Lung distribution, quantification, co-localization and speciation of silver nanoparticles after lung exposure in mice

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Lung distribution, quantification, co-localization and speciation of silver nanoparticles after lung exposure in

mice

Stijn Smulders, Camille Larue, Géraldine Sarret, Hiram Castillo-Michel, Jeroen Vanoirbeek, Peter H.M. Hoet

To cite this version:

Stijn Smulders, Camille Larue, Géraldine Sarret, Hiram Castillo-Michel, Jeroen Vanoirbeek, et al..

Lung distribution, quantification, co-localization and speciation of silver nanoparticles after lung ex- posure in mice. Toxicology Letters, Elsevier, 2015, 238 (1), pp.1-6. �10.1016/j.toxlet.2015.07.001�.

�hal-02325190�

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Lung distribution, quanti ﬁ cation, co-localization and speciation of silver nanoparticles after lung exposure in mice

Stijn Smulders

^a,1

, Camille Larue

^b,1

, Geraldine Sarret

^b

, Hiram Castillo-Michel

^c

, Jeroen Vanoirbeek

^a

, Peter H.M. Hoet

^a,

*

aCenterforEnvironmentandHealth,KULeuven,Leuven,Belgium

bISTerre,UniversitéGrenobleAlpes,CNRS,F-38041Grenoble,France

cEuropeanRadiationSynchrotronFacility,Grenoble,France

HIGHLIGHTS GRAPHICAL ABSTRACT

LocalAgdistribution,andco-localiza- tionwithFe,CuandS,wasdetermined.

XRF,m^XANES,^andm^PIXE^techniques wereused.

Aquarterofallmacrophagesinthe lumenoftheairwayscontainedENPs.

A large part of the ENPs was dis- solvedandcomplexedtothiol-containingmolecules.

Ag,S-richspotswereenrichedinFe andCu,suggestiveformetallothio- neins(MTs).

ARTICLE INFO

Articlehistory:

Received14May2015

Receivedinrevisedform30June2015 Accepted2July2015

Availableonline7July2015

Keywords:

Nanoparticles Silver

MicroX-rayﬂuorescence(m^XRF) MicroX-rayabsorptionnearedgestructure spectroscopy(m^XANES)

Microproton-inducedX-rayemission (mPIXE)

ABSTRACT

Largeknowledgegapsstillexistonthetoxicologicalmechanismsofsilver(Ag)engineerednanoparticles (ENPs);acomprehensiveunderstandingofthesources,biodistribution,toxicityandtransformationofAg ENPsalongtheirlifecycleandaftertransferinlivingorganismsisneeded.Inapreviousstudy,micewere pulmonaryexposedtoAgENPsandlocal(lung)andsystemictoxiceffectstogetherwithbiodistribution toorgansincludingheart,liver,spleenandkidneywereinvestigated.Here,Aglungdistribution,local concentration,co-localizationwithotherelementssuchasFe,CuandS,andspeciationweredetermined afterlungexposuretoAgENPsusingmicroX-rayfluorescence(m^XRF),^micro^X-rayâbsorption^nearêdge structurespectroscopy(m^XANES)ând^microproton-inducedX-rayemission(m^PIXE)techniques.We foundthatapproximatelyaquarterofallmacrophagesinthelumenoftheairwayscontainedENPs.High localconcentrationsofAgwerealsodetectedinthelungtissue,probablyphagocytizedbymacrophages.

ThelargestpartoftheENPswasdissolvedandcomplexedtothiol-containingmolecules.Increased concentrations of Fe and Cu observed in the Ag-rich spots suggest that these molecules are metallothioneins(MTs).TheseresultsgivemoreinsightsonthebehaviorofAgENPsinthelungin vivoandwillhelpintheunderstandingofthetoxicologicalmechanismsofAgENPs.

ã2015ElsevierIrelandLtd.Allrightsreserved.

* Correspondingauthorat:DepartmentofPublicHealthandPrimaryCareCenterforEnvironmentandHealth,Herestraat49mailbox706,B-3000Leuven,Belgium.

E-mailaddresses:stijn.smulders@med.kuleuven.be(S.Smulders),camille.Larue@ruhr-uni-bochum.de(C.Larue),geraldine.sarret@ujf-grenoble.fr(G.Sarret), hiram.castillo-michel@esrf.fr(H.Castillo-Michel),jeroen.vanoirbeek@med.kuleuven.be(J.Vanoirbeek),peter.hoet@med.kuleuven.be(P.H.M. Hoet).

1 Theseauthorscontributedequallytothismanuscript.

http://dx.doi.org/10.1016/j.toxlet.2015.07.001

0378-4274/ã2015ElsevierIrelandLtd.Allrightsreserved.

ContentslistsavailableatScienceDirect

Toxicology Letters

j o u r n al h o m e p a g e :w w w . el s e v i e r . c o m / l o c a t e / t o x l e t

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1.Introduction

Due totheirantimicrobialproperties, silver(Ag) engineered nanoparticles(ENPs)areoneofthemostwidelyusedENPswith applicationsinpaintsandcoatings,cosmetics,electronics,water disinfection,foodpackagingandtextiles(HanusandHarris,2013;

Sometal.,2011;Lietal.,2008).Furthermore,AgENPscanbefound inbiomedicalapplicationsincludingprostheses,wounddressings andsurgicalinstruments(ChenandSchluesener,2008; Ahamed etal.,2010).

The growing use of Ag ENPs in commercial products will inevitablyleadtoincreasedexposuretoAgintheenvironmentand thegeneralpopulation(Larueetal.,2014).Topredicttheriskon human health, a comprehensive understanding of the source, biodistribution,toxicityandtransformationofAgENPsalongtheir lifecycleandaftertransferintolivingorganismsisneeded.The debateis going onabout the sourceof AgENP toxicityand it remainsuncleartowhatextentAgENPsorthereleasedAgionsare responsibleforthetoxiceffects(Yuetal.,2013).Inparticular,Ag ionsreleasedfromthe ENPswereshown tointeract withvital enzymes and proteins, affecting cellular respiration and ion transport,ﬁnally resultingin thedeathofbacteria, virusesand fungi(Levardetal.,2012).

Several studies have been conducted investigating the dissolution of Ag ENPs in artificial body fluids such as fluids representingtheenvironmentofthestomach,bloodandairways (Stebounova et al., 2011a; Leo et al., 2013). Leo et al. (2013) studiedtheeffectofamodelpulmonarysurfactantonAgENPs, andshowed a decrease in the kinetics of Ag ENP dissolution andof theiraggregation. Althoughtherearepublisheddataon Ag localization and speciation in plants exposed to Ag ENPs (Larueetal., 2014),equivalentdataonanimalcellsand tissues afterAgENPexposureislacking.Bothinvitroandinvivotoxicity of Ag ENPs has already been studied; toxic effects such as glutathionedepletion, mitochondrial deviations and damage to cellmembraneswereobservedinvitro(Wijnhovenetal.,2009).

Long-term or high-dose exposure to Ag ENPs in vivo leads to inﬂammation,smallgranulomatouslesionsandchangesin lung function(Sungetal.,2008;Sungetal.,2009;Stebounovaetal., 2011b).

Inapreviousstudy,weevaluatedthelocal(lung)andsystemic toxiceffectsalongwithbiodistributiontoorgansincludingheart, liver,spleenand kidneyafterlung exposuretoAgENPsinmice (Smuldersetal.,2014).Weobservedsometoxiceffectsasseenby anincreasedneutrophilcountanda 2-foldincrease inthepro- inﬂammatory cytokines keratinocyte chemoattractant (KC) and interleukin-1ß(IL-1ß)inthelungtissue,withnosystemictoxicity.

Moreover,extrapulmonarydistributionofAgtotheliver,spleen andkidneywasseen.Inthisstudy,micewereexposedtoAgENPs byoropharyngeal aspiration.Aglung distribution,localconcen- tration,co-localizationwithotherelementssuchasFe,CuandS, andspeciationweredeterminedusingseveraltechniquesinclud- ingmicroX-rayﬂuorescence(

m

^XRF),^microproton-inducedX-ray emission(

m

^PIXE),^Rutherford backscatteringspectroscopy(RBS) and micro X-ray absorption near edge structure (

m

^XANES)

spectroscopy.Thisstudygivesmoreinsights onthebehaviorof AgENPsinthelunginvivoandwillhelpintheunderstandingofthe toxicologicalmechanismsofAgENPs.

2.MaterialsandMethods 2.1.Materials

AgENPswereprovidedbyindustrialprojectpartnersinvolved intheEuropeanFP7projectNanohouse.ThesameAgENPswere alreadyusedinnanotoxicologicalstudies(Smuldersetal.,2014;

Kaiseretal.,2013),ecotoxicologicalstudies(Larueetal.,2014)and a studyassessing therelease ofAg ENPsfromnano-containing paints(Zuinetal.,2013).Isoﬂurane(Forene¹)wasobtainedfrom Abbott Laboratories (S.A. Abbott N.V., Ottignies, Belgium) and pentobarbital (Nembutal¹) from Sanoﬁ Santé Animale (CEVA, Brussels,Belgium).

2.2.Particlecharacterization

Dynamic light scatteringand zeta potential measurement wereperformed witha nanoZSinstrument (Malvern).Ag ENPs were suspended in saline (0.9% NaCl) at a concentration of 0.8mg/ml.

2.3.Mice

Male BALB/cOlaHsd mice(6weekold) wereobtainedfrom Harlan (Horst, The Netherlands). The mice were kept in a conventionalanimalhousewith12-hdark/lightcycles.Theywere housedinﬁltertopcagesandreceivedlightlyacidiﬁedwaterand pelletedfood (TrouwNutrition,Ghent, Belgium)ad libitum. All experimental procedures were approved by the local Ethical CommitteeforAnimalExperiments.

2.4.Experimentalprotocol

Ondays0,7,14,21and28,micereceivedunderlightisoﬂurane anesthesia an oropharyngeal aspiration (25

m

^l) ôf Âg ÊNPs

(0.8mg/ml)orvehicle(saline(0.9%NaCl)),thisresultsinatotal doseof100

m

^g^per^mouse⁽⁴^mg/kg).^Mice^were^sacriﬁcedbyan intraperitonealinjectionofpentobarbital(90mg/kgbodyweight) 2 days after the last exposure (day 30) and an autopsy was performed.

2.5.Bronchoalveolarlavage

Thelung waslavaged,insitu,three timeswith0.7mlsterile saline (0.9% NaCl), and the recovered ﬂuid was pooled. The bronchoalveolar lavage (BAL) ﬂuid was centrifuged (1000g, 10min) and 250

m

^l ^of ^the resuspended pellets (100,000 cells/

ml)werespun(300g,6min)(Cytospin3,Shandon,TechGen,Zellik, Belgium)ontomicroscopeslides,air-driedandstained(Diff-Quik¹ method, Medical Diagnostics, Düdingen, Germany) for macro- phageandneutrophilcellcounts.AftertakingBALﬂuid,thelungs wereinstilledwith4%formaldehydeuntilfullinﬂationofalllobes, asjudgedvisually,forfurtheranalyses.

2.6.

m

XRFand

m

^XANES

m

^XRFândÂg^LÎII^-edge

m

^XANESmeasurementswereperformed onthescanningX-raymicroscopeonID21beamlineattheESRF (European Synchrotron Radiation Facility, France) in cryo-con- ditionsusinga vibration-free cryo-stage,passivelycooledbya liquidnitrogendewar.

Asmallpieceofthelungwasﬂash-frozeninliquidnitrogen, embeddedinOCT¹resinandthencutinthinsections(20

m

^m)

usingacryomicrotomeanddirectlytransferredtothecryo-stage.

m

^XRF^maps^were^recorded^with^various^step^sizes^(from^0.3

m

^m^x

0.3

m

^m ^to ³

m

^m ^x ³

m

^m) ^with încident ênergy ôf ^3.42 ^keV,

and dwell time of 200ms.

m

^XRF ^data ^were ^processed ^using

PyMcasoftware (Soleetal.,2007)asin(Larueet al.,2014).Ag LIII-edge

m

^XANES^spectra^were^recordedⁱⁿ^regionsôfînterestôf

the maps (Ag-rich regions).

m

^XANES ^data ^processing ^and

linearcombinationﬁttingwereachieved withAthenasoftware using reference compounds acquired previously (Larue et al., 2014).

2 S.Smuldersetal./ToxicologyLetters238(2015)1–6

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2.7.

m

PIXEandRBS

Distributionofendogenouselements(P,S,K,FeandCu)andAg wasmappedby

m

^PIXE^coupled^to^RBS^recordedsimultaneouslyon theAIFIRAnuclearmicroprobe(ApplicationsInterdisciplinairesde Faisceauxd’IonsenRégionAquitaine,CENBG,France).A2.5MeV protonbeam was focused to2.5

m

^m2.5

m

^m ^with^an ^average

intensityof1000pA.Freeze-driedcross-sectionscuttedfromthe samples prepared during the synchrotron experiment were analyzed.DatawereprocessedusingSIMNRA(Mayer,1999)and Gupix(Campbelletal.,2000)softwares.

2.8.Statisticalanalysis

DatainTable1arepresentedasmeanandstandarddeviations (SD).Alldatawereanalyzedusingthenon-parametricKruskal– Wallis test followed by a Dunn’s multiple comparison test (GraphpadPrism4.01,Graphpad SoftwareInc.,SanDiego,USA).

Alevelofp<0.05wasconsideredsigniﬁcant.

3.Results

AnalysisoftheENPsbydynamiclightscattering(DLS)showeda polydisperse suspensie with 3 populations: 130nm (11% in number),506nm(89%innumber),and5180nm(0.1%innumber).

The particles werenegatively charged witha zeta potential of 31mV.Inductivelycoupledplasmaopticalemissionspectrosco- py(ICP-OES)measurementsshowedimpuritiesofCa(216.8

m

^g/g),

Fe(23.8

m

^g/g)^and^Cu^(0.6

m

^g/g).Transmissionelectronmicroscopy (TEM)analysisofAgENPshasbeenpublishedbeforeby(Smulders et al., 2012) and showed a heterogenous composition with spherical(25nm)androd-shaped(80–90nm)ENPs(Fig.S1).

Increasesintotalcells(88.7(14.6)10³vs38.2(15.4)x10³), macrophages (75.8 (17.4)10³ vs 37.9 (16.6)10³) and neutrophils (12.9 (10.4)10³ vs 0.3 (0.3)10³) in the BAL ﬂuidwereseeninAg-exposedmicecomparedtocontrolmice.In theAg-exposedgroup26%oftheBALmacrophagescontainedENPs asjudgedvisuallyusinglightmicroscopy(Fig.1).Notethatthis visualexaminationdetectslargeagglomeratesorprecipitatesofAg only.NoincorporationofENPswasobservedinneutrophilsofthe BALﬂuid.

TheelementaldistributionofAginthelungafterlavagewas studiedby

m

^XRFôn^thin^sections.^No^spotsôfÂg^wereôbservedⁱⁿ

non-exposedcontrolanimals(Fig.S2).Lungslicesofmiceexposed to Ag ENPs showed clear round-shaped hot spots of Ag;

magniﬁcation of theAg-rich regionsshowspotsizesof around 5

m

^m^(Fig.²^and^Fig.^S3).^LIII-edge

m

^XANES^spectra^were^recorded

onthreeAg-richspotsidentiﬁedby

m

^XRF^and^Ag^speciation^was

determinedbylinearcombinationﬁts(Fig.3).Intwospots,Agwas stillpartiallypresentaselementalAg(spot2:37%,spot3:55%).

Spot1showed100%oxidativedissolutionandrecomplexationof Ag⁺ ions to thiol-containing molecules. These molecules may correspondtocysteine,glutathioneand/ormetallothionein(MT),

although XANES cannot distinguish among them. Spot 2 and 3showedrespectively63%and45% dissolutionandrecomplex- ationtothesametypeofligands.

m

^PIXE ând ^RBS ânalyses ^were ûsed ^toôbtain ^local ^concen-

trationsofAg.Threeoutofeightanalyzedregions(approximately 200

m

^m200

m

^m)ⁱⁿ^the^lung^tissue^showed^the^presence^of^Ag

spotswithanaveragesizeof5

m

^m.^Two^of^these^regions^are^shown

inFig.S4.TheconcentrationofdifferentelementsinAg-richspots weredeterminedandcompared toconcentrationsin thewhole lungsection.SigniﬁcanthigherconcentrationsofCu,FeandSwere foundintheAg-rich spots(Table1).TheCu/Ag,Fe/AgandS/Ag ratiosweremuchhigherthanintheoriginalNPs,sotheseelements didnotcomefromtheimpuritiesdetectedinthem.

4.Discussion

AsforallENPs,nano-Agmayenterthehumanbodythrough inhalation,skincontactandingestion(food,drinkingwater),and havethepotentialtopasstheair–bloodandeventheblood–brain barrier(Yangetal.,2010).InhalationofdustcontainingAgENPs occursprimarilyinoccupationalsettingsincludingmanufacturing and application of Ag-containing products. Inhalation studies showed distribution of Ag toextrapulmonary organs including liver,kidney,spleen,brainandheart;however,thesestudiesdid not reveal whether Ag reached these organs as ions or ENPs (Sungetal.,2009;Takenakaetal.,2001;Jietal.,2007).AgENP exposurecancausegenotoxiceffectsandcellulardamagethrough generation of oxidative stress, resulting in inﬂammation and apoptoticornecroticdeath.Thesetoxiceffectsstronglydependon thephysico-chemicalcharacteristicsoftheAgENPs.

In this study, we investigated the distribution, speciation, concentrationandco-localizationwithotherelementsofAgENPs in the lungs of mice. We observed uptake of Ag ENPs by approximatelyaquarterofallmacrophagesinthelumenofthe airways (BALﬂuid),whilenoneutrophilscontainingENPswere found. Furthermore,Ag was also foundin thelung tissue; we assumethattheseAg-richspotsrepresentmacrophagesﬁlledwith ENPspresentinthelumen(notremovedbylavage)andinthelung tissue.However,uptakebylungepithelialcellscannotbeexcluded.

Table1

Elementalconcentrations(mg/gdryweight)inlungsectionsandAg-richspotsasdeterminedbythecombinationofmicroproton-inducedX-rayemission(m^PIXE)^with Rutherfordbackscatteringspectroscopy(RBS).ConcentrationsofAg,Cu,Fe,P,S,KandweredeterminedbothinwholelungsectionsandmorespeciﬁcinAg-richspots.

Element Wholelungsection Mean

Ag-richspots

Ag-richspot1 Ag-richspot2 Ag-richspot3

Ag 0.20.3 14.812.2^* 27.9 13.0 3.6

Cu 0.100.03 0.230.04^* 0.27 0.19 0.23

Fe 0.970.07 1.180.06^* 1.12 1.22 1.19

P 27540 35527 325 376 364

S 20440 36260^* 365 421 302

K 10833 14241 121 115 190

* p<0.05comparedtowholelungsection.n.d.:notdetermined.

Fig.1.Representative imageofmacrophagewithoutandwithAgENPs inits cytoplasm.

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Otherinhalationandinstillationstudiesalreadyshowedthatthe largestpartof theENPs werephagocytized byalveolarmacro- phagesandtransportedtothedraininglymphnodesorremoved byrespiratorymucociliaryclearance(GeiserandKreyling,2010).

Onceremovedfromthelungs,theycan reachthestomachand gastrointestinaltract,canbetakenupinthegutorwillﬁnallybe excreted. Depending on the physicochemical properties of the particles(size,aggregationstatus,solubility,shape, ...),aminor fractionoftheparticlesthatisnotclearedfromthelungortaken upbymacrophagescantranslocatethroughtheair–bloodbarrier intothecirculationandwillaccumulateinsecondaryorgans(Luyts et al., 2013). In a previous Ag ENP biodistribution study, we hypothesizedthattheAgENPseasilydissolveandionscrossthe air–bloodbarrier(Smuldersetal.,2014).Here,

m

^XANES^analyses

showedthat theadministeredAg ENPsweremostlypresent in macrophages,eitherpartiallyortotallydissolvedandchelatedby thiol-containingligandssuchascysteine,glutathioneorMT.The increasedamountofSobservedinthesespotswasconsistentwith the presence of thiol-containing molecules. The persistence of elementalAginmacrophagessuggeststhatsome(ifnotall)Agis takenupasENPbythesecells,andthatdissolutionandchelation occurshereafter.

MTs are a family of cysteine-rich proteins that cooperate with glutathione in maintaining the cellular redox state (Nordberg and Arner, 2001). Localized in the cell cytoplasm andin someorganellesincludingmitochondriaandlysosomes,

they play a role in a number of functions including (toxic) metal detoxiﬁcation and protecting the cell against damage from reactive oxygen species (ROS) (Sutherland et al., 2010;

Namdarghanbari et al., 2011). In natural conditions, themetal binding siteconsisting of 20 cysteine residues is occupied by Zn ions (Babula et al., 2012). However, these ions can be substitutedforothermetalionssuchasAg,Cu,Cd,Hg,Pband Fe and protect cells againstthe toxicityof these metals. It is known that cysteine, and more generally thiol-containing molecules, are extremely strong binding ligands for Ag ions (AdamsandKramer,1999;Liuetal.,2012).

It has been shown that expression of MTs is induced in inflammatorylungdiseases(Inoueetal.,2008);Kaewamatawong et al. (2014) demonstrated the expression of MTs after lung exposure tocolloidalAgENPsin mice.In a previousstudy, we showedtheinflammatoryeffects ofpulmonaryadministeredAg ENPs,the sameENPs asused in this study, withinfiltrationof neutrophils and expression of the pro-inflammatory cytokine IL-1

b

^(Smulders êt âl., ^2014), â ^known înducer ôf ^MT ^gene

expression(Deetal.,1990).InadditionMTproductioncanalsobe up regulatedvia MTF-1 bindingtometal-response elementsin thepromoterregions ofMTgenesasshown inthepresenceof Ag-ionsin Drosophila (Atanesyanet al.,2011)In this study, we demonstrated that the administered Ag ENPs are partially or completely dissolved with chelation of the released Ag ions byMTsorotherthiol-containingmolecules.Furthermore,higher Fig.2.DistributionofAginthelungsusingmicroX-rayﬂuorescence(m^XRF).

m^XRFîmagesôfÂgând^Sdistributionswithinlungtissue2daysafterthelastoropharyngealaspirationofAgENPs.a)Imagesofthescannedlungtissueasseenunderlight microscope.b)Rightmapdisplaysm^XRFîmageôf^the^regionîndicatedônîmageâ.Ûpper^maps^showdistributionofAg(red)andS(green),lowermapsshowaheatmapofAg distribution.Theleftandmiddlemapsrepresentmagnificationsoftwospotsindicatedontherightmap.

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concentrationsofthemetalsFeandCuwereobservedintheAg- richspots.AlthoughtheAgENPscontainimpuritiesofFeandCu, theconcentrationoftheseimpuritiesistoolowtofullyexplainthe observedincrease.Ashypothesizedbefore,weassumethathigher amountofMTsarepresentin theAg-richspots,explaining the co-localizationofotherendogenousmetalsincludingFeandCu.

5.Conclusion

Inconclusion,wedemonstratedthattheadministeredAgENPs are taken up by macrophages and are partially to completely dissolved and recomplexed to thiol-containing molecules

includingMTs.AsyntheticschemeinFig.4 gathersthepresent ﬁndingsincombinationwiththoseobtainedduringourprevious study.TheseresultsgivemoreinsightsonthebehaviorofAgENPs in vivoand willhelp in theunderstanding of the toxicological mechanismsofAgENPs.

Acknowledgements

ThisworkwassupportedbytheSeventhFrameworkProgramof the European Commission NanoHouse-Grant (Agreement No.

207816). We acknowledgethe EuropeanSynchrotronRadiation Facilitysynchrotronforprovisionofbeamtime(ID21beamline).

Fig.3.SpeciationbyAgLIII-edgemicroX-rayabsorptionnearedgestructure(m^XANES)spectroscopy.

AgspeciationbymXANESofthreeAg-richspots(lowerlines)comparedtothereferences(upperlines).Forthethreeanalyzedspots,plainlinesshowexperimentaldataand dottedlinesrepresentthelinearcombinationﬁt.Spot1and2correspondtotheindicatedspotsinFig.2;spot3correspondstotheindicatedspotinFig.S2.

Fig.4.SyntheticschemeofthepotentialpathwaysoccurringinthelungsafterAgENPexposure.

LungexposuretoAgENPsinducesaninflammatoryresponsewithincreasesinmacrophages,neutrophilsandthecytokineIL-1b^.ÊNPsârephagocytizedbymacrophages,and willpartiallytototallydissolve.TheAg⁺ionsarechelatedbythiol-containingmoleculessuchascysteine,glutathioneandmetallothionein(MT).ThescaleoftheAgENPsand ionsisnotrepresentative.

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The authors are grateful toAIFIRA for providing access tothe nuclear microprobe. ISTerre is part of Labex OSUG@2020 (ANR10LABX56) and Labex Serenade (ANR-11-LABX-0064). We alsothankequipexNanoID(ANR-10-EQPX-39).

AppendixA.Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.

toxlet.2015.07.001.

References

Adams,J.R.,Kramer,N.W.H.,1999.Determinationofsilverspeciationinwastewater andreceivingwatersbycompetitiveligandequilibration/solventextraction.

Environ.Toxicol.Chem.18,2674–2680.

Ahamed,M.,Alsalhi,M.S.,Siddiqui,M.K.,2010.Silvernanoparticleapplicationsand humanhealth.Clin.Chim.Acta411,1841–1848.

Atanesyan,L.,Günther,V.,Celniker,S.E.,Georgiev,O.,Schaffner,W.,2011.

CharacterizationofMtnE,theﬁfthmetallothioneinmemberinDrosophila.J.

Biol.Inorg.Chem.16,1047–1056.

Babula,P.,Masarik,M.,Adam,V.,Eckschlager,T.,Stiborova,M.,Trnkova,L.,Skutkova, H.,Provaznik,I.,Hubalek,J.,Kizek,R.,2012.Mammalianmetallothioneins:

propertiesandfunctions.Metallomics4,739–750.

Campbell, T.L., Maxwell, J.A., Nejedly, Z., 2000. The Guelph PIXE software packageIII:alternativeprotondatabase.Nucl.Instrum.MethodsPhys.Res.

170,193–204.

Chen,H.J.,Schluesener,X.,2008.Nanosilver:ananoproductinmedicalapplication.

Toxicol.Lett.176,1–12.

De,S.K.,McMaster,M.T.,Andrews,G.K.,1990.Endotoxininductionofmurine metallothioneingeneexpression.J.Biol.Chem.265,15267–15274.

Geiser,W.G.,Kreyling,M.,2010.Depositionandbiokineticsofinhalednanoparticles.

Part.FibreToxicol.7,2.

Hanus,M.J.,Harris,A.T.,2013.Nanotechnologyinnovationsfortheconstruction industry.Prog.Mater.Sci.58,1056–1102.

Inoue,K.,Takano,H.,Kaewamatawong,T.,Shimada,A.,Suzuki,J.,Yanagisawa,R., Tasaka,S.,Ishizaka,A.,Satoh,M.,2008.Roleofmetallothioneininlung inﬂammationinducedbyozoneexposureinmice.FreeRadic.Biol.Med.45, 1714–1722.

Ji,J.H.,Jung,J.H.,Kim,S.S.,Yoon,J.U.,Park,J.D.,Choi,B.S.,Chung,Y.H.,Kwon,I.H., Jeong,J.,Han,B.S.,Shin,J.H.,Sung,J.H.,Song,K.S.,Yu,I.J.,2007.Twenty-eight-day inhalationtoxicitystudyofsilvernanoparticlesinSprague–Dawleyrats.Inhal.

Toxicol.19,857–871.

Kaewamatawong,T.,Banlunara,W.,Maneewattanapinyo,P.,Thammachareon,C., Ekgasit,S.,2014.Acuteandsubacutepulmonarytoxicitycausedbyasingle intratrachealinstillationofcolloidalsilvernanoparticlesinmice:

pathobiologicalchangesandmetallothioneinresponses.J.Environ.Pathol.

Toxicol.Oncol.33,59–68.

Kaiser,J.P.,Roesslein,M.,Diener,L.,Wick,P.,2013.Humanhealthriskofingested nanoparticlesthatareaddedasmultifunctionalagentstopaints:aninvitro study.PLoSOne8,e83215.

Larue,C.,Castillo-Michel,H.,Sobanska,S.,Cecillon,L.,Bureau,S.,Barthes,V., Ouerdane,L.,Carriere,M.,Sarret,G.,2014.FoliarexposureofthecropLactuca sativatosilvernanoparticles:evidenceforinternalizationandchangesinAg speciation.J.Hazard.Mater.264,98–106.

Leo,B.F.,Chen,S.,Kyo,Y.,Herpoldt,K.L.,Terrill,N.J.,Dunlop,I.E.,McPhail,D.S., Shaffer,M.S.,Schwander,S.,Gow,A.,Zhang,J.F.,Chung,K.F.,Tetley,T.D.,Porter, A.E.,Ryan,M.P.,2013.Thestabilityofsilvernanoparticlesinamodelof pulmonarysurfactant.Environ.Sci.Technol.47,11232–11240.

Levard,C.,Hotze,E.M.,Lowry,G.V.,BrownJr.,G.E.,2012.Environmental transformationsofsilvernanoparticles:impactonstabilityandtoxicity.

Environ.Sci.Technol.46,6900–6914.

Li,Q.,Mahendra,S.,Lyon,D.Y.,Brunet,L.,Liga,M.V.,Li,D.,Alvarez,P.J.,2008.

Antimicrobialnanomaterialsforwaterdisinfectionandmicrobialcontrol:

potentialapplicationsandimplications.WaterRes.42,4591–4602.

Liu,J.,Wang,Z.,Liu,F.D.,Kane,A.B.,Hurt,R.H.,2012.Chemicaltransformationsof nanosilverinbiologicalenvironments.ACSNano6,9887–9899.

Luyts,K.,Napierska,D.,Nemery,B.,Hoet,P.H.M.,2013.Howphysico-chemical characteristicsofnanoparticlescausetheirtoxicity:complexandunresolved interrelations.Environ.Sci.Process.Impacts15,23–38.

Mayer,M.,1999.SIMNRA,asimulationprogramfortheanalysisofNRA,RBSand ERDA.TheFifteenthInternationalConferenceontheApplicationofAccelerators inResearchandIndustry541–544.

Namdarghanbari,M.,Wobig,W.,Krezoski,S.,Tabatabai,N.M.,Petering,D.H.,2011.

Mammalianmetallothioneinintoxicology,cancer,andcancerchemotherapy.J.

Biol.Inorg.Chem.16,1087–1101.

Nordberg,E.S.,Arner,J.,2001.Reactiveoxygenspecies,antioxidants,andthe mammalianthioredoxinsystem.FreeRadic.Biol.Med.31,1287–1312.

Smulders,S.,Kaiser,J.P.,Zuin,S.,VanLanduyt,K.L.,Golanski,L.,Vanoirbeek,J.,Wick, P.,Hoet,P.H.,2012.Contaminationofnanoparticlesbyendotoxin:evaluationof differenttestmethods.Part.FibreToxicol.9,41.

Smulders,S.,Luyts,K.,Brabants,G.,Van,L.K.,Kirschhock,C.,Smolders,E.,Golanski, L.,Vanoirbeek,J.,Hoet,P.H.,2014.Toxicityofnanoparticlesembeddedinpaints comparedtopristinenanoparticlesinmice.Toxicol.Sci.141,132–140.

Sole,V.A.,Papillon,E.,Cotte,M.,Walter,P.,Susini,J.,2007.Amultiplatformcodefor theanalysisofenergy-dispersiveX-rayﬂuorescencespectra.Spectrochim.Acta PartB-AtomicSpectrosc.62,63–68.

Som,C.,Wick,P.,Krug,H.,Nowack,B.,2011.Environmentalandhealtheffectsof nanomaterialsinnanotextilesandfacadecoatings.Environ.Int.37(6),1131–

1142.

Stebounova,L.V.,Guio,E.,Grassian,V.H.,2011a.Silvernanoparticlesinsimulated biologicalmedia:astudyofaggregation,sedimentation,anddissolution.J.

Nanopart.Res.13,233–244.

Stebounova,L.V.,Mcakova-Dodd,A.,Kim,J.S.,Park,H.,O’Shaughnessy,P.T.,Grassian, V.H.,Thorne,P.S.,2011b.Nanosilverinducesminimallungtoxicityor inﬂammationinasubacutemurineinhalationmodel.Part.FibreToxicol.8,5.

Sung,J.H.,Ji,J.H.,Yoon,J.U.,Kim,D.S.,Song,M.Y.,Jeong,J.,Han,B.S.,Han,J.H.,Chung, Y.H.,Kim,J.,Kim,T.S.,Chang,H.K.,Lee,E.J.,Lee,J.H.,Yu,I.J.,2008.Lungfunction changesinSprague–Dawleyratsafterprolongedinhalationexposuretosilver nanoparticles.Inhal.Toxicol.20,567–574.

Sung,J.H.,Ji,J.H.,Park,J.D.,Yoon,J.U.,Kim,D.S.,Jeon,K.S.,Song,M.Y.,Jeong,J.,Han,B.

S.,Han,J.H.,Chung,Y.H.,Chang,H.K.,Lee,J.H.,Cho,M.H.,Kelman,B.J.,Yu,I.J., 2009.Subchronicinhalationtoxicityofsilvernanoparticles.Toxicol.Sci.108, 452–461.

Sutherland,D.E.,Willans,M.J.,Stillman,M.J.,2010.Supermetalationofthebeta domainofhumanmetallothionein1a.Biochemistry49,3593–3601.

Takenaka,S.,Karg,E.,Roth,C.,Schulz,H.,Ziesenis,A.,Heinzmann,U.,Schramel,P., Heyder,J.,2001.Pulmonaryandsystemicdistributionofinhaledultraﬁnesilver particlesinrats.Environ.HealthPerspect.109(Suppl.4),547–551.

Wijnhoven,S.W.P.,Peijnenburg,W.J.G.M.,Herberts,C.A.,Hagens,W.I.,Oomen,A.G., Heugens,E.H.W.,Roszek,B.,Bisschops,J.,Gosens,I.,VandeMeent,D.,Dekkers, S.,DeJong,W.H.,VanZijverden,M.,Sips,A.J.A.M.,Geertsma,R.E.,2009.Nano- silver—areviewofavailabledataandknowledgegapsinhumanand environmentalriskassessment.Nanotoxicology3,109–138.

Yang,Z.,Liu,Z.W.,Allaker,R.P.,Reip,P.,Oxford,J.,Ahmad,Z.,Ren,G.,2010.Areview ofnanoparticlefunctionalityandtoxicityonthecentralnervoussystem.J.R.

Soc.Interface7(Suppl.4),S411–S422.

Yu,S.J.,Yin,Y.G.,Liu,J.F.,2013.Silvernanoparticlesintheenvironment.Environ.Sci.

Process.Impacts15,78–92.

Zuin,M.,Ferrari,A.,Golanski,L.,2013.Leachingofnanoparticlesfromexperimental water-bornepaintsunderlaboratorytestconditions.J.Nanopart.Res.16.