ContentslistsavailableatScienceDirect
Journal
of
Trace
Elements
in
Medicine
and
Biology
j ou rn a l h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / j t e m b
Are
iron
oxide
nanoparticles
safe?
Current
knowledge
and
future
perspectives
Vanessa
Valdiglesias
a,1,
Natalia
Fernández-Bertólez
a,b,1,
Gözde
Kilic¸
c,
Carla
Costa
d,e,
Solange
Costa
d,e,
Sonia
Fraga
d,e,
Maria
Joao
Bessa
d,e,
Eduardo
Pásaro
a,
João
Paulo
Teixeira
d,e,
Blanca
Laffon
a,∗aDICOMOSAGroup,DepartmentofPsychology,AreaofPsychobiology,UniversidadedaCoru˜na,EdificiodeServiciosCentralesdeInvestigación,Campus
Elvi˜nas/n,ACoru˜na15071,Spain
bDepartmentofCellandMolecularBiology,UniversidadedaCoru˜na,FacultaddeCiencias,CampusAZapateiras/n,ACoru˜na15071,Spain cDivisionofMolecularToxicology,InstituteofEnvironmentalMedicine,KarolinskaInstitutet,Stockholm17177,Sweden
dDepartmentofEnvironmentalHealth,PortugueseNationalInstituteofHealth,RuaAlexandreHerculano,321,Porto4000-055,Portugal eEPIUnit—InstituteofPublicHealth,UniversityofPorto,RuadasTaipas,135,Porto4050-600,Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received29February2016
Receivedinrevisedform29March2016 Accepted30March2016
Keywords:
Ironoxidenanoparticles Invivostudies Invitrostudies Epidemiologicalstudies Toxicity
a
b
s
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Duetotheiruniquephysicochemicalproperties,includingsuperparamagnetism,ironoxidenanoparticles (ION)haveanumberofinterestingapplications,especiallyinthebiomedicalfield,thatmakethemone ofthemostfascinatingnanomaterials.Theyareusedascontrastagentsformagneticresonanceimaging, intargeteddrugdelivery,andforinducedhyperthermiacancertreatments.Togetherwiththesevaluable uses,concernsregardingtheonsetofunexpectedadversehealtheffectsfollowingexposurehavebeen alsoraised.Nevertheless,despitethenumerousIONpurposesbeingexplored,currentlyavailable infor-mationontheirpotentialtoxicityisstillscarceandcontroversialdatahavebeenreported.AlthoughION havetraditionallybeenconsideredasbiocompatible–mainlyonthebasisofviabilitytestsresults– influ-enceofnanoparticlesurfacecoating,size,ordose,andofotherexperimentalfactorssuchastreatment timeorcelltype,hasbeendemonstratedtobeimportantforIONinvitrotoxicitymanifestation.Invivo studieshaveshowndistributionofIONtodifferenttissuesandorgans,includingbrainafterpassingthe blood-brainbarrier;neverthelessresultsfromacutetoxicity,genotoxicity,immunotoxicity, neurotoxic-ityandreproductivetoxicityinvestigationsindifferentanimalmodelsdonotprovideaclearoverviewon IONsafetyyet,andepidemiologicalstudiesarealmostinexistent.Muchworkhasstilltobedonetofully understandhowthesenanomaterialsinteractwithcellularsystemsandwhat,ifany,potentialadverse healthconsequencescanderivefromIONexposure.
©2016ElsevierGmbH.Allrightsreserved.
Contents 1. Introduction...54 2. Invitrostudies...54 2.1. Cellulareffects...54 2.1.1. ROSgeneration ... 55 2.1.2. Ionrelease...55 2.2. Geneticeffects...56 3. Invivostudies...56
3.1. Toxicokineticsandacutetoxicity...57
3.2. Genotoxicity...57
∗ Correspondingauthor.
E-mailaddress:blaffon@udc.es(B.Laffon).
1 Theseauthorscontributedequallytothiswork.
http://dx.doi.org/10.1016/j.jtemb.2016.03.017
3.3. Neurotoxicity...58 3.4. Immunotoxicity...58 3.5. Reproductivetoxicity...59 4. Epidemiologicalstudies ... 59 5. Concludingremarks ... 59 Conflictofinterest...60 Acknowledgements ... 60 References...60 1. Introduction
Nanotechnologyisrapidlyexpanding.Withtheincreased appli-cations of nanotechnology products, especially for biomedical purposes, concerns regarding the onset of unexpected adverse healtheffects followingexposurehavebeenalsoraised. Under-standingoftoxicologicalprofilesofengineerednanomaterialsis necessaryinordertoensurethatthesematerialsaresafeforuse andaredevelopedresponsibly,withoptimizationofbenefitsand minimizationofrisks.However,developmentandproductionof engineerednanomaterials are increasingfaster than generation oftoxicologicalinformation.Thislackofinformationonpossible adverseeffectsofnanomaterialshasbeentakenintoconsideration bymanyorganizationsworldwidesuchastheUSEnvironmental ProtectionAgency(EPA),theWorldHealthOrganization(WHO), the US National Institute for Occupational Safety and Health (NIOSH),theEuropeanCommission(EC)andtheOrganizationfor EconomicCo-operation andDevelopment(OECD).Official docu-mentshavebeenpreparedbytheseorganizationsaddressingthe needofdedicatedresearchonappropriatemethodologicalassays forassessingengineerednanomaterialstoxicity[1].Consequently, startingintheearly2000s,concernsaboutthepotentialhuman and environmental health effects of nanomaterials were being expressedbymanyscientists,regulators,andnon-governmental agencies.Indeed,asaproofofthegrowinginterestonthistopic, thenumberofscientificarticlespublishedon‘nanotoxicity’or ‘nan-otoxicology’increasedprogressively in thelast decade (around 1700sofar,accordingtoPubMeddatabase);before2005itwas almostnegligible.
Among engineered nanomaterials magnetic nanoparticles – madeofiron,cobalt,ornickeloxides–offerpromisingpossibilities inbiomedicalfieldmainlyduetotheirspecialphysicochemical fea-tures,includingtheirprovenbiocompatibilityandtheirmagnetic propertiesthatallowthemtobemanipulatedbyanexternal mag-neticfieldgradient[2].Particularly,nanoparticlesmadeofa ferro-orferromagneticmaterial,i.e.,ironoxidenanoparticles(ION),can exhibitauniqueformofmagnetismcalledsuperparamagnetism, whichappearswhentheIONsizeisbelowacriticalvalue– depend-ingonthematerial,buttypicallyaround10–20nm–,andwhen thetemperatureisabovetheso-calledblockingtemperature[3]. Thissuperparamagneticbehaviourishighlyusefulinbiomedicine foranumberofapplicationsmainlyrelatedtodiagnosis,tumour imaging,imagingofthecentralnervoussystemforneurovascular, neurooncologicalorneuroinflammatoryprocesses,anddrug deliv-ery[4,5].Indeed,clinicaluseofseveralIONascontrastagentsfor imagingwerealreadyapprovedbytheUSFoodandDrug Admin-istrationsince1996(USFDA)[6–8].Therefore,duetothecurrent andpromisingbiomedicalusesofIONinvolvingthedirectcontact withdifferenttissuesandorgans,studiesaddressingtheirpotential toxicityareespeciallyrelevant.
IONareusuallymadeofacrystallinecoreandasurface coat-ingforstabilizingthecorepropertiesandoptionallyforpreventing theaggregation.ThecrystallinecoreofION,madeofferri-(Fe3+)or
ferro-(Fe2+)magneticmaterial,isgenerallysynthesizedthrough
protocolswithcontrolledprecipitationofironoxidesinorganic
solution[9],orinaqueoussolutionbyaddingabase[10].Among theeightiron oxidesknown,magnetite(Fe3O4),maghemite (
␥-Fe2O3)andhematite(␣-Fe2O3)arethemostcommonlyuseddue
totheirpolymorphisminvolvingtemperature-inducedphase tran-sition; they have unique biochemical, magnetic, catalytic, and otherproperties which provide suitabilityfor specifictechnical andbiomedicalapplications[9].Surfaceofcommerciallyavailable nanoparticlesisnormallymodifiedbycoatingwithdifferent mate-rialsinordertostabilizethem,modifytheirbiodistribution,and enhancetheirbiocompatibility.Thiscoatingisappliedbyaddinga stabilizingcoatingmaterial[e.g.,citrate,dextran,carboxydextran, chitosan, pullulan, polyethylene glycol (PEG), polyvinyl alcohol (PVA),polyethylenimine(PEI),polyethyleneoxide(PEO), polysac-charide,albumin,lipids,etc.]tomonocrystalline(uniformIONwith closeparticlesizedistribution)orpolycrystalline(withsignificant sizevariance)ION[11].Furthermore,particlecoatingmaybe fur-thermodified,especiallyincaseofmedicaluses,withfluorescent dyesforimaging[12,13],targetingmolecules[13,14],drugs[15]or nucleicacids[16,17].Thisgreatvarietyofcoatingsleadstomany diversetypesofIONwithdifferentpotentialactionmechanisms andtoxicpatterns.
IONhavebeenreportedinmanystudiestobehighly biocom-patiblenanomaterialswithnoneorlowtoxicitywhichdonotpose aseriousthreattotheorganism[18–21].Despitebeing consid-eredasgenerallysafe,potentialIONtoxicitycannotbecompletely discardedsinceresultsfromstudiesonthisregardareoften con-tradictoryandIONeffectsatparticularlevels,suchasgeneticor carcinogenic,have beenpoorlyaddressed.Also, theireffects on wholeorganismsand,specially,humanhealthrisksrelatedto occu-pationalandenvironmentalexposuretoIONhavebeenscarcely evaluated.Onthisbasis,andinordertoimprovetheknowledgein thisfield,theaimofthisreviewwastocompiletheinvitro,invivo andepidemiologicalstudiesonIONtoxicitypublishedtodate.Thus, theresultsandconclusionsfromthemainIONtoxicologystudies wereanalysed,providingageneralviewofthecurrent informa-tiononIONsafetyavailableaswellashighlightingthemaingaps ofknowledgeinthefieldthatmustbefurtheraddressed.
2. Invitrostudies
2.1. Cellulareffects
MoststudiesanalysingIONtoxicity arefocusedoncytotoxic effectsofthesenanoparticlesoncellcultures.Anumberof differ-entcelllinesandtestingconditionshavebeenassessedreporting IONcellulareffectsatdifferentlevels,mainlydecreaseinviability, ROSproduction, andiron ion release,but alsoapoptosis induc-tion,cellcyclealterations,cellmembranedisruptions,cytoskeleton modifications, etc. An exhaustive revision of the former works canbefoundin somepreviouspapers[22,23].Since then, sev-eralstudiespublishedaddressing thepotentialIONcytotoxicity showingeneralnoneorlowcytotoxiceffectsofthese nanopar-ticles. For instance, no adverse cellular effects were found in primaryratcerebellarcortexastrocytestreated withPEI-coated ION (magnetite) [24], in cultured rat astrocytes treated with
citrate-ordimercaptosuccinate(DMSA)-coatedION(maghemite)
[25,26],inmurinebonemarrowcellstreatedwithbareor citrate-modified ION (Fe2O3) [21], in human T lymphocytes exposed
topolyacrylicacid(PAA)-coatedornon-coatedION (magnetite)
[27], in human mesenchymal stem cells treated with ferucar-botran (magnetite/maghemite-carboxydextran) [28], in human amnioticfluid cells(hAFC)[29]incubatedwith carboxydextran-coatednanomagnetite,inculturedprimaryratcerebellarneurons treatedwithDMSA-coatedION(maghemite)[30],or,ingeneral, afterexposureofmurinemicroglialcellstodifferentION[31–33].
Nevertheless,otherinvitrostudieshavereportedpositive cyto-toxicity outcomes after ION exposure. Szalay et al. [34] found thatION(magnetite)inducedmoderatetime-and concentration-dependentdecreaseinviabilityofVerocellsafter24hexposure, andlowgenerationofcytotoxicitywasobservedinL-929 fibrob-laststreatedwithIONmodifiedwithdifferentfunctionalgroups
[35–37]. Similarly, decreasesin viabilitywere foundin human alveolarepithelial A549 cells treated withION (Fe2O3)[38], in
culturedratastrocytesexposedtoaminosilane-orstarch-coated ION(magnetite)atphysiologicaltemperatures(34–40◦C)[39],in humanglioblastoma(T98GandU251MG)andurinarybladder car-cinoma(ECV304)celllinesaftertreatmentwithrhamnose-coated ION(magnetite)[40]andinhumanneuronal(SHSY5Y)andglial (A172)cellsexposedtooleicacid-coatedorsilica-coatedmagnetite nanoparticles[41].
Onthebasisofmostofthesestudies,IONseemtobeinitially safeforbiomedicalusesincetheirpotentialcytotoxiceffects,if any,areusuallyslightorlimitedtospecificconditions(e.g.,highest dosesand/orlongestexposuretimes),andcytotoxicityis consid-eredonly asadecreaseincellviability.However,otherreports havedemonstratedthatthesenanoparticlescanexertotherdrastic effectsonthecellwellbeing(reviewedinRef.[42]).Indeed, dif-ferentcellulareffects–mainlydependentonIONconcentration, timeofexposure,presenceandtypeofcoating,andcelltype eval-uated–havebeenreportedafterIONexposureininvitrostudies. Thoseeffectsincludeplasmaticmembraneimpairment[43,44],cell cyclealterations[45,46],cytoskeletondisruption[47],autophagy
[48,49],changesinmitochondrialmembranepotential[50,51],and alterationsincellmotility[52]andincellintegrity[53].
In summary, someauthorshave suggestedthat inconsistent resultsonIONcellulareffectsmayberelatedtonanoparticle fea-tures,mainlysizeandsurfacecoating[54,55].Thus,Lietal.[56]
carriedoutacomparativestudymeasuringsomecytotoxiceffects, namely intracellular enzymatic activity and membrane disrup-tion,inhumancervicalcancer(HeLa)celllineandimmortalized normalhumanretinalpigmentepithelial(RPE)celllineexposed toION(magnetite).ResultsobtainedshowedthatuncoatedION resultedtoxictobothHeLaandRPEcellsatahighconcentration (0.40mg/ml); however, atlow concentrations, cytotoxicitywas cell-typespecific,beingRPEcellsmoresusceptiblethanHeLacells. Witha similarapproach,Soenenetal.[57]testedfourdifferent IONtypes(dextran-coatedEndorem,carboxydextran-coated Reso-vist,lipid-coatedmagnetoliposomesandcitrate-coatedverysmall ironoxideparticles)onc17.2neuralprogenitorcells,and differ-entcytotoxicpotential relating tothetype of coatingwasalso observedinthiscase.Thus,citrate-coatedIONresultedthemost toxicand lipid-coatedonesweretheless toxic.MoreoverRivet etal.[55]investigatedtheresponseofprimarycorticalneuronsto aminosilane-,dextran-andpolydimethylamine-coatedION (mag-netite),andobserveddifferenteffectsdependingonnanoparticle doseandcoating. Furthermore,uncoatedION(magnetite)were found tobe non-cytotoxic to human lymphoblastoidTK6 cells and primary humanblood cells, while oleate-coated magnetite nanoparticleswerecytotoxicina dose-dependentmanner[58]. Similarly,uncoatedION(magnetite)prevented–whilePAA-coated ION prevented – apoptotic signalling and apoptosis in human
neutrophils in vitro [59]. In another study, aminosilane-coated nanomagnetitewasfoundtodecreaseviabilityofprimarycortical culturedneurons,butinadiversegradedependingonwhetherION werepositivelyornegativelycharged[60].Morerecently,Schütz etal.[48]reportedthatthestressresponseofHT29andCaco2cells wascell-andnanoparticle-specific.However,otherauthors sug-gestedthatIONconcentrationmaybeanevenmorecriticalfactor forcytotoxicitythansurfacemodificationorsize[36,61].
AstheION-inducedcytotoxiceffectsreportedintheliterature arefrequently relatedtoreactiveoxygenspecies (ROS) genera-tionandironionrelease,thesespecificoutcomesareaddressed separatelyinthefollowingsubsections.
2.1.1. ROSgeneration
Anumberofinvitrostudieshaveassociatedcytotoxicityinduced byIONwithoxidativestressandROSproduction[62–64].Thus, anincreasedgenerationofROSbyIONexposurewaspreviously observedin Chinesehamsterovary(CHO-K1) cells[65],murine macrophage J774 cells [66], different vascular endothelial cells
[67,68],Chinesehamsterlungcells [69],humanlungA549cells
[43,70],brainmicrogliacells[71],andglialT98GandU251MGand bladderECV304cells[40].However,otherstudiesreported nega-tiveresultsonROSproductionafterIONtreatment[72,73].Several authorshavesuggestedthatoxidativestressandROSgeneration inducedbyIONcanbeassociatedwiththepresenceandtypeof surfacecoating[65,72].Inanycase,thispresenceofhigh intracel-lularROSlevelsmay[63,68]ormaybenot[43,70]associatedwith ION-inducedcytotoxicity.
SincethebrainisparticularlyvulnerabletoROSdamage,a num-ber of studieshave evaluated oxidative stress consequences of treatmentwithdifferentIONonculturedneuralcells.WuandSun
[45]foundoxidativestress,decreaseinneuronviability,and activa-tionofJNK-andp53-mediatedpathwaystoregulatethecellcycle andapoptosisinPC12cellstreatedwithION(Fe3O4).Wangetal.
[74]observedthattreatmentwithION(␣-and␥-Fe2O3)ledto
gen-erationofROSandnitricoxide,cellproliferation,andphagocytosis inmousemicroglialBv2cells,andROSformationwasalsofoundin humanbrain-derivedendothelialcells(amodeloftheblood-brain tumourbarrier)treatedwitholeicacid-andpolyvinylamine-coated ION(Fe3O4)[75].Recently,Pettersetal.[71]comparedtheeffects
ofDMSA-coatedION(␥-Fe2O3)exposureondifferentbraincells,
namely,microglialcells,neuronsandastrocytes.Resultsshowed thatIONtreatmentinducedincreasesiniron content,ROS gen-erationandcelltoxicityin microglialcells butnotinastrocytes andneurons.Similarlytothesenegativeresultsinastrocytesand neuronalcells,Hohnholtetal.[76,77]reportedneithersubstantial ROSproductionnoranyalterationinthecellularthiolreduction potentialafterexposureofoligodendroglialOLN-93cellstoION (␥-Fe2O3).
2.1.2. Ionrelease
Duetoiron capacitytoswitchbetweenferric(Fe3+)and
fer-rous(Fe2+)ionicformsbyeasilyacceptinganddonatingelectrons
(reduction-oxidationreactions),itplaysacriticalroleinimportant organicmetabolicpathwayssuchascytochrome P450function, mitochondrialoxidativephosphorylation,oxygentransport,DNA synthesis,andforenergyproduction[78].Nevertheless,asexcess of thismetal canbeverytoxic, iron levelsintheorganismare strictlycontrolled.FreeironreleasedfromIONmetabolisationcan beincorporatedtothenormalcellularironpoolfromtheendocytic compartment[22].Thus,IONexposureinavarietyofcellscaused elevatedintracellularironconcentrations,dependentonthedose
[25,32,40,79].Therefore,thenormalbodycapacitytomanageiron shouldbetakenintoaccountwhenconsideringadministrationof highorfrequentlyrepeateddosesofION[20].
Apartfromnanoparticleexposurecharacteristics,alsocell fea-turescaninfluenceIONeffectssince,dependingoncelltype,iron ionsreleasedfromIONcanbeharmlessforcells[25,32,79],induce cytotoxicity[80],orevenbeusedbycellsfortheirownmetabolism, asitwasobservedforoligodendroglialOLN-93cells[76,77].A pos-sibleexplanationisthat,undernormalconditions,ironreleased fromION canbeaccumulated in cells where it isstored as an iron-ferritincomplextoannulthehightoxicity associatedwith freeiron[80,81].Hence,thisstoragelikelycontributestohighcell resistancetoiron toxicity andis especially relevantin the ner-voustissue,sinceeventheprolongedpresenceoflargeamounts ofaccumulatedIONdoesnotharmthesecells.Onthisregard,a recentreviewonIONuptakeandmetabolisminbrainastrocytes suggeststhattheefficientuptakeofextracellulariron(liberated slowlyfromION)byastrocytes,aswellastheirstrongup-regulation ofthesynthesisofferritincontributetothehighresistanceofthese cellstoirontoxicity.Soastrocytesdealwellwithanexcessofiron andprotectthebrainagainstiron-mediatedtoxicity [81].These resultsaresupportedbyrecentfindingsshowingthatastrocytes, andalsoneurons,are moreresistantagainstacuteIONtoxicity, likely due to a slow transfer of internalized nanoparticles into thelysosomal compartment, requiredfor iron ion release from ION[71].However,underpathologicalconditions(suchascancer, atherosclerosis,hypertensionorarthritis)ironmayeffectivelybe releasedfromferritinleadingtoincreasedoxidativedamageand causingcellulartoxicity[82,83].
2.2. Geneticeffects
AnumberofinvitrostudieshaveevaluatedtheeffectsofION exposureongeneticmaterial,mainlyby meansofcometassay and micronucleus(MN) approach. Still, there is lackof consis-tence in ION genotoxicity results, even at similar doses. Thus, cometassayevaluationofmurineL-929fibroblaststreatedwith ION (magnetite) coated with (3-aminopropyl)trimethoxysilane (APTMS),tetraethylorthosilicate(TEOS)-APTMS,orcitrateshowed aconcentration-dependentincreaseinDNAdamageregarding con-trolcells[36].SimilargenotoxiceffectsweredescribedafterION (magnetite)treatmentinalveolarA549and bronchialepithelial BEAS-2Bcells[84],embryonickidneyHEK-293cellsand periph-eralbloodlymphocytes[85],andinskinepithelialA431cells[86], andalsoinhumanlymphoblastoidTK6cellsandprimaryhuman leukocytesexposedtooleate-coatednanomagnetite[58].Usingthe samemethodology,Bhattacharyaetal. [87]foundDNAdamage inductionin human lung IMR-90fibroblasts and human BEAS-2Bcellstreatedwithnanohematite,andRajivetal.[44]observed DNAbreaksandchromosomeaberrationsinhumanlymphocytes exposed to ION (Fe2O3).Also MN production wasobserved in
humanlymphoblastoidMCL5 cells treated withdextran-coated maghemitenanoparticlesfor24h[88].Similarly,positiveresponse wasobservedinA549cellstreatedwithbarenanomagnetiteboth in thecomet assay and MN test, but the damagingeffect was reducedbysimultaneousexposuretoN-acetylcysteineorby pre-treatmentwithbutylated hydroxyanisole, both ROS scavengers
[70].However,itwasrecentlyreportedthatoxidativestressplays, atmost, only a marginal role in genotoxicity induction (evalu-atedbycometassay)bysurface-modifiedmagnetitenanoparticles
[89].
Still,studiesshowingnegativeresultsforIONgenotoxicityare even morefrequent. Karlssonet al. [38,90]observed no induc-tionofprimaryDNAdamage(cometassay)inA549cellsexposed toION(Fe2O3 and Fe3O4),but oxidativeDNAdamage was
pro-ducedbymagnetite nanoparticles.Also, MNfrequency wasnot foundtobe altered in humanlymphoblastoid cells after treat-mentwithuncoatednanomaghemiteorwithnanomagnetite,both uncoatedanddextran-coated[88].Likewise,Guichardetal.[91]
obtainedneither increase in DNA damage, evaluated bycomet assay, nor induction of MN formation using Fe2O3 (primarily
maghemite) and magnetite nanoparticles to treat Syrian ham-ster embryo cells. Genotoxicity caused by exposureof Chinese hamster lung cells toglutamic acid-coatedFe2O3 nanoparticles
wasassessedbyusingthesametestsandnosignificantpositive responsewasobtained,althoughcellredoxstatuswasslightly dis-turbed[69].Liuetal.[92]foundnoincreaseintheincidenceof chromosomeaberrationsinChinesehamsterlungcellstreatedwith ION(10nm)withpositivelychargedPEIsurfaceorwithneutral non-functionalPEG-coatedION(10and 30nm).Besides,normal humanfibroblastsexposed tomeso-2,3-dimercaptosuccinicacid (DMSA)-coatedmaghemitenanoparticlesshowedalsonoincreases inDNA damageattributedin parttotheinhibition ofpotential toxicity by the DMSA coating, which acts as a barrier avoid-ingdirectcontactbetweenfibroblastsandthenanoparticlecore
[93].DNAdamagewasneitherobservedaftertreatmentofL-929 fibroblastswithbareorTEOS-coatedION(magnetite)[36]orin lymphoblastoidTK6cellsandprimaryhumanleukocytestreated withuncoatednanomagnetite[58].Morerecently,Coutoetal.[27]
alsodemonstratedabsenceofIONeffects ongeneticmaterialof humanT-lymphocytesreportingnochromosomeaberrations in cellstreatedwithPAA-coatedandnon-coatednanomagnetitefor 48h.Inagreementwiththesestudies,Paolinietal.[40]reported absenceofgenotoxicandcarcinogeniceffectsofrhamnose-coated ION (magnetite)on mouse fibroblast Balb/c-3T3 cells. Further-more,anumberofstudiesevaluatingthepotentialmutagenicity inducedbydifferentIONbymeansofAmestest, withor with-outmetabolicactivation,werealsoreportedwithnegativeresults
[34,94,95].However,Gomaaetal.[85]describedbothpositiveand negativeresultsintheAmestestforION(magnetite)depending ontheadministereddoseandthepresenceofmetabolic activa-tion.Supportingthisobservation,Liuetal.[92]concludedrecently thatIONmutagenicitycouldbedependentonnanoparticlesizeand surfacecoatingafterhavingfoundapositivemutagenicresponse (Amestest)incellstreatedwithPEG-coatedION(10nm)butnot incells treatedwithPEI-coatedION(10nm)orPEG-coatedION (30nm).
3. Invivostudies
Since nanomaterial studies based oncell cultures are often inconsistent and might underestimate their effects, toxicity of nanomaterialsneedstobeexaminedinwholeanimalsystems[96]. Besides,nanomaterialuptakeanddistributioninthebodyare com-plexprocessesthatcannotbeproperlyaddressedinculturedcells, andinvitroparticlesizecanchangewhenusedinvivoduetothe additionaldepositionofsalts,opsonizationwithplasmaproteins, lipidsorcarbohydrates,dependingonthesurfacechargesofthe coatingmolecules,orduetotheclusteringofnanoparticlestoform conglomerates[5].Thus,invivostudiesonnanomaterialstoxicity areessentialandhaveanobviousadvantageoverinvitrotests, pro-vidingactualinformationaboutoveralleffectsonalivingorganism, includingthefinalcellortissuetargets.
However,invivostudies onIONtoxicity arestill scarceand alsoshowedcontroversialresults.Thus,negativeortrivialtoxicity resultswerereportedfordifferentIONinseveralorgansofWistar ratstreatedintravenously[97],intratracheally[34]ororally[98], inmiceexposedorally[99]orsubcutaneously[100],ingrowing chickensafteroraladministration[101],andinmonkeysanddogs intravenouslytreated[95].Onthecontrary,positivetoxicity out-comeswereobservedinseveralrodentmodelsexposedtoIONby pulmonary[64,102],intraperitoneal[47,103,104]orintravenous
3.1. Toxicokineticsandacutetoxicity
Itisgenerallywellacceptedthatthetoxicokineticsof metal-licnanoparticlesdependsontheparticletype,size,surfacecharge, surfacecoating,proteinbinding,exposureroute,dose,andspecies
[106]. Bourrinet et al. [95] investigated ION toxicokinetics in rats and monkeys intravenously treated with ferumoxtran-10. The highest ION uptake was obtained in spleen, followed by central lymph nodes, peripheral lymph nodes, liver, and bone marrow,withageneraleliminationhalf-lifeofabout8-10days. StudiesdescribingIONtoxicokineticsinotherinvivomodels (usu-allyrodents)arefrequentlyaccompaniedbyassessmentofacute toxicityoutcomes.Severaloftheseworksreportedabsenceof tox-icityafter IONadministration. Thus, differentconcentrations of magnetite-zincoxidecore-shellnanoparticleswereinjected sub-cutaneously,weeklyforfourweeks,toC57BL/6micetoexamine tissuedistribution,excretionpatternandsystemictoxicity[100]. Nodistributionwasobservedtobrain,spleen,lung,kidneyorliver, andneitherchanges inzinc concentrationsin urineand faeces. Besides,absenceofsignificantmodificationsinmortality,clinical observations,bodyweight,foodintake,waterconsumption, uri-nalysis,haematology,serumbiochemistry,ororganweightswas reported.Justadose-dependentincreaseingranulomatous inflam-mationwasfoundattheinjectionsiteoftreatedanimals,butno otherhistopathological lesion in anyorgan couldbeattributed tothenanoparticleexposure.Moreover,nosystemicdistribution ofFe2O3 nanoparticlesorsignificantchanges intoxicity
param-eterswereobservedinSprague-Dawleyratsorallyadministered, dailyovera13-weekperiod[99].Ansciauxetal.[107] function-alizedseveralultrasmalliron oxideparticleswithpeptidesthat presentanaffinityforamyloid-peptide,forbeingusedinearly diagnosisofAlzheimer’sdisease.Theparticlescoupledtopeptide C-IPLPFYN-Cdemonstratedabilitytocrosstheblood-brainbarrier inmicewithoutanyfacilitatingstrategy,andaccumulatedinbrain 90minaftertheirintravenousinjection.However,notoxiceffects wereobservedafterexposureandnoneofthederivativestested wasfoundinanyorganoneweekafteradministration; elimina-tionhalf-lifewasabout3h.Recently,Yangetal.[108]investigated thesize-dependentinvivokinetics,toxicityandgeneexpression changescausedbycarboxyl-coatedION(magnetite,diameters10, 20, 30, and 40nm). They demonstrated that ION accumulated primarilyinliver and spleenof Kunmingmiceonthefirst day post-intravenous injection, following a size-dependent pattern, with10nmnanoparticlesshowingthehighestuptakebyliverand 40nmnanoparticlesthehighestuptakebyspleen.Moreover,10nm IONwereclearedfasterfromliverandkidneys,butenteredmore readilybrainanduterus,whereas40nmIONaccumulatedmore readilybutwereeasilyeliminatedinspleen.Noapparentsignsof acutetoxicitywereobserved,butIONexposurewasabletochange theexpressionlevelofsensitivegenesrelatedtooxidativestress, irontransport,metabolicprocesses,andapoptosis,amongothers. Likewise,Chamorroetal.[101]reportedaccumulationof nanopar-ticlesinliver,spleen,orduodenumofgrowingchickenschronically exposedtolowdosesofION(␥-Fe2O3)byoralroute;faeceswere
themainexcretionroute.Itwasalsonoticedthatironionswere releasedasaconsequenceofthepartialIONtransformationbythe acidgastricenvironment,buttheywereabsorbedenhancingthe ferricoverferrouspathway.Besides,nomortalityoradversesigns orsymptomswereobserved.Lackoftoxiceffectswasalsoreported inratstreatedwithION(II,III)sincenohistopathologicalalterations werefoundintheseanimalsafterasingleintratrachealinstillation
[34].
Nevertheless,oppositetoallthesestudies,anumberofworks havedescribedacuteanimaltoxicityafterIONexposure.Kumari etal.[109]reportedthatWistarratsexposedorallytoION(Fe2O3)
for28 days showeddistributionofthe nanoparticlestoseveral
organs,includingbrain,andtoxicsignssuchasdullness,irritation and moribundconditions,but nomortality.Increasediron con-tent,changesinhomeostasisoftraceelementsandimmunological alterationswereobservedinseveralorgansofmicethatreceived asingleION(magnetite)doseinjectedthroughthetailvein[110]. Nanoparticleswereprimarilydistributedtoliverat1week post-injection,and ironlevelsincreasedremarkablyin thymus,lung, heart,liver,andspleenat4weekspost-injection;at13weeks post-injection,ironlevelswerethehighestinthespleen.Furthermore, Kwonetal.[111]studiedthebiodistributionandbiomodificationof ION(Fe3O4and␣-Fe2O3)inDaphniamagnaandfoundanumberof
morphologicalchanges(e.g.,irregularshapedmicrovilli,epithelial cellprotrusion,anddilatationofcytoplasmicinclusion)inthegut tissuesofthesecrustaceans,along withbacterialcolonizationof thegutlumen,afterIONexposure,evenwithoutpenetratingthese tissues.TheauthorssuggestedthattheseeffectsmaybeduetoION biomodificationsprobablyinvolvingoxidativedissolutionofFe3O4
followedbyarapidprecipitationofferricoxideorhydroxide. Tworecentindependent studiesreportedalsorelevanttoxic effectsinmouseandratlungsafterIONadministration.RaduBalas etal.[112]evaluatedbiochemicalandhistopathologicalchangesin CD-1miceexposedtoIONcoatedwithphospholipid-based poly-mericmicellesbyintravenousinjection.Alterationsinactivityof severalenzymes–includingcatalase,glutathionereductase,lactate dehydrogenase,superoxidedismutaseandglutathioneperoxidase –werefoundintreatedanimalsregardingthecontrols. Further-more,histopathological modifications,dose-dependentdecrease ofthemouselungcapacityandmajorchangesintheexpression ofapoptosismarkerswerealsohighlighted.AndSadeghietal.[64]
evaluatedtheeffectsofIONonlungtissueofadultmaleWistar ratsafterpulmonaryinhalation.Administerednanoparticles pen-etratedthecirculation,rapidlyreachedliver,andcausedserious inflammationinlungandlivertissues.Resultsalsoshowed signif-icantincreaseoffreeradicalsandreductionofglutathioneinlung tissue,togetherwithpulmonaryemphysemaandinterstitial hyper-emiainlungs,andhepaticinjuriesthatledtoreleaseofhepatic enzymestothebloodserum.
In addition, Baratliet al.[113] examinedthe effects of ION (Fe3O4)onmitochondrialrespiratorychaincomplexactivitiesand
mitochondrial coupling in young (3 months) and middle-aged (18months)ratlivers,findinginterestingdifferencesdepending onanimalage.In youngindividuals,IONexposuredidnotalter mitochondrialfunction;however,nanoparticlesdose-dependently impairedallcomplexesofthemitochondrialrespiratorychainin middle-agedratliver.AndGustafssonetal.[114]investigatedthe inflammatoryandimmunologicalresponsestoIONinhealthy non-sensitizedmice,andinsensitizedmicewithanestablishedallergic airwaydisease.Animalswereexposedtohematitenanoparticles forupto7daysanddifferenttoxicresponses–including alter-ationsinwhitebloodcelllevelsandreductionofalveolarspace– wereobservedhighlydependentontheinitialmouserespiratory features.
Takingtheresultsfromallthesestudiestogether,itseemsthat differencesfoundintheliteratureregardingIONtoxicokineticsand associatedtoxicityarerelatedtonanoparticlesize[108],crystalline phaseanddissolutionrate[111],administereddose[101],andage
[113]orpreexistentpathologicalstate[114]ofthestudyanimals. 3.2. Genotoxicity
In vivo studies addressingthe potential genotoxic effects of IONarescarceintheliterature,andresultsobtainedfromthem are notconclusive yet.Different typesof DNA alterations were foundin a number ofanimal studiesafter IONexposure. Thus, DNA-protein crosslinksandoxidative DNAdamage (8-hydroxy-deoxyguanosine)were observedin hepatic and renal tissuesof
Kunmingmicetreateddailyfor1weekwithmagnetite nanoparti-clesviaintraperitonealinjection[103].AnincreaseinMNfrequency wasalsodetectedinbonemarrowcellsofmiceintraperitoneally exposed to magnetite nanoparticles[104], or after intravenous administrationofpolyasparticacid-coatedION(magnetite)[105]. Totsuka et al. [115] examined genotoxic effects of magnetite nanoparticlesinmiceintratracheallyinstilled,obtainingsignificant increasesinDNAadductlevels,oxidativestressandDNAbreaksin treatedanimalsascomparedwithcontrols.Inaddition, inflamma-torycellinfiltrationandfocalgranulomatousformationswerealso observedinlungsofexposedmice,suggestingthatan inflamma-toryresponsemightbeinvolvedingenotoxicityinducedbyIONin micelungs[116].Recently,AlFarajetal.[102]performeda lon-gitudinalstudyonBalb/cmiceintrapulmonaryadministeredION (PEG-coatedmagnetitemodifiedwithnegative[carboxyl]or pos-itive[amine]terminal)acutelyandsub-acutely.Accumulationof IONwasdetectedintheliverafterhigh-doseadministration. Fur-ther,asignificantincreaseinlipidperoxidation,DNAdamageand geneexpressionofCCL-17andIL-10biomarkerswasobservedin bothacuteandsub-acutesets.Theeffectsobservedresultedsurface coating-dependent,showingIONwithcarboxylterminalaslightly prominenteffectcomparedtotheaminemodification.
Despite these works, severalin vivo studies reported nega-tiveresultsongenotoxic effectsafterION exposure.Estevanato etal.[117]evaluatedgenotoxiceffectsofmaghemitenanoparticles encapsulatedwithinalbumin-basednanospheresonfemaleSwiss miceintraperitoneallyinjectedbyperformingMNtest systemat-icallyfrom30minuntil30daysafterinjection,andnoevidence ofMNproductionwasfoundinanycase.AlsonoincreaseinMN frequencywasobservedinbonemarrowcellsfromKunmingmice exposedbyabdominalinjectiontomagnetitenanoparticleseither naked[118]orcombinedwithdaunorubicin[47].Similarly,single andrepeatedintravenous(bolus)administrationof ferumoxtran-10(anultrasmallsuperparamagneticIONcontrastagent)didnot produceincreaseofMNfrequencyinmicebonemarrowafter24 or48hofexposure[95].Inthesamestudy,genotoxicitywas addi-tionallymeasuredbyassessingDNArepairasunscheduledDNA synthesis(UDS)inprimaryhepatocyteculturesfromtreatedrats, butnegativeresultswereobtainedalsointhiscase.Moreover,Liu etal.[92]foundnegativegenotoxicresponse,evaluatedbymeans ofMNtest,inmaleNIHmiceintraperitoneallyexposedtoPEG-and PEI-coatedION.Besides,Fe2O3(primarilymaghemite)
nanoparti-clesorallyadministeredinasingledosetoWistarratswereeasily abletopass acrosstheintestinalbarrier and,eventhoughthey weremainlyaccumulatedinliver,spleen,kidney,heartandbone marrow,theydidnotcauseDNAdamage(accordingcometassay results)andMNproductioninperipheralleukocytesor chromo-someaberrationsinbonemarrowcells[98].
TogetherwiththehighvariabilityinIONtypeanddosetested, experimentaldesignmay alsohelpexplain thenon-concordant resultsobtainedfromgenotoxicityassessmentofthese nanopar-ticles.Hence,someauthorshighlightedtheneedofcarryingouta rangeofgenotoxicityassaysinthesamestudyinordertocover allthepotentialformsof DNAdamageandaccordingly provide properconclusionsonthegenotoxicpotentialof nanomaterials
[119,120].Furthermore,possibleinterferenceofnanoparticleswith standardgenotoxicitymethodologiesisusuallynotconsideredin thesestudiesand itshouldbe,since interferencehasbeen pre-viouslydemonstratedtobelikelypresent,altering theobtained results[121,122].
3.3. Neurotoxicity
IONhavebeenshowntodisplaytheabilitytocrossthe blood-brainbarrierafteroral[123],inhalatory[111],andintraperitoneal
[124]administration,andtodirectlyreachthebrainthroughthe
olfactorynerveafterintranasalinstallation[125].Thisabilitymakes themespeciallyeligibleformedicalpurposesonnervoussystem, suchasdrugdeliveryandimagingdiagnostics,butalsopotentially harmfulforthissystem.Hence,aspecialattentionmustbepayedto thenervoustissuephysiologyandbehaviouraloutcomesinanimal studies.Nevertheless,unliketheconsiderableamountofstudies addressinginvitroeffectsofIONonneuralcells,thenumberof invivostudiesonpotentialneurotoxicityofthesenanoparticlesis quiterestricted.
Most of the in vivo studies on ION neurotoxicity employed ratsasexperimentalmodel.Hence,Kumarietal.[109]observed dullnessand irritationinWistar ratsafter28 daysof oraldaily exposuretoION(Fe2O3).Moreover,asignificantdose-dependent
inhibition of total, Na+-K+, Mg2+ and Ca2+-ATPases in brain, as
wellas acetylcholinesterase in brain and red blood cells, were foundinexposedanimals,suggestingthatIONexposuremayaffect synaptictransmissionandnerveconduction.Similarly,Bourrinet et al.[95] observed differentphysiological responses,including signs of polypnea, exophthalmos and mydriasis in this species afterintravenoustreatmentofION(ferumoxtran-10),althoughno neurobehavioural,neurovegetative,orpsychotropiceffectswere detected.Morerecently,Kimetal.[126]treatedSprague-Dawley rats with different ION (DMSA-coated maghemite, and DMSA-, PEG- and PEG-Au-coated magnetite) by intraneural injection (sciaticnerve);IONcausedimmunecellinfiltration,neural inflam-mationand apoptosis,andinducedneuralantioxidantresponse. Thesameyear,Wuetal.[127]detectedaregionaldistributionof ION(magnetite)inbrainofratsintranasallyinstilledforsevendays. IONinducedoxidativedamageinstriatumbutnotin hippocam-pus,despitethepresenceofnanoparticlesinbothregionsresulted particularlyhigh.
Agreeingwiththesestudiesonrats,neurotoxicityofIONhas beenalsoreportedinmiceand fish.Inmice,intranasal admin-istrationofFe2O3 nanoparticlesinducedpathologicalalterations
in olfactory bulb, hippocampusand striatum; microglial prolif-eration,activationandrecruitmentwerealsoobservedinthese areas,especiallyintheolfactorybulb[74].Inaddition,micetreated withmagnetitenanoparticlesbyintragastricadministrationwere reportedtoshowlessactivityandaslightlossofappetite[123].In fish,dextran-coatedFe3O4nanoparticlesintraperitoneally
admin-istered to adult zebrafish were found to accumulate in brain inducingapoptosisandinhibitionofacetylcholinesteraseinthis tis-sue.Moreover,althoughnoalterationsintheexpressionofgenes associatedwithinflammationwereobserved,increasedlevelsof ferricironandenhancedmRNAlevelsofcaspase-8,caspase-9and transcriptionalfactorAP-1inbrainoftreatedanimalswerealso detected[129].
3.4. Immunotoxicity
ImmunotoxiceffectsrelatedtoIONexposurewerealsoreported some recent in vivo studies. Park et al. [130] investigated the tissue distribution and immunotoxicity of ION (Fe2O3) in
six-week-oldmaleIRCmiceafterintravenousinjection.Increasediron levelsregardingthecontrolwerefoundinalltissuesevaluated, notablein liver,spleen andthymus, and alsoalterations inthe immunesystemwereobservedintreatedanimals.Thoseincluded increasedlevelsofwhitebloodcellsandneutrophils,interleukin (IL)-8secretionandlactatedehydrogenaserelease.Likewise, sys-temic administration of dextran-stabilized IONalso resulted in enhancedproliferationofmitogen-stimulatedspleen-derived lym-phocytesandsecretionofIL-1inmaleWistarratsafter7days ofintravenousadministration[131].Sadeghietal.[64]observed serious inflammation in lungs and liver of Wistar rats treated withION(Fe2O3)bypulmonaryadministrationaswellas
Similarly, Gustafsson et al. [114] observed an immunological response in mice exposed to ION (hematite). They described increasedlevelsof neutrophils,eosinophils,andlymphocytesin theairwaysofhealthymiceondays1and2post-exposure,but decreaseoflymphocytesinsensitized(withanestablished aller-gicairway disease)mice.Opposite totheseresults, Wangetal.
[128]foundnosignificantdifferencesinsplenocyteproliferation or cytokine release in mice fed with magnetite nanoparticles. However,despiteproportionsofT-lymphocytesubsetswerenot alteredafterlowIONexposures,CD3(+)CD4(+)andCD3(+)CD8(+) T-lymphocytesubsetswerehigherinanimalsexposedtomedium andhighIONdosesthatinthecontrolgroup.Finally,Parketal.
[110]foundthatmagnetitenanoparticlesintravenously adminis-teredtomicecandisturbhomeostasisoftheimmuneregulation. Particularly,after13weekspost-injection,theyobservedincreased percentagesofneutrophilsandeosinophils,enhanced releaseof lactate dehydrogenase, and elevated secretion of IL-8 and IL-6 inthebloodoftreatedanimals.Furthermore,expressionof anti-genpresentationrelated-proteinsandmaturationofdendriticcells resultedinhibitedafterIONexposurewhereasexpressionofseveral chemokineswasenhancedinsplenocytes.
3.5. Reproductivetoxicity
SimilartothereportsonIONneurotoxicityorimmunotoxicity, thenumberofstudiesaddressingreproductivetoxicityofIONis scarce.Initially,IONwereconsideredtoshowlowdevelopmental toxicityorteratogeniceffectsafterhavingexposedXenopus lae-visembryos(FrogEmbryoTeratogenesisAssayXenopus,FETAX assay)toION(Fe2O3)for48handobservingnomortalityor
sig-nificantmalformation; slightharmfuleffects, namelyvariations intotal body lengthandsnout ventlength,wereonly notedat thehighestconcentrationtested[132].Thesameyear,Noorietal.
[133]evaluatedIONeffectsonreproductionandoffspringofmice treatedwithDMSA-coatedmagnetitenanoparticles.Eventhough noadverseeffectsongestationandfoetalgrowthwereobserved, asignificantdecreaseintheoffspringgrowthandmaturationafter birthandabout70%deathbeforereachingpubertywerereported. Besides,maleoffspringshoweddecreaseinthelevelsof spermato-gonia,spermatocytes,spermatidsandmaturesperms,suggesting thatembryoandfoetalmousedevelopmentmightbedisruptedby placentaandfoetusexposuretoION.Agreeingwiththeseresults, alaterstudyonzebrafish(Daniorerio)showedthatuncoatedION (␣-Fe2O3)induceddevelopmentaltoxicityinearlylifestagesofthis
species,includingmortality,hatchingdelay,andembryonic malfor-mation[134].AndalsoFe2O3nanoparticles(primarilymaghemite)
werefoundtoinducecytotoxicityandROSproductioninSyrian hamsterembryocellsexposedfor72h[91].However,Piccinetti etal.[135]recentlyobservedthatsilica-coatedmagnetite nanopar-ticlesdonotinduceanytoxicityinzebrafishlarvaeexposedthrough foodforupto15days.Inthiscase,nanoparticleswereexcreted throughfaeces,andtheydidnotactivatedetoxificationprocesses orpromotetissue/cellinjuryinlarvaeoradultindividuals.
IONeffectsonfertility,reproductiveperformance, embryotoxic-ity,foetotoxicity,andteratogenicitywerealsoevaluatedinratsand rabbitsexposedtoferumoxtran-10[95].Althoughnoeffectswere generallyobservedonfertilityorearlyembryonic development, mildlymaternaltoxicityandmajorfoetalskeletalmalformations weredescribed in both species. Recently, developmental toxic-ityandbiodistributionofa singledoseversusmultipledosesof IONwithpositiveornegativesurfacecharges(PEI-coatedFe2O3
orPAA-coatedFe2O3,respectively)werealsoinvestigatedinvivo
in pregnant CD-1 mice [136]. In this case, multiple doses of positively-chargednanoparticlesgivenoverseveraldaysresulted insignificantlyincreasedfoetaldeathsandaccumulationofiron inthefoetalliverandplacenta.Thesameauthorsalsoevaluated
theeffectsofprenatalexposuretotheseIONonmiceduring criti-calstagesoforganogenesis[137].AlowdoseofIONdidnotinduce toxicity,butfoetallossesandmorphologicalalterationsoftheuteri andtestesofsurvivingoffspringwereobservedafterhighIONdose exposure.Furthermore,theeffectsatshort-orlong-termvaried dependingonthetypeofIONemployed.
4. Epidemiologicalstudies
Epidemiologicalstudies in thenanotoxicology field are very scarce. A limited number of nanomaterials, including titanium dioxide(TiO2)nanoparticles,carbonnanotubes(CNT)or
inciden-talultrafinenanoparticles,havebeenevaluatedsofartodetermine thehealthrisksassociatedwiththeiroccupationalexposure[138]. Properpopulationstudiesonenvironmentalnanomaterial expo-sureareevenmorelimited,almostinexistent.Thissignificantlack ofstudiesis likelyrelated tothedifficultyof characterisingthe exposurepatterns.For instance,thenanoparticle componentof theenvironmentalairparticulatepollutionhasnotbeen specifi-callymeasuredevermainlybecauseitisnotpossibletoseparate nanoparticlerelatedeffectsfromtheeffectsoflargerparticulates thatwereomnipresent[139].
Inthisregard,fieldstudiesontheexposurecharacteristicsof manufacturednanoparticlesarerelevant,buttheyarealsolimited thusfar.InthecaseofION,anddespitethegreatrelevanceoftesting thepotentialharmfuleffectsoftheseparticularnanoparticleson humanhealth,todatejusttworecentstudieshaveaddressedthis issue.Xingetal.[140]studiedtheexposurecharacteristics (par-ticlenature,metric-dependentconcentrationandparticlesize)of airborneIONgeneratedbythemanufacturingprocessesofFe2O3
nanomaterialsinafactoryinZhejiangprovince(EastChina).Inthis study,relevantbaselinedataonthecharacteristicsofIONexposure whichcouldbeusedforfurtherepidemiologicalstudieswere estab-lished. Thus, theyobservedthat nanoparticle generationability relatedtothedifferentworkingactivities(powderscreening, mate-rialfeedingandpackaging)varieddependingontheirnatureand thattheparticleconcentrationexhibitedperiodicityandactivity relevance.Althoughnopotentialadversehealthoutcomes associ-atedwithIONexposurewereaddressedinthisstudy,itsfindings highlightedtherelevanceofevaluatingthepotentialhealthrisks relatedtoIONinworkplaceswhereexposuretothesenanoparticles isespeciallynoticeable.Additionally,Pelclovaetal.[141]analysed differentoxidativestressbiomarkersin exhaledbreath conden-sate(EBC)andurinesamplesof14workersoccupationallyexposed toiron oxideaerosol (with more than80% of particles smaller than100nmindiameter)duringironoxidepigmentproduction foranaverageof10±4years,and14matchedcontrols.Almost alltheoxidativestressbiomarkersevaluated,includingmarkers oflipid,nucleicacidandproteinoxidation,resultedincreasedin EBCofworkersregardingthecontrolindividuals.Nodifferences inoxidativemarkersofbothgroupswerefoundinurinesamples. Theseresultsemphasizethenecessitytoperiodicallymonitorfor potentialadversehealtheffectsallIONexposedemployees, includ-ingresearchworkerswhoaredirectlyexposedbyhandlingthese nanoparticlesinthelab.
5. Concludingremarks
Dueto theiruniquephysicochemical properties, IONhave a numberof interestingcurrentand potentialfuture applications, especiallyinthebiomedicalfield,thatmakethemoneofthemost fascinatingnanomaterials.Amongotheruses,theyarecurrently employedincelllabelling,drugtargeting,genedelivery,biosensors, hyperthermiatherapy,anddiagnostics,andtheyhavepromising futureusesintherapiesagainstcancerandotherdiseases.However,
allthesemedicalapplicationsrequireinternalizationof IONfor efficientdiagnosisortreatment,leadingtopotentialrisks associ-atedwithexposure.Astheseapplicationsareincreasinginnumber andinbenefits,thereisanimperativeneedtocomprehensively investigateandelucidatethebiologicalundesirableconsequences ofexposuretothesenanomaterials.Still,despitethenumerousION purposesbeingexplored,insufficientand/orcontroversial informa-tionisavailableontheirpotentialtoxicity.
ThispaperrevisedthetoxiceffectsofIONreportedsofarby dif-ferentinvitro,invivoandepidemiologicalstudies.Theanalysisofall datacollectedhighlightsthelackofconsensusinestablishingthe toxicitymechanismassociatedwithIONexposure,mainlydueto thehighvariabilityofparticlespresentamongthedifferentstudies. Thepresence[48],chemicalcomposition[65],andcharge[60]of surfacecoatingseemtobecriticalfactorsforIONtoxicity;usually barenanoparticlesaremoretoxicthanthecoatedones.But experi-mentalconditions,suchascell/tissuetype,concentration,exposure time,administrationpathwayandpresenceofproteincorona,can alsoinfluencetoxicityresults. Specialattentionmustbepaidto thetechnicalproceduresemployedfor toxicityevaluation,since IONhavebeenrecentlyproventointerferewiththeapproaches commonlyusedforcytotoxicity[41,58]andgenotoxicity[121,122]
evaluation,leadingtoobtainfalsepositiveresults.
Togetherwiththelownumberofstudiespublished,especially thoseperformedinwholeorganisms,thereisindeedamarked dif-ficultytosetupcomparisonsandestablishatoxicitypatternfor IONmainlybecauseofthedifferentnanoparticlestested,butalso tothelackofmethodologicalstandardization.Duetothevarietyof mechanismsthatcouldleadtonanomaterialinducedcelltoxicity,a batteryofharmonizedtestingsystemsisrequiredtoestablishthe presumptivetoxicpotentialofIONatdifferentlevels.Moreover, inordertomakeresultsobtainedintheseinvestigationsonION withdifferentcoatingsandcharacteristicscomparable,theuseof standardizedmethods,includingpropertestingofpotential inter-ferenceswithstandardprotocols,ishighlydesirableineachcase.
Insummary,IONtoxicity,althoughsuspectedtobelow, can-notbeproperlyestablishedyetsinceresultsfrominvitrostudies are often contradictory, in vivo studies are scarce, and human epidemiologicalstudiesarealmostinexistent.Hence,inviewof theextremelyusefulpresentandpromisingfutureapplicationsof ION,especiallythoserelatedtobiomedicalpurposeswhichinvolve theirdirectintroductioninthehumanbody, theinteractions of thesenanomaterialswithcellularsystems,aswellasthepotential adversehealthconsequencesofIONexposure,requiretobefully understood,andmuchworkhasstilltobedoneinthisfield.
Conflictofinterest
Theauthorsdidnotreportanyconflictofinterest.
Acknowledgements
ThisworkwassupportedbyXuntadeGalicia(EM2012/079),the projectNanoToxClass(ERAERASIINN/001/2013),andbyTD1204 MODENACOSTAction.
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