Silica Nanoparticles Inhibit Responses to ATP in Human Airway Epithelial 16HBE Cells

Abstract

Because of their low cost and easy production, silica nanoparticles (SiNPs) are widely used in multiple manufacturing applications as anti-caking, densifying and hydrophobic agents. However, this has increased the exposure levels of the general population and has raised concerns about the toxicity of this nanomaterial. SiNPs affect the function of the airway epithelium, but the biochemical pathways targeted by these particles remain largely unknown. Here we investigated the effects of SiNPs on the responses of 16HBE14o- cultured human bronchial epithelial (16HBE) cells to the damage-associated molecular pattern ATP, using fluorometric measurements of intracellular Ca2+ concentration. Upon stimulation with extracellular ATP, these cells displayed a concentration-dependent increase in intracellular Ca2+, which was mediated by release from intracellular stores. SiNPs inhibited the Ca2+ responses to ATP within minutes of application and at low micromolar concentrations, which are significantly faster and more potent than those previously reported for the induction of cellular toxicity and pro-inflammatory responses. SiNPs-induced inhibition is independent from the increase in intracellular Ca2+ they produce, is largely irreversible and occurs via a non-competitive mechanism. These findings suggest that SiNPs reduce the ability of airway epithelial cells to mount ATP-dependent protective responses.

Full text

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Int.J.Mol.Sci.2021,22,10173.https://doi.org/10.3390/ijms221810173www.mdpi.com/journal/ijms
Article
SilicaNanoparticlesInhibitResponsestoATPinHuman
AirwayEpithelial16HBECells
AlinaMilici,AliciaSanchezandKarelTalavera*
LaboratoryofIonChannelResearch,DepartmentofCellularandMolecularMedicine,VIBCenterfor
Brain&DiseaseResearch,Herestraat49,3000Leuven,Belgium;alina.milici@kuleuven.be(A.M.);
alyciasl@gmail.com(A.S.)
*Correspondence:karel.talavera@kuleuven.be;Tel.:+32‐1633‐0469
Abstract:Becauseoftheirlowcostandeasyproduction,silicananoparticles(SiNPs)arewidelyused
inmultiplemanufacturingapplicationsasanti‐caking,densifyingandhydrophobicagents.How‐
ever,thishasincreasedtheexposurelevelsofthegeneralpopulationandhasraisedconcernsabout
thetoxicityofthisnanomaterial.SiNPsaffectthefunctionoftheairwayepithelium,butthebio‐
chemicalpathwaystargetedbytheseparticlesremainlargelyunknown.Hereweinvestigatedthe
effectsofSiNPsontheresponsesof16HBE14o‐culturedhumanbronchialepithelial(16HBE)cells
tothedamage‐associatedmolecularpatternATP,usingfluorometricmeasurementsofintracellular
Ca
2+
concentration.UponstimulationwithextracellularATP,thesecellsdisplayedaconcentration‐
dependentincreaseinintracellularCa
2+
,whichwasmediatedbyreleasefromintracellularstores.
SiNPsinhibitedtheCa
2+
responsestoATPwithinminutesofapplicationandatlowmicromolar
concentrations,whicharesignificantlyfasterandmorepotentthanthosepreviouslyreportedfor
theinductionofcellulartoxicityandpro‐inflammatoryresponses.SiNPs‐inducedinhibitionisin‐
dependentfromtheincreaseinintracellularCa
2+
theyproduce,islargelyirreversibleandoccursvia
anon‐competitivemechanism.ThesefindingssuggestthatSiNPsreducetheabilityofairwayepi‐
thelialcellstomountATP‐dependentprotectiveresponses.
Keywords:silica;nanoparticles;ATP;purinergicreceptor;airway;epithelialcell;intracellularCa
2+
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1.Introduction
Nanoparticles(NPs)designatesmall‐sizednaturalorengineeredparticulatematter
withdimensionsoflessthan100nminatleastonedimensionandphysicalandchemical
propertiesthatdifferfromthoseofthebulkmaterial.Theyarecomposedofawiderange
ofmaterials,suchasmetals,inorganiccarbon,andorganiccompounds.Inthelastdec‐
ades,NPshavegainedpopularityinmanyindustries,astheiruniquepropertiesmake
themsuitableforuseincosmetics,biotechnology,food,pharmaceuticalandchemicalin‐
dustries[1–7].
HumanexposuretoNPshasincreasedconsiderablysincethenano‐technological
revolution.Theirsmalldimensionsanduniquephysico‐chemicalpropertiesfacilitatethe
abilityofnanoparticlestocrossdifferentbarriersandtoreachdistalorgans[8–12].They
canpenetratetheplasmamembraneanddepositintosubcellularstructuressuchasmito‐
chondria,endoplasmicreticulumandlysosomes,therebyposingpotentialhealththreats
[13–17].This,togetherwiththeirextensiveuseinconsumerproducts,hasraisedconcerns
aboutthesafetyofNPs[8,18].Agrowingbodyofevidencepointstowardsthedeleterious
effectsofNPs[19–22].However,notallNPsareharmful,andsomeofthemareengi‐
neeredtobeusedinmedicineasdrugnanocarriersduetotheirspecificinteractionwith
targetorgans[6,23].NPscanenterourbodiesbyinhalation,ingestion,injectionor
throughtheskin,butthemainentryrouteistheairwayepithelium[8,24].
Besidesitsroleingasexchange,theairwayepitheliumprotectsthebodyfromforeign
substances.Thecellsliningtherespiratorytracthavekeyrolesinmucociliaryclearance
Citation:Milici,A.;Sanchez,A.;
Talavera,K.SilicaNanoparticles
InhibitResponsestoATPinHuman
AirwayEpithelial16HBECells.Int.
J
.Mol.Sci.2021,22,10173.https://
doi.org/10.3390/ijms221810173
AcademicEditors:UdayKishore
andGiorgioPelosi
Received:31August2021
Accepted:16September2021
Published:21September2021
Publisher’sNote:MDPIstaysneu‐
tralwithregardtojurisdictional
claimsinpublishedmapsandinstitu‐
tionalaffiliations.

Copyright:©2021bytheauthors.Li‐
censeeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsandcon‐
ditionsoftheCreativeCommonsAt‐
tribution(CCBY)license(http://crea‐
tivecommons.org/licenses/by/4.0/).

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andcontributetotheinitiationofairwayinflammation[25–28].Intheairways,NPsmay
interactwithepithelialcellsandaltertheirexocrineandparacrinefunctions,andwith
sensoryneurons,whichinitiatethecoughreflexandneurogenicinflammation[29].Ithas
beenshownthatuponinhalation,NPsimpairthenormalfunctioningoftheairwaysby
inducinginflammation,increasingthesecretionofmucusandinhibitingtheciliarybeat
frequency[30–34].
Silicaparticles(SiNPs),thesubjectofthisstudy,areamongthemostproducedsyn‐
theticnanomaterialsworldwideduetotheirtunablephysico‐chemicalproperties,stabil‐
ity,lowcostandeasyproduction[7,35,36].SiNPshavebeenshowntoinducelunginflam‐
mation,cytotoxicresponsessuchasapoptosis,DNAdamageandendoplasmicreticulum
stress;theyincreasethereactiveoxygenspecies(ROS)productionbyalteringCa2+home‐
ostasis,disrupttheplasmamembraneuponuptakeandaltermembranefluidityandin‐
tegrity[7,10,15,20,21,37,38].PreviousstudiesindicatethattheeffectsinducedbySiNPs
aresize‐andconcentration‐dependentanddifferacrosscelltypes.Morepronouncedtoxic
effectshavebeenreportedforsmallerparticles,increaseddosesandprolongedexposure
times[7,15,39].Veryrecentstudiesusingthree‐dimensionalmodelairwayandlungprep‐
arationshaveshownthatSiNPsinducegenotoxicity[40],asymmetricchangesinpro‐in‐
flammatorycytokineandchemokineexpressionacrosstheairwaybarrierandtheincrease
inadhesionmoleculesintheapicalcompartment[41].
DespitetheextensiveresearchonthetoxicityofSiNPs,themechanismsunderlying
theirdeleteriouseffectsarepoorlyunderstood.Forinstance,mostofthestudiesrelatedto
SiNPsarefocusedonchroniceffectsforinvitroexposuretimesrangingfromseveral
hoursuptodays[42].Ontheotherhand,recentresearchhasillustratedtheimportance
ofstudyingalsotheacuteinteractionofSiNPswithspecificcellularstructuresandsignal‐
ingpathways.Forinstance,SiNP‐inducedmodulationinthetimescalesofsecondsand
minuteshavebeenreportedforcation‐permeablechannelsthatplaycrucialrolesincell
signaling[30,37,39,43,44].Inthissense,ATP‐mediatedsignalingisconsideredtobein‐
volvedininflammatoryresponsestoSiNPsinmultiplecelltypes[45–50].
ATPreceptorsareligand‐gatedcationchannels(P2X,ionotropic)orGprotein‐cou‐
pledreceptors(P2Y,metabotropic)[51–54].ActivationofP2Xreceptorsleadstoextracel‐
lularCa2+influx,resultinginplasmamembranedepolarizationanddirectincreaseinin‐
tracellular[Ca2+].Incontrast,activationofP2Yreceptorsactivatesinositoltriphosphate
(IP3)‐mediatedsignaling,whichopensIP3receptorCa2+channelsinthemembraneofen‐
doplasmicreticulum(ER),leadingtoCa2+releasefromthesestores[52,55].Intheairways,
extracellularATPsignalstissuedamageinducedbyparticulatesorpathogens,andis
thereforeconsideredasadamage‐associatedmolecularpattern(DAMP)[56].ATPrecep‐
torsareexpressedalongtherespiratorytractinepithelialcellsandinsensorynervesin
upper(trigeminalganglia)andlowerairways(nodoseanddorsalrootganglia)[57–59].It
hasbeenreportedthatthesetypesofcellsexpressP2X,aswellasP2Yreceptors.However,
airwayinjuryinducedbyparticulatematterinhalationhasbeenpredominantlyassociated
withP2Y‐mediatedsignalinginepithelialcellsleadingtoenhancedmucociliaryclearance.
Ontheotherhand,P2Xreceptorshaveaprominentroleinsensorynerves,astheiracti‐
vationtriggersdefensiveresponsessuchascough[56,60–65].Mostofthestudiesaddress‐
ingATP‐mediatedsignalingarerelatedtoATPreleaseuponcelldamage[66].
ThepurposeofthisstudywastodeterminewhetherSiNPsaffecttheresponsesof
airwayepithelialcellstoATP.Usingculturedhumanbronchialepithelial(16HBE)cellsas
amodel,wefoundthatacuteextracellularapplicationofcommerciallyavailableSINPs
potentlyreducestheresponsestoATPinaconcentration‐dependentmanner.Ourresults
unveiltheATPsignalingpathwayasadirectcellulartargetofSiNPs.Inthebroadcontext,
thissuggeststhatSiNPsaffecttheabilityofairwayepithelialcellstorespondtoenviron‐
mentalandendogenousstimulithatrequireATP‐mediatedsignaling.
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2.Results
2.1.IntracellularCa2+Responseof16HBECellstoExtracellularATP
Wefirstcharacterizedtheresponseof16HBEcellstoextracellularapplicationofATP,
underconstantperfusionofstandardKrebssolution(containing1.5mMCa2+,seeMateri‐
alsandMethods).WedeterminedtheamplitudeoftheincreaseinintracellularCa2+con‐
centrationsinducedbythreeconsecutiveapplicationsofATP,atvariousconcentrations
(from0.3to100μM;Figure1a–c).Theresponsesverycommonlyshowedasharpinitial
upstrokephase,followedbyaslowdecreasein[Ca2+],typicaloftheactivationofATP
receptors[67].Asexpected,theresponseinintracellular[Ca2+]wasconcentration‐depend‐
ent(Figure1d).

Figure1.ExtracellularATPinducesconcentration‐dependentresponsesin16HBEcells.(a–c)Exam‐
plesofintracellular[Ca2+]tracesshowingtheresponsestoextracellularATPappliedat1μM(a),10
μM(b)or100μM(c),usingastandardKrebssolution.(d)Concentrationdependenceoftheampli‐
tudeoftheresponsestothe1st(black),2nd(blue)and3rd(purple)applicationsofATP.Thedata
arerepresentedasmean±s.e.m.(n=179,59,75,96,278and128,forATP0.3,1,3,10,30and100
μM,respectively).
ThistypeofstimulationprotocolallowedustoevokeconsecutiveresponsestoATP
inareliablemanner,withnodesensitization.Thus,wecoulduseavariantofthisprotocol
todetermineifSiNPsaffecttheresponsestoATP,bycomparingtheamplitudesofthe
responsestoATPintheabsenceandinthepresenceofSiNPsinthesamecells.
2.2.TheCa2+ResponsestoATPAreMediatedbyReleasefromIntracellularStores
TheintracellularCa2+responsestoATPmaybemediatedbymetabotropic(P2Y)
and/orbyionotropic(P2X)receptors.TodeterminetheoriginoftheCa2+increaseupon

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ATPapplicationinourexperimentalconditions,weperformedmeasurementsintheab‐
senceofextracellularCa2+.Forthis,cellswereallowedtostabilizeinthestandardKrebs
solutionbeforetheexperimentsandthenperfusedwithaCa2+‐freeextracellularsolution
(seeMaterialsandMethods).Inthelattercondition,afirstapplicationof10μMATPelic‐
itedsizeableintracellularCa2+responses,whoseamplitudeswerenotsignificantlydiffer‐
entfromtheamplitudesoftheresponsesrecordedinthepresenceofextracellularCa2+
(Figure2a,b,columnsATP1st).Incontrast,thesecondATPapplicationinducedatransi‐
entincreaseinintracellular[Ca2+]onlyinaverylimitednumberofcells(Figure2a,b,col‐
umnsATP2nd),andathirdapplicationfailedtotriggeranyresponse(Figure2a,b,col‐
umnsATP3rd).

Figure2.ATPmobilizesCa2+fromintracellularstores.(a)Examplesoftracesof[Ca2+]recordedin
16HBEcellsshowingtheeffectsofrepetitiveextracellularapplicationof10μMATPintheabsence
ofextracellularCa2+.(b)Amplitudeoftheresponsestothe1st,2ndand3rdATPapplicationsinthe
presenceandintheabsenceofextracellularCa2+.Thedataarerepresentedasmean±s.e.m.(n=207,
482,97,249,97and247forthecolumnsfromlefttoright).Theasterisks(**)denotestatistically
significantdifferenceofthedataobtainedintheCa2+‐freecondition(−Ca2+)withrespecttothecor‐
respondingcontroldata(+Ca2+)(p<0.01;Kolmogorov–Smirnovtest).
Theseresultsshowthat16HBEcellscansupportfull‐sizedresponsesatleasttoafirst
applicationofATPintheabsenceofextracellularCa2+.Thisdemonstratesthatthere‐
sponsesaremediatedbyreleasefromintracellularstores.
2.3.Concentration‐DependentInhibitionofResponsestoATPbySiNPs
TodetermineifSiNPsaltertheresponsestoATP,weusedthesameprotocolde‐
scribedforFigure1,butapplyingSiNPs7minpriorandduringthesecondstimulation
with10μMATP,allduringperfusionofthestandardKrebssolution(Figure3a–c).Nota‐
bly,SiNPsincreasedthebaseline[Ca2+]priortothesecondATPapplication,inaccordance
withourpreviousstudy[30].Thisincreasein[Ca2+]appearednottobereversibleafter
washoutofthenanoparticlesforatleastthenext9minofdurationoftheexperiment
(Figure3a–c).The7minpre‐applicationofSiNPswasrequiredtoassesstheeffectsofATP
withoutinterferingwiththeincreaseinintracellular[Ca2+]elicitedbytheparticles.SiNPs
wereappliedatseveralconcentrations(1,3,10,or100μg/mL).Toquantifytheeffectsof
SiNPs,wedeterminedtheratiobetweentheamplitudesoftheresponsestothesecondor
thirdapplicationofATPandtheamplitudeoftheresponsestothefirstapplicationof
ATP.SiNPsreducedtheamplitudeofresponsestoATPinaconcentration‐dependent
manner.TheamplitudeofthesecondresponsetoATPwasnotsignificantlyaffecteddur‐
ingtheapplicationof1μg/mLSiNPs(Figure3a,d),butwasreducedathigherconcentra‐
tions(Figure3b–d).TheinhibitoryeffectofSiNPswasmorepronouncedonthethirdATP
application,aftertheremovaloftheparticlesfromtheextracellularsolution(Figure3d).

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Figure3.SiNPsinduceaconcentration‐dependentinhibitionoftheresponsetoATP.(a–c)Examples
ofintracellular[Ca2+]tracesshowingtheeffectsofSiNPswhenappliedat1μg/mL(a),3μg/mL(b)
or10μg/mL(c),beforeandduringthe2ndapplicationof10μMATP.Thearrowspointtothe
increaseinintracellular[Ca2+]inducedbytheapplicationoftheSiNPs.(d)Ratiobetweentheampli‐
tudesoftheresponsestothe2ndand3rdapplicationsofATPandtheamplitudeoftheresponseto
the1stapplicationofATP,asafunctionoftheconcentrationofSiNPs.Thedataarerepresentedas
mean±s.e.m.(n=135,135,52and92forSiNPsat1,3,10and100μg/mL,respectively.Theasterisks
(**)denotestatisticallysignificantdifferencewithrespecttothecorrespondingcontroldata[SiNPs]
=0μg/mL(p<0.01;Kolmogorov–Smirnovtest).
Viabilitytestsusingfluorescenceactivatedcellsortingwithpropidiumiodideasa
markerofdeadcellsshowedonlyamarginalincreaseinthenumberofnon‐viablecells
after10minexposureto100μg/mLSiNPs(3.2%abovethecontrollevel;FigureS1).
2.4.ProlongedInhibitoryEffectsofSiNPsontheResponsestoATP
TheaboveresultsshowthatSiNPsnotonlyinhibittheresponsetoATPwhenthey
areappliedsimultaneously,butalsoaffecttheresponsetoasubsequentATPapplication
9minapart.TodetermineifthereductioninATPresponsecausedbySiNPspersistsafter
alongertime,wemodifiedthepreviousprotocolbydelayingthethirdATPapplication
by30min.WefoundthattheresponsestoathirdapplicationofATPafteralmost1hof
keepingthecellsinKrebssolutionwerelargelypreserved(Figure4a,c).

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Figure4.PersistentinhibitoryactionofSiNPsonresponsestoATP.(a)Examplesofintracellular
[Ca2+]tracesshowingtheeffectsofextracellularATPappliedat10μM.(b)Examplesofintracellular
[Ca2+]tracesshowingtheeffectsofSiNPswhenappliedat100μg/mL,beforeandduringasecond
applicationof10μMATP.(c)AmplitudeoftheresponsestothreeconsecutiveapplicationsofATP
forthecontrolseriesofexperiments(blackbars,n=164)andforaseriesofexperimentsinwhich
100μg/mLSiNPswasapplied9minbeforeandduringthesecondapplicationofATP(whitebars,
n=140).Thedataarerepresentedasmean±s.e.m.Theasterisks(**)denotestatisticallysignificant
differenceofthedataobtainedinSiNPswithrespecttothecorrespondingcontroldataintheab‐
senceofSiNPs(p<0.01;Kolmogorov–Smirnovtest).The#symboldenotesstatisticallysignificant
differencebetweenATP2nd+SiNPsandATP3rdafterSiNPs(p<0.01;Kolmogorov–Smirnovtest).
ThisallowedustoconfidentlyassesstheeffectofSiNPsonadelayedexposureto
ATP.Usingthisprotocol,weobservedthatSiNPsapplicationresultedinanincreasein

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theintracellular[Ca2+]throughouttheremainingmeasurements,i.e.,longafterSiNPs
washout(Figure4b).However,morecriticallytothecurrentstudy,wefoundthatthe
responsestoATPdidnotrecoverafter35minofwashoutoftheSiNPs(Figure4b,c).
2.5.EffectsofIntracellularCa2+OverloadontheResponsestoATP
SiNPsontheirowninduceanincreasein[Ca2+]that,hypothetically,mightleadto
inhibitionoftheresponsetoATP.Asawaytoassessthispossibility,wetestedwhether
theinhibitionoftheresponsetoATPcorrelatedwiththeincreaseinintracellular[Ca2+]
inducedbythepreviousapplicationof100μg/mLSiNPs.Forthis,weusedthedatacor‐
respondingtothepoint100μg/mLSiNPspresentedinFigure3d.Wefoundthattheratio
oftheamplitudesoftheresponsestothesecondandfirstapplicationsofATP,whichisa
directmeasureoftheeffectoftheSiNPs,didnotcorrelatewitheitherthemaximalincrease
ortheaverageincreaseinducedbytheapplicationofSiNPsprevioustothesecondATP
application(Figure5a,b).Thelackofcorrelationwasevenmoreobviousbetweentheef‐
fectsoftheSiNPsonthethirdATPapplicationandthepreviousintracellularCa2+levels,
i.e.,maximalormeanvalues(Figure5c,d).

Figure5.LackofcorrelationbetweenthereductionintheresponsetoATPandtheincreaseinin‐
tracellular[Ca2+]inducedbySiNPs.(a,b)Plotsoftheratiooftheamplitudesoftheresponsestothe
2ndand1stapplicationsof10μMATPversusthemaximalincrease(a)andmeanincrease(b)in
intracellular[Ca2+]inducedby100μg/mLSiNPs.(c,d)Equivalentplotstothoseshowninpanels(a)
and(b),butusingtheratiooftheamplitudesoftheresponsestothe3rdand1stapplicationsof10
μMATP.Thelinesrepresentthecorrespondinglinearfits,withR2valuesof0.04,0.04,0.002and
0.004,forpanels(a–d),respectively.

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Wefurtherprobedforarelationbetweenanincreaseintheintracellular[Ca2+]and
theamplitudeoftheresponsetoATPbytestingwhetheranincreasein[Ca2+]inducedby
amechanismdistinctfromthatoftheSiNPshasasimilarinhibitoryeffectontheresponses
toATP.WedidthisbyperfusingthecellswiththestandardKrebssolutionandtesting
theeffectsofaddingtheCa2+ionophoreionomycin(1μM).Aseriesofparallelcontrol
experimentsshowedthatconsecutiveATPapplicationstriggeredsizeableintracellular
Ca2+responses(Figure6a,c;blackbars).

Figure6.ResponsetoATPin16HBEcellsoverloadedwithCa2+.(a)Exampleofcontrol[Ca2+]traces
recordedin16HBEcellsshowingtheresponsestorepetitiveextracellularapplicationof10μMATP.
(b)Examplesoftracesshowingtheeffectsof1μMionomycinwhenappliedbeforeandduringthe
secondapplicationof10μMATP.(c)Amplitudeoftheresponsestothreeconsecutiveapplications
ofATPforthecontrolseriesofexperiments(blackbars,n=36)andforaseriesofexperimentsin
which1μMionomycinwasapplied2minbeforeandduringthesecondapplicationofATP(white
bars,n=42).Thedataarerepresentedasmean±s.e.m.Theasterisksdenotestatisticallysignificant
differencebetweenATP3rdandATP3rdafterionomycin(p<0.01;Kolmogorov–Smirnovtest).(d)
Comparisonoftheamplitudeofthe[Ca2+]responsesinducedby100μg/mLSiNPsandby1μM
ionomycin(n=92and42,respectively).Thedataarerepresentedasmean±s.e.m.Theasterisks(**)
denotestatisticallysignificantdifferencebetweenthetwobars(p<0.01;Kolmogorov–Smirnovtest).
Asexpected,ionomycininducedafastandrobustincreasein[Ca2+],whichwasin
factmuchlargerthanthatinducedbySiNPs(Figure6b,d).Incontrasttotheincreaseob‐
servedwiththeSiNPs,applicationofATPduringtheperfusionofionomycindidelicitan
additionalincreasein[Ca2+](Figure6b).Theamplitudesoftheseresponseswerenotsig‐
nificantlydifferentfromtheamplitudesobservedinthecontrolexperiments(Figure6c).
Wedidobserve,however,thattheresponsestoATPdevelopedmoreslowlyinthe

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presenceofionomycinthanincontrolcondition(Figure6b).Thiseffectwasnotfurther
characterized.TheresponsetothethirdapplicationofATPappearedtobeaffectedbythe
priortreatmentwithionomycin(Figure6b,c),butneverthelessthiseffectwassmallerthan
thatcausedby100μg/mLSiNPs(seedataforATP10μMinFigure2d).
2.6.EffectsofSiNPsontheConcentrationDependenceoftheResponsetoATP
InordertogainfurtherinsightintothemechanismofactionofSiNPs,wedetermined
howtheyaltertheconcentrationdependencyoftheresponsetoATP.Forthis,weused
thesameATPapplicationprotocoldescribedabove(Figure1),butperfusing100μg/mL
SiNPs7minpriorandduringthesecondstimulationwithATP(inthestandardKrebs
solution).WeusedSiNPsatthisconcentrationbecausetheeffectsweremostrobustboth
fortheincreaseinintracellular[Ca2+]andfortheinhibitionoftheresponsestoATP,which
wasexpectedtoresultinlessdatavariance.WeconfirmedtheresponsestoATPtobe
significantlyinhibitedinthepresenceofSiNPs(Figure7a–d),andthattheresponsetothe
thirdapplicationofATPwasalsoinhibited,furtherdemonstratingthatSiNPshadanin‐
hibitoryeffectevenaftertheywereremovedfromtheextracellularsolution(Figure7a–
d).

Figure7.EffectsofSiNPsontheconcentrationdependencyoftheresponsetoATP.(a–c)Examples
ofintracellular[Ca2+]tracesshowingtheeffectsof100μg/mLSiNPsontheresponsestoextracellular
ATPappliedat1μM(a),10μM(b)or100μM(c).(d)Concentrationdependenceoftheamplitude
oftheresponsestoATP,forthe1st(control),2nd(inthepresenceofSiNPs)and3rd(afterwashout
ofSiNPs)applications.Thedataarerepresentedasmean±s.e.m.(n=213,68,130,109,169and146
forATP0.3,1,3,10,30and100μM,respectively).
BycomparingthedatashowninFigure7dwiththoseinFigure1d,itcanbenoticed
that,apartfromtheobviousdifferencesinmaximalvalues,theshapesoftheconcentration

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dependenciesdeterminedintheabsenceandinthepresenceof100μg/mLSiNPsap‐
pearedtobesimilar.Totestwhetherthiswasindeedthecase,wereplottedthedataby
normalizingeachcurvetotherespectivevalueobtainedatATP100μM(Figure8a,b).

Figure8.Non‐competitiveinhibitoryeffectofSiNPsontheresponsetoATP.(a,b)Normalizedcon‐
centrationdependenceoftheamplitudeoftheresponsestoATP,forthe2ndapplication(control
andinthepresenceofSiNPs)andforthe3rdapplication(controlandafterwashoutofSiNPs).The
normalizationwasperformedbydividingthedatasetsofFigures1dand5dbythecorresponding
valuesobtainedfor100μMATP.(c,d)Lineweaver–BurkplotsofthedatashowninFigures1dand
5d.Thelinesrepresentfitswithlinearfunctions.Thedataarerepresentedasmean±s.e.m.Then
numbersarethesameasintheoriginalfigures.
Thecomparisonofthenormalizedcurvesrevealedalargeoverlapofthedataob‐
tainedinthepresenceandduringwashoutoftheSiNPswiththerespectivenormalized
controlcurves.Finally,werepresentedthesedataasLineweaver–Burkplots(1/Δ[Ca2+]vs.
1/[ATP])andfittedthemwithlinearfunctions(Figure8c,d).Thefitsyieldedlinearfunc‐
tionswithslopesthatweredifferentbetweencontrol(2.45±0.11and2.08±0.24forsecond
andthirdATPapplications,respectively)andSiNPs(9.92±0.9and12.9±3.3forsecond
andthirdATPapplications,respectively).Incontrast,theresultinginterceptswiththe
1/[ATP]axis,whichrelatetotheinverseoftheapparentequilibriumconstant(–1/KM),
werenearlyidenticalforcontrolandSiNPsconditions(secondATPapplication:–0.44±
0.08μM−1and‐0.47±0.17μM−1,respectively,andthirdATPapplication:–0.52±0.08μM−1
and–0.52±0.21μM−1,respectively).ThecorrespondingKMvaluesforthesecondATP
applicationweretherefore2.27±0.4μMand2.2±0.8μMforcontrolandSiNPs,respec‐
tively,andforthethirdATPapplication,1.9±0.3μMand1.9±0.8μMforcontroland
SiNPs,respectively.ThisanalysisshowsthattheSiNPsonlydecreasedthemaximal

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response(efficacy)andnotthesensitivitytoATP,suggestingthattheyhaveanon‐com‐
petitiveinhibitoryaction.
3.Discussion
ExtracellularATPisakeysignalingmoleculeforthephysiologyoftheairwayepi‐
thelium,asitmediatesresponsessuchastheincreaseinciliarybeatfrequencyandmucus
productionandtriggersthecoughreflex.Moreover,ATPisakeymessengerofcellular
damage,anditsdetectionplaysakeyroleinactivatingprotectivemechanismsinneigh‐
boringhealthycells.Therefore,aninsufficientabilitytotriggerafullresponsetoATPis
expectedtoleadtoreducedprotectiveresponses.Previousstudieshaveestablishedthe
implicationofATP‐mediatedsignalinguponexposuretonanomaterials,includingsilica.
However,thesehavemainlyreportedtheinductionofATPreleaseuponlong‐termexpo‐
sure,from30–40min[47–49]toseveralhours[46,68,69].Incontrast,theacuteeffectsof
thenanoparticlesonATP‐mediatedsignaling,thatis,ontheresponsetoATPitself,re‐
mainedtobeinvestigated.Tocoverthisinformationgap,inthisstudyweassessedthe
acuteeffectsof10nmSiNPsontheresponsesofculturedhumanairwayepithelialcellsto
ATP.
Weusedthe16HBEcellline,whichhasbeenreferredtoasanexcellentmodelfor
studyingATP‐inducedintracellularCa2+transientsinhumanairwayepithelialcells[70].
Accordingly,wefoundthesecellstorespondconsistentlytoextracellularapplicationof
ATPintherangebetween0.3and100μM.Theseresponseshadamplitudesthatincreased
withtheATPconcentrationanddisplayedthetypicalmorphology,withafastupstroke
phasefollowedbyabiphasicdecay[70].Wefurtherfoundthatcellsrespondedtoafirst
applicationofATPwithCa2+transientsofnormalamplitudeinfreeextracellularCa2+so‐
lutions.Thisshowsthatinourexperimentalconditions,16HBEcellsrespondtoATPvia
activationofP2Yreceptors,leadingtoIP3‐inducedCa2+releasefromintracellularstores
viatype3IP3receptors,aspreviouslyreported[70,71].Interestingly,thesecondandthird
applicationsofATPinCa2+‐freesolutionfailedtoevokeresponses,whichmayhavebeen
duetoastrongdepletionoftheintracellularstoresinducedbythefirstATPapplication.
Inturn,thisindicatesthecrucialimportanceofthereplenishingmechanismsforthe
maintenanceofresponsivenesstorepetitivestimulationwithATPinphysiologicalcondi‐
tions(inthepresenceofextracellularCa2+).Weindeedfoundthatinthelattercondition,
consecutiveapplicationsofATPseveralminutesapartinducedreproducibleresponses.
ThisallowedustoassesstheeffectsofSiNPsbycomparingtheamplitudesofthere‐
sponsestoATPintheabsenceandinthepresenceofSiNPsinthesamecells.
Usingthisexperimentalparadigm,wefoundthatSiNPsreducetheresponsetoATP
inaconcentration‐dependentmanner.Thisinhibitoryeffectwasnotonlyobservedinthe
presenceoftheSiNPs,butalsofoundtopersistandtobeactuallystrongerupto35min
aftertheremovalofSiNPsfromtheextracellularsolution(Figures3,4and6).Futureex‐
perimentsmayservetodeterminewhethertheinhibitionoftheresponsestoATPisre‐
versibleoverlongerperiodsoftime.Nevertheless,theprolongedirreversibilityofthein‐
hibitoryactionofSiNPsdemonstratesthattheprimaryunderlyingmechanismisnotme‐
diatedbySiNPsactingfromtheextracellularsideofthemembrane.Importantly,thevia‐
bilitytestsshowedonlyaverysmallincreaseinthenumberofnon‐viablecellsfollowing
exposuretothehighestSiNPsconcentrationtested(100μg/mL).Thisresultagreeswith
thestabilityoftheintracellular[Ca2+]recordingsinallseriesofexperiments,indicating
thatnomajorchangesincellfunctionalityleadingtocompromisedplasmamembrane
integrityunderlietheobservedacuteinhibitoryeffectofSiNPsonATPresponse.
AnotherhypothesistoconsiderregardingthemechanismofactionoftheSiNPsis
that,becauseoftheirnegativesurfacecharge,thenanoparticlesmightchelateintracellular
cationsandtherebydecreasetheintracellularCa2+signalsdetectedwithFura2.However,
thismechanismcanbediscarded,asitisexpectedtooperateforallintracellularCa2+sig‐
nals,andwehavepreviouslydemonstratedthattheSiNPsactuallyenhancetheresponse
ofTRPV1totheagonistcapsaicin[30].

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Alternatively,itcouldbeenvisagedthatSiNPsinhibittheCa2+responsebydirectly
targetingcomponentsoftheATPsignalingpathway.Forinstance,SiNPsmayalterthe
mechanicalpropertiesofcellularmembranes[38],andthiscouldaffectthefunctionof
ATPreceptors.Furthermore,previousTEMstudieshaveshownthatSiNPscanbeinter‐
nalizedviaendocytosisthroughplasmamembranevesiclescontainingseveralnanopar‐
ticlesandthatthisuptakeprocessisaccompaniedbyplasmamembraneconsumptionand
disruption[15].ThismayleadtoadecreaseinthenumberofATPreceptorsandtothe
formationofCa2+‐permeableporesintheplasmamembrane,whichinturnmayexplain
thereductionoftheresponsestoATPandtheSiNPs‐inducedincreaseinintracellular
Ca2+],respectively.
However,thehypothesisconsideringthatSiNPsdirectlytargetcomponentsofthe
ATPsignalingpathwayhasalimitation.ThecellularconcentrationofSiNPsandthereby
anyputativedirecteffectonasignalingcomponentwouldbeexpectedtodecay,notto
increase,uponwashout.Thus,itremainsdifficulttoexplainwhytheconcentrationde‐
pendenciesoftheSiNPsactionshowamorepotenteffectontheATPresponsesduring
washoutthanduringtheapplicationoftheparticles.
AmoreplausibleexplanationisthattheSiNPstriggeraninhibitorymechanismthat
outlivesandisfurtherenhancedaftertheceasingoftheaccumulationofthenanoparticles
inthecellularcompartments.Inthisdirection,theincreaseinintracellularCa2+emerged
asacandidatefactortobeinvolved,asweobservedthatSiNPsinducedsucheffectina
ratherirreversiblemanner,inagreementwithourpreviousreport[30].However,we
foundthattheinhibitoryeffectsoftheSiNPsontheATPresponseswerenotcorrelatedto
theincreasein[Ca2+]inducedbytheparticles.Furthermore,theexperimentsinwhichwe
usedionomycinrevealedthatcellsundergoingintracellularCa2+overloadwereableto
respond,albeitmoreslowly,toafirstapplicationofATP.Takentogether,ourdata
stronglyindicatethattheincreaseinCa2+inducedbytheSiNPsisnottheprimaryfactor
underlyingthedecreaseintheresponsestoATP.
Althoughourpresentexperimentsdidnotallowpinpointingaprecisetargetof
SiNPsaction,theydidletusconcludethatSiNPsactviaanon‐competitiveinhibitory
mechanism.Thus,theseparticlesmayaffectanyoftheelementsofATPsignalingcascade
thatdeterminethemaximalintracellularCa2+response,butdonotactonanyfactorsde‐
terminingthesensitivitytoATP.Forinstance,SiNPsmaydecreasethenumberofATP
receptorsavailableforactivationattheplasmamembraneand/ortheirmaximallevelof
activation,butdonotdecreasetheiraffinityforATP.Thelatterisinformative,asitindi‐
catesthatSiNPsdonotinterferewiththebindingandunbindingofATPtoandfromP2Y
receptors.Furthermore,becauseweassessedtheresponsestoATPfromtheamplitudeof
theCa2+increase,noneoftheeventsthatdeterminethesensitivitytoATPmaybeaffected.
Futureexperiments,complementedbymathematicalmodeling,mayhelpindiscerning
whatelementsofthepathwaymaybeaffectedornotbytheSiNPs.
AnothercuriousresultwasthattheinhibitoryeffectofSiNPsappearedtoreacha
plateauatconcentrationsabovearound10μg/mL,indicatingthattheyareunabletoin‐
hibittheresponsestoATPcompletelywhenappliedatconcentrationsupto100μg/mL.
Interestingly,wepreviouslyfoundaqualitativelysimilareffectfortheinhibitionof
TRPV4in16HBEcells,buttheplateauformaximalinhibitionwasreachedatconcentra‐
tionshigherthanaround300μg/mL[30].Thedifferenceintheconcentrationsforreaching
theplateauphaseofthetwoeffectssuggeststhataconcentration‐dependentchangein
propertiesoftheSiNPsisnotthecauseofthisphenomenon.Alternatively,SiNPsmight
affectdifferentlydistinctpoolsofATPreceptorsorotherelementsofthissignalingpath‐
way,ortheparticlesmightnothavetheabilitytofullyabrogatetheirmaximalactivation.
TheeffectsofSiNPsmustbeundoubtedlylinkedtotheirphysicalandchemicalprop‐
erties.Thespecificcontributionsofeachofthesepropertiestothecellularactionsofthe
particlescouldbestudiedbycomparingtheeffectsofparticlesdifferingonlyinoneprop‐
erty—forinstance,particleshavingexactlythesamesize,texture,etc.,butdifferentzeta
potential.However,wedonothavesuchparticlesatourdisposal,andatthispointwe

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
cannotmakeanyinferenceonhowspecificparticlepropertiesdeterminetheiractionson
theresponsestoATP.
AkeyaspectofourfindingsisthattheSiNPsconcentrationsrequiredfortheinhibi‐
tionoftheresponsestoATP(1–3μg/mL)aremuchlowerthanthoseneededtoinduce
cytotoxicityortoobservecytokinereleaseinvitro(25–6000μg/mL)[72–74].Furthermore,
thetimescalefortheeffectswereportedherewasbetween3‐to150‐foldshorterthan
thoserequiredforotherreportedSiNPseffects.ThissuggeststhattheATPsignalingpath‐
wayisaprimaryandverysensitivetargetofSiNPs.Finally,itshouldbenotedthatthe
inhibitoryactionsofSiNPswereportedhereandpreviouslyonTRPV4werespecific,as
thesameparticlesenhancedtheactivationofthecapsaicinreceptorTRPV1[30].
OurresultsdemonstratethatSiNPsinduceanacutenon‐competitiveinhibitionof
theP2Y‐mediatedintracellularCa2+responsesofculturedhumanairwayepithelialcells
toATP.Thiseffectoccurssignificantlyfasterandatconcentrationslowerthanthosepre‐
viouslyreportedfortheinductionofcellulartoxicityandpro‐inflammatoryresponses.
Futureresearchshouldbeconductedtodeterminethemolecularmechanismsofthese
actions,totestwhetherSiNPsaregeneralinhibitorsofpurinergicsignalingpathwaysin
othercelltypes,andtodeterminewhetherSiNPsreducetheabilityofairwayepithelial
cellstomountprotectiveresponsesviatheATPsignalingpathway,i.e.,theincreasein
mucociliaryclearance.
4.MaterialsandMethods
4.1.Ludox®SiNPs
SiNPsusedinthisstudywerepurchasedfromSigma‐Aldrich(Bornem,Belgium)as
thecommercialsourceof30%wtsuspensioninwater.Theirbasicpropertieswerechar‐
acterizedinpreviousstudiesbyourgroup[30]andcanbefoundinTable1.
Table1.PropertiesofSiNPs.
PropertyValue
Size10.2nm(P10=8.1nmandP90=11.8nm)
ShapeSpherical
SolidstructureAmorphous
Zetapotential−20±3mV
DispersionMonodispersed
Endotoxincontent<0.05EU/mL
Fortheexperiments,thenanoparticlesuspensionwasdilutedtothedesiredconcen‐
trationsinKrebsorCa2+‐freeKrebssolutions(seebelowReagentsandSolutions).
4.2.CellCulture
Humanbronchialepithelialcells(16HBE)weregrowninDulbecco’smodifiedEa‐
gle’smedium:nutrientmixtureF‐12(DMEM/F‐12)containing5%(v/v)fetalcalfserum
(FCS),2mML‐glutamine,2U/mLpenicillinand2mg/mLstreptomycinat37°Cinahu‐
midity‐controlledincubatorwith5%CO2andwereseededon18mmcoverslipscoated
with0.1mg/mLpoly‐L‐lysine.
4.3.RatiometricIntracellularCa2+Imaging
Ca2+‐imagingexperimentswereconductedwiththeratiometricfluorescentdyeFura‐
2AMester(Biotium,Hayward,CA,USA)asanindicatorforfreeintracellularcalcium.
Cellswereincubatedwith2μLFura‐2for30minat37°C.Solutionswereappliedusing
amulti‐barrelperfusionsystem.Theintracellular[Ca2+]wascalculatedfromtheratioof
fluorescencemeasureduponalternatingilluminationat340and380nm.Experiments
wereperformedusinganinvertedmicroscopewithMT‐10illuminationsystemandthe

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xCellenceProsoftwareofthemicroscope(Olympus,Planegg,Germany).Allmeasure‐
mentswereperformedat35°C.Fluorescenceintensitieswerecorrectedforbackground
signal,andintracellularCa2+concentrationswerecalculatedasdescribedpreviously[75].
DatawereanalyzedanddisplayedusingOrigin(OriginLabCorporation,Northampton,
MA,USA).
4.4.DataandStatisticalAnalysis
Todeterminetheamplitudeoftheresponseinducedbythecompoundsofinterest,
wesubtractedthebaseline[Ca2+]priortotheapplicationfromthepeakvaluereached
duringchallenging(valuedenotedbyΔ[Ca2+]).Thebaselinewascalculatedbydetermin‐
ingthemean[Ca2+]duringthelast20sbeforetheapplicationofthecompound.Dataare
givenas±standarderrorofthemean.
4.5.ReagentsandSolutions
AllchemicalswerepurchasedfromSigma‐Aldrich(Bornem,Belgium).Thesolutions
containingATPwerepreparedbyaddingtheappropriateamountsofa50mMNa2ATP
stocksolutiontothecorrespondingextracellularsolution(standardKrebsorCa2+‐free
Krebs).Ionomycin,anionophorethattriggersCa2+influx,wasusedtoshowthattheeffect
observedduringtheapplicationofSiNPsisnotcalcium‐mediatedandthusisaresultof
theinteractionofSiNPswithsubcellularstructures.Ionomycinat1μMconcentrationwas
obtainedbydilutionof2mMstockinKrebs.Solutionsusedinmeasurementsperformed
intheabsenceofextracellularCa2+werepreparedwithKrebstitratedtopH7.4with
NaOH(Table2).
Table2.Compositionofthesolutionsusedintheexperiments.
Krebs
150mMNaCl
6mMKCl
1.5mMCaCl2×2H2O
1mMMgCl×6H2O
10mMglucose
10mM4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonicacid(HEPES)
Ca2+‐FreeKrebs
150mMNaCl
6mMKCl
1mMMgCl×6H2O
10mMglucose
10mMHEPES
10mMethyleneglycol‐bis(β‐aminoethylether)‐N,N,N’,N’‐tetraacetic
acid(EGTA)
SupplementaryMaterials:Thefollowingareavailableonlineatwww.mdpi.com/arti‐
cle/10.3390/ijms221810173/s1.
AuthorContributions:Conceptualization,A.M.,A.S.andK.T.;formalanalysis,A.M.,A.S.andK.T.;
investigation,A.M.andA.S.;writing—originaldraftpreparation,A.M.andK.T.;writing—review
andediting,A.M.,A.S.andK.T.;visualization,A.M.andK.T.;supervision,K.T.;projectadministra‐
tion,K.T.;fundingacquisition,A.M.andK.T.Allauthorshavereadandagreedtothepublished
versionofthemanuscript.
Funding:ThisresearchwassupportedbygrantsfromtheResearchFoundationFlandersFWO
(G076714)andbytheResearchCounciloftheKULeuven(GOA/14/011).A.M.isfundedbyEras‐
mus+2019–2021oftheEuropeanCommission(KA103‐061892).
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.

Int.J.Mol.Sci.2021,22,1017315of18
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DataAvailabilityStatement:DataiscontainedwithinthearticleorSupplementaryMaterials.
Acknowledgments:TheauthorsthankM.B.forthemaintenanceofthecellcultures.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
References
1. Kreyling,W.G.;Semmler‐Behnke,M.;Chaudhry,Q.Acomplementarydefinitionofnanomaterial.NanoToday2010,5,165–168,
doi:10.1016/j.nantod.2010.03.004.
2. Bleeker,E.A.J.;deJong,W.H.;Geertsma,R.E.;Groenewold,M.;Heugens,E.H.W.;Koers‐Jacquemijns,M.;vandeMeent,D.;
Popma,J.R.;Rietveld,A.G.;Wijnhoven,S.W.P.;etal.ConsiderationsontheEUdefinitionofananomaterial:Sciencetosupport
policymaking.Regul.Toxicol.Pharmacol.2013,65,119–125,doi:10.1016/j.yrtph.2012.11.007.
3. Naito,M.;Yokoyama,T.;Hosokawa,K.;Nogi,K.NanoparticleTechnologyHandbook;Elsevier:Hoboken,NJ,USA,2018.
4. Liu,D.;Gu,N.NanomaterialsforFresh‐KeepingandSterilizationinFoodPreservation.RecentPat.FoodNutr.Agric.2009,1,
149–154,doi:10.2174/2212798410901020149.
5. Fan,Z.;Zhang,L.;Di,W.;Li,K.;Li,G.;Sun,D.Methyl‐graftedsilicananoparticlestabilizedwater‐in‐oilPickeringemulsions
withlow‐temperaturestability.J.ColloidInterfaceSci.2021,588,501–509,doi:10.1016/j.jcis.2020.12.095.
6. Qhobosheane,M.;Santra,S.;Zhang,P.;Tan,W.Biochemicallyfunctionalizedsilicananoparticles.Analyst2001,126,1274–1278,
doi:10.1039/b101489g.
7. Murugadoss,S.;Lison,D.;Godderis,L.;VanDenBrule,S.;Mast,J.;Brassinne,F.;Sebaihi,N.;Hoet,P.H.Toxicologyofsilica
nanoparticles:anupdate.Arch.Toxicol.2017,91,2967–3010,doi:10.1007/s00204‐017‐1993‐y.
8. Wang,J.;Asbach,C.;Fissan,H.;Hülser,T.;Kuhlbusch,T.A.J.;Thompson,D.;Pui,D.Y.H.Howcannanobiotechnologyoversight
advancescienceandindustry:Examplesfromenvironmental,health,andsafetystudiesofnanoparticles(nano‐EHS).J.
Nanopart.Res.2011,13,1373–1387,doi:10.1007/s11051‐011‐0236‐z.
9. Vishwakarma,V.;Samal,S.S.;Manoharan,N.SafetyandRiskAssociatedwithNanoparticles‐AReview.J.Miner.Mater.Charact.
Eng.2010,9,455–459,doi:10.4236/jmmce.2010.95031.
10. Dubes,V.;Parpaite,T.;Ducret,T.;Quignard,J.F.;Mornet,S.;Reinhardt,N.;Baudrimont,I.;Dubois,M.;Freund‐Michel,V.;
Marthan,R.;etal.Calciumsignallinginducedbyinvitroexposuretosiliciumdioxidenanoparticlesinratpulmonaryartery
smoothmusclecells.Toxicology2017,375,37–47,doi:10.1016/j.tox.2016.12.002.
11. Zhang,Y.N.;Poon,W.;Tavares,A.J.;McGilvray,I.D.;Chan,W.C.W.Nanoparticle–liverinteractions:Cellularuptakeand
hepatobiliaryelimination.J.ControlRelease2016,240,332–348,doi:10.1016/j.jconrel.2016.01.020.
12. Du,Z.;Zhao,D.;Jing,L.;Cui,G.;Jin,M.;Li,Y.;Liu,X.;Liu,Y.;Du,H.;Guo,C.;etal.Cardiovasculartoxicityofdifferentsizes
amorphoussilicananoparticlesinratsafterintratrachealinstillation.Cardiovasc.Toxicol.2013,13,194–207,doi:10.1007/s12012‐
013‐9198‐y.
13. Mortezaee,K.;Najafi,M.;Samadian,H.;Barabadi,H.;Azarnezhad,A.;Ahmadi,A.Redoxinteractionsandgenotoxicityof
metal‐basednanoparticles:Acomprehensivereview.Chem.Biol.Interact.2019,312,108814,doi:10.1016/j.cbi.2019.108814.
14. Sun,L.;Li,Y.;Liu,X.;Jin,M.;Zhang,L.;Du,Z.;Guo,C.;Huang,P.;Sun,Z.Cytotoxicityandmitochondrialdamagecausedby
silicananoparticles.Toxicol.Vitr.2011,25,1619–1629,doi:10.1016/j.tiv.2011.06.012.
15. Messerschmidt,C.;Hofmann,D.;Kroeger,A.;Landfester,K.;Mailänder,V.;Lieberwirth,I.Onthepathwayofcellularuptake:
Newinsightintotheinteractionbetweenthecellmembraneandverysmallnanoparticles.BeilsteinJ.Nanotechnol.2016,7,1296–
1311,doi:10.3762/bjnano.7.121.
16. Geiser,M.;Rothen‐Rutishauser,B.;Kapp,N.;Schürch,S.;Kreyling,W.;Schulz,H.;Semmler,M.;ImHof,V.;Heyder,J.;Gehr,
P.Ultrafineparticlescrosscellularmembranesbynonphagocyticmechanismsinlungsandinculturedcells.Environ.Health
Perspect.2005,113,1555–1560,doi:10.1289/ehp.8006.
17. Zhang,S.;Gao,H.;Bao,G.PhysicalPrinciplesofNanoparticleCellularEndocytosis.ACSNano2015,9,8655–8671,
doi:10.1021/acsnano.5b03184.
18. Hamra,G.B.;Guha,N.;Cohen,A.;Laden,F.;Raaschou‐Nielsen,O.;Samet,J.M.;Vineis,P.;Forastiere,F.;Saldiva,P.;Yorifuji,
T.;etal.Outdoorparticulatematterexposureandlungcancer:Asystematicreviewandmeta‐analysis.Environ.HealthPerspect.
2014,122,doi:10.1289/ehp.1408092.
19. Xia,T.;Kovochich,M.;Brant,J.;Hotze,M.;Sempf,J.;Oberley,T.;Sioutas,C.;Yeh,J.I.;Wiesner,M.R.;Nel,A.E.Comparisonof
theabilitiesofambientandmanufacturednanoparticlestoinducecellulartoxicityaccordingtoanoxidativestressparadigm.
NanoLett.2006,6,1794–1807,doi:10.1021/nl061025k.
20. Corbalan,J.J.;Medina,C.;Jacoby,A.;Malinski,T.;Radomski,M.W.Amorphoussilicananoparticlestriggernitric
oxide/peroxynitriteimbalanceinhumanendothelialcells:inflammatoryandcytotoxiceffects.Int.J.Nanomed.2011,6,2821–
2835,doi:10.2147/ijn.s25071.
21. Liu,Y.;Yoo,E.;Han,C.;Mahler,G.J.;Doiron,A.L.Endothelialbarrierdysfunctioninducedbynanoparticleexposurethrough
actinremodelingviacaveolae/raft‐regulatedcalciumsignalling.NanoImpact2018,11,82–91,doi:10.1016/j.impact.2018.02.007.
22. Chen,E.Y.;Garnica,M.;Wang,Y.C.;Mintz,A.J.;Chen,C.S.;Chin,W.C.AmixtureofanataseandrutileTiO2nanoparticles
induceshistaminesecretioninmastcells.Part.FibreToxicol.2012,9,2,doi:10.1186/1743‐8977‐9‐2.

Int.J.Mol.Sci.2021,22,1017316of18


23. Selvan,S.T.;YangTan,T.T.;KeeYi,D.;Jana,N.R.Functionalandmultifunctionalnanoparticlesforbioimagingandbiosensing.
Langmuir2010,26,11631–11641,doi:10.1021/la903512m.
24. Lee,K.I.;Su,C.C.;Fang,K.M.;Wu,C.C.;Wu,C.T.;Chen,Y.W.Ultrafinesilicondioxidenanoparticlescauselungepithelialcells
apoptosisviaoxidativestress‐activatedPI3K/Akt‐mediatedmitochondria‐ andendoplasmicreticulumstress‐dependent
signalingpathways.Sci.Rep.2020,10,9928,doi:10.1038/s41598‐020‐66644‐z.
25. Marchiando,A.M.;Graham,W.V.;Turner,J.R.Epithelialbarriersinhomeostasisanddisease.Annu.Rev.Pathol.Mech.Dis.2010,
5,119–144,doi:10.1146/annurev.pathol.4.110807.092135.
26. Ganesan,S.;Comstock,A.T.;Sajjan,U.S.Barrierfunctionofairwaytractepithelium.TissueBarriers2013,1,e24997,
doi:10.4161/tisb.24997.
27. Legendre,M.;Zaragosi,L.E.;Mitchison,H.M.Motileciliaandairwaydisease.Semin.CellDev.Biol.2021,110,19–33,
doi:10.1016/j.semcdb.2020.11.007.
28. Hewitt,R.J.;Lloyd,C.M.Regulationofimmuneresponsesbytheairwayepithelialcelllandscape.Nat.Rev.Immunol.2021,21,
347–362,doi:10.1038/s41577‐020‐00477‐9.
29. Fariss,M.W.;Gilmour,M.I.;Reilly,C.A.;Liedtke,W.;Ghio,A.J.Emergingmechanistictargetsinlunginjuryinducedby
combustion‐generatedparticles.Toxicol.Sci.2013,132,253–267,doi:10.1093/toxsci/kft001.
30. Sanchez,A.;Alvarez,J.L.;Demydenko,K.;Jung,C.;Alpizar,Y.A.;Alvarez‐Collazo,J.;Cokic,S.M.;Valverde,M.A.;Hoet,P.H.;
Talavera,K.SilicananoparticlesinhibitthecationchannelTRPV4inairwayepithelialcells.Part.FibreToxicol.2017,14,43,
doi:10.1186/s12989‐017‐0224‐2.
31. Yu,H.;Li,Q.;Kolosov,V.P.;Perelman,J.M.;Zhou,X.Regulationofparticulatematter‐inducedmucinsecretionbytransient
receptorpotentialvanilloid1receptors.Inflammation2012,35,1851–1859,doi:10.1007/s10753‐012‐9506‐x.
32. Memon,T.A.;Nguyen,N.D.;Burrell,K.L.;Scott,A.F.;Almestica‐Roberts,M.;Rapp,E.;Deering‐Rice,C.E.;Reilly,C.A.Wood
SmokeParticlesStimulateMUC5ACOverproductionbyHumanBronchialEpithelialCellsThroughTRPA1andEGFR
Signaling.Toxicol.Sci.2020,174,278–290,doi:10.1093/toxsci/kfaa006.
33. Kim,B.G.;Park,M.K.;Lee,P.H.;Lee,S.H.;Hong,J.;Aung,M.M.M.;Moe,K.T.;Han,N.Y.;Jang,A.S.Effectsofnanoparticleson
neuroinflammationinamousemodelofasthma.Respir.Physiol.Neurobiol.2020,271,103292,doi:10.1016/j.resp.2019.103292.
34. Shapiro,D.;Deering‐Rice,C.E.;Romero,E.G.;Hughen,R.W.;Light,A.R.;Veranth,J.M.;Reilly,C.A.Activationoftransient
receptorpotentialankyrin‐1(TRPA1)inlungcellsbywoodsmokeparticulatematerial.Chem.Res.Toxicol.2013,26,750–758,
doi:10.1021/tx400024h.
35. Piccinno,F.;Gottschalk,F.;Seeger,S.;Nowack,B.Industrialproductionquantitiesandusesoftenengineerednanomaterialsin
Europeandtheworld.J.Nanopart.Res.2012,14,1109,doi:10.1007/s11051‐012‐1109‐9.
36. Breysse,P.N.ToxicologicalProfileforSilica;ATSDR:Atlanta,GA,USA,2019.
37. Gilardino,A.;Catalano,F.;Ruffinatti,F.A.;Alberto,G.;Nilius,B.;Antoniotti,S.;Martra,G.;Lovisolo,D.InteractionofSiO2
nanoparticleswithneuronalcells:Ionicmechanismsinvolvedintheperturbationofcalciumhomeostasis.Int.J.Biochem.Cell
Biol.2015,66,101–111,doi:10.1016/j.biocel.2015.07.012.
38. Wei,X.;Jiang,W.;Yu,J.;Ding,L.;Hu,J.;Jiang,G.EffectsofSiO2nanoparticlesonphospholipidmembraneintegrityandfluidity.
J.Hazard.Mater.2015,287,217–224,doi:10.1016/j.jhazmat.2015.01.063.
39. Ariano,P.;Zamburlin,P.;Gilardino,A.;Mortera,R.;Onida,B.;Tomatis,M.;Ghiazza,M.;Fubini,B.;Lovisolo,D.Interactionof
sphericalsilicananoparticleswithneuronalcells:Size‐dependenttoxicityandperturbationofcalciumhomeostasis.Small2011,
7,766–774,doi:10.1002/smll.201002287.
40. Haase,A.;Dommershausen,N.;Schulz,M.;Landsiedel,R.;Reichardt,P.;Krause,B.C.;Tentschert,J.;Luch,A.Genotoxicity
testingofdifferentsurface‐functionalizedSiO2,ZrO2andsilvernanomaterialsin3Dhumanbronchialmodels.Arch.Toxicol.
2017,91,3991–4007,doi:10.1007/s00204‐017‐2015‐9.
41. Skuland,T.;Låg,M.;Gutleb,A.C.;Brinchmann,B.C.;Serchi,T.;Øvrevik,J.;Holme,J.A.;Refsnes,M.Pro‐inflammatoryeffects
ofcrystalline‐ Andnano‐sizednon‐crystallinesilicaparticlesina3Dalveolarmodel.Part.FibreToxicol.2020,17,13,
doi:10.1186/s12989‐020‐00345‐3.
42. Sharma,N.;Jha,S.Amorphousnanosilicainducedtoxicity,inflammationandinnateimmuneresponses:Acriticalreview.
Toxicology2020,441,152519,doi:10.1016/j.tox.2020.152519.
43. Distasi,C.;Dionisi,M.;Ruffinatti,F.A.;Gilardino,A.;Bardini,R.;Antoniotti,S.;Catalano,F.;Bassino,E.;Munaron,L.;Martra,
G.;etal.TheinteractionofSiO2nanoparticleswiththeneuronalcellmembrane:Activationofionicchannelsandcalciuminflux.
Nanomedicine2019,8,575–594,doi:10.2217/nnm‐2018‐0256.
44. Milici,A.;Talavera,K.Trpchannelsascellulartargetsofparticulatematter.Int.J.Mol.Sci.2021,22,2783,
doi:10.3390/ijms22052783.
45. Dekali,S.;Divetain,A.;Kortulewski,T.;Vanbaelinghem,J.;Gamez,C.;Rogerieux,F.;Lacroix,G.;Rat,P.Cellcooperationand
roleoftheP2X7receptorinpulmonaryinflammationinducedbynanoparticles.Nanotoxicology2013,7,1302–1314,
doi:10.3109/17435390.2012.735269.
46. Kojima,S.;Negishi,Y.;Tsukimoto,M.;Takenouchi,T.;Kitani,H.;Takeda,K.PurinergicsignalingviaP2X7receptormediates
IL‐1βproductioninKupffercellsexposedtosilicananoparticle.Toxicology2014,321,13–20,doi:10.1016/j.tox.2014.03.008.
47. Nagakura,C.;Negishi,Y.;Tsukimoto,M.;Itou,S.;Kondo,T.;Takeda,K.;Kojima,S.InvolvementofP2Y11receptorinsilica
nanoparticles30‐inducedIL‐6productionbyhumankeratinocytes.Toxicology2014,322,61–68,doi:10.1016/j.tox.2014.03.010.

Int.J.Mol.Sci.2021,22,1017317of18


48. Hofmann,F.;Bläsche,R.;Kasper,M.;Barth,K.Aco‐culturesystemwithanorganotypiclungsliceandanimmortalalveolar
macrophagecelllinetoquantifysilica‐inducedinflammation.PLoSONE2015,10,e0117056,doi:10.1371/journal.pone.0117056.
49. Nakanishi,K.;Tsukimoto,M.;Tanuma,S.ichi;Takeda,K.;Kojima,S.SilicananoparticlesactivatepurinergicsignalingviaP2X7
receptorindendriticcells,leadingtoproductionofpro‐inflammatorycytokines.Toxicol.Vitr.2016,35,202–211,
doi:10.1016/j.tiv.2016.06.003.
50. Braun,K.;Stürzel,C.M.;Biskupek,J.;Kaiser,U.;Kirchhoff,F.;Lindén,M.Comparisonofdifferentcytotoxicityassaysforin
vitroevaluationofmesoporoussilicananoparticles.Toxicol.Vitr.2018,52,214–221,doi:10.1016/j.tiv.2018.06.019.
51. Qi,Z.;Murase,K.;Obata,S.;Sokabe,M.ExtracellularATP‐dependentactivationofplasmamembraneCa2+pumpinHEK‐293
cells.Br.J.Pharmacol.2000,131,370–374,doi:10.1038/sj.bjp.0703563.
52. Dubyak,G.R.;el‐Moatassim,C.SignaltransductionviaP2‐purinergicreceptorsforextracellularATPandothernucleotides.
Am.J.Physiol.1993,265,C577–C606,doi:10.1152/ajpcell.1993.265.3.c577.
53. Burnstock,G.Historicalreview:ATPasaneurotransmitter.TrendsPharmacol.Sci.2006,27,166–176,
doi:10.1016/j.tips.2006.01.005.
54. Ralevic,V.;Burnstock,G.Receptorsforpurinesandpyrimidines.Pharmacol.Rev.1998,50,119–244,doi:10.1007/978‐3‐642‐28863‐
0_5.
55. Sherwood,C.L.;ClarkLantz,R.;Boitano,S.Chronicarsenicexposureinnanomolarconcentrationscompromiseswound
responseandintercellularsignalinginairwayepithelialcells.Toxicol.Sci.2013,132,222–234,doi:10.1093/toxsci/kfs331.
56. vanderVliet,A.;Bove,P.F.Purinergicsignalinginwoundhealingandairwayremodeling.Subcell.Biochem.2015,55,139–157,
doi:10.1007/978‐94‐007‐1217‐1_6.
57. Lazarowski,E.R.;Boucher,R.C.Purinergicreceptorsinairwayepithelia.Curr.Opin.Pharmacol.2009,9,262–267,
doi:10.1016/j.coph.2009.02.004.
58. Burnstock,G.P2Xreceptorsinsensoryneurones.Br.J.Anaesth.2000,84,476–488,doi:10.1093/oxfordjournals.bja.a013473.
59. Nakamura,F.;Strittmatter,S.M.P2Y1purinergicreceptorsinsensoryneurons:Contributiontotouch‐inducedimpulse
generation.Proc.Natl.Acad.Sci.USA1996,93,10465–10470,doi:10.1073/pnas.93.19.10465.
60. Burnstock,G.;Brouns,I.;Adriaensen,D.;Timmermans,J.P.Purinergicsignalingintheairways.Pharmacol.Rev.2012,64,834–
868,doi:10.1124/pr.111.005389.
61. Burnstock,G.PurinergicSignallingandNeurologicalDiseases:AnUpdate.CNSNeurol.Disord.‐DrugTargets2017,16,257–265,
doi:10.2174/1871527315666160922104848.
62. Burnstock,G.Purinesandsensorynerves.Handb.Exp.Pharmacol.2009,194,333–392,doi:10.1007/978‐3‐540‐79090‐7_10.
63. Fischer,W.;Wirkner,K.;Weber,M.;Eberts,C.;Köles,L.;Reinhardt,R.;Franke,H.;Allgaier,C.;Gillen,C.;Illes,P.
CharacterizationofP2X3,P2Y1andP2Y4receptorsinculturedHEK293‐hP2X3cellsandtheirinhibitionbyethanoland
trichloroethanol.J.Neurochem.2003,85,779–790,doi:10.1046/j.1471‐4159.2003.01716.x.
64. Vilotti,S.;Marchenkova,A.;Ntamati,N.;Nistri,A.B‐typenatriureticpeptide‐induceddelayedmodulationofTRPV1andP2X3
Receptorsofmousetrigeminalsensoryneurons.PLoSONE2013,8,e81138,doi:10.1371/journal.pone.0081138.
65. Droguett,K.;Rios,M.;Carreño,D.V.;Navarrete,C.;Fuentes,C.;Villalón,M.;Barrera,N.P.AnautocrineATPrelease
mechanismregulatesbasalciliaryactivityinairwayepithelium.J.Physiol.2017,595,4755–4767,doi:10.1113/JP273996.
66. Wirsching,E.;Fauler,M.;Fois,G.;Frick,M.P2purinergicsignalinginthedistallunginhealthanddisease.Int.J.Mol.Sci.2020,
21,4973,doi:10.3390/ijms21144973.
67. Carew,M.A.;Wu,M.L.;Law,G.J.;Tseng,Y.Z.;Mason,W.T.ExtracellularATPactivatescalciumentryandmobilizationvia
P2U‐purinoceptorsinratlactotrophs.CellCalcium1994,16,227–235,doi:10.1016/0143‐4160(94)90025‐6.
68. Riteau,N.;Baron,L.;Villeret,B.;Guillou,N.;Savigny,F.;Ryffel,B.;Rassendren,F.;LeBert,M.;Gombault,A.;Couillin,I.ATP
releaseandpurinergicsignaling:Acommonpathwayforparticle‐mediatedinflammasomeactivation.CellDeathDis.2012,3,
e403,doi:10.1038/cddis.2012.144.
69. Baron,L.;Gombault,A.;Fanny,M.;Villeret,B.;Savigny,F.;Guillou,N.;Panek,C.;LeBert,M.;Lagente,V.;Rassendren,F.;et
al.TheNLRP3inflammasomeisactivatedbynanoparticlesthroughATP,ADPandadenosine.CellDeathDis.2015,6,e1629,
doi:10.1038/cddis.2014.576.
70. Sienaert,I.;Huyghe,S.;Parys,J.B.;Malfait,M.;Kunzelman,K.;DeSmedt,H.;Verleden,G.M.;Missiaen,L.ATP‐inducedCa2+
signalsinbronchialepithelialcells.Pflug.Arch.Eur.J.Physiol.1998,436,40–48,doi:10.1007/s004240050602.
71. Communi,D.;Paindavoine,P.;Place,G.A.;Parmentier,M.;Boeynaems,J.M.ExpressionofP2Yreceptorsincelllinesderived
fromthehumanlung.Br.J.Pharmacol.1999,127,562–568,doi:10.1038/sj.bjp.0702560.
72. Kasper,J.;Hermanns,M.I.;Bantz,C.;Maskos,M.;Stauber,R.;Pohl,C.;Unger,R.E.;Kirkpatrick,J.C.Inflammatoryandcytotoxic
responsesofanalveolar‐capillarycoculturemodeltosilicananoparticles:Comparisonwithconventionalmonocultures.Part.
FibreToxicol.2011,8,6,doi:10.1186/1743‐8977‐8‐6.
73. Rabolli,V.;Badissi,A.A.;Devosse,R.;Uwambayinema,F.;Yakoub,Y.;Palmai‐Pallag,M.;Lebrun,A.;DeGussem,V.;Couillin,
I.;Ryffel,B.;etal.ThealarminIL‐1αisamastercytokineinacutelunginflammationinducedbysilicamicro‐andnanoparticles.
Part.FibreToxicol.2014,11,69,doi:10.1186/s12989‐014‐0069‐x.


Int.J.Mol.Sci.2021,22,1017318of18


74. Skuland,T.;Øvrevik,J.;Låg,M.;Schwarze,P.;Refsnes,M.Silicananoparticlesinducecytokineresponsesinlungepithelialcells
throughactivationofap38/TACE/TGF‐α/EGFR‐pathwayandNF‐κΒ signalling.Toxicol.Appl.Pharmacol.2014,279,76–86,
doi:10.1016/j.taap.2014.05.006.
75. Grynkiewicz,G.;Poenie,M.;Tsien,R.Y.AnewgenerationofCa2+indicatorswithgreatlyimprovedfluorescenceproperties.J.
Biol.Chem.1985,260,3440–3450,doi:10.1016/s0021‐9258(19)83641‐4.

