hpf,oxygenconsumptionissignificantlydecreased.Uponhatching(>96hpf),larvaeexposedtoPVP-AgNPsarereleasedintonormoxiabutarealsosmallerrelativetocontrolspecimens.B.ProposedmolecularmechanismofvasculartoxicitybyPVP-AgNPs.Attheembryonicstage,thechorioniscoveredbyPVP-AgNPs,blockingoxygenintake.Hypoxia-inducedexpressionofVEGFpathwaygenesincreasesrapidly;atthesametime,intracellularPVP-AgNPsentertheendoplasmicreticulum(ER)andblockproteinsynthesis.Thelattereffectnotonlyblockshypoxia-inducedangiogenesis,butcanalsoincreasethemortalityofzebrafishembryosatalaterdevelopmentstage.

64

3.6APPENDIX

TableA.3.1Biologicalfunctionofgenesandprimersinformation.F:Forwardsequence;R:

Reversesequence.

Gene PrimerSequence BiologicalFunction Accession

No.

vegf-Aa F-TGCTCCTGCAAATTCACACAA Angiogenesisendothelialcell

proliferation,patterningblood

vessels.

XM_0092

92018.1R-ATCTTGGCTTTCACATCTGCAA

vegfr-

kdr

F-ATAAGAGCCCGCCAAAAGAG MediatesVEGF-induced

endothelialproliferation,

survival,migration,tubular

morphogenesisandsprouting.

NM_0010

24653.2R-AGACACCAGACAGGGAATGA

plc-

gamma

F-ACGGTGCTTTCCTTGTTAGG Mediatesdistinctaspectsof

arterydevelopmentandcontrols

thestrengthofheartbeating.

NM_1944

07.1R-AACTCCGAGGTTCCCAAAAC

pkc-α F-TTGAGGCAGAAAAACGTCCA Receptordesensitization,

membranestructureevents,

transcriptionandcellgrowth.

NM_0012

56241.1R-TGGCACTGAAATCCTTGCTT

pi3k

F-GTGAAGTTTGAGGGCAGTGA Regulatecellularproliferation,

survival,degranulation,vesicular

migration.

NM_2011

99.1R-AGGCTGTAGTCCTCTGGTTT

bcl2 F-ATGGCGTCCCAGGTAGATAAT Regulatesprogrammedcell

deathorapoptosis.

NM_0010

30253.2R-CAAGCCGAGCACTTTTGTTAG

hif1a F-GATGTCATGTTGCCCTCTTC Playrolesincellularresponseto

systemicoxygenlevels.

NM_0013

08559.1R-GGGAATTGGTCGTGTTGTAG

65

ef1a F-ATGGGAAAGGAAAAGACCCAC Enzymaticdeliveryofaminoacyl

tRNAstoribosome.

NM_1312

63.1R-TCCACCGCATTTGTAGATCAG

66

FigureA.3.1PVP-AgNPsparticlesizecharacterizationusingNanoTrackingAnalysis(NTA).Mean

sizeofPVP-AgNPsinfishembryomediumwas53.3nm.

FigureA.3.2MortalityrateofzebrafishembryosexposedtoPVP-AgNPsoverexposuretime.

0

20

40

60

80

100

1 21 41 61 81 101 121 141

Concen

tration(Particles/m

L)

x1000000

ParticleSize(nm)

PVP-AgNPsParticleSizeinFreeWater

PVP-AgNPsParticleSizeinFishEmbryoMedium

0102030405060708090

24 48 72 96

Mortality%

Developmentalstage(hpf)

Control 0.1mg/L 1mg/L 10mg/L

67

FigureA.3.3HeatmapsummarizinggeneexpressionprofilesforthePVP-AgNPs,AgNO3andPVP-

coatedgoldnanoparticles(PVP-AuNPs)treatedgroups.A.PVP-AgNPs(0.1,1and10mg/L)

treatedgroupsat2,4,6,12,24and72hpf;B.AgNO3(0.01,0.1and1mg/L)treatedgroupsat2,4,

6,12and24hpf.C.PVP-AuNPs(10mg/L)treatedgroupsat12,24,48and72hpf.

B.AgNO3A.PVP-AgNPs

C.PVP-AuNPs

68

CHAPTER4 NANOSILVERCOATEDSOCKSANDTHEIRTOXICITYTOZEBRAFISH(DANIORERIO)EMBRYOS

Reproducedfrom:Gao,J.,Sepúlveda,M.S.,Klinkhamer,C.,Wei,A.,Gao,Y.,&Mahapatra,C.T.(2015).Nanosilver-coatedsocksandtheirtoxicitytozebrafish(Daniorerio)embryos.Chemosphere,119,948-952(Gaoetal.,2015).

4.0Summary

Silvernanoparticles(AgNPs)arebeingincorporatedandareknowntobereleasedfromvarious

consumerproductssuchastextiles.However,nodataareavailableonthetoxicityofAgNPs

releasedfromanyofthesecommercialproducts.Inthisstudy,wequantifiedtotalsilver

releasedfromsocksintowashwaterbyinductivelycoupledplasmamassspectrometry(ICP-MS)

anddeterminedthepresenceofAgNPsusingtransmissionelectronmicroscopy(TEM).Wethen

exposedzebrafish(Daniorerio)embryosfor72htoeitherthisleachate(‘‘sock-AgNP’’)ortothe

centrifugate(‘‘spun-AgNP’’)freeofAgNPsandcomparedtheirtoxicitytothatofionicsilver

(Ag+).OurdatasuggestthatAgNPsdogetreleasedintothewashwater,andcentrifugation

eliminatedAgNPsbutdidnotdecreasetotalsilverconcentrations,indicatingthatmostofthe

silverinthesock-AgNPsolutionwasintheionicform.Allembryosdiedduringthefirst24h

whenexposedtoundilutedsock-AgNPandspun-AgNPsolutionsresultinginsignificantlylower

LC50values(0.14and0.26mg/L)comparedtoAgNO3(0.80mg/L).Similarly,at72hpf,bothsock-

derivedsolutionsweremorepotentataffectinghatchingandinducingabnormaldevelopment.

Theseresultssuggestthatbothsock-AgNPandspun-AgNPsolutionsweremoretoxicthan

AgNO3.Previousstudieshaveconsistentlyshowntheopposite,i.e.,AgNPsareabout10times

lesstoxicthatAg+.Alltogetherourresultsshowthatthehightoxicityinducedbytheleachateof

thesesocksislikelynotcausedbyAgNPsorAg+.Morestudiesareneededtoevaluatethe

toxicityofthemyriadofAgNP-coatedcommercialproductsthatarenowestimatedtobeclose

to500.

69

4.1Introduction

Silvernanoparticles(AgNPs)arebeingappliedasbactericidalagentsindifferentconsumer

products(Moronesetal.,2005;Duran,2007;Kimetal.,2007;Shahverdietal.,2007;Kokuraet

al.,2010;Lansdown,2010).AccordingtotherecentNanotechnologyConsumerProducts

Inventoryonemergingnanotechnology,thereare�425consumerproductscontainingAgNPs,

a26%increasesince2011(http://www.nanotechproject.org).Fromthissamereport,theuseof

AgNPsintextilesrepresentsanimportantemergingapplication.StudieshaveshownthatAgNPs

canbereleasedfromtextilesintowashwater.Forinstance,AgNP-coatedsocksreleasedas

muchas650μgofionicsilver(Ag+)into500mLofdistilledwater(BennandWesterhoff,2008).

ThesamegroupalsoreportedthatAg+wasreleasedinquantitiesofupto45μg/g(ppm)from

shirts,medicalmasks,toothpaste,shampoo,anddetergent(Bennetal.,2010).Althoughno

studieshavequantifiedtheconcentrationofAgNPsintheenvironment,concentrationsranging

from0.09to320ng/Linsurfacewaterhavebeenestimated(Blaseretal.,2008;Gottschalket

al.,2009).

DespitethehighpotentialofAgNP-coatedcommercialproductstoleachAgNPsandAg+into

washwaterandfromthereintoaquaticsystems,nostudieshaveevaluatedthetoxicityofthese

leachates.Incontrast,severalrecentstudieshaveevaluatedthetoxicityofdifferenttypesof

AgNPsonavarietyofanimalmodels.Inzebrafish(Daniorerio)embryos,ithasbeen

demonstratedthatAgNPscaninducesublethaleffectssuchasdevelopmentalabnormalities

includingbentnotochordandpericardialedema(Leeetal.,2007;Asharanietal.,2008;Bar-Ilan

etal.,2009)anddelayedhatching(Asharanietal.,2011;Georgeetal.,2011;Powersetal.,

2011)atconcentrationsrangingfrom1to50mg/LdependingonthetypeandsizeofAgNPs.

Effectsarealsoage-dependent,withhighestsensitivityobservedduringearlystages(64-128cell

stage)(Asharanietal.,2008).

TheobjectiveofthepresentstudywastoevaluatethetoxicityofleachatesfromAgNP-coated

sockstozebrafishembryosusingacombinationofstandardtoxicityassaysandmolecular

endpointsandtocomparetheseresultstopreviousreportsonAgNPtoxicityinthisanimal

model.WehypothesizedthatleachatesfromcommercialsocksimpregnatedwithAgNPswould

elicittoxiceffectstozebrafishembryosinamannersimilartothoseobservedwithpureAgNPs.

70


4.2.1Preparationofexposuresolutions

WefollowedthemethodusedbyBennandWesterhoff(2008)topreparethesocks’leachate.

Onepairofsocks(FoxRiverMills,X-StaticOdorFreeCrewSocks,Osage,IA,USA)coatedwith

AgNPswasplacedin3LultrapureMilliporeQ(MQ)waterandagitatedfor24honanorbital

shakerat50rpm(BennandWesterhoff,2008).Sockswerethenremoved,theleachate

collected(hereinreferredtoas“sock-AgNP”)andimmediatelycentrifugedat17,000rpmfor1h

toseparateAgNPsfromthesolution(hereinreferredtoas“spun-AgNP”)(Chaoetal.,2011).A

silvernitrate(AgNO3)(Sigma-Aldrich)stocksolution(10g/mLtotalsilver)waspreparedby

dissolvingitindeionizedwater.Allsolutionswerestoredat4˚C.

4.2.2Embryoexposureandtoxicitytesting

AdultABwild-typezebrafishweremaintainedattheAquaticEcologyLaboratory(Purdue

University)withaphotoperiodof14L:10Dandatemperatureof28±1°C.Fishwerefedad

libitumtwicedailywithacombinationofhatchedArtemianaupliiandcommercialfood

(Tetramin).Maleandfemalezebrafishwerehousedseparatelyforaweekandthedaybefore

eggswererequired,theywereplacedinbreedingtanksata2:1maletofemaleratio.Fishwere

leftundisturbedovernightandeggscollected1hafterthelightwasturnedonthenextmorning.

Eggswererinsedin0.0002%methyleneblueand20blastula-stageembryos(6hourspost

fertilization,hpf)wererandomlyplacedinplasticPetridishescontaining25mLofeachofthe

exposuresolutionsandexposeduntil72hpf.Treatmentsolutionswerediluted2,25,50,100

and200timesusingROwaterandevery100mLofthissolutioncontained15µLofembryo

media(13-14%calcium,1%magnesium,0.12%potassium,and0.6-0.7%sodium;Replenish,

SeachemLaboratoriesInc.,Madison,GA,USA).Thesedilutionscorrespondedto0.01,0.02,0.03,

0.04,0.05,0.09,0.4,0.8mg/Loftotalsilverinsocksolution;and0.01,0.03,0.04,0.06,0.07,

0.08,0.18,0.36mg/Linspunsolution.Halfofthetestsolutionwasreneweddaily.Eachtest

consistedofthreereplicatesandeachexperimentwasrepeatedatleastthreetimesfrom

embryoscollectedfromdifferentmatingevents.Numberofdeadembryos,hatchedlarvae,and

developmentalabnormalitieswererecordedusingaNikonSMZ1500stereomicroscope

equippedwithaNikonDigitalSightcamera.Arepresentativenumberofsurvivinglarvaewere

71

randomlycollectedfromeachofthetreatmentsandimmediatelyfrozenandstoredat−80˚Cfor

geneexpressionassays.

4.2.3QuantificationoftotalsilverandcharacterizationofAgNPs

Totalsilverwasquantifiedinthesock-AgNPandspun-AgNPsolutionsusinginductivelycoupled

plasma-massspectrometry(ICP-MS)asdescribedbyStensbergetal.(2014a).Briefly,8mLof

eachofthesesampleswasplacedin15mLfalcontubesand2mLof2%nitricacidaddedto

enhancedissolution.ConcentrationofdissolvedAginthesesampleswasquantifiedusingAgNO3

asastandardwithconcentrationsrangingfrom0.5to500µg/L.ForTEManalysis,a25times

moreconcentratedsampleofsock-AgNPandspun-AgNPsolutionwaspreparedseparatelywith

aCentrifanTMPEPersonalevaporator/condenser.Theconcentratedtestsolutionswerethen

placedonacoppergridcoatedwithacontinuouscarbonsupportfilm,air-driedatroom

temperatureandanalyzedunderaPhilipsCM100transmissionelectronmicroscope.Thecore

sizeofAgNPswasmeasuredat92,000-timemagnification(acceleratingvoltage100kV)andthe

detectionofAg+wasperformedusingelectrondispersiveX-rayanalysis(EDX)(OxfordINCA250

X-MAX80silicondrift).Dynamiclightscattering(DLS)andzetapotentialmeasurementswere

conductedusingaMalvernZetasizerNanoZS(Malvern,UK)at25°Cinembryomedium(15μL

Replenishin100mLtestsolution)(EPA,2002).Eachsamplewasanalyzedintriplicate.

Table4.1Primersequencesforsodandactingenes.

Gene PrimerSequence AccessionNo.

sod F-GACTGGTGAAATTACTGGCCTTAC NM131294.1

R-GGTCTCCGACGTGTCTAACACTAT

b-actin F-CTAAAAACTGGAACGGTGAAGG NM181601.4

R-AGGCAAATAAGTTTCGGAACAA

4.2.4Geneexpressionanalysis

IthasbeenpreviouslyreportedthatamainmechanismoftoxicityofAg+isoxidativestress(Choi

etal.,2010).Superoxidedismutases(sod)catalyzethedismutationofsuperoxide(O2−)

intooxygenandhydrogenperoxideandinthatwayprotectcellsfromoxidativedamagecaused

72

byreactiveoxygenspecies.Therefore,sodexpressioninzebrafishembryoswasanalyzedby

quantitativeRT-PCR(qRT-PCR).Foreachtestreplicate,three72hpfzebrafishlarvaewere

pooledfortotalRNAextractionusingTriSure(Bioline)reagentfollowingre-suspensionofRNAin

DEPC(diethylpyrocarbonate)treatedwater.TotalRNAwasquantifiedusingaNanodrop2000

spectrophotometer(ThermoFisherScientific)atabsorbance260nmandonlysampleswithratio

ofabsorbance260/280nm>1.8wereusedforfurtheranalysis.Samplesweretreatedwith

DNaseI(Fermentas)at37˚Cfor30minutestoremovegenomicDNAcontaminationandcDNA

wassynthesizedusingahigh-capacityreverse-transcriptionkit(AppliedBiosystems)according

tothemanufacturer’sinstructions.Primersforsodandthereferencegenebeta-actin(ß-actin)

werepurchasedthroughIntegratedDNATechnologies(Table4.1)andgeneexpressionwas

0

0.5

1

1.5

2

2.5

A B C D

Totalsilvercon

centration

(m

g/L)

Sock-AgNPsSpun-AgNPs

A.

A-sock

B-sock

C-sock

D-sockA-spun

B-spun

C-spun

-50

0

50

100

150

200 Avgdiameter(nm)Zetapotential(mV)

B.

Figure4.1A.Measuredconcentrationsoftotalsilverinsocks-AgNPandspun-AgNPsolutionsusinginductivelycoupledplasmamassspectrometry(ICP-MS)fromfourindependentexperiments.B.Particlesizedistributionofsilvernanoparticlesinsocks-AgNPandspun-AgNPsolutionsfromthreeindependentexperiments.Valuesweremeasuredatthestartoftheexperiment.

73

quantifiedusingaBio-RadCFX96system(Bio-RadLaboratories).Eachreaction(20µLtotal

volume)contained10µLof2XiQ™SYBR®GreenSupermix(Bio-RadLaboratories170-8880),10

µMgene-specificprimers(forwardandreverse),60ngofcDNAtemplate,andnucleasefree

water.AllquantitativePCRswereperformedinduplicatewithnotemplatenegativecontrols.

Thethresholdcycle(Ct)valuesbetweenreplicateswereaveraged.Aminimumofthree

biologicalreplicates(i.e.embryosfromthreeindependentexposuretests)wereusedper

exposureconcentration.Targetgeneexpressionwasnormalizedtothatofß-actinandgene

expressionlevelsdeterminedbythecomparativeCTmethod(LivakandSchmittgen,2001).

4.2.6Dataanalysis

AllstatisticalanalyseswereconductedusingSPSS16.0.ResultsofexposureassaysandqPCRare

expressedasmeans±SE(standarderror)ofthreeormoreindependentdeterminations.Tow-

200nm 100nm 200nm

A B D

C E

01234567891000.511.522.533.5

Figure4.2Transmissionelectronmicrograph(TEM)andEnergy-dispersiveX-rayspectroscopy(EDX)ofsilvernanoparticles:(A&B)Silvernanoparticles(<20nm)foundinthesock-AgNPsolution.AgglomerationofAgNPsisseenonthefabricsurfaceofAgNP-coatedsocks(arrows).(C)EDXanalysisconfirmsthepresenceofsilver.(D)Fiberresiduefoundinspun-AgNPsolution.NoAgNPswerefoundinthespun-AgNPsolution.(E)EDXanalysisshowsthatnosilverpeakswerepresentinthefiber.

X-rayenergy(KeV)X-rayenergy(KeV)

74

wayanalysisofvariance(ANOVA)followedbyposthocTukey’smultiplecomparisontestwas

usedtocomparemeansacrosstreatmentsanddoses.Probitanalysiswasusedtoquantify

effectsonsurvival.Significancewasdeclaredatp<0.05.

4.3Resultsanddiscussion

4.3.1Quantificationandcharacterizationofsilvernanoparticles

TheconcentrationoftotalAg+inthetestsolutions(sock-AgNPandspun-AgNP)issummarizedin

Fig.4.1A.Althoughthesameprocedurewasusedtopreparetheleachate,theamountoftotal

Ag+quantifiedinthesock-AgNPleachaterangedfrom0.83−2.24mg/Lacrossdifferent

replicates.ThisdifferencecouldsuggestanunevenadditionofAgNPsintothecommercialsocks

usedinthepresentstudy.ToinvestigatetheextenttowhichAgNPsaccountedfortheoverall

toxicityobserved,AgNPswereremovedbycentrifugationandthesupernatant(spun-AgNP)was

usedtoexposeembryos.TheamountoftotalAg+inthespun-AgNPsolutionswasalsovariable,

rangingfrom0.36−1.95mg/L(Fig.4.1A).Furthermore,therewerenosignificantdifferencesin

meantotalAg+betweenthesetwosolutions(1.49±0.60mg/Land1.34±0.69mg/L,forsock-

AgNPandspun-AgNP,respectively).

ThehydrodynamicdiameteroftheAgNPsbyDLSrangedfrom37to165nminthesock-AgNP

solution(mean±SE,78±11nm)andfrom14to50nminthespun-AgNPsolution(39±17nm).

Themeanzetapotentialvaluesinthesock-AgNPandspun-AgNPsolutionswere-15.8±8.06mV

and-18.5±9.69mV,respectively(Fig.4.1B),implyingthatparticlesinbothsolutionswere

1mm A

1mm a

b B 1mm

c

C

Figure4.3Exampleofthemajortypesofabnormalitiesobservedinsilvernanoparticlesexposedzebrafishembryosafter72hrs.(A)Normallarvae(B&C)deformedlarvaea.Pericardialedema.b.Yolksacedema.c.Spinalcurvature.

75

relativelystable.TEMandEDXanalysiswereperformedtoconfirmthepresenceofAgNPs(Fig.

4.2).TEMresultsindicatedthatAgNPswerereleasedintowashwater(Fig.4.2A)andalthough

notquantified,themajorityoftheparticleswereobservedstucktothefibers(Fig.4.2B).EDX

analysisconfirmedtheseparticlescontainedsilver(Fig.4.2C).Incontrast,noAgNPswere

observedinthespun-AgNPsolution(Figs.4.2D-E),althoughsomefibersremained.Eventhough

itispossiblesomeAgNPsremainedaftercentrifugation,theabsenceofdetectable

nanoparticlesintheTEManalysesstronglysuggestwewereabletosignificantly

decrease/eliminatethemfromthetestsolutions.Theparticulatematterdetectedinthespun-

AgNPsolutionbyDLSbuttheabsenceofAg+suggeststhepresenceofotherunknownparticles

andnotAgNPs.Altogether,thisdatasuggeststhat(i)AgNPsdogetreleasedintothewash

water,and(ii)centrifugation(17,000rpm)eliminatedAgNPsbutdidnotdecreasetotalsilver

concentrations.Thisimpliesthatmostofthesilverinthesock-AgNPsolutionwasintheionic

form,inagreementwithapreviousstudybyBennandWesterhoff(2008)usingthesamebrand

ofsocksandsimilarwashmethod.

Table4.272h-LC50andEC50responsesinzebrafishembryosexposedtosock-AgNP,spun-AgNPandAgNO3.Concentrationsareinmg/L.

Lethality(%) Hatchability(%) Abnormalities(%)

LC50 95%C.I. EC50 95%C.I. EC50 95%C.I.

Sock-AgNP 0.26 0.12-1.3 0.05 0.02-0.11 0.15 0.08-0.75

Spun-AgNP 0.14 0.07-0.8 0.04 0.005-0.22 0.12 0.08-0.78

AgNO3 0.80b 0.47-1.9 0.14c 0.03-0.41 -a -

a Novaluesareprovided sincenoabnormalitieswere inducedby theAgNO3 treatment; b SignificantdifferencewithSock-AgNPandSpun-AgNP(Pvalue<0.005);cSignificantdifferencewithSock-AgNPandSpun-AgNP(Pvalue=0.05).

76

4.3.2Toxicitytests

Acutetoxicitytestswereconductedwithdifferentconcentrations(range0.010−0.83mg/L)of

sock-AgNP,spun-AgNPandAgNO3.Zebrafishembryos(6hpf)wereexposedtothesesolutions

until72hpfandlethalandeffectiveconcentrationsresultingin50%deathoreffects(percent

hatching)werecalculated(72h-

LC50and72h-EC50,Table4.2).All

embryosdiedduringthefirst24h

whenexposedtoundilutedsock-

AgNPandspun-AgNPsolutions

resultinginsignificantlylowerLC50

values(0.14and0.26mg/L)

comparedtoAgNO3(0.80mg/L;

~anorderofmagnitudelower

comparedtoanotherstudythat

testedthesameagezebrafish

embryos,Griffittetal.,2008).

Similarly,at72hpf,bothsock-

derivedsolutionsweremorepotentataffectinghatchingandinducingabnormaldevelopment

(Table4.2).Further,noabnormalitieswereobservedwithAgNO3(seeFig.4.3forthemost

commonlyobservedabnormalities).Altogether,theseresultssuggestthatsock-AgNPandspun-

AgNPsolutionsweremoretoxicrelativetoAgNO3.Theseresultsareindisagreementwith

previousstudiesinzebrafishandotherorganisms,whichhaveconsistentlyshownthatAgNPs

arelesstoxiccomparedtoAg+(Asharanietal.,2008;Bar-Ilanetal.,2009;Bilbergetal.,2012;

Massarskyetal.,2013).Itisworthnotingthatintheseearlierstudiestheinvestigatorstested

pureAgNPs,whereasoursourceofAgNPswasderivedfromacommercialtextileproduct.These

resultssuggestthetoxicityisbeingelicitedbyotherelementsorcompoundsaddedduringthe

manufacturingprocess,ratherthanAgNPs.

4.3.3Oxidativestressgeneexpression

Sodcontrolsagroupofcriticalproteinsthatprotectcellsfromoxidativedamagecausedby

reactiveoxygenspecies.WetestedsodmRNAlevelafterexposuretothesock-derivedsolutions

0

2

4

6

8

10

0 0.1 0.4 0.8 1.2

FoldCha

nge

TotalAgConcentration(mg/L)

Sock Spun AgNO3

*

Figure4.4Relativesuperoxidedismutase(sod)mRNAlevelsinzebrafishembryosexposedtodifferentconcentrationsofsock-AgNP,spun-AgNPandAgNO3solutionsquantifiedbyqRTPCR(n=3pertreatment).Onlyspun-AgNPcausedamarkedup-regulationofsod(*p=0.007)

77

andtoAgNO3andfoundasignificantup-regulationonlyatthehighestconcentration(1.2mg/L)

ofspun-AgNP(Fig.4.4).Therearepreviousstudiesthathavemeasuredsodexpressionafter

exposuretoAgNPs,whichhavereportedeitheranupregulation(soilnematode,<100nmat

0.05-0.5mg/L),nochanges(adultzebrafishlivers,5-20nmat120mg/L,zebrafishembryos)

(Choietal.,2010)(Massarskyetal.,2013)ordown-regulation(earthworms,10-50nmat100-

500mg/kg)(Tsyuskoetal.,2012).Studiesidentifiedthattherewerenosignificantsod

expressionchangescausedbyAgNO3exposure(soilnematodes,0.05-0.5mg/L;zebrafish

embryos,0.03-1.55mg/L)(Rohetal.,2009,Massarskyetal.,2013).Wedidnotseeany

differentialexpressionofsodmRNAuponsock-AgNPorAgNO3exposure.Therefore,the

oxidativestressobservedwiththespun-AgNPsisnotlikelycausedbyAgNPsorAg+presentin

thewashwater.

4.4Conclusions

Thisstudyisthefirsttodeterminethetoxicityofcommercialproducts(socks)coatedwith

AgNPs.Ourresultsshowthatthehightoxicityinducedbytheleachateofthesesocksislikely

notcausedbyAgNPsorAg+.MorestudiesareneededthatevaluatethetoxicityofAgNP-coated

commercialproducts.

78

CHAPTER5 RESEARCHSUMMARY,GLOBALCONCLUSIONS&FUTUREDIRECTIONS

5.1ProteincoronaofAgNPsinfishplasma

5.1.1Impacttofield

InChapter2,wedemonstratedforthefirsttime,theuseoffish(smallmouthbass)plasmafor

thestudyofproteincorona(PC)formationandcompositiononthesurfaceofPVP-AgNPs.We

showdifferencesinthesizeandcompositionofPCthatweretimeandgender-dependent.Over

300differentproteinswereidentified,withthemajoritybeing<75kDainsize.Themost

commonproteinsincludedimmunoglobulins,complementcomponents,hemoglobin,blood

coagulationproteinsandapolipoproteins.Inaddition,female-specificeggproteins(vitellogenin

andzonapellucidaglycoproteins)werediscoveredbindingtotheparticlesonlyinfemale

plasma.WeproposethatfishplasmacanbeusedforfuturePCstudies.TheimplicationsofPC

formationontoxicityandbiodistributionofPVP-AgNPsremainunknown.

5.1.2Futureresearchneeds

PreviousresearchhasshownthatNPsize,shape,surfacecharge,functionalizationandcore

materialcanimpactthecompositionofPCs.Therefore,itisofimportancetostudyhowchanges

onanyofthesephysico-chemicalparameterscanaffectPCformationusingfishplasma.For

instance,differentsizeAgNPs(5,10,20,100nm)andcoating(citrate)couldbetested.

Additionalcorematerials,suchasgoldandtitanium,shouldalsobestudiedsinceprevious

researchhasshownthatthesizedistributionofproteinsishighlydependentonthisimportant

factor.AnotherareathatneedsexploringrelatestotheimplicationsofPCformationonAgNP

toxicityandorgandistribution.AlthoughwedetectedeggproteinsonthesurfaceofAgNPs,we

didnotquantifythemovementoftheseparticlesintotheovary.Itwouldalsobeofinterestto

studypotentialmaternaltransferofAgNPsintothedevelopingembryos.Morestudiesare

neededthatevaluatetheoverallimpactofPCformationontoxicity.Finally,aninteresting

hypothesisthatneedsfurtherstudyistherelationshipbetweenthetypesofproteinsthat

79

associatetoNPsandtheorgansweretheymoveto.Thisareaofresearchcouldalsobeusefulin

thefieldofnanomedicinethatseekstotargetspecificcellsandorgansfordiagnosticand

treatmentpurposes.

5.2VasculartoxicityofAgNPs

5.2.1Impacttofield

InChapter3,wereportedthatearlyacuteexposure(0–12hourspost-fertilization,hpf)of

zebrafishembryostoPVP-AgNPs(50nm)at1mg/Lorhigherresultsinatransient,dose-

dependentinductioninvascularendothelialgrowthfactor(VEGF)-relatedgeneexpressionthat

returnstobaselinelevelsathatching(72hpf).Hatchingresultsinnormoxia,negatingtheeffects

ofAgNPsonvasculardevelopment.Interestingly,increasedgenetranscriptionwasnotfollowed

bytheproductionofassociatedproteinswithintheVEGFpathway,whichweattributetoNP-

inducedstressintheendoplasmicreticulum(ER).Theimpairedtranslationmayberesponsible

fortheobserveddelaysinvasculardevelopmentatlaterstages,andforsmallerlarvaesizeat

hatching.Silverion(Ag+)concentrationswere<0.001mg/Latalltimes,withnosignificant

effectsontheVEGFpathway.WeproposethatPVP-AgNPstemporarilydelayembryonic

vasculardevelopmentbyinterferingwithoxygendiffusionintotheegg,leadingtohypoxic

conditionsandERstress.


AdditionalstudiesshouldbeconductedtocorroborateNP-inducedstresstotheERsincethis

couldimpactthetranslationofadditionalproteinswithinandoutsidetheVEGFpathway.

Anotherareathatneedsfurtherresearchrelatestothelong-termimpactofadelayinvascular

developmentinzebrafishembryos.Althoughourdatasuggestschangesaretransitoryand

reversible,wedidfindadecreaseinlarvaesizewhichcouldnegativelyaffectlarvaeandjuvenile

survival.

5.3ToxicityofcommercialproductscontainingAgNPs

5.3.1Impacttofield

InChapter4,weconfirmedthereleaseofAgNPsfromcommercialproducts(socks)intowash

wateranddemonstratedthattoxicityofthisleachatetozebrafishembryoswasnotduetosilver

intheNPformasweremoveditusingultracentrifugationandconfirmedtheirabsenceusing

80

transmissionelectronmicroscopy.Centrifugation,however,didnotdecreasetotalsilver

concentrations,indicatingthatmostofthesilverinthesock-AgNPsolutionwasintheionic

form.Further,thetoxicityofthisleachatewasmuchhigher(LC500.14-0.26mg/L)comparedto

thatofionicsilver(0.80mg/L).Alltogetherourresultsshowedthatthehightoxicityinducedby

theleachateofthesesockswaslikelynotcausedbyAgNPsorAg+,butbyunknownchemical(s).

Atthetimethisstudywaspublished,therewasnodataonthetoxicityofAgNPsreleasedfrom

anycommercialproducts.


Consideringthelargenumberofcommercialproducts(severalhundred)thatnowcontain

AgNPs,thereisarealneedtodeterminemovementofNPsand/orAg+todifferent

environmentalmedia.Additionally,morestudiesareneededthatevaluatethetoxicityofthese

products.ThemerepresenceofAgNPsinacommercialproductdoesnotnecessarilyimplythat

silverwillbereleasedandadverselyimpactorganisms.Inthecaseoftextiles,itwouldbeof

interesttobetterunderstandtheadditionaltypesofchemicalsthatareaddedinorderto

incorporateAgNPsintothem.Asweobservedwiththesocksexamined,thesechemicalscould

havehighertoxicitythansilver.

LISTOFREFERENCES

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VITA

91

VITA

JIEJUNGAO

Forestry&NaturalResourcesDepartment

PurdueUniversity

EDUCATION

Ph.D.,ForestryScienceandBiology,PurdueUniversity,WestLafayette,IN,USA.12/2016

• MajorConcentration:Fisheries&AquaticSciences• Thesis:NanoparticleToxicityandMolecularMechanismsinFish:ACaseStudywith

SilverNanoparticles.M.S.,PlantProtection(Pesticide),ChinaAgricultureUniversity,Beijing,China.05/2012

• Thesis:Bioconcentrationofmyclobutanilinadultzebrafishandtoxicityinzebrafish.B.S.,Chemistry,ChinaAgricultureUniversity,Beijing,China.05/2009

• MinorinMarketingofManagementPROFESSIONALEXPERIENCE

ResearchAssistant,AquaticmolecularbiologyandanalyticalLaboratory(Advisor:Dr.MariaS.Sepúlveda),PurdueUniversity,WestLafayette,IN08/2012-Present

ProteincoronaofNanoparticlesandcytotoxicology

• CompositionofproteinsbindingtothesurfaceofnanoparticlesafterincubatingwithsmallmouthbassplasmausingLC-MS/MS

SilverNanoparticlecardiovasculartoxiceffectsonzebrafishembryos

• Phenotypeeffectsofsilvernanoparticlesonvasculardevelopmentofzebrafish(Transgeniclines,fli:1a)embryos

• Heartdevelopmenteffectsofsilvernanoparticlesofzebrafish(Wildtype)embryos• Molecularmechanismsinvestigationofnanoparticlesvasculareffectsonzebrafish

embryos

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Nanosilver-coatedsocksandtheirtoxicitytozebrafishembryos

• TrackingnanoparticlesreleasefromcommercialproductsusingTEMandICP-MS• Washingwaterofcommercialproductsthatincludednanoparticlestoxicityteston

zebrafishembryosUndergraduateResearchFellow

• Ledtestingdifferentheartfunctionendpointsofsilvernanoparticlesacutetoxiceffectsonzebrafishembryos

Researchextention,PurdueUniversity,WestLafayette,IN,USA

• PurdueNanoDay:IntroducingNanotoxicologytohighschoolstudents05/2013• TheFamiliarFacesProjectFieldDay:Introducingecotoxicologyandzebrafishmodelto

highschoolstudents09/2016

Researchassistant,BiologicalAssayLab(Advisor:Dr.XuefengLi),ChinaAgricultureUniversity,Beijing,China2009-2012

Bioconcentrationandacutetoxictestofpesticideonaquaticorganisms

• Acutetoxicitytestofmyclobutanilondifferentstagesofzebrafish(Adult,yolk-saclarvae,embryos)

• BioconcentrationofmyclobutanilindifferenttissuesofadultzebrafishUndergraduateResearchFellow

• DevelopedmethodofdetectingconcentrationofmyclobutanilinfishmediumusingGC-ECD

• LedtooptimizetheextractionmethodofmyclobutanilfromfishtissuesResearchassistant,ChinesePesticidesRegistrationToxicityContractLab(Advisor:Dr.LihongQu),ChinaAgricultureUniversity,Beijing,China2009-2010

AcutetoxictestsofpesticideproductsonScenedesmusobliquus

• Acutetoxicitytestofaround100commercialpesticideproductsforregistrationinChinesemarket

• WritingofficialtoxicreportsforallofthetestedpesticideproductsInternship,QuzhouChinaAgricultureUniversityResearchStation,Heibei,China08/2008

InvestigatingpesticidesusageinChineseCountry

PUBLICATION

Gao,J.,Sepúlveda,M.S.,Klinkhamer,C.,Wei,A.,Gao,Y.,&Mahapatra,C.T.(2015).Nanosilver-coatedsocksandtheirtoxicitytozebrafish(daniorerio)embryos.Chemosphere,119,948-952.doi:10.1016/j.chemosphere.2014.08.031

Gao,J.,Mahapatra,C.T.,Mapes,C.D.,Khlebnikova,M.,Wei,A.,&Sepúlveda,M.S.(2016).Vasculartoxicityofsilvernanoparticlestodevelopingzebrafish(Daniorerio).Nanotoxicology,1-10.doi:10.1080/17435390.2016.1214763

93

Mu,X.,Pang,S.,Sun,X.,Gao,J.,Chen,J.,Chen,X.,Li,X.,&Wang,C.(2013).Evaluationofacuteanddevelopmentaleffectsofdifenoconazoleviamultiplestagezebrafishassays.EnvironmentalPollution,175,147-157.Doi:10.1016/j.envpol.2012.12.029

Gao,J.,Pang,S.,Wang,C.,Mu,X.,Li,X.,&Sepúlveda,M.S.Acutetoxicity,developmentaleffectsandbioconcentrationoffungicidemyclobutanilinzebrafish(Deniorerio).(Underreview)

Gao,J.,&Sepúlveda,M.S.Vasculotoxicityofmetal-basednanoparticles:Amini-review.(manuscript)

Gao,J.,Lin,L.,Wei,A.,&Sepúlveda,M.S.GenderspecificproteincoronainSmallmouthbassplasmaofsilvernanoparticles.(Manuscript)

PRESENTATIONS

Gao,J.,Mahapatra,C.T.,Mapes,C.D.,Wei,A.,Sepúlveda,M.S.(November2016).Proteincoronapredictsbiologicresponseandtoxicitymechanismsofsilvernanoparticlesinfish.Plateformpresentedatthe37thConferenceoftheSocietyofEnvironmentalToxicologyandChemistry,Orlando,FL,USA.

Gao,J.,Mahapatra,C.T.,Mapes,C.D.,Wei,A.,Sepúlveda,M.S.(March2016).Vasculartoxicityofnanoparticlestozebrafish(Daniorerio)embryos.PosterpresentedatPurdueUniversityForestryandNaturalResourcesSymposium,WestLafayette,IN,USA.

Gao,J.,Mahapatra,C.T.,Mapes,C.D.,Wei,A.,Sepúlveda,M.S.(March2016).Vasculartoxicityofnanoparticlestozebrafish(Daniorerio)embryos.Posterpresentedatthe55thConferenceoftheSocietyofToxicology,NewOrleans,LA,USA.

Gao,J.,Mahapatra,C.T.,Mapes,C.D.,Wei,A.,Sepúlveda,M.S.(March2016).Vasculartoxicityofnanoparticlestozebrafish(Daniorerio)embryos.PosterpresentedatSigmaXipostercompetition,PurdueUniversity,WestLafayette,IN,USA.

Gao,J.,Mahapatra,C.T.,Mapes,C.D.,Wei,A.,Sepúlveda,M.S.(November2015).Cardiovasculartoxicityofnanoparticlestozebrafish(Daniorerio)embryos.Plateformpresentedatthe36thConferenceoftheSocietyofEnvironmentalToxicologyandChemistry,SaltLakeCity,UT,USA.

Gao,J.,MahapatraC.T.,MapesC.D.,WeiA.,&Sepúlveda,M.S.(November2014).SilverNanoparticleEffectsonVasculogenesisandCardiovascularDevelopmentinZebrafishEmbryos.Posterpresentedatthe35thConferenceoftheSocietyofEnvironmentalToxicologyandChemistry,Vancouver,Canada.

Gao,J.,Sepúlveda,M.S.,Klinkhammer,C.,Wei,A.,Gao,Y.,&Mahapatra,C.T.(September2014)Nanosilver-CoatedSocksandtheirToxicitytoZebrafish(Daniorerio)Embryos.PosterpresentedatPurdueUniversityCenterfortheEnvironment,WestLafayette,IN,USA.

Gao,J.,Sepúlveda,M.S.,Klinkhammer,C.,Wei,A.,Gao,Y.,&Mahapatra,C.T.(April2014)NanosilverCoatedSocksandtheirToxicitytoZebrafish(Daniorerio)Embryos.PosterpresentedatPurdueUniversityForestryandNaturalResourcesSymposium,WestLafayette,IN,USA.

94

Gao,J.,Klinkhammer,C.,Sepúlveda,M.S.,Wei,A.,Gao,Y.,&Mahapatra,C.T.(September2013)TheEffectsofSilverNanoparticlesReleasedfromCommercialProductsonZebrafish(Daniorerio)Embryos.PosterpresentedatSocietyofToxicology-OhioValley,Louisville,KY,USA.

Gao,J.,Mahapatra,C.T.,Klinkhammer,C.,&Sepúlveda,M.S.(April2013).Theeffectsofsilvernanoparticlesreleasedfromcommercialproductsonzebrafish(Daniorerio)embryos.PosterpresentedatPurdueUniversityForestryandNaturalResourcesSymposium,WestLafayette,IN,USA.

Abdel-moneim,A.,Degan,D.,Gao,J.,&Sepúlveda,M.S.(April2016).Vitellogenin-Abiomarkerforgonadalintersexshowingseasonalvariabilityinsmallmouthbass.PosterpresentedatPurdueUniversityForestryandNaturalResourcesSymposium,WestLafayette,IN,USA.

Mapes,C.,Gao,J.,&Sepúlveda,M.S.(April2015).Studyingtheeffectsofsilvernanoparticlesonfishembryosdevelopment.PosterpresentedatPurdueUniversityUndergraduateStudentResearchSymposium,WestLafayette,USA.

AWARDS

SocietyofToxicology

• 2016AnnualMeetingGraduateStudentTravelFund2016SocietyofEnvironmentalToxicologyandChemistry

• NorthAmerican36thAnnualMeetingGraduateStudentTravelfund2015PurdueGraduateStudentGovernment

• GraduateStudentTravelGrant2015ChinaScholarshipCouncil,Beijing,China

• ChinaGovernmentScholarship2012-2016ChinaAgricultureUniversity,Beijing,China.

• ThirdPrizeScholarship2005-2006• SecondPrizeScholarship;JinZhengdaScholarship;Tri-ExcellentStudentHonor

2006-2007• ThirdPrizeScholarship2007-2008

VOLUNTEERSERVICES

FamiliarFacesProjectFieldDay

• Interactivetablewithinformationofecotoxicologyto100HighSchoolstudentsfromMerrillvilleHighSchool,WestLafayette,IN,USA.09/2016

SocietyofEnvironmentalToxicologyandChemistry

• NorthAmerican36thAnnualMeeting,SLC,UT,USA.11/2015PurdueUniversity,WestLafayette,IN,USA.

• PurdueResearchRoundtable11/2015• Nano-Days05/2013• SpringFestival04/2013

95

ChinaAgricultureUniversity,Beijing,China.

• ChinaBiomassEnergyExhibition03/2011

Documents

Nanoparticle toxicity and molecular mechanisms in fish: A