Original
article
Effects
of
titanium
dioxide
nanoparticles
in
human
gastric
epithelial
cells
in
vitro
Monica
Catarina
Botelho
a,b,*
,
Carla
Costa
c,
Susana
Silva
a,
Solange
Costa
a,
Alok
Dhawan
d,e,
Paula
A.
Oliveira
b,f,
Joa˜o
P.
Teixeira
aa
INSANationalInstituteofHealth,DepartmentofHealthPromotion,Porto,Portugal
b
CECACentrefortheStudyofAnimalScience,ICETA,PortoUniversity,Porto,Portugal
c
ISPUPInstituteofPublicHealth-UniversityofPorto,Porto,Portugal
dNanomaterialToxicologyGroup,IndianInstituteofToxicologyResearch,Lucknow,India
e
InstituteofLifeSciences,SchoolofScienceandTechnology,AhmedabadUniversity,Gujarat,India
f
UTADDepartmentofPathology,UniversityofTra´s-os-MonteseAltoDouro,VilaReal,Portugal
1. Introduction
Nanotechnologyis oneof thefastestgrowing sectors of the high-techeconomy.Therearemorethan200separateconsumer products aloneusing nanomaterials withpersonal,commercial, medical,andmilitaryuses[1,2].Engineerednanomaterialswith dimension of 100nm orless provide usa widerange of novel applicationsintheelectronics,healthcare,cosmetics,technologies andengineeringindustries.Theexploitationofpropertiesinherent tomaterialsatthenanoscalehasinitiatedinnovativeapproaches totechnologieswhichshapeourworld.Lackoftoxicologicaldata onnanomaterialsmakesitdifficulttodetermineifthereisarisk associatedwithnanomaterialsexposure.Thus,thereisanurgent needtodeveloprapid,accurateandefficienttestingstrategiesto assesshealtheffectoftheseemergingmaterials[3].
Nano-sizedorultrafineTiO2(<100nm)isusedincreasinglyin other industrial products, such as toothpastes, sunscreens,
cosmetics, pharmaceuticals, and food products [4]. Human exposure may occur during both manufacturing and use.Such widespreaduseanditspotentialentryinthebodythroughdermal, ingestion,and inhalationroutessuggest thatTiO2nanoparticles pose a potential exposure risk to humans, livestock, and the ecosystem[4–9].
However, it hasbeen difficult toestablish a comprehensive mechanism of nanoparticlecytotoxicitybased on previous, and rather inconsistent, observations. For instance, some reports indicatedthatexposureofcellstoTiO2leadstolipidperoxidation, DNAdamage,caspaseactivationfollowedbymicronuclei forma-tion, chromatin condensation and eventual cell death via apoptosis.However,otherinvestigatorshavereportedthat TiO2 nanoparticleexposureinsteadcausesplasmamembranedamage anddecrementsinmitochondrialfunction.Thereareevenreports thatTiO2exposuredoesnotleadtomembranedamage,caspase activationorcelldeath[10].
These conflicting results are likely caused by variations in experimental procedures. Further differences such as protein adsorptionpriortocellexposureandparticle dispersion/agglom-eration havealsobeenrecentlyshown toplay importantroles. These input variables are likely related to varied toxicological outputs.Itisofparamountimportancetoidentifythemechanistic
ARTICLE INFO Articlehistory: Received24July2013 Accepted10August2013 Keywords: TiO2nanoparticles
Gastricepithelialcells
Proliferation Apoptosis
Oxidativestress
Genotoxicity
ABSTRACT
Manufacturingorusingnanomaterialsmayresultinexposureofworkerstonanoparticles.Potential
routesofexposureincludeskin,lungandgastrointestinaltract.Thelackofhealth-basedstandardsfor
nanomaterials combined with their increasing use in many different workplaces and products
emphasizetheneedforareliabletemporaryriskassessmenttool.Therefore,theaimofthisworkwasto
exploretheeffectsofdifferentdosesoftitaniumdioxidenanoparticlesonhumangastricepithelialcells
invitro.WeanalyzedproliferationbyMTTassay,apoptosisbyTunel,migrationbyinjuryassay,oxidative
stressbydeterminingGSH/GSSGratioandDNAdamagebyCometassayonnanoparticle-treatedAGS
human gastricepithelialcell linein comparisontocontrols. We showand discussthetumor-like
phenotypes of nanoparticles-exposed AGS cells in vitro, as increased proliferation and decreased
apoptosis.Ourresultsdemonstrateforthefirsttimethatnanoparticlesinducetumor-likephenotypesin
humangastricepithelialcells.
ß2013ElsevierMassonSAS.Allrightsreserved.
* Correspondingauthor.HealthPromotion Department,NationalInstituteof
Health, Rua Alexandre Herculano, 321, 4000-055 Porto, Portugal. Tel.:+351
223401185;fax:+351223401149.
E-mailaddresses:monicabotelho@hotmail.com,
monica.botelho@insa.min-saude.pt(M.C.Botelho).
Available
online
at
www.sciencedirect.com
0753-3322/$–seefrontmatterß2013ElsevierMassonSAS.Allrightsreserved.
responseofexposure-pronecellstonanomaterialsastheyarenot onlypotentialenvironmentalexposurehazards,butare continu-ously employed in biomedical applications in many different tissuesandcompartmentsinsidethebody[10].
We have therefore carried out a comparative study on the cytotoxiceffectsof common,widelyusedTiO2nanoparticleson gastricepithelialcells. Twodispersionmediawereusedforthis purpose:oneproteinrichandtheotherwithonetypeofprotein alone.Weevaluatedproliferation,apoptosis,oxidativestressand genotoxicityofexposedcells.Tothebestofourknowledge,thisis thefirstreportaddressingcytotoxicityofnanomaterialsongastric epithelialcells.
2. Materialsandmethods 2.1. Nanoparticles
TiO2 nanoparticles (commercial grade, Aeroxide TiO2 P-25, primary size 21nm, 80/20 anatase/rutile) were obtained from DegussaCorp. (Parsippany,NJ).TiO2 nanopowder 637254 (tita-nium(IV) oxideanatase,<25nm)werepurchased from Sigma-Aldrich(St.Louis,MO).
2.2. Particlepreparationandcharacterization
TiO2 NPsweresuspendedintwodifferentdispersionmedia: Milli-QwaterandRPMIsupplementedwith10%FBSor2%BSAin phosphate-bufferedsaline(PBS)andprobesonicatedat30Wfor 5min(1.5minpulseonand1minpulseofffortwotimesanda finalpulseof2min).
The average hydrodynamic size, size distribution and zeta potentialofTiO2NPsinwaterweredeterminedbydynamiclight scattering(DLS)and phase analysislightscatteringrespectively usingaZetasizerNano-ZSequippedwith4.0mW,633nmlaser (ModelZEN3600,MalvernInstrumentsLtd.,Malvern,UK).
Beforeuse,TiO2NPsstocksuspension(150
m
g/mL)inmedium wasseriallydilutedtodesiredconcentrationsinfreshsuspension medium.Allsampleswerepreparedundersterileconditions. 2.3. ParticletreatmentThe treatment experimental design consisted of serial con-centrationsofTiO2NPssuspendedintwodifferentmedia:RPMI supplemented with 10% FBS or 2% BSA in phosphate-buffered saline(PBS),appliedtocells froma singlepassagetominimize confoundingof comparisonsby passage-to-passagevariation of the cultured cells. Each multiwell cell culture plate included negativecontrols.
2.4. Celllineandcellculture
AGS (gastric epithelial cancer) cells were cultured and maintainedat378Cina5%CO2humidifiedatmosphereinRPMI medium (Sigma) with 10% FBS (Sigma) and 1% penicillin/ streptomycin(Sigma). Cellswerepassaged every5days. Before treatments with nanoparticles suspended in BSA, cells were allowedtoreach80%confluencebeforeserum-starvedfor16h. 2.5. Trypanblueexclusionassay
The trypan blue exclusion method was used to assess cell viability. AGS cells were plated and incubated until 80% confluency.ThecellsweretreatedwithTiO2-nanoparticles.After treatment,thecellswereharvestedbytrypsinizationandcounted undermicroscopeaftertrypanbluestaining.Three independent
experimentswerecarriedoutbasedonthefollowingformula:cell viabilitypercentage=number of cells in drugtreatment group/ numberofcellsincontrolgroup100%.
2.6. Proliferationassay
The CellTiter 96 AQ nonradioactive cell-proliferation assay (Promega)wasusedtoassesscellviability.Theassayiscomposed of the tetrazolium compound MTS and an electron coupling reagent,PMS.MTSisreducedbyviablecellstoformazan,which can be measured witha spectrophotometer by the amount of 490nmabsorbance.Formazanproductionistime-dependentand proportionaltothenumberofviablecells.AGScellswerecultured in0.1mLRPMImediain96-wellflat-bottomedplates. Cultures wereseededat1104cells/wellandallowedtoattachovernight. After the indicated time of incubation with the appropriate medium, 20
m
L reagent was added per well, and cells were incubated 1h before measuring absorbance at 490nm. Back-groundabsorbancefromthecontrolwellswassubtracted.Studies wereperformedintriplicateforeachexperimentalcondition. 2.7. ApoptosisApoptosis in cellcultures wasassessed withthein situ cell death detection kit, fluorescein (TUNEL technology) (Roche), analyzed by fluorescence microscopy. TUNEL assay (Terminal deoxynucleotidyl transferase-mediated deoxyuridine tripho-sphate nick endlabeling) was performed according to the manufacturer’s instructions. Nuclei were counter-stained with DAPI(Roche Diagnostics,Basel,Switzerland).Thepercentage of TUNEL-stained nuclei wasevaluated in relationtoevery DAPI-stained nuclei observed. Immunofluorescence was visualized under a fluorescence microscope (Olympus, BH-2, UK). The percentageof stainedcells wasevaluatedbycountingthecells stainedwithTUNELdividedbythetotalnumberofnucleistained withDAPIatamagnification200field.Onethousandnucleiwere evaluated.Threeindependentexperimentswereperformed. 2.8. Oxidativestressassay
OxidativestresswasanalysedbyevaluationofGSHt,GSHand GSSG levelsThe intracellular levelsof GSHand GSSGin TiO2 -nanoparticle-treatedAGScellswereevaluatedbytheDTNB-GSSG reductaserecyclingassay,aspreviouslydescribed[11].Aftera3-h treatment with150
m
g/mLTiO2 nanoparticles,thetreated cells werelysedand proteinswereprecipitatedwith5%HClO4.After centrifugation(16,000g,10min,488C),thesupernatantobtained was used for the determination of GSHt, GSH and GSSG by spectrophotometryat412nm.2.9. Cometassay
Aftertreatment,cellswerewashedtwicewithprechilledPBS (Mg2+ and Ca2+-free), centrifuged at 78g for 5min and resuspended inPBS. Cell viabilitywasover 85%forthe tested doseinthisstudyasassessedusingtrypanblue dye-exclusion staining.Thealkalineversionofthecometassaywasperformedas describedby[12]withminormodifications.Briefly,cellscollected bycentrifugation(9000rpmfor3min)andsuspendedin60
m
Lof 0.6% low-melting-point agarose (LMA) in PBS (pH 7.4) were droppedontoafrostedslideprecoatedwithalayerof1%normal meltingpointagarose.Slideswereplacedonicefor4minand allowedtosolidify.Coverslipswerethenremovedandslideswere immersed in freshly prepared lysing solution (2.5M NaCl, 100mMNa2EDTA,10mMTrisBase,0.25MNaOH,pH10)for1h at48C,inthedark.Afterlysis,slideswereplacedonahorizontalelectrophoresis tank in an ice bath. The tank was filled with freshlymadealkalineelectrophoresissolution(1mMNa2EDTA, 300mMNaOH,pH13)tocovertheslides,andtheywereleftfor 20mininthedarktoallowDNAunwindingandalkali-labilesite expression.
Electrophoresiswascarriedoutfor20minat30Vand300mA (1.2V/cm).Theslideswerethenwashedfor10minwith1mLof neutralizingsolution(0.4MTrisBase,pH7.5).Afterneutralization, gels were stained with 100
m
L of ethidium bromide solution (20m
g/mL)andcoveredwithcoverslipsfor20min.Afterstaining, theslideswerewashedtwice withice-coldbidistilledwaterfor 20min.Slides werecoded and examined bya ‘blind’ scorerusing a magnificationof400.Onehundredrandomlyselectedcells(50 perreplicate)wereexamined foreach dose.Imagecapture and analysiswereperformedwithCometAssayIVsoftware(Perceptive Instruments);percentageoftailDNA(%T)wastheDNAdamage parameterevaluatedaccordingtowhathasbeenrecommendedby Kumaraveletal.[13].ThepercentageDNAinthetailisthefraction of DNA in the tail divided by the amount of DNA in the cell multipliedby100.
2.10. Statisticalanalysis
DatawereexpressedasmeanSD.t-testwasusedtoassessthe statistical significance of differences. P<0.05 was considered statisticallysignificant.
3. Results
3.1. Nanoparticlescharacterization
ThemeanhydrodynamicdiameterofSigmaTiO2NPsinMilliQ waterasmeasuredbyDLSwas420.7nmandthezetapotential was 9.96mV.ResultsofsizeandzetapotentialofDegussaTiO2 NPswererespectively,160.5nmand–27.8mV.
3.2. FBSimpairstheeffectsofTiO2nanoparticlesoncellproliferation Generally,serumisaddedtothecellculturemediumsuchas RPMIbecausetheserumcomponentisimportantfornormalcell growth. Therefore, we used RPMI supplemented with FBS for nanoparticlessuspension.Thegrowthlevelsshowthatnoeffectis observedbythistreatmentwithincreasingconcentrationsofTiO2 -nanoparticles(Fig.1AandB).
3.3. TiO2nanoparticlesincreasedtheproliferationofepithelialcellsin vitro
Weused a simple ‘‘FBS mimic’’ proteincocktail containing similarconcentrationsofBSAandPBSfornanoparticle suspen-sion[14].TobegininvestigatingtheeffectofTiO2nanoparticles oncellviabilityandproliferation,AGScellswereseededon 96-wellplates,starvedovernight,treatedwithincreasing concen-trationsofTiO2nanoparticlesfor3h,6hand24h,cultivatedfor 24hoursandthen analyzedby MTSassay(Fig. 2AandB).The growthlevels showthattreated cellsproliferatedsignificantly fasterandmorethancontrolcells.Weconfirmedtheincreasein cell viability using the concentration of 150
m
g/mL of TiO2 nanoparticles for 3h. This concentration and time were confirmed by trypan blue assay and used forthe subsequent assays.Ourresultssuggestthattheincreaseofbothproliferation and overall survival in AGS cells is a consequence of nanoparticletreatment.Theexperimentsweredoneintriplicate (P<0.01).3.4. TiO2nanoparticlesdecreasedtheapoptosisofgastricepithelial cellsinvitro
Toanalyzeapoptosis,AGScellswereseededon96-wellplates, starvedo.n.,treatedwith150
m
g/mLofTiO2nanoparticles(Sigma)A
B
0 20 40 60 80 100 120 140 150 120 100 80 60 40 20 cPercentage of proliferang cells
Treatments Cell Proliferaon 3h 6h 24h 0 20 40 60 80 100 120 140 160 150 120 100 80 60 40 20 c
Perecentage of proliferang cells
Treatments Cell Proliferaon
3h 6h 24h
Fig.1.CellproliferationassayofAGS cells treated with TiO2nanoparticles suspension in
RPMIsupplementedwithFBS.Thegrowthlevelsshownodifferencesbetweencontrol
andtreatedAGScells.A.AGScellswereseededon96-wellplates,starvedo.n.,treated
withincreasingconcentrationsofTiO2nanoparticles(Sigma)suspension,for3,6and
24h.B.AGScellswereseededon96-wellplates,starvedo.n.,treatedwithincreasing
concentrationsofTiO2nanoparticles(Degussa)suspension,for3,6and24h.
A
B
0 50 100 150 200 250 300 350 150 120 100 80 60 40 20 cPercentage of proliferang cells
Treatments Cell Proliferaon 3h 6h 24h 3h+24h 6h+24h 0 50 100 150 200 250 300 350 400 450 500 150 120 100 80 60 40 20 c
Percentage of proliferang cells
Treatments Cell Proliferaon 3h 6h 24h 3h+24h 6h+24h
Fig.2.CellproliferationassayofAGSTiO2nanoparticlessuspensioninBSAwithPBS.
Thegrowthlevelsshowthattreatedcellsproliferatedsignificantlyfasterandmore
thancontrolcells(P<0.01;controlvs.150mg/mL).A.AGScellswereseededon
96-well plates, starved o. n., treated with increasing concentrations of TiO2
nanoparticles(Sigma)suspension,for3,6and24h,cultivatedfor24handthen
analyzedbyMTSassay.B.AGScellswereseededon96-wellplates,starvedo.n.,
treatedwithincreasingconcentrationsofTiO2nanoparticles(Degussa)suspension,
for3handthenanalyzedbyTUNELassay.Apoptoticlevelsshow thattreatedcellsmarkedlydecreasedapoptosisincomparisonto controlcells.Theexperimentsweredoneintriplicate(P<0.01). AGScellsshowedanincreasingnumberofapoptoticcellsperfield inthecontrol(Fig.3A)comparedtocellstreatedwith150
m
g/mL ofTiO2nanoparticles(Fig.3B).Cellcountsshowthattreatedcells markedly decreased apoptosis in comparison to control cells (Fig.3C).3.5. TiO2nanoparticlesincreasedoxidativestressofgastricepithelial cellsinvitro
Oxidative stress was determined by measuring oxidized glutathione(GSSG).AGScellsshowanincreaseinGSSGlevelsin thecellstreatedwith150
m
g/mLofTiO2nanoparticlescomparedtocontrols.InFig.4,thelevelsofGSSGinAGS cellsaftera3-h incubationperiodwithTiO2nanoparticlesandincontrolcellscan beobserved.AsignificantdifferencecanbeobservedinGSSGlevels when compared with controlgroup, as shown in Fig. 4. These resultsshowthatTiO2nanoparticlestreatmentwithwasableto elicitthealterationsinglutathionestatusobserved.
3.6. InducedgenotoxicityofgastricepithelialcellsinvitrobyTiO2 nanoparticles
GenotoxicitywasdetectedbyCometassay.AGScellsshowan increase in tail intensity in 150
m
g/mL of TiO2 nanoparticles (Sigma)-treated cells compared tocontrol. AGS cells showless damaged nuclei in the control compared to cells treated with 150m
g/mLof TiO2 nanoparticles(Fig.5A and B). In thecomet assay,therewasa1.88-foldsignificant(P<0.05)increasein%Tail DNAwhenthecellsweretreatedwithTiO2nanoparticlesatadose of 150m
g/mL for 3h exposure, i.e. 47.349% for treatedcells versus25.195%foruntreatedcells(Fig.5C).4. Discussion
Thepurposeofthisstudywastoexaminegastricepithelialcell responsestoTiO2nanoparticles.Toourknowledge,thisisthefirst study addressing these effects in a gastric epithelial cell line. Nanoparticleswereevaluatedovera rangeofconcentrations.In thepresentstudy,wecharacterizedtheeffectofTiO2nanoparticles inhumangastriccellsusingcellbiologicalapproachesnormally used in carcinogenesis studies, like proliferation, apoptosis, oxidativestressandgenotoxicity.
Fig.3.ApoptosisofTiO2nanoparticles-treatedcellsanalysedbyTUNEL.Theexperimentsweredoneintriplicate(A)microphotographsofapoptoticcellsofcontrolsand(B)
TiO2nanoparticles-treatedcells.Bothpanelshavethesamemagnification.AGScells,showinganincreasingnumberofapoptoticcellsperfieldinthecontrolcomparedtocells
treatedwith150mg/mLofTiO2nanoparticles(Sigma).C.Thegrowthcurveshowsthattreatedcellsmarkedlydecreasedapoptosisincomparisontocontrolcells(P<0.01;
controlvs.150mg/mL). 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 TiO2 D TiO2 S Control mM GSSG Oxidave stress
Fig.4.Oxidativestresswasdeterminedbymeasuringoxidizedglutathione(GSSG).
AGScellsshowanincreaseinGSSGlevelsintreatedcellswith150mg/mLofTiO2
nanoparticlescomparedtocontrolcells.Asignificantdifferencecanbeobservedin
First,weusedRPMIsupplementedwithFBSforthesuspension ofTiO2nanoparticles.Thistreatmentcausednoalterationoncell proliferation. These results are explained by the effect of the protein adsorption ability of metal oxide nanoparticles on the cytotoxicity.First,theadsorptionofthecomponentsoftheculture mediaonto themetal oxide nanoparticlesinduces a starvation state and subsequent enervation of cells in vitro. Therefore, a correcttoxicityassessment maybeimpairedbythis adsorption effect.Second,thecoatingofthemetaloxidenanoparticleswith proteins may change their biological activities. Generally, the prepared cell culture mediacontain proteins from the supple-mentedFBS.Theadsorptionofmediumcomponentsbyultrafine metaloxideparticles,especiallyproteinadsorption,affectsthecell growthandmetabolism;therefore,theadsorptionabilityaffects anaccuratecytotoxicityassessment[14].
In this investigation, TiO2 nanoparticles were selected as a modelnanoparticleandbovineserumalbumin(BSA)wasselected asamodelproteinforstudyingtheeffectofTiO2nanoparticleina gastricepithelialcellline.Fetalbovineserum(FBS)isaneffective dispersingagentforTiO2nanoparticlesduetosynergisticeffectsof itsmultiple proteincomponents[14]. Jiet al. [14]successfully reproduced FBS using a simple ‘‘FBS mimic’’ protein cocktail containingsimilarconcentrationsofBSAandPBSthatweincluded inthepresentstudy[14].Withthisnanoparticlesuspensionwe observedincreasedproliferationofgastricepithelialcells.Wehave no references so far in the literature to compare our results concerningnanoparticlesandgastricepithelialcellproliferation. Nevertheless, Knaapen et al. [15] found that TiO2-nano drive deregulatedcellproliferationandanchorage-independentgrowth inlungepithelialcells[15].
Apoptotic cell loss in carcinogenesis has been examined by TUNELmethod[16].Weusedthismethodtoanalyzeapoptosisin thepresentworkandfounditdramaticallydecreasedinthecells aftertreatmentwithTiO2nanoparticles.Inagreement,alterationsin thebalancebetweencellproliferationandapoptosismayreflectan important mechanism of carcinogenesis [17]. Our findings are furthersupportedbymicroarraygeneexpressionprofilesindicating rolesofTiO2nanoparticlesinmodulatingnumerousgene expres-sionsinvolvedincellcycleandapoptosis[18],indicatingthatTiO2 nanoparticlescanmodulateintracellularphysiologicalprocesses.
One of the most discussed mechanisms behind the health effectsinducedbyambientparticlesistheabilityofparticlesto causeoxidativestress.Thismechanismisbelievedtobeimportant for thetoxicity ofmanufactured nanoparticlesaswell. In vitro studieshavegenerallysupportedthepathophysiologicalresponses foundinanimalmodels,includingincreasedgenerationofROSin cellsexposedtovariousnanomaterials.Manyinvitrostudieshave identified increased ROS generation as an initiating factor of toxicityinnanoparticleexposedcells.Theinteractionsofparticles withcellmembranesresultinthegenerationofreactiveoxygen species (ROS), and the generated oxidative stress maycause a breakdown of membrane lipids, an imbalance of intracellular calciumhomeostasis,andDNAbreakage[19–21].In thepresent study,wefoundincreasedoxidativestressinTiO2 -nanoparticles-treated cells. In accordance, previous studies have shown that nano-TiO2inducesoxidativestress-mediatedtoxicityinmanycell types[22–24]andthatnano-TiO2exposureinducesROStocause DNAlesions[4,23,25,26].
TheDNAdamageresponseistriggeredbythedetectionofDNA lesions.This responseconsistsofan orderlysequence ofsignal
Fig.5.EffectofTiO2nanoparticlesongenotoxicityofAGScellsanalyzedbyCometassay.(A)microphotographsofcometsofcontrolsand(B)TiO2nanoparticles-treatedcells.
Bothpanelshavethesamemagnification.NotetheincreasedtailofcometsinTiO2nanoparticles-treatedcells.C.TiO2nanoparticlesinducedgenotoxicityoftreatedcells5
transductioneventsthatcaninducetheaccumulationofgenetic errors,thatplayacriticalroleinrespondingtovariousstressesthat cause DNA damage, especially ROS [27]. We confirmed the genotoxic effects of nano-TiO2 on gastric epithelial cells using alkalinesingle-cellgelelectrophoresis(Comet). Kangetal. [28]
foundthesameeffectsonlymphocytes.
Studying thegenotoxic molecular mechanism of TiO2 nano-particleshashelpedelucidatepathwaysrelatedtoits tumourigen-esis.Thecentralhypothesisbasedonourstudiesisthatgenotoxic events and sustained signaling pathway stimulation drive deregulatedcellproliferationandanchorage-independentgrowth, the processes both required for mutations and progression towards neoplastic lesions play a role in TiO2 nanoparticles-inducedmutagenesisandcarcinogenicity.Thewell-known biolo-gicalmechanisms,suchasthealterationofcell-signalingpathways and induction of DNA damage, play a vital role in neoplasia induction. The initiation stage of carcinogenesis is mainly characterized by genotoxic processes, which may lead to irreversiblechangesinthestructureofcellulargeneticmaterials. AlthoughDNArepairpathwaysexistforDNArestoration,however, erroneousrepairandextensiveDNAdamagemaycausemutations andultimatelyleadtocelltransformation[29].Furthermore,since there is a link between DNA damage, mutations, and cancer, particlesthatarepotentincausingDNAdamagecanberegardedas morelikelytohaveaneffectoncancerdevelopment.Inagreement, Knaapenetal.[15]suggestedthatROSgenerationinducedbyTiO2 nanoparticlesmightdirectlyorindirectlydamageDNA tocause genotoxicity and impact on cellular signaling pathways to modulatecellproliferation,resultingirreversiblecell transforma-tion[15]. Inaddition, theInternationalAgencyforResearch on Cancer(IARC)recentlyclassifiedTiO2 asaGroup2Bcarcinogen (possibly carcinogenic to humans) based on mechanistic and animalstudies[30].
Taken together, the effects observed in TiO2 -nanoparticles-treatedcellsseemtobeinterconnected.Althoughwedonotyetfully understandthephysiologicalfunctionsofnanoparticles,thepresent studyrevealednovelaspectsingastricepithelial,onthepotential routes of exposure to nanoparticles. Through its effects in cell biology,TiO2nanoparticlesarelikelytoparticipateinanumberof carcinogenesis-mediatedprocesses,suchas increasedcell prolif-eration, decreased apoptosis, increased oxidative stress and increasedgenotoxicity,allofwhichareprocessesneededforcancer cellsurvival. Theeffects of nanoparticles on the cellcyclemay contributetothehighproliferationrateandaccumulationofgenetic changes.Oxidativestressmaybethereasonfortheuncontrolled proliferationseeninTiO2nanoparticles-treatedcellsandcouldbe involvedincancer-associatedpathways.Additionalworkneedsto beundertakentounderstandthemechanismsofdamage. Disclosureofinterest
The authors declare that they have no conflicts of interest concerningthisarticle.
Acknowledgements
WewouldliketoshowourdeepestappreciationtoPr.Victor Costaforthecollaborationandvaluableinsightonoxidativestress. WewouldalsoliketoacknowledgeNanoLINENnetworkproject fundingforthiswork.
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