View
4
Download
0
Category
Preview:
Citation preview
Purdue UniversityPurdue e-Pubs
Open Access Dissertations Theses and Dissertations
12-2016
Nanoparticle toxicity and molecular mechanismsin fish: A case study with silver nanoparticlesJiejun GaoPurdue University
Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations
Part of the Surgery Commons, and the Toxicology Commons
This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact epubs@purdue.edu foradditional information.
Recommended CitationGao, Jiejun, "Nanoparticle toxicity and molecular mechanisms in fish: A case study with silver nanoparticles" (2016). Open AccessDissertations. 925.https://docs.lib.purdue.edu/open_access_dissertations/925
Graduate School Form30 Updated ����������
PURDUE UNIVERSITYGRADUATE SCHOOL
Thesis/Dissertation Acceptance
This is to certify that the thesis/dissertation prepared
By
Entitled
For the degree of
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):
Approved by:Head of the Departmental Graduate Program Date
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
3.2Materialsandmethods.......................................................................................................50
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
4.2Materialsandmethods.......................................................................................................70
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
5.2.2Futureresearchneeds..................................................................................................79
5.3ToxicityofcommercialproductscontainingAgNPs............................................................79
5.3.1Impacttofield..............................................................................................................79
5.3.2Futureresearchneeds..................................................................................................80
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)
39 HCMEC 1-100mg/L↑permeability,
inflammatoryresponse,
expressionvcam-1,icam-1,mcp-
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
andROSinterferingwithLH,
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
andROSinterferingwithLH,
CTSDandCTSB
(Halamoda
Kenzaouietal.,
2012)
Yttrium
oxide(Y2O3)
20-60 HAEC ≥10mg/L↑inflammatory
response,expressionicam-1,il-
8,mcp-1
(Gojovaetal.,
2007)
Zincoxide
(ZnO)
45 HCMEC 1-100mg/L↑permeability,
expressionvcam-1,icam-1,mcp-
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
3.2Materialsandmethods
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
2hpf 4hpf 6hpf 12hpf 24hpf 72hpf
vegfrC 0.1mg/L1mg/L 10mg/L
* * **
*
0
2
4
6
2hpf 4hpf 6hpf 12hpf 24hpf 72hpf
Foldchange
plc
**
*
2hpf 4hpf 6hpf 12hpf 24hpf 72hpf
pkc
** *
*
*
0
2
4
6
2hpf 4hpf 6hpf 12hpf 24hpf 72hpf
Foldchange
pi3k
**
*
2hpf 4hpf 6hpf 12hpf 24hpf 72hpf
bcl2
*
2hpf 4hpf 6hpf 12hpf 24hpf 72hpf
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
4.2Materialsandmethods
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.
5.2.2Futureresearchneeds
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.
5.3.2Futureresearchneeds
Consideringthelargenumberofcommercialproducts(severalhundred)thatnowcontain
AgNPs,thereisarealneedtodeterminemovementofNPsand/orAg+todifferent
environmentalmedia.Additionally,morestudiesareneededthatevaluatethetoxicityofthese
products.ThemerepresenceofAgNPsinacommercialproductdoesnotnecessarilyimplythat
silverwillbereleasedandadverselyimpactorganisms.Inthecaseoftextiles,itwouldbeof
interesttobetterunderstandtheadditionaltypesofchemicalsthatareaddedinorderto
incorporateAgNPsintothem.Asweobservedwiththesocksexamined,thesechemicalscould
havehighertoxicitythansilver.
LISTOFREFERENCES
81
LISTOFREFERENCES
Adams,R.H.,Alitalo,K.,2007.Molecularregulationofangiogenesisandlymphangiogenesis.NatRevMolCellBiol,8,464-478.Aillon,K.L.,Xie,Y.,El-Gendy,N.,Berkland,C.J.,Forrest,M.L.,2009.Effectsofnanomaterialphysicochemicalpropertiesoninvivotoxicity.AdvDrugDelivRev,61,457-466.Apopa,P.L.,Qian,Y.,Shao,R.,Guo,N.L.,Schwegler-Berry,D.,Pacurari,M.,Porter,D.,Shi,X.,Vallyathan,V.,Castranova,V.,Flynn,D.C.,2009.Ironoxidenanoparticlesinducehumanmicrovascularendothelialcellpermeabilitythroughreactiveoxygenspeciesproductionandmicrotubuleremodeling.PartFibreToxicol,6,1.Asharani,P.V.,LianWu,Y.,Gong,Z.,Valiyaveettil,S.,2008.Toxicityofsilvernanoparticlesinzebrafishmodels.Nanotechnology,19,255102.Asharani,P.V.,Lianwu,Y.,Gong,Z.,Valiyaveettil,S.,2011.Comparisonofthetoxicityofsilver,goldandplatinumnanoparticlesindevelopingzebrafishembryos.Nanotoxicology,5,43-54.Austin,C.A.,Umbreit,T.H.,Brown,K.M.,Barber,D.S.,Dair,B.J.,Francke-Carroll,S.,Feswick,A.,Saint-Louis,M.A.,Hikawa,H.,Siebein,K.N.,Goering,P.L.,2012.Distributionofsilvernanoparticlesinpregnantmiceanddevelopingembryos.Nanotoxicology,6,912-922.Bakkers,J.,2011.Zebrafishasamodeltostudycardiacdevelopmentandhumancardiacdisease.CardiovascRes,91,279-288.Bar-Ilan,O.,Albrecht,R.M.,Fako,V.E.,Furgeson,D.Y.,2009.Toxicityassessmentsofmultisizedgoldandsilvernanoparticlesinzebrafishembryos.Small,5,1897-1910.Beck,L.,D'Amore,P.A.,1997.Vasculardevelopment:cellularandmolecularregulation.FASEBJ,11,365-373.Bello,S.M.,Heideman,W.,Peterson,R.E.,2004.2,3,7,8-Tetrachlorodibenzo-p-dioxininhibitsregressionofthecommoncardinalveinindevelopingzebrafish.ToxicolSci,78,258-266.Benn,T.,Cavanagh,B.,Hristovski,K.,Posner,J.D.,Westerhoff,P.,2010.Thereleaseofnanosilverfromconsumerproductsusedinthehome.JEnvironQual,39,1875-1882.Benn,T.M.,Westerhoff,P.,2008.Nanoparticlesilverreleasedintowaterfromcommerciallyavailablesockfabrics.EnvironSciTechnol,42,4133-4139.Bilberg,K.,Hovgaard,M.B.,Besenbacher,F.,Baatrup,E.,2012.InVivoToxicityofSilverNanoparticlesandSilverIonsinZebrafish(Daniorerio).JToxicol,2012,293784.Blaser,S.A.,Scheringer,M.,Macleod,M.,Hungerbühler,K.,2008.Estimationofcumulativeaquaticexposureandriskduetosilver:contributionofnano-functionalizedplasticsandtextiles.SciTotalEnviron,390,396-409.Borm,P.J.,Robbins,D.,Haubold,S.,Kuhlbusch,T.,Fissan,H.,Donaldson,K.,Schins,R.,Stone,V.,Kreyling,W.,Lademann,J.,Krutmann,J.,Warheit,D.,Oberdorster,E.,2006.Thepotentialrisksofnanomaterials:areviewcarriedoutforECETOC.PartFibreToxicol,3,11.Buzea,C.,Pacheco,I.I.,Robbie,K.,2007.Nanomaterialsandnanoparticles:sourcesandtoxicity.Biointerphases,2,MR17-71.
82
Card,J.W.,Zeldin,D.C.,Bonner,J.C.,Nestmann,E.R.,2008.Pulmonaryapplicationsandtoxicityofengineerednanoparticles.AmJPhysiolLungCellMolPhysiol,295,L400-411.Carlson,C.,Hussain,S.M.,Schrand,A.M.,Braydich-Stolle,L.K.,Hess,K.L.,Jones,R.L.,Schlager,J.J.,2008.Uniquecellularinteractionofsilvernanoparticles:size-dependentgenerationofreactiveoxygenspecies.JPhysChemB,112,13608-13619.Casals,E.,Pfaller,T.,Duschl,A.,Oostingh,G.J.,Puntes,V.,2010.Timeevolutionofthenanoparticleproteincorona.ACSNano,4,3623-3632.Chang,J.,Ichihara,G.,Shimada,Y.,Tada-Oikawa,S.,Kuroyanagi,J.,Zhang,B.,Suzuki,Y.,Sehsah,R.,Kato,M.,Tanaka,T.,Ichihara,S.,2015.CopperOxideNanoparticlesReduceVasculogenesisinTransgenicZebrafishThroughDown-RegulationofVascularEndothelialGrowthFactorExpressionandInductionofApoptosis.JNanosciNanotechnol,15,2140-2147.Chao,J.B.,Liu,J.F.,Yu,S.J.,Feng,Y.D.,Tan,Z.Q.,Liu,R.,Yin,Y.G.,2011.Speciationanalysisofsilvernanoparticlesandsilverionsinantibacterialproductsandenvironmentalwatersviacloudpointextraction-basedseparation.AnalChem,83,6875-6882.Chatterjee,N.,Bhattacharjee,B.,2016.RevelationofZnSNanoparticlesInducesFollicularAtresiaandApoptosisintheOvarianPreovulatoryFolliclesintheCatfishMystustengara(Hamilton,1822).Scientifica(Cairo),2016,3927340.Chen,R.,Huo,L.,Shi,X.,Bai,R.,Zhang,Z.,Zhao,Y.,Chang,Y.,Chen,C.,2014.Endoplasmicreticulumstressinducedbyzincoxidenanoparticlesisanearlierbiomarkerfornanotoxicologicalevaluation.ACSNano,8,2562-2574.Childs,S.,Chen,J.N.,Garrity,D.M.,Fishman,M.C.,2002.Patterningofangiogenesisinthezebrafishembryo.Development,129,973-982.Choi,J.E.,Kim,S.,Ahn,J.H.,Youn,P.,Kang,J.S.,Park,K.,Yi,J.,Ryu,D.Y.,2010.Inductionofoxidativestressandapoptosisbysilvernanoparticlesintheliverofadultzebrafish.AquatToxicol,100,151-159.Choi,O.,Hu,Z.,2008.Sizedependentandreactiveoxygenspeciesrelatednanosilvertoxicitytonitrifyingbacteria.EnvironSciTechnol,42,4583-4588.Christen,V.,Capelle,M.,Fent,K.,2013.Silvernanoparticlesinduceendoplasmaticreticulumstressresponseinzebrafish.ToxicolApplPharmacol,272,519-528.Cleaver,O.,Tonissen,K.F.,Saha,M.S.,Krieg,P.A.,1997.NeovascularizationoftheXenopusembryo.DevDyn,210,66-77.Covassin,L.D.,Siekmann,A.F.,Kacergis,M.C.,Laver,E.,Moore,J.C.,Villefranc,J.A.,Weinstein,B.M.,Lawson,N.D.,2009.AgeneticscreenforvascularmutantsinzebrafishrevealsdynamicrolesforVegf/Plcg1signalingduringarterydevelopment.DevBiol,329,212-226.Crosera,M.,Bovenzi,M.,Maina,G.,Adami,G.,Zanette,C.,Florio,C.,FilonLarese,F.,2009.Nanoparticledermalabsorptionandtoxicity:areviewoftheliterature.IntArchOccupEnvironHealth82,1043-1055.DeJong,W.H.,Borm,P.J.,2008.Drugdeliveryandnanoparticles:applicationsandhazards.IntJNanomedicine,3,133-149.DeSmet,F.,Segura,I.,DeBock,K.,Hohensinner,P.J.,Carmeliet,P.,2009.Mechanismsofvesselbranching:filopodiaonendothelialtipcellsleadtheway.ArteriosclerThrombVascBiol,29,639-649.Decuzzi,P.,Ferrari,M.,2007.Theroleofspecificandnon-specificinteractionsinreceptor-mediatedendocytosisofnanoparticles.Biomaterials,28,2915-2922.Delov,V.,Muth-Köhne,E.,Schäfers,C.,Fenske,M.,2014.TransgenicfluorescentzebrafishTg(fli1:EGFP)y¹fortheidentificationofvasotoxicitywithinthezFET.AquatToxicol,150,189-200.
83
Ding,F.,Radic,S.,Chen,R.,Chen,P.,Geitner,N.K.,Brown,J.M.,Ke,P.C.,2013.Directobservationofasinglenanoparticle-ubiquitincoronaformation.Nanoscale,5,9162-9169.Docter,D.,Distler,U.,Storck,W.,Kuharev,J.,Wünsch,D.,Hahlbrock,A.,Knauer,S.K.,Tenzer,S.,Stauber,R.H.,2014.Quantitativeprofilingoftheproteincoronasthatformaroundnanoparticles.NatProtoc,9,2030-2044.Duan,J.,Yu,Y.,Li,Y.,Huang,P.,Zhou,X.,Peng,S.,Sun,Z.,2014.Silicananoparticlesenhanceautophagicactivity,disturbendothelialcellhomeostasisandimpairangiogenesis.PartFibreToxicol,11,50.Duan,J.,Yu,Y.,Li,Y.,Sun,Z.,2013a.Cardiovasculartoxicityevaluationofsilicananoparticlesinendothelialcellsandzebrafishmodel.Biomaterials,34,5853-5862.Duan,J.,Yu,Y.,Li,Y.,Zhou,X.,Huang,P.,Sun,Z.,2013b.ToxiceffectofsilicananoparticlesonendothelialcellsthroughDNAdamageresponseviaChk1-dependentG2/Mcheckpoint.PLoSOne,8,e62087.Duran,N.,2007.Antibacterialeffectofsilvernanoparticlesproducedbyfungalprocessontextilefabricsandtheireffluenttreatment.in:Marcato,P.D.,DeSouza,G.I.H.,Alves,O.L.,Esposito,E.(Eds.).J.Biomed.Nanotechnol.,203–208.Ehrhart,F.,Evelo,C.,Willighagen,E.,2015.Currentsystemsbiologyapproachesinhazardassessmentofnanoparticles.bioRxiv,028811.Eigenheer,R.,Castellanos,E.,Nakamoto,M.,Gerner,K.,Lampe,A.,Wheeler,K.,2014.Silvernanoparticleproteincoronacompositioncomparedacrossengineeredparticlepropertiesandenvironmentallyrelevantreactionconditions.EnvironmentalScience:Nano,238-247.Ellertsdóttir,E.,Lenard,A.,Blum,Y.,Krudewig,A.,Herwig,L.,Affolter,M.,Belting,H.G.,2010.Vascularmorphogenesisinthezebrafishembryo.DevBiol,341,56-65.EPA,2002.Methodsformeasuringtheacutetoxicityofeffluentsandreveivingwaterstofreshwaterandmarineorganisms.http://www.epa.gov/waterscience/methods/wet/disk2/atx.pdf.Flynn,T.,Wei,C.,2005.Thepathwaytocommercializationfornanomedicine.Nanomedicine,1,47-51.Fröhlich,E.,2012.Theroleofsurfacechargeincellularuptakeandcytotoxicityofmedicalnanoparticles.IntJNanomedicine,7,5577-5591.Gao,J.,Sepúlveda,M.S.,Klinkhamer,C.,Wei,A.,Gao,Y.,Mahapatra,C.T.,2015.Nanosilver-coatedsocksandtheirtoxicitytozebrafish(Daniorerio)embryos.Chemosphere119,948-952.Gao,J.,Mahapatra,C.T.,Mapes,C.D.,Khlebnikova,M.,Wei,A.,Sepúlveda,M.S.,2016.Vasculartoxicityofsilvernanoparticlestodevelopingzebrafish(Daniorerio).Nanotoxicology,1-10.Garg,A.D.,Kaczmarek,A.,Krysko,O.,Vandenabeele,P.,Krysko,D.V.,Agostinis,P.,2012.ERstress-inducedinflammation:doesitaidorimpedediseaseprogression?TrendsMolMed,18,589-598.George,S.,Xia,T.,Rallo,R.,Zhao,Y.,Ji,Z.,Lin,S.,Wang,X.,Zhang,H.,France,B.,Schoenfeld,D.,Damoiseaux,R.,Liu,R.,Bradley,K.A.,Cohen,Y.,Nel,A.E.,2011.Useofahigh-throughputscreeningapproachcoupledwithinvivozebrafishembryoscreeningtodevelophazardrankingforengineerednanomaterials.ACSNano,5,1805-1817.Gerhardt,H.,Golding,M.,Fruttiger,M.,Ruhrberg,C.,Lundkvist,A.,Abramsson,A.,Jeltsch,M.,Mitchell,C.,Alitalo,K.,Shima,D.,Betsholtz,C.,2003.VEGFguidesangiogenicsproutingutilizingendothelialtipcellfilopodia.JCellBiol,161,1163-1177.
84
Gojova,A.,Guo,B.,Kota,R.S.,Rutledge,J.C.,Kennedy,I.M.,Barakat,A.I.,2007.Inductionofinflammationinvascularendothelialcellsbymetaloxidenanoparticles:effectofparticlecomposition.EnvironHealthPerspect,115,403-409.Gore,A.V.,Monzo,K.,Cha,Y.R.,Pan,W.,Weinstein,B.M.,2012.Vasculardevelopmentinthezebrafish.ColdSpringHarbPerspectMed,2,a006684.Gottschalk,F.,Sonderer,T.,Scholz,R.W.,Nowack,B.,2009.Modeledenvironmentalconcentrationsofengineerednanomaterials(TiO(2),ZnO,Ag,CNT,Fullerenes)fordifferentregions.EnvironSciTechnol,43,9216-9222.Gottschalk,F.,Sun,T.,Nowack,B.,2013.Environmentalconcentrationsofengineerednanomaterials:reviewofmodelingandanalyticalstudies.EnvironPollut,181,287-300.Griffitt,R.J.,Luo,J.,Gao,J.,Bonzongo,J.C.,Barber,D.S.,2008.Effectsofparticlecompositionandspeciesontoxicityofmetallicnanomaterialsinaquaticorganisms.EnvironToxicolChem,27,1972-1978.Gurunathan,S.,Lee,K.J.,Kalishwaralal,K.,Sheikpranbabu,S.,Vaidyanathan,R.,Eom,S.H.,2009.Antiangiogenicpropertiesofsilvernanoparticles.Biomaterials,30,6341-6350.HalamodaKenzaoui,B.,ChapuisBernasconi,C.,Guney-Ayra,S.,Juillerat-Jeanneret,L.,2012.Inductionofoxidativestress,lysosomeactivationandautophagybynanoparticlesinhumanbrain-derivedendothelialcells.BiochemJ,441,813-821.Han,Y.,Li,S.,Cao,X.,Yuan,L.,Wang,Y.,Yin,Y.,Qiu,T.,Dai,H.,Wang,X.,2014.Differentinhibitoryeffectandmechanismofhydroxyapatitenanoparticlesonnormalcellsandcancercellsinvitroandinvivo.SciRep,4,7134.Hedrick,V.E.,LaLand,M.N.,Nakayasu,E.S.,Paul,L.N.,2015.Digestion,Purification,andEnrichmentofProteinSamplesforMassSpectrometry.CurrProtocChemBiol,7,201-222.Herbert,S.P.,Stainier,D.Y.,2011.Molecularcontrolofendothelialcellbehaviourduringbloodvesselmorphogenesis.NatRevMolCellBiol,12,551-564.Huang,H.,Lai,W.,Cui,M.,Liang,L.,Lin,Y.,Fang,Q.,Liu,Y.,Xie,L.,2016.AnEvaluationofBloodCompatibilityofSilverNanoparticles.SciRep,6,25518.Huang,X.,Tan,C.,Yin,Z.,Zhang,H.,2014.25thanniversaryarticle:hybridnanostructuresbasedontwo-dimensionalnanomaterials.AdvMater,26,2185-2204.Iruela-Arispe,M.L.,Davis,G.E.,2009.Cellularandmolecularmechanismsofvascularlumenformation.DevCell,16,222-231.Isogai,S.,Lawson,N.D.,Torrealday,S.,Horiguchi,M.,Weinstein,B.M.,2003.Angiogenicnetworkformationinthedevelopingvertebratetrunk.Development,130,5281-5290.Iversen,N.K.,Frische,S.,Thomsen,K.,Laustsen,C.,Pedersen,M.,Hansen,P.B.L.,Bie,P.,Fresnais,J.,Berret,J.-F.,Baatrup,E.,Wang,T.,2013.Superparamagneticironoxidepolyacrylicacidcoatedγ-Fe2O3nanoparticlesdonotaffectkidneyfunctionbutcauseacuteeffectonthecardiovascularfunctioninhealthymice.ToxicologyandAppliedPharmacology,266,276-288.Kalishwaralal,K.,Banumathi,E.,RamKumarPandian,S.,Deepak,V.,Muniyandi,J.,Eom,S.H.,Gurunathan,S.,2009.SilvernanoparticlesinhibitVEGFinducedcellproliferationandmigrationinbovineretinalendothelialcells.ColloidsSurfBBiointerfaces,73,51-57.Kamei,M.,Saunders,W.B.,Bayless,K.J.,Dye,L.,Davis,G.E.,Weinstein,B.M.,2006.Endothelialtubesassemblefromintracellularvacuolesinvivo.Nature,442,453-456.Karlsson,H.L.,Gustafsson,J.,Cronholm,P.,Möller,L.,2009.Size-dependenttoxicityofmetaloxideparticles--acomparisonbetweennano-andmicrometersize.ToxicolLett,188,112-118.
85
Karthikeyan,B.,Kalishwaralal,K.,Sheikpranbabu,S.,Deepak,V.,Haribalaganesh,R.,Gurunathan,S.,2010.GoldnanoparticlesdownregulateVEGF-andIL-1β-inducedcellproliferationthroughSrckinaseinretinalpigmentepithelialcells.Experimentaleyeresearch,91,769-778.Kerativitayanan,P.,Carrow,J.K.,Gaharwar,A.K.,2015.NanomaterialsforEngineeringStemCellResponses.AdvHealthcMater,4,1600-1627.Khandoga,A.,Stampfl,A.,Takenaka,S.,Schulz,H.,Radykewicz,R.,Kreyling,W.,Krombach,F.,2004.Ultrafineparticlesexertprothromboticbutnotinflammatoryeffectsonthehepaticmicrocirculationinhealthymiceinvivo.Circulation,109,1320-1325.Kim,J.S.,Kuk,E.,Yu,K.N.,Kim,J.H.,Park,S.J.,Lee,H.J.,Kim,S.H.,Park,Y.K.,Park,Y.H.,Hwang,C.Y.,Kim,Y.K.,Lee,Y.S.,Jeong,D.H.,Cho,M.H.,2007.Antimicrobialeffectsofsilvernanoparticles.Nanomedicine,3,95-101.Kokura,S.,Handa,O.,Takagi,T.,Ishikawa,T.,Naito,Y.,Yoshikawa,T.,2010.Silvernanoparticlesasasafepreservativeforuseincosmetics.Nanomedicine,6,570-574.Laban,G.,Nies,L.F.,Turco,R.F.,Bickham,J.W.,Sepúlveda,M.S.,2010.Theeffectsofsilvernanoparticlesonfatheadminnow(Pimephalespromelas)embryos.Ecotoxicology,19,185-195.Lansdown,A.B.,2010.Apharmacologicalandtoxicologicalprofileofsilverasanantimicrobialagentinmedicaldevices.AdvPharmacolSci,2010,910686.Lee,K.J.,Nallathamby,P.D.,Browning,L.M.,Osgood,C.J.,Xu,X.H.,2007.Invivoimagingoftransportandbiocompatibilityofsinglesilvernanoparticlesinearlydevelopmentofzebrafishembryos.ACSNano,1,133-143.Lee,Y.,Choi,J.,Kim,P.,Choi,K.,Kim,S.,Shon,W.,Park,K.,2012.Atransferofsilvernanoparticlesfrompregnantrattooffspring.ToxicolRes,28,139-141.Lesniak,A.,Fenaroli,F.,Monopoli,M.P.,Åberg,C.,Dawson,K.A.,Salvati,A.,2012.Effectsofthepresenceorabsenceofaproteincoronaonsilicananoparticleuptakeandimpactoncells.ACSNano,6,5845-5857.Lettiero,B.,Andersen,A.J.,Hunter,A.C.,Moghimi,S.M.,2012.Complementsystemandthebrain:selectedpathologiesandavenuestowardengineeringofneurologicalnanomedicines.JControlRelease,161,283-289.Liang,D.,Chang,J.R.,Chin,A.J.,Smith,A.,Kelly,C.,Weinberg,E.S.,Ge,R.,2001.Theroleofvascularendothelialgrowthfactor(VEGF)invasculogenesis,angiogenesis,andhematopoiesisinzebrafishdevelopment.MechDev,108,29-43.Lin,J.H.,Walter,P.,Yen,T.S.,2008.Endoplasmicreticulumstressindiseasepathogenesis.AnnuRevPathol,3,399-425.Liu,J.,Stainier,D.Y.,2012.Zebrafishinthestudyofearlycardiacdevelopment.CircRes,110,870-874.Liu,X.,Sun,J.,2010.EndothelialcellsdysfunctioninducedbysilicananoparticlesthroughoxidativestressviaJNK/P53andNF-kappaBpathways.Biomaterials,31,8198-8209.Liu,X.Q.,Zhang,H.F.,Zhang,W.D.,Zhang,P.F.,Hao,Y.N.,Song,R.,Li,L.,Feng,Y.N.,Hao,Z.H.,Shen,W.,Min,L.J.,Yang,H.D.,Zhao,Y.,2016.Regulationofneuroendocrinecellsandneuronfactorsintheovarybyzincoxidenanoparticles.ToxicolLett,256,19-32.Liu,Y.,Cox,S.R.,Morita,T.,Kourembanas,S.,1995.Hypoxiaregulatesvascularendothelialgrowthfactorgeneexpressioninendothelialcells.Identificationofa5'enhancer.CircRes,77,638-643.Livak,K.J.,Schmittgen,T.D.,2001.Analysisofrelativegeneexpressiondatausingreal-timequantitativePCRandthe2(-DeltaDeltaC(T))Method.Methods,25,402-408.
86
Long,Y.M.,Zhao,X.C.,Clermont,A.C.,Zhou,Q.F.,Liu,Q.,Feener,E.P.,Yan,B.,Jiang,G.B.,2016.Negativelychargedsilvernanoparticlescauseretinalvascularpermeabilitybyactivatingplasmacontactsystemanddisruptingadherensjunction.Nanotoxicology,10,501-511.Lundqvist,M.,Stigler,J.,Elia,G.,Lynch,I.,Cedervall,T.,Dawson,K.A.,2008.Nanoparticlesizeandsurfacepropertiesdeterminetheproteincoronawithpossibleimplicationsforbiologicalimpacts.ProcNatlAcadSciUSA,105,14265-14270.Mahdieh,Y.,Mahsa,S.,Andishe,K.,Parinaz,A.,Melika,T.,Sajad,S.,Mehrdad,M.,2015.TheeffectsofTiO2nanoparticlesonwhitebloodcellsinmice.DerPharmaciaLettre,153-156.Maiorano,G.,Sabella,S.,Sorce,B.,Brunetti,V.,Malvindi,M.A.,Cingolani,R.,Pompa,P.P.,2010.Effectsofcellculturemediaonthedynamicformationofprotein-nanoparticlecomplexesandinfluenceonthecellularresponse.ACSNano,4,7481-7491.Mangini,V.,Dell'Aglio,M.,DeStradis,A.,DeGiacomo,A.,DePascale,O.,Natile,G.,Arnesano,F.,2014.Amyloidtransitionofubiquitinonsilvernanoparticlesproducedbypulsedlaserablationinliquidasafunctionofstabilizerandsingle-pointmutations.Chemistry,20,10745-10751.Massarsky,A.,Dupuis,L.,Taylor,J.,Eisa-Beygi,S.,Strek,L.,Trudeau,V.L.,Moon,T.W.,2013.Assessmentofnanosilvertoxicityduringzebrafish(Daniorerio)development.Chemosphere,92,59-66.Milani,S.,Bombelli,F.B.,Pitek,A.S.,Dawson,K.A.,Rädler,J.,2012.Reversibleversusirreversiblebindingoftransferrintopolystyrenenanoparticles:softandhardcorona.ACSNano,6,2532-2541.Miquerol,L.,Langille,B.L.,Nagy,A.,2000.Embryonicdevelopmentisdisruptedbymodestincreasesinvascularendothelialgrowthfactorgeneexpression.Development,127,3941-3946.Monopoli,M.P.,Aberg,C.,Salvati,A.,Dawson,K.A.,2012.Biomolecularcoronasprovidethebiologicalidentityofnanosizedmaterials.NatNanotechnol,7,779-786.Morishita,Y.,Yoshioka,Y.,Takimura,Y.,Shimizu,Y.,Namba,Y.,Nojiri,N.,Ishizaka,T.,Takao,K.,Yamashita,F.,Takuma,K.,Ago,Y.,Nagano,K.,Mukai,Y.,Kamada,H.,Tsunoda,S.,Saito,S.,Matsuda,T.,Hashida,M.,Miyakawa,T.,Higashisaka,K.,Tsutsumi,Y.,2016.DistributionofSilverNanoparticlestoBreastMilkandTheirBiologicalEffectsonBreast-FedOffspringMice.ACSNano,10,8180-8191.Morones,J.R.,Elechiguerra,J.L.,Camacho,A.,Holt,K.,Kouri,J.B.,Ramírez,J.T.,Yacaman,M.J.,2005.Thebactericidaleffectofsilvernanoparticles.Nanotechnology,16,2346-2353.Mueller,N.C.,Nowack,B.,2008.Exposuremodelingofengineerednanoparticlesintheenvironment.EnvironSciTechnol,42,4447-4453.Mukherjee,P.,Bhattacharya,R.,Wang,P.,Wang,L.,Basu,S.,Nagy,J.A.,Atala,A.,Mukhopadhyay,D.,Soker,S.,2005.Antiangiogenicpropertiesofgoldnanoparticles.ClinCancerRes,11,3530-3534.Nasevicius,A.,Larson,J.,Ekker,S.C.,2000.DistinctrequirementsforzebrafishangiogenesisrevealedbyaVEGF-Amorphant.Yeast,17,294-301.Nel,A.E.,Mädler,L.,Velegol,D.,Xia,T.,Hoek,E.M.,Somasundaran,P.,Klaessig,F.,Castranova,V.,Thompson,M.,2009.Understandingbiophysicochemicalinteractionsatthenano-biointerface.NatMater,8,543-557.Neufeld,G.,Cohen,T.,Gengrinovitch,S.,Poltorak,Z.,1999.Vascularendothelialgrowthfactor(VEGF)anditsreceptors.FASEBJ,13,9-22.Oberdörster,G.,Oberdörster,E.,Oberdörster,J.,2005.Nanotoxicology:anemergingdisciplineevolvingfromstudiesofultrafineparticles.EnvironHealthPerspect,113,823-839.
87
Osborne,O.J.,Johnston,B.D.,Moger,J.,Balousha,M.,Lead,J.R.,Kudoh,T.,Tyler,C.R.,2013.EffectsofparticlesizeandcoatingonnanoscaleAgandTiO₂exposureinzebrafish(Daniorerio)embryos.Nanotoxicology,7,1315-1324.Pan,M.L.,Bell,W.J.,Telfer,W.H.,1969.Vitellogenicbloodproteinsynthesisbyinsectfatbody.Science,165,393-394.Pan,Y.,Neuss,S.,Leifert,A.,Fischler,M.,Wen,F.,Simon,U.,Schmid,G.,Brandau,W.,Jahnen-Dechent,W.,2007.Size-dependentcytotoxicityofgoldnanoparticles.Small,3,1941-1949.Pan,Y.,Wu,Q.,Qin,L.,Cai,J.,Du,B.,2014a.GoldnanoparticlesinhibitVEGF165-inducedmigrationandtubeformationofendothelialcellsviatheAktpathway.BiomedResInt,2014,418624.Pan,Y.,Wu,Q.,Qin,L.,Cai,J.,Du,B.,2014b.GoldNanoparticlesInhibitVEGF165-InducedMigrationandTubeFormationofEndothelialCellsviatheAktPathway.BioMedResearchInternational,p.11.Peters,A.,Dockery,D.W.,Muller,J.E.,Mittleman,M.A.,2001.Increasedparticulateairpollutionandthetriggeringofmyocardialinfarction.Circulation,103,2810-2815.Powers,C.M.,Slotkin,T.A.,Seidler,F.J.,Badireddy,A.R.,Padilla,S.,2011.Silvernanoparticlesalterzebrafishdevelopmentandlarvalbehavior:distinctrolesforparticlesize,coatingandcomposition.NeurotoxicolTeratol,33,708-714.Pozzi,D.,Caracciolo,G.,Capriotti,A.L.,Cavaliere,C.,LaBarbera,G.,Anchordoquy,T.J.,Laganà,A.,2015.Surfacechemistryandserumtypebothdeterminethenanoparticle-proteincorona.JProteomics,119,209-217.Quaife,N.M.,Watson,O.,Chico,T.J.,2012.Zebrafish:anemergingmodelofvasculardevelopmentandremodelling.CurrOpinPharmacol,12,608-614.Raldúa,D.,Piña,B.,2014.Invivozebrafishassaysforanalyzingdrugtoxicity.ExpertOpinDrugMetabToxicol,10,685-697.Roh,J.Y.,Sim,S.J.,Yi,J.,Park,K.,Chung,K.H.,Ryu,D.Y.,Choi,J.,2009.EcotoxicityofsilvernanoparticlesonthesoilnematodeCaenorhabditiselegansusingfunctionalecotoxicogenomics.EnvironSciTechnol,43,3933-3940.Rosas-Hernández,H.,Jiménez-Badillo,S.,Martínez-Cuevas,P.P.,Gracia-Espino,E.,Terrones,H.,Terrones,M.,Hussain,S.M.,Ali,S.F.,González,C.,2009.Effectsof45-nmsilvernanoparticlesoncoronaryendothelialcellsandisolatedrataorticrings.ToxicolLett,191,305-313.Scown,T.M.,vanAerle,R.,Tyler,C.R.,2010.Review:Doengineerednanoparticlesposeasignificantthreattotheaquaticenvironment?CritRevToxicol,40,653-670.Serbedzija,G.N.,Flynn,E.,Willett,C.E.,1999.Zebrafishangiogenesis:anewmodelfordrugscreening.Angiogenesis,3,353-359.Setyawati,M.I.,Tay,C.Y.,Docter,D.,Stauber,R.H.,Leong,D.T.,2015.Understandingandexploitingnanoparticles'intimacywiththebloodvesselandblood.ChemSocRev,44,8174-8199.Shahverdi,A.R.,Fakhimi,A.,Shahverdi,H.R.,Minaian,S.,2007.SynthesisandeffectofsilvernanoparticlesontheantibacterialactivityofdifferentantibioticsagainstStaphylococcusaureusandEscherichiacoli.Nanomedicine,3,168-171.Shalaby,F.,Ho,J.,Stanford,W.L.,Fischer,K.D.,Schuh,A.C.,Schwartz,L.,Bernstein,A.,Rossant,J.,1997.ArequirementforFlk1inprimitiveanddefinitivehematopoiesisandvasculogenesis.Cell,89,981-990.
88
Shalaby,F.,Rossant,J.,Yamaguchi,T.P.,Gertsenstein,M.,Wu,X.F.,Breitman,M.L.,Schuh,A.C.,1995.Failureofblood-islandformationandvasculogenesisinFlk-1-deficientmice.Nature,376,62-66.Shannahan,J.H.,Fritz,K.S.,Raghavendra,A.J.,Podila,R.,Brown,J.M.,2016.Disease-InducedDisparitiesinFormationoftheNanoparticle-BiocoronaandtheToxicologicalConsequences.ToxicolSci.Shannahan,J.H.,Lai,X.,Ke,P.C.,Podila,R.,Brown,J.M.,Witzmann,F.A.,2013.Silvernanoparticleproteincoronacompositionincellculturemedia.PLoSOne,8,e74001.Sheikpranbabu,S.,Kalishwaralal,K.,Lee,K.-j.,Vaidyanathan,R.,Eom,S.H.,Gurunathan,S.,2010.Theinhibitionofadvancedglycationend-products-inducedretinalvascularpermeabilitybysilvernanoparticles.Biomaterials,31,2260-2271.Sheikpranbabu,S.,Kalishwaralal,K.,Venkataraman,D.,Eom,S.H.,Park,J.,Gurunathan,S.,2009.SilvernanoparticlesinhibitVEGF-andIL-1beta-inducedvascularpermeabilityviaSrcdependentpathwayinporcineretinalendothelialcells.JNanobiotechnology,7,8.Shirinifard,A.,McCollum,C.W.,Bolin,M.B.,Gustafsson,J.,Glazier,J.A.,Clendenon,S.G.,2013.3Dquantitativeanalysesofangiogenicsproutgrowthdynamics.DevDyn,242,518-526.Simeonova,P.P.,Erdely,A.,2009.Engineerednanoparticlerespiratoryexposureandpotentialrisksforcardiovasculartoxicity:predictivetestsandbiomarkers.InhalToxicol,21Suppl1,68-73.Simkó,M.,Mattsson,M.O.,2010.Risksfromaccidentalexposurestoengineerednanoparticlesandneurologicalhealtheffects:acriticalreview.PartFibreToxicol,7,42.Stainier,D.Y.,Lee,R.K.,Fishman,M.C.,1993.Cardiovasculardevelopmentinthezebrafish.I.Myocardialfatemapandhearttubeformation.Development,119,31-40.Stapleton,P.A.,Nichols,C.E.,Yi,J.,McBride,C.R.,Minarchick,V.C.,Shepherd,D.L.,Hollander,J.M.,Nurkiewicz,T.R.,2015.MicrovascularandmitochondrialdysfunctioninthefemaleF1generationaftergestationalTiO2nanoparticleexposure.Nanotoxicology,9,941-951.Stensberg,M.C.,Madangopal,R.,Yale,G.,Wei,Q.,Ochoa-Acuña,H.,Wei,A.,McLamore,E.S.,Rickus,J.,Porterfield,D.M.,Sepúlveda,M.S.,2014a.Silvernanoparticle-specificmitotoxicityinDaphniamagna.Nanotoxicology,8,833-842.Stensberg,M.C.,Zeitchek,M.A.,Inn,K.,McLamore,E.S.,Porterfield,D.M.,Sepúlveda,M.S.,2014b.Comparativestudyofnon-invasivemethodsforassessingDaphniamagnaembryotoxicity.EnvironSciPollutResInt,21,10803-10814.Stone,J.,Itin,A.,Alon,T.,Pe'er,J.,Gnessin,H.,Chan-Ling,T.,Keshet,E.,1995.Developmentofretinalvasculatureismediatedbyhypoxia-inducedvascularendothelialgrowthfactor(VEGF)expressionbyneuroglia.JNeurosci,15,4738-4747.Su,L.,Han,L.,Ge,F.,Zhang,S.L.,Zhang,Y.,Zhao,B.X.,Zhao,J.,Miao,J.Y.,2012.Theeffectofnovelmagneticnanoparticlesonvascularendothelialcellfunctioninvitroandinvivo.JHazardMater,235-236,316-325.Sun,J.,Wang,S.,Zhao,D.,Hun,F.H.,Weng,L.,Liu,H.,2011.Cytotoxicity,permeability,andinflammationofmetaloxidenanoparticlesinhumancardiacmicrovascularendothelialcells:cytotoxicity,permeability,andinflammationofmetaloxidenanoparticles.CellBiolToxicol,27,333-342.Tabatabaei,S.R.,Moshrefi,M.,Askaripour,M.,2015.PrenatalExposuretoSilverNanoparticlesCausesDepressionLikeResponsesinMice.IndianJPharmSci,77,681-686.
89
Tal,T.L.,McCollum,C.W.,Harris,P.S.,Olin,J.,Kleinstreuer,N.,Wood,C.E.,Hans,C.,Shah,S.,Merchant,F.A.,Bondesson,M.,Knudsen,T.B.,Padilla,S.,Hemmer,M.J.,2014.Immediateandlong-termconsequencesofvasculartoxicityduringzebrafishdevelopment.ReprodToxicol,48,51-61.Tenzer,S.,Docter,D.,Kuharev,J.,Musyanovych,A.,Fetz,V.,Hecht,R.,Schlenk,F.,Fischer,D.,Kiouptsi,K.,Reinhardt,C.,Landfester,K.,Schild,H.,Maskos,M.,Knauer,S.K.,Stauber,R.H.,2013.Rapidformationofplasmaproteincoronacriticallyaffectsnanoparticlepathophysiology.NatNanotechnol,8,772-781.Treuel,L.,Malissek,M.,Gebauer,J.S.,Zellner,R.,2010.Theinfluenceofsurfacecompositionofnanoparticlesontheirinteractionswithserumalbumin.Chemphyschem,11,3093-3099.Treuel,L.,Nienhaus,G.U.,2012.Towardamolecularunderstandingofnanoparticle–proteininteractions.BiophysicalReviews,4,137-147.Tsyusko,O.V.,Hardas,S.S.,Shoults-Wilson,W.A.,Starnes,C.P.,Joice,G.,Butterfield,D.A.,Unrine,J.M.,2012.Short-termmolecular-leveleffectsofsilvernanoparticleexposureontheearthworm,Eiseniafetida.EnvironPollut,171,249-255.Vance,M.E.,Kuiken,T.,Vejerano,E.P.,McGinnis,S.P.,Hochella,M.F.,Rejeski,D.,Hull,M.S.,2015.Nanotechnologyintherealworld:Redevelopingthenanomaterialconsumerproductsinventory.BeilsteinJNanotechnol,6,1769-1780.Walkey,C.D.,Olsen,J.B.,Song,F.,Liu,R.,Guo,H.,Olsen,D.W.,Cohen,Y.,Emili,A.,Chan,W.C.,2014.Proteincoronafingerprintingpredictsthecellularinteractionofgoldandsilvernanoparticles.ACSNano,8,2439-2455.Wallace,R.A.,1985.Vitellogenesisandoocytegrowthinnonmammalianvertebrates.DevBiol(NY1985),1,127-177.Wen,Y.,Geitner,N.,Chen,R.,Ding,F.,Chen,P.,Andorfer,R.,Govindanb,P.,Ke,P.C.,2013.Bindingofcytoskeletalproteinswithsilvernanoparticles.RSCAdvances,22002–22007.Wu,Y.,Zhou,Q.,2012.Dose-andtime-relatedchangesinaerobicmetabolism,chorionicdisruption,andoxidativestressinembryonicmedaka(Oryziaslatipes):underlyingmechanismsforsilvernanoparticledevelopmentaltoxicity.AquatToxicol,124-125,238-246.Xia,X.R.,Monteiro-Riviere,N.A.,Riviere,J.E.,2010.Anindexforcharacterizationofnanomaterialsinbiologicalsystems.NatNanotechnol,5,671-675.Yu,T.,Greish,K.,McGill,L.D.,Ray,A.,Ghandehari,H.,2012.Influenceofgeometry,porosity,andsurfacecharacteristicsofsilicananoparticlesonacutetoxicity:theirvasculatureeffectandtolerancethreshold.ACSNano,6,2289-2301.Zhang,R.,Piao,M.J.,Kim,K.C.,Kim,A.D.,Choi,J.Y.,Choi,J.,Hyun,J.W.,2012.Endoplasmicreticulumstresssignalingisinvolvedinsilvernanoparticles-inducedapoptosis.IntJBiochemCellBiol,44,224-232.Zhang,W.-D.,Zhao,Y.,Zhang,H.-F.,Wang,S.-K.,Hao,Z.-H.,Liu,J.,Yuan,Y.-Q.,Zhang,P.-F.,Yang,H.-D.,Shen,W.,Li,L.,2016.Alterationofgeneexpressionbyzincoxidenanoparticlesorzincsulfateinvivoandcomparisonwithinvitrodata:Aharmoniouscase.Theriogenology,86,850-861.Zhang,X.D.,Wu,H.Y.,Wu,D.,Wang,Y.Y.,Chang,J.H.,Zhai,Z.B.,Meng,A.M.,Liu,P.X.,Zhang,L.A.,Fan,F.Y.,2010.Toxicologiceffectsofgoldnanoparticlesinvivobydifferentadministrationroutes.IntJNanomedicine,5,771-781.Zhao,Y.,Sun,X.,Zhang,G.,Trewyn,B.G.,Slowing,I.I.,Lin,V.S.,2011.Interactionofmesoporoussilicananoparticleswithhumanredbloodcellmembranes:sizeandsurfaceeffects.ACSNano,5,1366-1375.
90
Zhu,M.T.,Wang,Y.,Feng,W.Y.,Wang,B.,Wang,M.,Ouyang,H.,Chai,Z.F.,2010.Oxidativestressandapoptosisinducedbyironoxidenanoparticlesinculturedhumanumbilicalendothelialcells.JNanosciNanotechnol,10,8584-8590.Zhu,X.,Tian,S.,Cai,Z.,2012.Toxicityassessmentofironoxidenanoparticlesinzebrafish(Daniorerio)earlylifestages.PLoSOne,7,e46286.Zucker,R.M.,Daniel,K.M.,Massaro,E.J.,Karafas,S.J.,Degn,L.L.,Boyes,W.K.,2013.Detectionofsilvernanoparticlesincellsbyflowcytometryusinglightscatterandfar-redfluorescence.CytometryPartA,83,962-972.
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
92
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
Recommended