Arora et al.

15 Pages·2012·0.63 MB·English

ToxicologyandAppliedPharmacology258(2012)151–165
ContentslistsavailableatSciVerseScienceDirect
Toxicology and Applied Pharmacology
journal homepage: www.elsevier.com/locate/ytaap
Review
Nanotoxicology and in vitro studies: The need of the hour
⁎
Sumit Arora, Jyutika M. Rajwade, Kishore M. Paknikar
CentreforNanobioscience,AgharkarResearchInstitute,G.G.Agarkarroad,Pune411004,India
a r t i c l e i n f o a b s t r a c t
Articlehistory: Nanotechnologyisconsideredasoneofthekeytechnologiesofthe21stcenturyandpromisesrevolutionin
Received6May2011 ourworld.Objectsatnanoscale,takeonnovelpropertiesandfunctionsthatdiffermarkedlyfromthoseseen
Revised12November2011 inthecorrespondingbulkcounterpartprimarilybecauseoftheirsmallsizeandlargesurfacearea.Studies
Accepted15November2011
have revealed that the same properties that make nanoparticles so unique could also be responsible for
Availableonline2December2011
theirpotentialtoxicity.Nanotechnologyisrapidlyadvancing,withmorethan1000nanoproductsalready
onthemarket.Consideringthefactthatintendedaswellasunintendedexposuretonanomaterialsisincreas-
Keywords:
ingandpresentlynoclearregulatoryguideline(s)onthetesting/evaluationofnanoparticulatematerialsare
Nanotoxicology
Invitrocytotoxicity available,theinvitrotoxicologicalstudiesbecomeextremelyrelevantandimportant.Thisreviewpresentsa
Bio-distributionofnanoparticles summaryofnanotoxicologyandaconciseaccountoftheinvitrotoxicitydataonnanomaterials.Fornanoma-
Genotoxicityofnanoparticles terialstomoveintotheapplicationsarena,itisimportantthatnanotoxicologyresearchuncoversandunder-
Moleculardeterminantsofnanotoxicology stands how these multiple factors influence their toxicity so that the ensuing undesirable effects can be
avoided.
©2011ElsevierInc.Allrightsreserved.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Nanotoxicology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Physicochemicalpropertiesofnanomaterials:biologicaleffects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Routesofexposure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Respiratorytract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Gastrointestinaltract(GIT). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Biodistribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Moleculardeterminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Genotoxicityandimmunogenicpotential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Regulatoryissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Methodsforassessingtoxicityofnanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Currentlyusedinvitromethodsinnanotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Hemolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Genotoxicityassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Newermethodstoassessnanomaterialtoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Eco-toxicology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Datagapsandresearchneeds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Concludingremarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Conflictofinterest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
⁎ Correspondingauthor.Fax:[phone].
E-mailaddress:[email protected](K.M.Paknikar).
0041-008X/$–seefrontmatter©2011ElsevierInc.Allrightsreserved.
doi:10.1016/j.taap.2011.11.010
152 S.Aroraetal./ToxicologyandAppliedPharmacology258(2012)151–165
Size/Shape
Surface area Surface chemistry
Physicochemical
determinants
Government Respiratory tract
Routes of
Industry Regulatory issue Skin
exposure
Academy Gastrointestinal Tract
Nanotoxicology
Mutagenesis Clearance
Genotoxicity Biodistribution
Chromosomal Opsonization
Aberrations
Molecular
determinants
Oxidative
Inflammation stress
Fig.1.Complexarrayofissuessurroundingtoxicityofnanoparticles.
Introduction Anyinvivouseofnanoparticlesentailsthoroughunderstandingof
thekineticsandtoxicologyoftheparticles(Lewinskietal.,2008),estab-
Theprefix“nano”isderivedfromtheGreekword“nanos”meaning lishmentofprinciplesandtestprocedurestoensuresafemanufacture
“dwarf”.Nanotechnologyinvolvesthemanipulationandapplicationof andusageofnanomaterials(Neletal.,2006),andcomprehensiveinfor-
engineeredparticlesorsystemsthathaveatleastonedimensionless mation about their safety and potential hazard (Nel et al., 2006;
than 100 nanometers (nm) in length (Hoyt and Mason, 2008). The Oberdorsteretal.,2005b).
term “nanoparticles” applies only to engineered particles (such as
metaloxides,carbonnanotubes,fullerenesetc.)anddoesnotapplyto Nanotoxicology
particles under 100nm that occur naturally or are by-products of
other processes such as welding fumes, fire smoke, or carbon black Nanotoxicologywasproposedasanewbranchoftoxicologytoad-
(HoytandMason,2008). dress the gaps in knowledge and to specifically address the adverse
Growingexplorationofnanotechnologyhasresultedintheidentifi- health effects likely to be caused by nanomaterials (Donaldson et al.,
cationofmanyuniquepropertiesofnanomaterialssuchasenhanced 2004).Intheoriginalarticleonnanotoxicology,Donaldsonetal.(2004)
magnetic,catalytic,optical,electrical,andmechanicalpropertieswhen quoted,“disciplineofnanotoxicologywouldmakeanimportantcontri-
comparedtoconventionalformulationsofthesamematerial(Ferrari, butiontothedevelopmentofasustainableandsafenanotechnology”.
2005;Qinetal.,1999;Vasiretal.,2005;Websteretal.,1999,2000). Nanotoxicologyencompassesthephysicochemical determinants,
Thesematerialsareincreasinglybeingusedforcommercialpurposes routes of exposure, biodistribution, molecular determinants, geno-
such as fillers, opacifiers, catalysts, water filtration, semiconductors, toxicity,andregulatoryaspects(Fig.1).Inaddition,nanotoxicology
cosmetics,microelectronicsetc.leadingtodirectandindirectexposure isinvolvedinproposingreliable,robust,anddata-assuredtestproto-
inhumans(Neletal.,2006).Apartfromtheuseofnanomaterialsin colsfornanomaterialsinhumanandenvironmentalriskassessment
consumerproducts,numerousapplicationsarebeingreportedinthe (Donaldsonetal.,2004;Lewinskietal.,2008).
biomedicalfield,especiallyasdrug-deliveryagents,biosensorsorimag-
ingcontrastagents(Ferrari,2005;Vasiretal.,2005).Theapplications Physicochemicalpropertiesofnanomaterials:biologicaleffects
pertainingtomedicineinvolvedeliberatedirectingestionorinjection
ofnanoparticlesintothebody.Nanomaterialsforimaginganddrugde- The unusual physicochemical properties of engineered nanoma-
liveryareoftenintentionallycoatedwithbiomoleculessuchasDNA, terialsareattributabletotheirsmallsize(surfaceareaandsizedistri-
proteins,andmonoclonalantibodiestotargetspecificcells(Lewinski bution), chemical composition (purity, crystallinity, electronic
et al., 2008). Materials in this size range may approach the length propertiesetc.),surfacestructure(surfacereactivity,surfacegroups,
scale at which some specific physical or chemical interactions with inorganicororganiccoatingsetc.),solubility,shapeandaggregation.
their environment can occur (Oberdorster et al., 2005a). Apart from Actually,theverysamepropertiesthatleadtothetechnicaladvan-
this,duetotheirextremelysmallsize,nanomaterialspossessextremely tages of nanotechnology also lead to unique biological effects (Nel
highsurfaceareatovolumeratiowhichrendersthemhighlyreactive. etal.,2006).InareviewbyNeletal.(2006)aquestion,“Donanoma-
Highreactivitypotentiallycouldleadtotoxicityduetoharmfulinterac- terials properties necessitate a new toxicological science?” was
tions of nanomaterials with biological systems and the environment raised.Itwasarguedthatthemaincharacteristicofnanomaterialsis
(Oberdorsteretal.,2005b). their size in the transitional zone between individual atoms or
S.Aroraetal./ToxicologyandAppliedPharmacology258(2012)151–165 153
Table1
Biologicaleffectsduetophysicochemicalpropertiesofnanomaterials.
Physicochemicalproperty Toxicokineticfindings Biologicaleffects References
Size 15nmgoldnanoparticles Mostwidespreadorgandistribution Biodistributionofthe Sonavaneet
(NPs) includingblood,liver,lung,spleen, nanoparticles al.(2008)
kidney,brain,heart,stomachinmice
15and50nmgoldNPs Passblood–brainbarrier(BBB) BloodBrainBarrier(BBB) Sonavaneet
inmice permeability al.(2008)
40–50nmgoldNPs Activationofmembranereceptorsin Jiangetal.
SK-BR-3cells (2008)
50nmgoldNPs MaximumuptakebyHelacells Chithraniet
al.(2006)
50nmquantumdots Efficientreceptor-mediatedendocytosis Osakietal.
inHelacells (2004)
1–10nmsilverNPs ExclusivelyattachtoHIV-1 Elechiguerra
etal.(2005)
1–10nmsilverNPs Penetrateinsidethebacteria Moroneset
al.(2005)
Shape Open-endedSingle-walled EfficientblockingofionchannelsinCHO Sphericalshapedclose-ended Parketal.
carbonnanotubes(SWNTs) cells SWNTsarecomparativelyless (2003)
reactive
SphericalgoldNPs HigheruptakebyHelacells Rrod-shapedgoldNPsshowed Chithraniet
lessuptake al.(2006)
Carbonparticles,except Stimulatedhumanplateletaggregation Biologicalreactivity:mixed Radomskiet
C60CS, invitroandacceleratedtherateof carbonnanoparticles al.(2005)
vascularthrombosisinratcarotid (MCNs)≥single-walledcarbon
arteries nanotubes(SWNTs)>multi-
walledcarbonnanotubes
(MWNTs)
Filomicelles(Filamentous Moreefficientfordrugdelivery Gengetal.
micelles) thantheirspherical (2007)
counterpartsinratsandmice
Surface TiO2(300cm2Surfacearea) Increasedlymph-nodeburdensand Morereactiveinratsas Tranetal.
area/ Inflammation comparedtoBaSO4(200cm2 (2000)
volume Surfacearea)
ratio TiO2andBaSO4Withsame Inflammatoryeffectsweresimilar Inflammation Tranetal.
surfacearea (2000)
Ultrafinecarbonblackparticles Causegreaterpulmonarytoxicity Increasedreactivityin Nikulaetal.
(270m2/gsurfacearea) inrats comparisonwithlarger-sized (1995),
carbonblackparticles(22m2/g Driscollet
surfacearea) al.(1996)
Chemical Incorporationof1%(w/w) IncreaseinUVAabsorptionand Wakefield
composition manganesedopinginto reductioninfreeradical etal.(2004)
titaniaparticles generationviasurfacereactions
Carbonnanomaterials Differentgeometricstructuresexhibit Thecytotoxicityfollowsa Guangetal.
quitedifferent sequenceorder: (2005)
cytotoxicityinvitro SWNTs>MWNTs>quartz>C60
onalveolarmacrophages
isolatedfromguineapigs
Metaltracesassociatedwiththe Adose-andtime-dependentincrease Morereactiveascomparedto Pulskampet
commercialcarbonnanotubes ofintracellularreactiveoxygenspecies purifiedcarbonnanotubes al.(2007)
andadecreaseofthemitochondrial
membranepotentialinratmacrophages
(NR8383)andhumanA549lungcells
Quantumdotscoremetalloid cancrosstheblood–brainbarrierand Aprobablecarcinogen Hardman
complexesofCadmium,Cd placenta,andissystemicallydistributed (2006)
toallbodilytissues,withliverandkidney
beingtargetorgansoftoxicity
Quantumdotscoremetalloid Amarkedimpactonthelocal Hardman
complexesofSelenium,Se ecosystemresultedfrom (2006)
elevatedenvironmental
concentrationsofSe
Ag,MoO3,Fe3O4,Al,MnO2andW Agwashighlytoxicwhereas,MoO3 Reducedcellproliferationand Hussainet
(Tungsten) moderatelytoxicandFe3O4,Al,MnO2and death al.(2005)
W(Tungsten)displayedlessornotoxicity
atthedosestestedoninvitroratliver
derivedcellline(BRL3A)
La0.7Sr0.3MnO3(LSMO) LowcytotoxicityinCe-dopedsamplesas Improvedcellproliferation Kaleetal.
nanoparticlesdopedwith wellasinsampleswithreducedLa/Srratio uponCedoping (2006)
cerium(La0.7-xCexSr0.3MnO3 asrevealedbyinvitrostudiesonHT-1080
where0≤x≤0.7)andLa1 (humanfibrosarcoma)andA431
−ySryMnO3nanoparticleswith (humanskin/carcinoma)cells
differentvaluesofy(La/Sr
ratio)
Surface NeutralNPsandlow Drugdeliveryapplicationsto Lockmanet
charge concentrationanionicNPs braininrats al.(2004)
CationicNPs
(continuedonnextpage)
154 S.Aroraetal./ToxicologyandAppliedPharmacology258(2012)151–165
Table1(continued)
Physicochemicalproperty Toxicokineticfindings Biologicaleffects References
Toxiceffectatthebloodbrain Lockmanet
barrierinrats al.(2004)
AnionicNPsatlower Superioruptakeratesas Lockmanet
concentrations comparedtoneutralorcationic al.(2004)
NPsatthesameconcentrations
inrats
Positivesurfacechargedpoly Depositionintotissuesishigherthanneutral Higherdepositionintissues Nigavekaret
(amidoamine)dendrimers surfacedendrimersinB16melanomaandDU al.(2004)
145humanprostatecancermousetumormodel
Coatingofrespirablequartz InhibitsDNAstrandbreakageandformationof Reductionintoxicity Schinsetal.
surfacewithaluminumlactateor 8-hydroxy-deoxyguanosine (2002)
polyvinyl-pyridine-N-oxide inhumanlungepithelialcells
(PVNO)
Aggregation Rope-likeagglomeratesofcarbon Inducedmorepronouncedcytotoxiceffects Cytotoxicity Wicketal.
state nanotubes thanwelldispersedcarbonnanotubesin (2007)
humanMSTO-211Hcells
moleculesandthecorrespondingbulkmaterials.Thiscanmodifythe Skin. Skinisthelargestprimarydefenseorganinourbodyanddi-
physicochemicalpropertiesofthematerialaswellascreatetheop- rectlycomesintocontactwithmanytoxicagents.Theskinisstruc-
portunityforincreaseduptakeandinteractionwithbiologicaltissues turedorgancomprisingthreelayers:theepidermis,thedermisand
(Chithranietal.,2006;Sonavaneetal.,2008).Thiscombinationofef- the subcutaneous layer. The strongly keratinized stratum corneum
fectscangenerateadversebiologicalresponsesinlivingcellsother- actsastheprimaryprotectinglayerandmaybetherate-limitingbar-
wisenotseenwiththesamematerialinlarger(bulk)form(Neletal., riertodefendagainstthepenetrationofmostmicronsizedparticlesand
2006).Theincreaseinsurfaceareadeterminesthepotentialnumber harmfulexogenetictoxicants.Skinexposuretonanomaterialscanalso
ofreactivegroupsontheparticlesurface.Table1summarizestheob- occur during the intentional application of topical creams and other
servedbiologicaleffectsvis-à-visphysicochemicalpropertiesandthe drugtreatments(Curtisetal.,2006;Hagensetal.,2007;Oberdorster
typesofnanomaterials.Shapeofthenanoparticleshasbeenshownto et al.,2005b).Accordingtoa studyby vander Merwe et al. (2009),
haveapronouncedeffectonthebiologicalactivity.Itisreportedthatsil- nanocrystalline magnesium oxide and titanium dioxide applied to
vernanoparticlesundergoshape-dependentinteractionwithE.coli(Pal dermatomed human skin (as dry powder, water suspension, and
etal.,2007);Chithranietal.(2006)reportedbetteruptakeofspherical water/surfactantsuspension)for8hdidnotshowdermalabsorption
goldnanoparticlesthangoldnanorodsinHeLacells.Incaseofanatase throughhumanskinwithintactfunctionalstratumcorneum.Inanother
TiO nanomaterial,itwasshownthatalterationtoafiberstructureof study,Gontieretal.(2008)testedpenetrationoftopicallyappliedtita-
2
greaterthan15μmcreatedahighlytoxicparticlethatinitiatedanin- niumdioxide(TiO )nanoparticles(sizerange20–100nm)inporcine-,
2
flammatory response by alveolar macrophages and that length may healthyhuman-,andhumangrafted-skinsamples.Itwasseenthatpene-
be an important determinant of nanomaterial biocompatibility trationofTiO nanoparticleswasrestrictedtothetopmost3–5corneocyte
2
(Hamiltonetal.,2009).AnotherstudybyJourneayetal.(2008)demon- layersofthestratumcomeum.Incontradistinctiontothisfinding,there
stratedthatwater-solublerosettenanotubestructuresdisplaylowpul- are many reports that show deeper penetration of nanoparticles.
monary toxicity due to their biologically inspired design and Lademannetal.(1999)showedthatTiO particlescouldgetthrough
2
self-assembled architecture. In a review on widely used metal oxide the human stratum corneum and reach epidermis and even dermis.
and carbon nanomaterials, Landsiedel et al. (2010) emphasized that Flexingmovementofnormalskinwasshowntofacilitatethepenetra-
physico-chemicalcharacterizationofnanomaterialsandtheirinterac- tion of micrometer-size fluorescent beads into the dermis (Tinkle et
tionwithbiologicalmediaareessentialforreliablestudies.Inastudy al.,2003).Oberdorsteretal.(2005b)demonstratedpenetrationofava-
with 1.5nm sized gold nanoparticles it was observed that surface rietyofnanoparticlesinthedermisandtranslocationtothesystemic
chargewasamajordeterminantoftheiractiononcellularprocesses; vasculature via lymphatic system and regional lymph. Further,
the charged NPs inducing cell death through apoptosis and neutral Ryman-Rasmussen et al. (2006) demonstrated that quantum dots
NPsleadingtonecrosisinHaCaTcells(Schaeublinetal.,2011).Consid- with diverse physicochemical properties could penetrate the intact
ering the physicochemical properties of various nanomaterials and stratumcorneumbarrierandgetlocalizedwithintheepidermalandder-
their interactions with the biological environment, Maynard et al. mallayers.Inaclinicalstudy,treatmentofburnsusingnanosilvercoat-
(2011)statethatthechallengespresentedbysimplenanoscalemate- eddressings(Tropetal.,2006)ledtoabnormalelevationofbloodsilver
rialssuchasTiO ,ZnO,Ag,carbonnanotubes,andCeO arenowbegin- levelsandargyria(blueorgraydiscolorationoftheskinduetosilverac-
2 2
ning to be appreciated. But these simple materials are merely the cumulation in the body over time which is a ‘cosmetic problem’).
vanguardofaneweraofcomplexmaterials,wherenovelanddynamic Thoughnanosilver-baseddressingsandsurgicalsutureshavereceived
functionality is engineered into multifaceted substances. Further, approvalforclinicalapplicationandgoodcontrolofwoundinfection
accordingtoMaynardetal.(2011),ifwearetomeetthechallengeof isachieved,theirdermaltoxicityisstillatopicofscientificdebateand
ensuringthesafeuseofthisnewgenerationofsubstances,itistime concern.Despitelaboratoryandclinicalstudiesconfirmingthedermal
tomovebeyond“nano”toxicologyandtowardanewtoxicologyofso- biocompatibility of nanosilver-based dressings (Chen et al., 2006a,
phisticatedmaterials. 2006b;Muangmanetal.,2006;Suppetal.,2005;Wrightetal.,2002)
Thusitisevidentthatphysicochemicalcharacteristicsofthema- severalotherresearchershavedemonstratedthecytotoxicityofthese
terials are very important with respect to the observed biological materials.Paddle-Ledineketal.(2006)exposedculturedkeratinocytes
effects. toextractsofseveraltypesofsilvercontainingdressings.Ofthese,ex-
tractsofnanocrystallinesilvercoateddressingsweremostcytotoxic.
Routesofexposure SimilarobservationswerealsoreportedbyLametal.(2004)inanother
study.Fullerene-basedpeptideswerealsoshowntobecapableofpen-
Thehumanbodyhasseveralsemi-openinterfacesfordirectsub- etratingintactskinandmechanicalstressorscouldfacilitatetheirtra-
stance exchange with the environment, i.e. the skin, respiratory versalintothedermis(Rouseetal.,2007).Intradermallyadministered
tractandgastrointestinaltract(GIT). quantum dots could enter subcutaneous lymphatics (Gopee et al.,
S.Aroraetal./ToxicologyandAppliedPharmacology258(2012)151–165 155
2007)andregionallymphnodes(Kimetal.,2004).Topicallyapplied Biodistribution
fineandultrafineberylliumparticlescanbephagocytosedbymacro-
phagesandLangerhanscellspossibly leadingto perturbationsofthe “Do nanoparticles show a different biodistribution profile than
immune system (Tinkle et al., 2003). Epidermal keratinocytes have largesizedparticles?Howlongdotheyaccumulateintissues/organs?
alsobeenshowntobecapableofphagocytosingavarietyofengineered Dotheyexhibitorganspecificity?Canclearanceofnanoparticlesbe
nanoparticles and setting off inflammatory responses (Monteiro- accurately assessed? Does chemical composition of nanomaterial
Riviere et al., 2005). It is worth noting that some other types of playanimportantroleinbiodistribution?”aresomeofthequestions
nanoparticles,i.e.single-/multi-wallcarbonnanotubes,quantumdots with reference to studies on in vivo interactions of nanoparticles.
withsurfacecoatingandnanoscaletitania,havebeenshowntohave Studiescarriedoutsofarpointatinvolvementofphysicalclearance
toxiceffectsonepidermalkeratinocytesandfibroblastsandareca- processes(viz.,mucociliarymovement,epithelialendocytosis,inter-
pableofalteringtheirgene/proteinexpression(Christieetal.,2006; stitialtranslocation,lymphaticdrainage,bloodcirculationtransloca-
Dingetal.,2005;Monteiro-Riviereetal.,2005;Ryman-Rasmussen tion and sensory neuron translocation) and chemical clearance
et al., 2006; Sarkar et al., 2007; Tian et al., 2006; Witzmann and processes such as dissolution, leaching and protein binding
Monteiro-Riviere,2006;Zhangetal.,2007). (Oberdorster et al., 2005b). Certain kinds of nanoparticles can pass
throughtheGITandarerapidlyeliminatedinfecesandinurineindi-
catingthatabsorptionacrosstheGITbarrierandentryintothesys-
Respiratorytract. Therespiratorysystemservesasamajorportal
temic circulation (Curtis et al., 2006; Oberdorster et al., 2005b).
forambientparticulatematerials.Pathologiesresultingfromairborne
However,somenanoparticulatescanaccumulateintheliverduring
particle materials, e.g. quartz, asbestos and carbon have long been
first-passmetabolism(Oberdorsteret al., 2005b). After intravenous
thoroughlyresearchedinoccupationalandenvironmentalmedicine
administration, nanoparticles get distributed to the colon, lungs,
(Alfaro-Moreno et al., 2007; Donaldson et al., 2001; Gillissen et al.,
bonemarrow,liver,spleen,andthelymphatics(Fabianetal.,2008;
2006;Kanjetal.,2006;Lametal.,2006;Ovreviketal.,2005;Parks
Hagensetal.,2007;Huangetal.,2008).Suchdistributionisfollowed
etal.,1999;Warheit,2001).Recently,thepathogeniceffectsandpa-
by rapid clearancefrom the systemic circulation, predominantly by
thologyofinhaledmanufacturednanoparticleshavereceivedatten-
actionoftheliverandspleenicmacrophages(Moghimietal.,2005).
tion (Donaldson et al., 2006; Lam et al., 2006; Nel et al., 2006;
Clearanceandopsonizationofnanoparticlesdependsonsizeandsur-
Oberdorsteretal.,2005a).Beingdifferentthanmicronsizedparticles
facecharacteristics(Curtisetal.,2006;Moghimietal.,2005).Differ-
thatarelargelytrappedandclearedbyupperairwaymucociliaryes-
ential opsonization translates into variations in clearance rates and
calatorsystem,particleslessthan2.5μmcangetdowntothealveoli.
macrophage sequestration of nanoparticles (Moghimi et al., 2005).
Thedepositionofinhaledultrafineparticles(aerodynamic-diameter
Toincreasethepassiveretentionofnanomaterialsinsystemiccircu-
b100nm) mainly takes place in the alveolar region (Curtis et al.,
lation,thesuppressionofopsonizationeventsisnecessaryatdesired
2006;Hagensetal.,2007).Afterabsorptionacrossthelungepitheli-
sitesoranatomicalcompartments.Forexampleincaseofhydropho-
um, nanomaterials can enterthe blood and lymph to reach cells in
bic particles, a coating with poly(ethylene) glycol (PEG), would in-
the bone marrow, lymph nodes, spleen and heart (Hagens et al.,
creasetheirhydrophilicity,henceincreasingthesystemiccirculation
2007; Oberdorster et al., 2005a). The latter could be of significance
time(GarnettandKallinteri,2006).InanotherstudywithPEGylated
since the association between inhaled ambient ultrafine particles
(Polyethylene glycol coated) gold nanoparticles Myllynen et al.
and cardiovascular events such as coagulation and cardiac rhythm
(2008)observedthat10–30nmsizedparticlesdidnotcrosstheper-
disturbances has been proven (Nurkiewicz et al., 2006; Yeates and
fusedhumanplacentaandwerenotdetectedinfetalcirculation.
Mauderly,2001).Othertargetsaftertranslocationincludethesensory
AstudybyTakenakaetal.(2001)carriedoutinratsrevealedthat
nerveendingsembeddedintheairwayepithelia,followedbyganglia
inhaledultrafinesilvernanoparticlesweredistributedinliver,lungs
andthecentralnervoussystemviaaxons(Oberdorsteretal.,2005b;
and brain. The authors have shown considerable amount of silver
OldforsandFardeau,1983).Takenakaetal.(2001)havedemonstrat-
couldbedetectedinratbrainfollowinginhalationofsilvernanopar-
edthatinbothinhalationandinstillationexperiments,ultrafinesilver
ticles.FewotherstudieswithInhalednanoparticlesdemonstratedis-
particlesweretakenupbyalveolarmacrophagesandaggregatedsil-
tribution of particles to the lungs, liver, heart, kidney, spleen and
ver particles persisted there for up to 7days. Aggregated silver
brain(BeruBeetal.,2007;Hagensetal.,2007;Medinaetal.,2007;
nanoparticlesandsomeothernanomaterialshavebeenshowntobe
Oberdorsteretal.,2002)andclearanceviaphagocytosisinthealveo-
cytotoxictoalveolarmacrophagecellsaswellasepitheliallungcells
larregionbymacrophages(Curtisetal.,2006;GarnettandKallinteri,
(Sotoetal.,2007).
2006;Oberdorsteretal.,2005b).Inaddition,atleastoneclinicalre-
porthasassociatedimpairedliverfunctiontosilvernanoparticlesre-
Gastrointestinaltract(GIT). NanomaterialscanreachtheGITafter leasedfromawounddressing(Tropetal.,2006).
mucociliary clearancefrom therespiratorytractthrough thenasal Jongetal.(2008)demonstratedsizedependenttissuedistribution
region,orcanbeingesteddirectlyinfood,water,cosmetics,drugs, ofgoldnanoparticleswiththesmallest(10nm)nanoparticlesshow-
and drug delivery devices (Hagens et al., 2007; Oberdorster et al., ing the most widespread distribution (blood, liver, spleen, kidney,
2005b).Theutilityofbiodegradablenanoparticlesinthedeliveryof testis, thymus, heart, lung and brain) whereas the larger particles
oralvaccineshasbeenproposedforantigensknowntobesuscepti- (50,100and250nm)weredetectedonlyinblood,liverandspleen.
bletoproteolysis(Russell-Jones,2000).Apparentlystudiesontoxic- In another study on biodistribution of gold nanoparticles, Niidome
ity of nanomaterials post oral ingestion are limited. Chen et al. et al. (2006), detected most of gold stabilized with hexadecyltri-
(2006a, 2006b) determined the acute toxicity of copper particles methylammoniumbromide(CTAB)intheliverwhereas54%ofPEG-
(bulk)andnanocopperinmiceandfoundthatnanocopperwassev- modifiedgoldnanoparticleswerefoundinbloodat0.5hafterintra-
eralfoldstoxicthanbulkcopper(LD fornanocopper413mg/kg; venousinjection.
50
bulkcopper>5000mg/kg).Nanocopperwasalsoreportedtocause Owingtocharacteristicinternalizationandsystemicdistribution
pathological damage to liver, kidney and spleen. Chung et al. ofinorganicandpolymericnanoparticles,thereisagrowinginterest
(2010)recentlyreportedoccurrenceofsystemicargyriaafteringes- inexploringtheirusesforimaging,systemicdeliveryofdrugs,target
tionofcolloidalnanosilverprovesitstranslocationfromtheintesti- specifickillingofcancerouscellsetc.Understandingtherelationship
nal tract. Earlier Smith et al. (1995) reported the uptake of between the physico-chemical properties (size, surface charge, hy-
fluorescently labeled polystyrene nanoparticles by intestinal lym- drophilicityetc.)ofnanoparticlesandtheirADME(absorption,distri-
phatictissue(Peyer'spatches). bution, metabolism and elimination) characteristics is critical to
156 S.Aroraetal./ToxicologyandAppliedPharmacology258(2012)151–165
Fig.2.Possiblemechanismsbywhichnanomaterialsinteractwithbiologicaltissue.Examplesillustratetheimportanceofmaterialcomposition,electronicstructure,bondedsurface
species(e.g.,metal-containing),surfacecoatings(activeorpassive),andsolubility,includingthecontributionofsurfacespeciesandcoatingsandinteractionswithotherenviron-
mentalfactors(e.g.,UVactivation)FromNeletal.(2006)Science311,622–627.ReprintedwithpermissionfromAAAS.
achieve desired biological effect (Li and Huang, 2008, Liang et al., bycatalaseorglutathioneperoxidase(GPx)(Fridovich,1995).Cata-
2008).Kunzmannetal.(2011)haveextensivelyreviewedthecom- laseisahighlyreactiveenzyme,whichconvertsH O toformwater
2 2
monly studied nanomaterials viz., iron oxide nanoparticles, dendri- and molecular oxygen (Mates and Sanchez-Jimenez, 1999).
mers, mesoporous silica particles, gold nanoparticles, and carbon
nanotubeswithreferencetotheirtoxicity,biocompatibility,biodistri-
butionandbiodegradation.Theauthorsre-emphasizetheimportance Table2
Possiblepathophysiologicaloutcomesduetovariousnanomaterials.
ofphysico-chemicalcharacteristicsofnanoparticlesaswellasensuing
immunologicalreactionsvis-a-visthetargetbiologicalapplication.Zhi ExperimentalNMeffects Possiblepathophysiologicaloutcomes
Yongetal.(2009)recommendtheuseofradiotracertechniquesforde-
ROSgeneration* Protein,DNAandmembraneinjury,*
terminingADMEcharacteristics. oxidativestress†
Oxidativestress* PhaseIIenzymeinduction,inflammation†,
Moleculardeterminants mitochondrialperturbation*
Mitochondrialperturbation* Innermembranedamage*,permeability
transition(PT)poreopening*,energy
When exposed to light or transition metals, nanoparticles may failure*,apoptosis*,apo-necrosis,
promote the formation of pro-oxidants which, in turn, destabilizes cytotoxicity
the delicate balance between the biological system's ability to pro- Inflammation* Tissueinfiltrationwithinflammatory
cells†,fibrosis†,granulomas†,
duce and detoxify the reactive oxygen species (ROS) (Curtis et al., atherogenesis†,acutephaseprotein
2006;Kabanov,2006).Size,shapeandaggregationarenanomaterial
expression(e.g.,C-reactiveprotein)
characteristics that can culminate in ROS generation (Shvedova Uptakebyreticulo-endothelial Asymptomaticsequestrationandstorage
etal.,2005a,2005b).Propertiessuchassurfacecoatingandsolu- system* inliver*,spleen,lymphnodes†,possible
bilitymaypossiblydecreaseoramplifythesizeeffectasillustrated organenlargementanddysfunction
Proteindenaturation,degradation* Lossofenzymeactivity*,auto-antigenicity
inFig.2.
Nuclearuptake* DNAdamage,nucleoproteinclumping*,
ROSincludefreeradicalssuchasthesuperoxideanion(O2•−),hy- autoantigens
droxylradicals(.OH)andthenon-radicalhydrogenperoxide(H O ), Uptakeinneuronaltissue* Brainandperipheralnervoussystem
2 2
whichareconstantlygeneratedincellsundernormalconditionsasa injury
Perturbationofphagocyticfunction,* Chronicinflammation†,fibrosis†,
consequenceofaerobicmetabolism.Whencellsareexposedtoany
“particleoverload,”mediator granulomas†,interferenceinclearanceof
insult (chemical/physical), it results in the production of ROS (Luo release* infectiousagents†
etal.,2002).Butcellsarealsoendowedwithanextensiveantioxidant Endothelialdysfunction,effectson Atherogenesis*,thrombosis*,stroke,
defensesystemtocombatROS,eitherdirectlybyinterceptionorindi- bloodclotting* myocardialinfarction
rectlythroughreversalofoxidativedamage.Cellularantioxidantscan Generationofneoantigens, Autoimmunity,adjuvanteffects
breakdowninimmunetolerance
bedividedintoprimary(superoxidedismutase,glutathioneperoxi-
Alteredcellcycleregulation Proliferation,cellcyclearrest,senescence
dase,catalaseandthioredoxinreductase)orsecondarydefense(re- DNAdamage Mutagenesis,metaplasia,carcinogenesis
duced glutathione) mechanisms (Stahl et al., 1998). Superoxide
Effectssupportedbylimitedexperimentalevidencearemarkedwithasterisks(*);
dismutase (SOD) converts the highly reactive radical superoxide effectssupportedbylimitedclinicalevidencearemarkedwithdaggers(†).
intothelessreactiveperoxide(H2O2)whichfurthercanbedestroyed FromNeletal.(2006)Science311,622–627.ReprintedwithpermissionfromAAAS.
S.Aroraetal./ToxicologyandAppliedPharmacology258(2012)151–165 157
Glutathioneperoxidasecatalyzesthereductionofavarietyofhydro- engineerednanomaterialsbytheimmunesystem,andtheoperat-
peroxides(ROOHandH O )usingGSH,therebyprotectingmamma- ingprimarycellulardefensemechanisms.
2 2
lian cells against oxidative damage and also reducing cellular lipid
hydroperoxides(Jornotetal.,1998).Undernormalconditions,more Regulatoryissues
than95%oftheglutathione(GSH)inacellisreducedandsothein-
tracellularenvironmentisusuallyhighlyreducing.However,deple- Asfarasthesafetyaspectsofnanomaterialsareconcerned;acade-
tion of GSH will lower the reducing capacity of the cell and can mia,industryandregulatorygovernmentalagenciesshouldconsider
therefore induce oxidative stress without the intervention of ROS. the unique biological properties of nanomaterials, and the related
Free radicals also attack free fatty acids in cell membranesforming potential risks (Curtis et al., 2006; Lanone and Boczkowski, 2006;
lipid hydroperoxides. Consequently, lipid peroxidation causes dam- Neletal.,2006).Multidisciplinarystudiesareencouragedtoestab-
age to cell membrane. Oxidative stress induced by nanoparticles is lish nanomaterials classification and testing procedures which
reported to enhance inflammation through upregulation of redox- wouldincludetoxicology,materialscience,medicine,molecularbiolo-
sensitive transcription factors including nuclear factor kappa β gy, and bioinformatics (Curtis et al., 2006; Lanone and Boczkowski,
(NFκβ),activatingprotein1(AP-1),extracellularsignalregulatedki- 2006).Regulatoryaspectsonthesynthesis,useanddisposalofnano-
nases (ERK) c-Jun, N-terminal kinases, JNK, and p38 mitogen- particlesarebeyondthescopeofthisreview.
activated protein kinases pathways (Curtis et al., 2006; Kabanov,
2006). The possible pathophysiological outcomes of effects due to Methodsforassessingtoxicityofnanomaterials
nanomaterials have been concisely complied and presented in
Table2.Generallyspeaking,biologicalsystemsareabletointegrate Aswithanyotherman-madematerials,bothinvitroandinvivo
multiple pathways of injury into a limited number of pathological studies on biological effectsof nanoparticles need to be performed.
outcomes,suchasinflammation,apoptosis,necrosis,fibrosis,hyper- Invitromodelsystemsprovidearapidandeffectivemeanstoassess
trophy, metaplasia, and carcinogenesis (Table 2). However, even if nanoparticles for a number of toxicological endpoints. They also
nanomaterials do not introduce new pathology, there could be allowdevelopmentofmechanism-drivenevaluationsandprovidere-
novelmechanismsofinjurythatrequirespecialtools,assays,andap- finedinformationonhownanoparticlesinteractwithhumancellsin
proachestoassesstheirtoxicity.Specificbiologicalandmechanistic many ways. Such studies can be used to establish concentration–
pathways can be elucidated under controlled conditions in vitro; effect relationships and the effect-specific thresholds in cells.
these,inconjunctionwithinvivostudieswouldrevealalinkofthe These assays are suited for high-throughput screening of an ever
mechanismofinjurytothepathophysiologicaloutcomeinthetarget increasing number of new engineered nanomaterials obviating
organ(Neletal.,2006). the need for in vivo testing of individual materials. They also
serve as well defined systems for studying the structure–activity
Genotoxicityandimmunogenicpotential relationshipsinvolvingnanomaterials.
Someofthedistinctadvantagesofinvitrosystemsusingvarious
Reactiveoxygenspecies(ROS),duetotheirhighchemicalreactiv- celllinesinclude;(1)revelationofprimaryeffectsoftargetcellsin
ity can react with DNA, proteins, carbohydrates and lipids in a de- theabsenceofsecondaryeffectscausedbyinflammation;(2)identifica-
structivemannercausingcelldeatheitherbyapoptosisornecrosis. tionofprimarymechanismsoftoxicityintheabsenceofthephysiolog-
Themostfrequentlyaffectedmacromoleculesarethosegenesorpro- ical and compensatory factors that confound the interpretation of
teins,whichhaverolesinoxidativestress,DNAdamage,inflamma- whole animal studies; (3) efficiency, rapidity and cost-effectiveness;
tion orinjurytotheimmunesystem.Forexample, sub-micronic to and (4) scope for improvements in design of subsequent expensive
nanometer-sizedpreparationsofSiO werefoundtoincreasearachi- wholeanimalstudies(Huangetal.,2010).Otheradvantagessuchasre-
2
donicacidmetabolismeventuallyleadingtolunginflammationand duction in variability between experiments; reduced requirement of
pulmonarydiseaseaswellasexpressioningenesdirectlyrelatedto test materials thereby leading to generation of limited amounts of
inflammation (Driscoll et al., 1996; Englen et al., 1990). Similar re- toxicwastes;possibilityofusingtransgeniccelllinescarryinghuman
sults were obtained by Ishihara et al. (1999) for nanometer sized genes etc. have been discussed in a review by Takhar and Mahant
TiO particlesandTiO whiskers(widthof140nm).Basedondetailed (2011).Utilityofsuchassayshasalsobeendemonstratedinassessment
2 2
analysesofstudieswhichinvestigatedthemechanismsofthesead- ofpulmonaryhazardsduetofineandnanoscalematerials(Sayesetal.,
verseeffects,severalresearchershaveputforththeconceptofprima- 2009;Warheitetal.,2009).
ryversussecondarygenotoxicity(Knaapenetal.,2004;MacNeeand The potential dangers of exclusive use of in vitro testing have
Donaldson,2003;VallyathanandShi,1997).Genotoxicitydirectlyre- been documented by Donaldson et al. (2009) and the authors
latedtotheexposureofthe‘substance’isreferredtoasprimarygen- statethatcellsinculturedonotexperiencetherangeofpathogen-
otoxicity. Secondary genotoxicity is the result of the ‘substance’ iceffectsthatarelikelytobeobservedinvivo;whicharepartlyre-
interactingwithcellsortissuesandreleasingfactors,which,inturn, lated to issues of translocation, toxicokinetics and coordinated
cause adverse effects such as inflammation and oxidative stress. tissue responses. The latter is the most under-researched area in
Mostinvestigationsongenotoxicityandcellularinteractionsofengi- toxicology. In another study, Monteiro-Riviere et al. (2009) have
neerednanomaterialsarelimitedtoscreeningforcytotoxicity.Afew observed that classical dye-based assays such as MTT and neutral
studies have focused on immunological responses of nanoparticles. red(NR) thatdeterminecell viability produce invalidresults with
Moghimi et al. (2005) showed that PEG-grafted liposome infusion some nanomaterials due to interaction and/or adsorption of the
triggered non-IgE-mediated signs of hypersensitivity whereas dye/dye products. Further, carbon nanomaterials interact with
peptide-functionalized carbon nanotubeswereshownto formim- assaymarkerstocausevariableresultswithclassicaltoxicologyas-
munogeniccomplexes,enhancingtheantibodyresponse(Curtiset saysandmaynotbesuitableforassessingnanoparticlescytotoxic-
al.,2006).Thesestudieshighlighttheneedforundertakingfurther ity. Thus the authors indicate the lower utility of in vitro assays
investigations on the antigenicity (capacity to evoke immune re- using human cell lines. The interaction of fluorimetric dyes with
sponse)ofnanoparticles per se andtheircomplexes(with cellular dextran coated SPIONS has been reported by Griffiths et al.
biomolecules) as well as the resulting specific immune responses (2011);suchinteractionsneedseriousconsiderationincytotoxici-
(Curtisetal.,2006;LanoneandBoczkowski,2006).Interactionsof ty assays. In a recent article by Dhawan and Sharma (2010) the
nanomaterials with eukaryotic cells have been recently reviewed methods for both in vitro and in vivo toxicity of nanomaterials
by Shvedova et al. (2010) with reference to recognition of have been reviewed. The authors discussed interferences in in
158 S.Aroraetal./ToxicologyandAppliedPharmacology258(2012)151–165
vitro assays (due to the unique physico-chemical properties of study mechanisms underlying toxicity a combination of assays is
nanomaterials), as well as major challenges for in vivo assays oftenrequired.
such as dosimetry, optimization of dispersion, evaluation of inter-
actions and biodistribution etc. Hence it is essential that multiple
Hemolysis. Invitrohemolysisisatesttoevaluatethebiocompati-
assaysbeemployeddependingonthetypeofnanomaterialinad-
bilityofnanoparticles. Inthisassaytheimpactofphysico-chemical
ditiontoimagingtechniquessuchastransmissionelectronmicros-
characteristics of nanoparticles viz., size, porosity and surface func-
copytovalidatechemicalmarker-basedviabilityassays.
tionalityonhumanredbloodcells(RBCs)isevaluatedbyquantifying
the release of hemoglobin. Mesoporous SiO and amine-modified
2
SiO were observed to exhibit reduced hemolysis in comparison
Currentlyusedinvitromethodsinnanotoxicology 2
withbareSiO (Yuetal.,2011).
2
Presently,in absenceofanyclear guideline(s) by theregulatory
agencies on the testing/evaluation of nanoparticulate materials, in Genotoxicityassays. Thecytotoxiceffectsforalmostallkindsofme-
vitro studies (using established cell lines and primary cells derived tallic,metaloxide,semiconductornanoparticles,polymericnanoparticles
from target tissues) become extremely relevant and important. In andcarbonbasednanomaterialsetc.havebeenreported.Forestablishing
general, all the current experimental techniques of cellular biology ‘safe’nanotechnologyitwouldbenecessarytoprovenon-genotoxicna-
and toxicology can be employed for nanotoxicological studies tureofthenanomaterialinquestion.Severalgenotoxicityassayscanbe
(Monteiro-RiviereandTran,2007).Thetechniquesthatcanbeused carriedoutinvitro.Forexample,inarecentarticlebyGonzalezetal.
toassesstoxicityofnanomaterialsinclude(1)invitroassaysforcell (2011)theapplicabilityofinvitromicronucleus(MN)assayasdescribed
viability/proliferation,mechanisticassays[ROSgeneration,apoptosis, inOECDguidelinefortestingnanomaterialsisreviewed.Severaltypesof
necrosis,DNAdamagingpotential](2)microscopicevaluationofin- nanomaterialswereshowntoinduceasignificantincreaseofMNfre-
tracellular localization [include SEM-EDS, TEM, AFM, Fluorescence quencies. Based on the micronucleus test (MNinv) data on 21
spectroscopy,MRI,VEDICmicroscopy](3)geneexpressionanalysis, nanomaterials,itwasproposedthattheinvitroMNtestisquiteappro-
high-throughputsystems(4)invitrohemolysisand(5)genotoxicity priate to screen nanoparticles for potential genotoxicity. However it
etc. wasrecommendedthatprotocolsshouldbeformulatedtoastoachieve
Thefirststeptowardsunderstandinghowanagentwillreactin maximumsensitivityandavoidfalsenegatives.Determinationofthecel-
the body often involves cell-culture studies. Compared to animal lulardose,cytochalasin-Btreatment,timeofexposure,serumlevelsand
studies,invitrostudiesarelessethicallyambiguous,areeasiertocon- choiceofcytotoxicityassaywasadvisedforabetterinterpretationofMN
trolandreproduceandarelessexpensive.Inthecaseofcytotoxicity, frequencyresults.
itisimportanttorecognizethatinadditiontotheconcentrationof Thecometassayisawidelyusedinvitroassayinfundamentalre-
thepotentiallytoxicagentbeingtested,cellsinculturearesensitive search for DNA damage and repair, in genotoxicity testing of novel
tochangesintheirenvironmentsuchasfluctuationsintemperature, chemicals and pharmaceuticals, environmental biomonitoring and
pH,nutrientandwasteconcentrations.Therefore,controllingtheex- humanpopulationmonitoring.Ithasbeenemployedfortoxicityas-
perimental conditions is crucial to ensure that the measured cell sessmentofnanoparticles.InthearticlebyKarlsson(2010)atleast
deathcorrespondstothetoxicityoftheaddednanoparticlesversus 46 cellular in vitro studies and several in vivo studies using the
theunstableculturingconditions. Inaddition,asnanomaterialscan cometassayhavebeenreviewed.Thesestudieshadusedthecomet
adsorbdyesandcanberedoxactive,itisimportantthatthechoice assaytoinvestigatethetoxicityofmanufacturednanoparticles.Find-
of the cytotoxicity assay is appropriate. Conductingmultipletestsis ingsindicatethatmajorityofthenanoparticlesexhibitedhighreac-
advantageoustoensurevalidconclusionsaredrawn(Lewinskietal., tivity and cause DNA strand breaks or oxidative DNA lesions.
2008). Consideringthesensitivityoftheassayitcanenabletheassessment
In vitro cytotoxicity studies of nanoparticles using different cell oftheirrelativepotency.However,theauthoralsostatesthat,addi-
lines,incubationtimesandcolorimetricassayswithdifferentnano- tional methods to measure DNA damage/genotoxicity should be
materials are increasingly being published. It should also be borne employedandmorestudiesinvestigatingmutagenicitywouldprove
inmindthatwhilethenumberofnanomaterialstypesandapplica- valuable.
tionscontinuestoincrease,studiestocharacterizetheireffectsafter AmesTest(orBacterialReversionMutationTest)isyetanotherin
exposure and to address their potential toxicity are comparatively vitro assay used to assess the genotoxic potential of nanomaterials.
few(Lewinskietal.,2008).Itcanbesaidthatrelativelyfewernumber Thetestemployshistidinedependent(auxotrophic)mutantstrains
ofassayshavebeenusedtoassessthecytotoxicpotentialofawhole ofSalmonellatyphimurium.Thistestisusuallyemployedasanadjunct
rangeofnanomaterialsfromcarbonnanotubestometallicnanoparti- techniquebecauseitisdifficulttointerpretthedatageneratedina
clestosemiconductornanoparticleswithcompletelydiverseapplica- prokaryoticsystemtoaeukaryoticgenotoxicitytesting.Furthermore
tions. As is clear from the literature, for nanomaterials, the major resultscouldbeambiguousinsomeinstanceswhencertainnanoma-
biologicaleffectsinvolveinteractionswithcellularcomponentssuch terialsarenotabletocrossthebacterialwallorinsituationswhere
astheplasmamembrane,organellesorgeneticmaterial.Itisimpor- thenanomaterialsarebactericidal.
tant to perform cytotoxicity studies for each nanomaterial type be- Singhetal.(2009)havereviewedtheabilitiesofmetalnanoparti-
cause of their unique biological response (Lewinski et al., 2008). cles,metal-oxidenanoparticles,quantumdots,fullerenes,andfibrous
SimilarobservationswerereportedbyKrolletal.(2011)for23engi- nanomaterials,withreferencetotheirpotentialtodamageorinteract
neerednanomaterialswhichweretestedusingtendifferentcelllines withDNA.Inthesestudieschromosomalfragmentation,DNAstrand
inthreedifferentassays.Accordingtotheauthors,invitrotoxicityof breakages, point mutations, oxidative DNA adducts and alterations
the analyzed engineered nanomaterials was not attributed to a de- in gene expression profiles have largely been assessed based on in
fined physicochemical property and the accurate identification of vitroassaysforthediversegroupofmaterialsstudied.Studiesonneu-
nanomaterial cytotoxicity would requirea matrixbased on a set of rologicaleffectsofnanoparticleshavebeenreviewedbyYangetal.
sensitivecelllinesandinvitroassaysmeasuringdifferentcytotoxicity (2010);moststudiesfocusontheinteractionbetweenCNSneuronal
endpoints. Table 3 summarizes the toxicity assays being currently lines(PC-12,CA1andCA3)andnanoparticles(includingCu,CuO,Zn
usedforseveralclassesofnanomaterials.Thereisnotasinglemethod andAg).Accordingtotheauthors,morestudiesshouldbefocusedon
that is satisfactory forobtaining all theinformation on thetoxicity. biologicalcellsofhippocampalmembrane.InarecentreviewBecker
Since differentnanoparticles elicit different biological responses; to et al. (2011) have stated that with the available tests/assays,
S.Aroraetal./ToxicologyandAppliedPharmacology258(2012)151–165 159
Table3
Summaryoftoxicityassaysfordifferentnanoparticles.
Assay Purpose Usedfornanoparticles References
Lightmicroscopy Morphologicalobservations Single-walledcarbonnanotubes(SWNTs) Fioritoetal.(2006)
Silvernanoparticles Aroraetal.(2008);
Aroraetal.(2009)
Neutralredassay Cellviability(lysosomalactivity) Carbonnanotubes, Flahautetal.(2006);
Monteiro-Riviereand
Inman(2006)
Silver,molybdenum,aluminum,ironoxide Hussainetal.(2005)
andtitaniumdioxidenanoparticles
Titaniumdioxidenanoparticles Shuklaetal.(2011)
Colonyformationassay Proliferativecapacity Carbonbasednanomaterials Herzogetal.(2007)
Trypanblue Cellviability/cellgrowth(membrane Goldnanoparticles Goodmanetal.(2004)
integrity) SWNTs Bottinietal.(2006)
Calceinacetoxymethyl(calceinAM)/ Cellviability(cellmetabolicactivity/ Fullerenes, Sayesetal.(2004)
ethidiumhomodimer membraneintegrity) Goldnanoshells Hirschetal.(2003)
Looetal.(2004)
Lactatedehydrogenase(LDH) Cellviability(membraneintegrity) Carbonnanoparticles Sayesetal.(2004);
Mulleretal.(2005),
Uoetal.(2005)
Tetrazoliumsalts(MTT,MTS,XTT,WST) Cellviability/cellgrowth(cell Fullerenes Sayesetal.(2004)
metabolicactivity) Carbonnanoparticles Flahautetal.(2006)
Monteiro-Riviereand
Inman(2006)
Silvernanoparticles Aroraetal.(2008),
Aroraetal.(2009)
TiO2,SiCnanoparticlesormulti-walled Barilletetal.(2010)
carbonnanotubes(MWCNT).
23engineerednanomaterialsincludingTiO2, Krolletal.(2011)
CeO2,carbonblack
AlOOH,Ti–Zr,Al–Ti–Zr,ZrO2,BaSO4,SrCO3
ResazurinorAlamarblue Cellviability/cellgrowth(cell Quantomdots, Selverstovetal.
metabolicactivity) (2006),Selvanetal.
(2005)
Propidiumiodide Cellviability/cellgrowth/apoptosis Carbonnanoparticles Pantarottoetal.
(membranepermeability) (2004),Kametal.
(2004),Kostareloset
al.(2007)
SiO2nanoparticles Yuetal.(10.1021/nn2013904
LDHassay Celldeath 23engineerednanomaterialsincludingTiO2, Krolletal.(2011)
CeO2,carbonblack
AlOOH,Ti–Zr,Al–Ti–Zr,ZrO2,BaSO4,SrCO3
DNAladdering Biochemicalhallmarkofapoptosis Silvernanoparticles Gopinathetal.(2008),
Aroraetal.(2008)
Acridineorange/ethidiumbromide Apoptosis/necrosis Silvernanoparticles Gopinathetal.(2008),
Aroraetal.(2008),
Aroraetal.(2009),
Jainetal.(2009)
Caspase-3activity Apoptosis Silvernanoparticles Aroraetal.(2008),
Aroraetal.(2009),
Jainetal.(2009)
Levelsofreduced(GSH)andoxidized Oxidativestress Polymericnanoparticles Fernandez-Urrusuno
(GSSG)glutathione,superoxide etal.(1997)
dismutase(SOD),glutathione
peroxidase(GPx),catalase(CT),ROS
production
ROSproductionandlevelsofGSH Oxidativestress Silver,molybdenum,aluminum,ironoxide Hussainetal.(2005)
andtitaniumdioxidenanoparticles,
VitaminE,levelsofGSHandlipid Oxidativestress SWNTs Shvedovaetal.(2003)
peroxidation
LevelsofGSHandlipidperoxidation Oxidativestress C60fullerenes Sayesetal.(2005)
LevelsofGSH,GPx,SOD,catalase(CT)and Oxidativestress Silvernanoparticles Aroraetal.(2008),
lipidperoxidation Aroraetal.(2009),
Jainetal.(2009)
ROSgeneration Oxidativestress Titaniumdioxidenanoparticles Shuklaetal.(2011)
DCFassay Oxidativestress 23engineerednanomaterialsincludingTiO2, Krolletal.(2011)
CeO2,carbonblack
AlOOH,Ti–Zr,Al–Ti–Zr,ZrO2,BaSO4,SrCO3
TiO2,SiC Barilletetal.(2010)
nanoparticlesormulti-walledcarbonnanotubes
(MWCNT).
Transmissionelectronmicroscopy Visualizationofintracellular Fullerenederivatives Foleyetal.(2002)
localization Ultrafineparticulates Lietal.(2003)
Silvernanoparticles Aroraetal.(2009),
Jainetal.(2009)
Titaniumdioxidenanoparticles Shuklaetal.(2011)
(continuedonnextpage)
160 S.Aroraetal./ToxicologyandAppliedPharmacology258(2012)151–165
Table3(continued)
Assay Purpose Usedfornanoparticles References
Synchrotronradiationbasedtechniques Biodistributionofnanoparticlesinvitro NanoscaleZerovalentiron,Titanium Wangetal.(2010)
andinvivo,interactionswithbiological dioxide,ZnO,CeO2etc.
systemsincludingROSgeneration,
chemicalspeciationetc.
Cellularuptakeusingradiolabelled Cellularuptake Cobaltnanoparticles Pontietal.(2009)
nanoparticles
Invitromicronucleustest Genotoxicity Severalclassesofnanoparticles Gonzalezetal.(2011)
Alliumcepachromosomedamagetest Genotoxicity Chitosan/Polymethylmethacrylate deLimaetal.(2010)
nanoparticles
Cometassay DNAdamage metalnanoparticles,carbonbased Karlsson(2010)
nanomaterials,magneticnanomaterials,
metaloxidenanoparticlesetc.
TiO2,SiC Barilletetal.(2010)
nanoparticlesormulti-walledcarbon
nanotubes
(MWCNT).
Colonyformingefficiencytest Cytotoxicity, Cobaltnanoparticles Pontietal.(2009)
Hemoglobinestimation Hemolysis SiO2nanoparticles Yuetal.(2011)
carcinogenicityofnanomaterialscanonlybeassessedonacase-by- (‘signatures’) for thousands of different nanomaterials in order to
casebasis. promote the safe development of such materials. The authors also
point out that, HTS will not replace conventional toxicology but
Newermethodstoassessnanomaterialtoxicity couldaidintheprioritizationofnanomaterialsforfurthertesting;in-
cluding animal testing. HTS may also allow for the development of
Based on measurements of certain physical parameters such as modelsthatpredictbehaviorofnanoparticlesinbiologicalsystems.
size,zetapotentialandbiologicalpropertysuchaslactatedehydroge- Similar to the above report, George et al. (2011) describe use of
naserelease,SayesandIvanov(2010)havedevelopedamathemati- multi-parametric,automatedscreeningassaythatincorporatessub-
cal model to provide insights on how engineered nanomaterial lethal and lethal cellular injury responses to perform high-
featuresinfluencecellularresponses.Thestudyprovesthatpredictive throughput analysis of a batch of commercial metal/metal oxide
computationalmodelsforbiologicalresponsescausedbyexposureto nanoparticles(nano-ZnO,Pt,Ag,SiO ,Al O )withtheinclusionofa
2 2 3
nanomaterialscanbedevelopedandappliedtoassessnanomaterial quantumdot(QD1).Thedataoninvitroassayswasco-relatedwith
toxicity. invivodatausingzebra-fishembryos.Theapproachwasusedtopre-
Withtheadventofnanotechnology,increasinglylargenumbersof dicttoxicityandprioritizenanomaterialsforinvivotesting.
compounds have been introduced in the environment and data on
toxicityofthesematerialsisrequired.Insuchcases,traditionaltoxic- Eco-toxicology
ity testing using animal models is often not possible because it is
oftentime-intensive,lowcapacity,expensiveandassessesonlyalim- Toensurea‘safe’nanotechnologyindustrytheneedforproactive
ited number of endpoints. North and Vulpe (2010) propose researchintheareaecotoxicologyofnanomaterialshasbeenempha-
mechanism-centered high-throughput testing as an alternative ap- sizedNeletal.(2006).Severalassaysforeco-toxicologicaltestingof
proach to meet this pressing need for analysis of responses due to nanomaterialshavebeendeveloped.Literatureonthetoxicityofme-
the large number and types of nanomaterials. According to the au- tallic nanoparticles to bacteria has been reviewed by Niazi and Gu
thorsthisapproachalongwithfunctionaltoxicogenomics(whichis (2009). Various mechanisms that govern toxicity as well as useful-
theglobalstudyofthebiologicalfunctionofgenesonthemodulation nessofbacterialsystemstostudytoxicityofmanufacturednanopar-
ofthetoxiceffectofacompound),canplayanimportantroleiniden- ticles have been explained. In another study, C suspensions have
60
tifying the essential cellular components and pathways involved in beenshowntobetoxictobacteria(Lyonetal.,2005,2006),fathead
toxicityresponse. minnows(Pimephalespromelas)(Zhuetal.,2006),andzebrafishem-
Genomearrayshavebeenusedtoassesstheeffectsofnanoparticles. bryos(Usenkoetal.,2007;Zhuetal.,2007).Toxicityofsingle-walled
AccordingtoLeeetal.(2010)theinhaledsilvernanoparticlescaused carbonnanotube(SWNT)-basednanomaterialstoanestuarinecope-
modulationoftheexpressionofseveralgenesassociatedwithmotor pod(Amphiascustenuiremis),Daphnia,andrainbowtrouthavebeen
neurondisorders,neurodegenerativediseaseandimmunecellfunction, reported (Roberts et al., 2007; Smith et al., 2007; Templeton et al.,
indicatingpotentialneuro-andimmune-toxicity.Accordingtotheau- 2006).Adamsetal.(2006)comparedtheecotoxicitiesofTiO ,ZnO,
2
thorsthesegenesmayassistinthedevelopmentofsurrogatemarkers and SiO nanoparticles suspended in water using Escherichia coli
2
forsilvernanoparticlesexposureand/ortoxicity. andBacillussubtilisastwomodelbacterialspeciesanditwasreported
Jin et al. (2010) have reported the utility of high-throughput thatZnOwastoxictoBacillussubtilis.Experimentsonembryoniczeb-
screening(HTS)methodsforscreeningtheeffectofsilvernanoparti- rafish demonstrated similar results; ZnO nanoparticles were more
clesonbacterialcells.Thishelpsformonitoringtheecologicaleffects toxicthanTiO orAl O nanoparticles(Zhuetal.,2008).Moreover,
2 2 3
ofnanoparticles.SimilarstudieswereperformedwithZnOandiron Hund-RinkeandSimon(2006)reportedthefirstresultsonthetox-
dopedZnOparticles(Lietal.,2011).Sadiketal.(2009)describepor- icityofTiO nanoparticlestoDaphnia (acommon freshwater zoo-
2
table, dissolved oxygen electrochemical sensor arrays capable of plankton) and green algae (Desmodesmus subspicatus). In a
detecting the engineered nanomaterials (quantum dots and fuller- comprehensive study on the 48-h acute toxicity of water suspen-
enes) as well as provide rapid nanotoxicological information. Such sions of six manufactured nanomaterials (i.e., ZnO, TiO , Al O ,
2 2 3
sensorswillbeofutilitybecauseoftheirportablenature.Feliuand C ,SWCNTs,andMWCNTs)toDaphniamagna,usingimmobiliza-
60
Fadeel(2010)haveextensivelyreviewedHTSmethodsdevelopedin tion and mortality as toxicological endpoints, a dose dependence
miniaturizeddevicesforscreeningofnanomaterialstoxicity.Theau- inacutetoxicitywasdemonstrated(Zhuetal.,2009).Withfishhe-
thorsclearlystatethegoalofHTS:toutilizerapid,automatedscreen- patocyteculturesasmodelsystemScownetal.(2010)havenoted
ing approaches to provide detailed and comparable toxicity data their suitability for studies investigating the cellular uptake of