Research Article Natural Transformation of Zinc Oxide Nanoparticles and Their Cytotoxicity and

13 Pages·2017·4.65 MB·English

Hindawi
Journal of Nanomaterials
Volume 2017, Article ID 8457960, 12 pages
https://doi.org/10.1155/2017/8457960
Research Article
Natural Transformation of Zinc Oxide Nanoparticles
and Their Cytotoxicity and Mutagenicity
MeiM.Wang,1,2JuanWang,3RuiCao,1SiY.Wang,1andHuaDu3
1DepartmentofPathophysiology,AnhuiMedicalUniversity,No.81,Mei-ShanRoad,Hefei,Anhui230032,China
2SchoolofPublicHealth,AnhuiMedicalUniversity,No.81,MeiShanRoad,Hefei,Anhui230032,China
3KeyLaboratoryofIonBeamBioengineering,HefeiInstitutesofPhysicalScience,ChineseAcademyofSciencesandAnhuiProvince,
Hefei,Anhui230031,China
CorrespondenceshouldbeaddressedtoSiY.Wang;[email protected];[email protected]
Received 2 May 2017; Revised 5 June 2017; Accepted 7 June 2017; Published 2 August 2017
AcademicEditor:KimberlyHamad-Schifferli
Copyright©2017MeiM.Wangetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
With rapid development of the nanoindustry, studies focusing on the transformation of nanoparticles (NPs) are required to
understand their stability and toxicity after being released into the environment. Here, we characterized the physicochemical
propertiesofZnONPsandfoundthattheyarenaturallyalkalizedinthepresenceofair(withouttheadditionofexogenousalkaline
substances).EnergydispersiveX-ray/X-raypowderdiffraction/Fouriertransforminfrared(EDX/XRD/FTIR)/Ramanspectroscopy
gaveevidencefortheformationofhydrozincite(Zn5(CO3)2(OH)6)andzinchydroxide(Zn(OH)2).Further,wecomparatively
evaluatedthecellulartoxicityofpristineandalkalizedZnONPs.Cellviabilitytesting(colonyformation)showedthatalkalization
time-dependentlydecreasedcytotoxicity.AlkalizedNPsexhibitedmutagenicityatmultipleconcentrations,asshownbyaCD59
2+
genelocimutationassay.VariationsintoxicitywereassociatedwiththechemicaltransformationofZnONPs,andZn playeda
keyroleinthemutagenicityofalkalizedNPs.TheseresultsindicatethatNPsarechemicallytransformedintheenvironment.These
transformationsresultinobviousvariationsintoxicity,suggestingthattheNPtransformationprocessshouldbeconsideredmore
thoroughlywhenevaluatingtoxicity.
1.Introduction and children’s products [11, 12]. It has been reported that a
variety of environmental transformations, including aggre-
Nanoparticles (NPs) are important materials that primarily gation [13], dissolution [14], sulfidation [15], and phospho-
differfromtheirmacroscopiccounterpartsinsizeandhave rylation[16],arepossibleforZnONPs.Forexample,recent
beenwidelyusedinmanydifferentfieldssuchaselectronics, studieshaveshownthatdissolutionconvertsZnONPsinto
dyestuff, coating, medicine, clothing, and cosmetics [1–5]. Zn2+ under suitable pH environments [17, 18]. Also, newly
As of October, 2013, the number of nanoproducts on the formed products such as hydrozincite (Zn5(CO3)2(OH)6),
marketreached1628accordingtoemergingnanotechnology smithsonite(ZnCO3),andzincsulfide(ZnS)arepossiblein
projectstatistics[6].Consequently,NPswillinevitablycon- the presence of an appropriate concentration of carbonate
tactenvironmentalmedia(water,soil,sediment,etc.)directly and sulfide and under suitable pH conditions [19–23]. An
orindirectly,withunpredictableimpactsonecosystemsand increasing number of studies have shown that ZnO NPs
humanhealth[7–10].Recently,issueshavearisenregarding inducevarioustoxiceffects,includingcytotoxicity,genotoxi-
thetypesofNPtransformationsandstableproductsthatmay city, inflammation,and oxidative stress [12, 24–28]. Among
occurinthenaturalsystemandhowtheymay,inturn,affect these documented adverse effects, mutagenicity is particu-
thenature,behavior,andadverseeffectsofNPs[10]. larlyimportantbecauseitisinheritableandcouldtherefore
ZnO NPs are engineered nanomaterials that are com- cause severe and far-reaching negative effects to human
monly used in various human applications such as sun- health [29]. Some studies have used the Ames test to show
screens,ceramics,rubberprocessing,wastewatertreatment, thatZnONPsinduceweakmutagenicpotentialinSalmonella
2 JournalofNanomaterials
typhimuriumTA98,TA1537,andEscherichiacoliWP2trpu- for20min.NPstocksuspensionsweresealedinbrownglass
vrA[19,25].However,moststudieshavereportedthatZnO reagentbottles.
∘
NPsarenotmutagenic[30,31].Thesediscrepanciesmaybe For air-exposed NPs, suspensions were stored at 25 C
attributabletotheinabilityofprokaryotestoperformendo- and processed manually every 5 days as follows: samples
cytosis;further,ZnONPsmayhaveantimicrobialproperties were vortexed for 2min, sonicated at 30W for 20min, and
[32], suggesting that bacteria are not a suitable model for exposed to air on the sterile workbench for 5min during
studyingtheirmutagenicpotential.Inaddition,thesestudies thesimulatedtransformationperiod(20,60,and120days).
only include analyses of pristine ZnO NPs; however, NPs Unless otherwise stated, the transformation process was
likelyundergotransformationsaftertheyarereleasedintothe modeledperthisprocedure.
environment, a phenomenon that has not been adequately Control(pristine,noairexposure)NPsuspensionswere
∘
considered.SeveralresearchgroupshaveconcludedthatZnO sealed and maintained in the dark at 25 C for the natu-
NP toxicity is primarily attributable to dissolved Zn2+ ions ral transformation period without any other manipulations
[24, 33]. Others have shown that both nanoparticles and beforebeingappliedtoAL cellsorphysicochemicalcharac-
Zn2+ionsarelikelytobemajorcontributors[34–36].While terization.
different,mostofthesetoxicitystudiesfocusonlyonpristine
ZnO NPs rather than the transformed products expected 2.3. Physicochemical Characterization of Pristine and Trans-
in the environment, which likely have different properties formed NPs. Pristine and naturally transformed ZnO NPs
∘
fromthoseofpristineNPs[35].Inaddition,existingstudies werecharacterizedwithseveraltechniquesat25 C.Transmis-
onNPtransformationsinvolvetheadditionofchemicalsor sionelectronmicroscopy(TEM,JEOL2100HT,Jeol,Japan)
adjustmentofphysicalparameters;fewstudieshavefocused wasusedtodetermineNPsize,shape,andmorphology.The
onnaturaltransformationprocesses. samples were prepared by dropping 10𝜇L aliquots of the
Here, we focus on possible natural transformations of particle suspensions (0.1mg/mL in ultrapure water) onto a
ZnO NPs in ultrapurewater without the addition of chem- coppergridandthenairdrying.AZetasizerNanoSeriesS90
icals or other substances. The human-hamster hybrid (AL) (MalvernInstruments)apparatusbasedonthedynamiclight
cell line, formed by fusion of the gly2A mutant of Chinese scattering (DLS) method was used to measure the hydro-
hamster ovary (CHO) cells with human fibroblasts, is sen- dynamic particle size of the NPs (0.1mg/mL suspensions).
sitive to large deletion mutations [37] and was used in the Analysesoftransformationproductsresultingfromexposure
presentstudytoevaluateZnONPmutagenicity.Weevaluated of ZnO NP stock solutions (10𝜇L, 1mg/mL in ultrapure
therelationshipbetweennaturallyoccurringtransformations water) to ultrapure water were conducted using FTIR and
in ultrapure water and the consequential changes in ZnO Ramanspectroscopy.FTIRspectraofNPs(pristineortrans-
NPtoxicity.TheNPswerecarefullycharacterized,andcyto- formed)werecollectedbetweenthespectralrangeof400and
−1
toxicityandmutagenicityofpristineandalkalizedNPswere 4000cm usingtheKBrpellettechnique.Ramanspectraof
compared in AL cells. Further, we measured the release of NPswereobtainedwitha532nmlaserandanOlympus50x
2+
Zn anddetermineditsroleinNPtoxicity.Possiblephysic- working distance lens using an Xplora Raman microspec-
ochemical mechanisms underlying the observed changes trometer (Horiba Jobin Yvon). Chemical and structural
in toxicity, as well as key factors influencing the natural characterizations of pristine or transformed ZnO NPs were
transformationofZnONPs,werealsostudiedanddiscussed. measured by synchrotron-based X-ray diffraction (XRD,
PANalyticalB.V.,Shanghai,China)andenergydispersiveX-
2.MaterialsandMethods rayspectrometry(EDX,JEOL2100HT,Jeol,Japan).
2.1. Particles and Reagents. Commercial ZnO NPs (90– 2.4.CellCulture,Treatment,Morphology,andViability. The
200nm) were purchased from NanoAmor (Houston, TX) comparative toxicity of pristine and transformed ZnO NPs
with a purity of 99.9+% and a specific surface area of was evaluated in the immortalized AL cell line derived
2
4.9–6.8m /g.Theparticleswereusedasreceived.Biological fromhybridizationofhamstergly-AmutantCHOcellswith
reagents used in the experiments involving cell culture, normal human fibroblasts. The cell line contains a set of
includingglycine,gentamicin,andHam’sF12mediumwere ChinesehamsterCHO-K1chromosomesandasinglecopyof
of reagent grade and were purchased from Sigma-Aldrich humanchromosome11.ALcellsaresensitivetothecytotoxic
(Shanghai,China).CrystalvioletwaspurchasedfromSangon effectsofmonoclonalE7.1antibodies,whicharespecifictothe
Biotech (Shanghai, China). Model compounds, including CD59antigenlocatedonhumanchromosome11[38].ALcells
Zn5(CO3)2(OH)6andZn(OH)2,werepurchasedfromQual- wereculturedinsupplementalHam’sF12mediumandseeded
ity Inspection Biotechnology (Beijing, China). Supplemen- atadensityof1.5×105cellsperPetridish(60-mmdiameter)
∘
tal Ham’s F12 medium was prepared by mixing 8% heat- andincubatedinahumidifiedincubator(37 C,5%CO2).The
inactivatedfetalbovineserum(Hyclone,GrandIsland,NY), cells attached to the bottom of the dishes within 2h; after
2×10−4Mglycine,and25mg/mLgentamicin.Allchemicals 24h, cells reached the exponential growth phase. ZnO NP
wereusedwithoutfurtherpurification. stocksolutionswerevortexedfor2minandsonicated(30W)
for30min,andthenaliquotsweretakentoprepareworking
2.2.NaturalTransformationofZnONPs. PristineZnONPs solutions in supplemented Ham’s F12 medium. In addition,
weresuspendedinMilli-Qwater(Millipore,18MΩcm)ata the working solutions were sonicated at 30W for 20s and
∘
concentrationof1mg/mLandsterilizedbyheatingto120 C then dispersed into cell culture dishes to ensure proper
JournalofNanomaterials 3
dispersionoftheNPs.Medium(4mL)containingpristineor lethal concentration (LC)100 of NPs, as previously deter-
transformedNPsuspensionsatvariousconcentrations(0,5, mined.NPsuspensionswithoutcentrifugationandfiltration
10,15,20,30,50,and60𝜇g/mL)wasimmediatelyaddedtothe were also applied to cells for comparison. Supplemented
testcellsandsupplementedmedium(4mL).Treatedcellsand medium without ZnO NPs was added to the control cells.
∘
controlcellswereculturedfor72hat37 C.Aftertreatment, Thedetailedproceduresfordeterminingcellviabilityandfor
the culture solution was removed and cell morphology was themutationassayaftertreatmentwerethesameasdescribed
observedusingamicroscope(NikonEclipseTi,Japan).For above.
evaluationofcellviability,thecontrolcellsandtreatedcells
werereplatedatadensityof300cellsperPetridish(60mm 2.7.EffectsofAirExposureonToxicity. After60daysofstor-
∘
diameter)infreshsupplementalmediumandculturedfor8 ageinthedarkat25 C,thetransformedZnONPsuspensions
days.Thecolonieswerethenfixedwithformaldehyde,stained (with air exposure) and control suspensions (without air
withcrystalvioletdye,andthenumberofsurvivingcolonies exposure) were subjected to morphology analysis by TEM
was recorded. Survival fraction was defined as percentage andcytotoxicityevaluationusingthecolonyformationassay.
of the plating efficiency of the treated group to the plating Thedetailedprocedureswerethesameasdescribedabove.
efficiencyofthecontrolgroup[38].
2.8. Data Analysis. All data in this study are represented
2.5.MutationPotential. Aftertreatment,cellswerereplated as the means ± SD of three to four replicates of identical
into 60mm Petri dishes at 1 × 105 cells/dish and cultured experiments.Significantdifferencesbetweentreatmentswere
for7–14consecutivedays.Thisexpressionperiodallowedthe determined by one-way analysis of variance (ANOVA).
cells to recover from the temporary growth lag after ZnO Resultswith𝑝<0.05wereconsideredstatisticallysignificant.
NP treatment and to multiply such that the progeny of the
mutated cells no longer expressed lethal amounts of CD59 3.Results
surfaceantigen.Duringthisperiod,themediumwaschanged
everyseconddayandthecellswerepassagedonceevery3-4 3.1. NP Properties. To analyze possible ZnO NP transfor-
days.Thecellswerethenharvestedand0.5×105 cellswere mations in ultrapure water, morphological changes were
seededin60mmPetridishescontaining2mLsupplemented observed with TEM. As shown in Figure1(a), pristine ZnO
Ham’s F12 medium. After incubation in a CO2 incubator NPs are a mixture of approximately spherical and rod-like
for 2h, freshly thawed antibodies (0.2%) and complement particles. After alkalization for 20 days, a subtle change in
(1.5%(V/V))wereadded.Eachexperimentconsistedofthe the nanocrystal microstructures was observed. When the
same batch for the control group, including the antibody- alkalization time was prolonged to 60 days, the number of
alone treatment group, the complement-alone treatment visible NPs in oneTEM captionsharply decreased and was
group, and the blank control group. Cultures were further replaced by sheets and a mesh material; NP morphology
incubatedfor7to10daysuntilmacroscopicallyvisibleclones also began to significantly change. For example, the sharp
were formed. The culture medium was then discarded and crystaledgesdisappearedandwerereplacedbyblurredand
cellswerefixedwithformaldehydeandstainedwithcrystal amphibolousboundaries,indicatingcorrosivedamageonthe
violet dye; the number of clones was then counted under NPsurface.WhenZnONPswerealkalizedinpurewaterfor
white light. The mutation frequency (MF) was calculated 120days,theoriginalNPmorphologybecamelessapparent,
as the number of surviving colonies divided by the total showingsignsofdissolutionduringthealkalizationprocess
number of cells plated after correcting for any nonspecific andbecoming“flaky”andlesssolidinappearance.Tofurther
cytotoxicity attributable to complement alone. The relative determine the transformation products, EDX was used to
increaseinmutationfrequencyisexpressedasfoldincrease analyzeZnONPsafterundergoingalkalizationfor20and120
overbackground. days.AsshowninFigure1(a),zincwasthemajorconstituent
of the crystalline suspension after 20 days and the lamellar
2.6. Quantification of Zn2+ and Its Contribution to Toxicity. substances present after 120 days. To further determine
2+
Zn released from pristine and alkalized ZnO NPs was the possible conversion products of ZnO NPs, we used
quantified by inductively coupled plasma optical emission FTIR and Raman spectroscopy, as well as XRD. FTIR and
spectrometry (ICP-OES). Specifically, 50𝜇g/mL sonicated RamandatashowtheformationofZn(OH)2 (characteristic
−1
stock suspension was centrifuged in a Beckman Airfuge peak at 1500cm in Figure1(b) and characteristic peaks
−1 −1
Ultracentrifugeat20,000rpmfor30minwithcoolingtopre- at 1153.7cm and 1508.9cm in Figure1(c)), which was
cipitatesolidparticles.Thesupernatantwasfilteredthrough thenconfirmedbyXRDspectroscopy(cardnumber00-003-
aWhatmanaluminaAnotopmembrane(0.02𝜇mporesize, 0888).Further,XRDdatashowtheneoformationofanother
47mm diameter, Maidstone, Kent, UK), which excluded Zn5(CO3)2(OH)6 solid phase (card number 00-011-0287)
particleslargerthan20nm.Afterhigh-speedcentrifugation (Figure1(d)).DLS,amethodcommonlyusedtocharacterize
andmembranefiltration,theconcentrationofelementalZn NPs,wasusedtodeterminethehydrodynamicsizeoftheson-
(206.200nm)inthesupernatantswasmeasuredusingICP- icatedpristineandtransformedNPs.Althoughthismethodis
OES(Optima7300DV,PerkinElmerCorporation,Norwalk, moreaccurateforassessingsphericalthannonsphericalNPs,
CT). itcanprovidetherelativesizeanddispersionofnonspherical
2+
To determine the contributionof released Zn to ZnO (irregular) NPs, including whether they are agglomerated.
NPtoxicity,weexposedALcellstothesupernatantof(nearly) The results show a certain degree of NP aggregation in the
4 JournalofNanomaterials
EDX
Zn 2-1-1
Zn
Cu
Zn
Zn
Cu
OCu Cu
C Cu Zn
Pristine 20 days
0 1 2 3 4 5 6 7 8 9 10 11 12 13
(keV)
Full scale 4173cts cursor:−0.085(26cts)
2-1-2
Zn
Cu
Zn
Zn
O Zn
Cu
CCu Cu Cu Zn
0.2m 60 days 120 days
0 1 2 3 4 5 6 7 8 9 10 11 12 13
(keV)
Full scale 1910cts cursor:−0.347(0cts)
(a)
70000
Pristine 60000
e
c
n 50000 1153.7
a
elative transmitt 2600 ddaayyss Intensity (cnt) 234000000000000 1508.9
R
120 days 10000
437.9
0
0 500 1000 1500 2000 2500 3000 3500 4000 4500 400 800 1200 1600 2000 2400 2800 3200
−1
Wave number (cm ) Raman shift (1/cm)
60-day ZnO NPs
Pristine ZnO NPs
Zn(OH)2
(b) (c)
Zn5(CO3)2(OH)6
u.)
a.
y ( Zn(OH)2
nsit
e
Int 60 days ZnO NPs
Pristine ZnO NPs
20 40 60 80
2𝜃(degrees)
(d)
Figure 1: Characterization of pristine and naturally transformed ZnO nanoparticles (NPs). (a) Low resolution transmission electron
microscopy(TEM)imagesofNPsandenergydispersiveX-ray(EDX)measurements(indicatedbytheredsquares)fromZnONPsalkalized
for20and120days.(b)Fouriertransforminfrared(FTIR)spectraofpristineandnaturallytransformedZnONPs.Orangesquaresindicate
thespecificpeaks.(c)RamanscatteringspectraofZnONPsandZn(OH)2.(d)X-raypowderdiffraction(XRD)patternsofZn5(CO3)2(OH)6,
Zn(OH)2,pristineNPs,andNPsalkalizedfor60days.
JournalofNanomaterials 5
Table1:ParticlesizeandzincconcentrationofpristineandalkalizedNPssuspension.
NPswithdifferentalkalizationtime Hydrodynamicsize(nm) ZnconcentrationinNPstocksuspension(𝜇g/mL)
Stocksuspension F-12/8%FBS
Pristine 324.4±81 117±33 4.8970
20days 394±141 123±67 9.1402
60days 582±70 157±116 13.8040
120days 633±97 191±108 28.1730
Nanoparticlesizewasexpressedasintensity-basedaveragehydrodynamicdiameter.Bars:±SD.ThedissolvedZnconcentrationwasmeasuredbyICP-OES:
∘
50mg/mLNPswassuspendedinMilli-Qwaterat25C.Afterhigh-speedcentrifugation(20,000rpmfor30minwithcooling)andmembranefiltration
(WhatmanaluminaAnotopmembrane,0.02𝜇mporesize,47mmdiameter),theconcentrationofelementalZn(206.200nm)inthesupernatantswasmeasured
usingICP-OES.
stocksuspensions(324.4±81nmforpristineNPsand633± Viability assays are essential for evaluating the cellular
97nmforNPsafter120daysofalkalization)(Table1),which responsetotoxicants.Tomeasuretotalcellviabilityfollowing
isconsistentwiththeTEMobservations(Figure1(a)).After NP exposure, we used a colony formation assay [40]. As
dilutioninsupplementalHam’sF12medium,thesizeofthe showninFigure3,allZnONPsexhibitedadose-dependent
NPagglomeratesdecreasedobviouslyandtherewasacertain increase in cytotoxicity regardless of the alkalization time.
degree of difference between pristine (117 ± 33nm) and After treatment for 72h, significant toxicity was recorded
transformed ZnO NPs (123 ± 67nm for NPs after 20 days, at 10𝜇g/mL and higher (𝑝 < 0.05) in cells incubated
157 ± 116nm for NPs after 60 days, and 191 ± 108nm for with pristine and alkalized NPs, with a widely variable
NPs after 120 days), as shown in Table1. These results are LC100 (pristine NPs: 15𝜇g/mL; NPs alkalized for 20 days:
likelyattributabletobovineserumalbumin(BSA)andother 15.5𝜇g/mL; NPs alkalized for 60 days: 32𝜇g/mL; and NPs
componentsofthecellculturemedium,whichstabilizethe alkalizedfor120days:60𝜇g/mL).Notably,atconcentrations
NPsandcontributetotheirdispersion[39]. between 10𝜇g/mL and the observed LC100, viability tended
AllthesecharacterizationresultsindicatedthatZnONPs to be higher in cells treated with alkalized NPs than that
were naturally alkalized (the process of making something of cells treated with pristine NPs (Figure3). These results
alkaline without the addition of alkaline substances) in suggest that alkalized ZnO NPs cause less cytotoxicity than
ultrapurewaterwithairexposure. thatofpristineZnONPs.Further,thedecreaseincytotoxicity
resulting from ZnO NP exposure was dependent on the
3.2.Cytotoxicity. NPtreatmentresultsinanoticeablechange durationofalkalization.
in cellular shape, or morphology, in vitro [28]. Therefore,
weexaminedcellularmorphologybymicroscopy.Asshown 3.3. Mutagenicity. We hypothesized that the lower toxicity
inFigure2,cellmorphologyinthecontrolgroupremained observed in cells treated with alkalized ZnO NPs would
normal; the cells adhered well, with most attaching within translate to a safer product. To confirm this idea and to
2h. Most cells were spindle-shaped or polygonal, with a efficiently determine the mutagenic potential of ZnO NPs
few newly dividing cells showing a more transparent cyto- during the alkalization process, we conducted a CD59 gene
plasmandbetterdispersionduringtheprocessofadhering. loci mutation assay in AL cells. AL cells were plated at 80%
After treatment with 10𝜇g/mL pristine ZnO NPs for 72h, confluency, with an average spontaneous mutation rate of
cell morphology significantly changed. Although most cells 67 ± 27 mutant cells per 1 × 105 surviving cells. As shown
adhered within 3–5h, they could not spread, and some in Figure4, treatment with pristine ZnO NPs at 5𝜇g/mL
became rounded and lost the polygonal shape. When the and 10𝜇g/mL did not result in a significant increase in
concentration of ZnO NPs was increased to 15𝜇g/mL, the themutationratio.However,somecellsthatwereadversely
treatedcellsatrophiedandcouldnotadhere,suggestingthat affected by treatment with pristine ZnO NPs (15𝜇g/mL)
cellviabilitywassignificantlylowerthanthatofthecontrol recovered from the temporary growth lag after culturingin
cells and cells treated with 10𝜇g/mL. These results indicate freshsupplementalmediumfor14days(Figures2and3);the
that the LC100 for pristine ZnO NPs is mostly like less mutationrate(2.34±0.16,𝑝<0.05)wassignificantlyhigher
than15𝜇g/mL.Incontrast,cellmorphologyaftertreatment in these cells than that of the control group. A significant
with ZnO NPs alkalized for 20 days (10𝜇g/mL) was not increaseinmutationratewasobservedincellstreatedwith
significantly different from that of the control group, and NPsalkalizedfor20daysat5𝜇g/mLandabove,with1.89±
most of the cells could adhere and spread; however, no 0.24 being the highest measured maximum mutation rate.
cells survived at a dose of 20𝜇g/mL. A similar trend was Further,asignificantincreaseinmutationratewasshownin
observed in cells treated with ZnO NPs alkalized for 60 cells treated with NPs alkalized for 60 and 120 days at ≥10
and120days;theLC100 inbothtreatmentgroupsincreased and ≥20𝜇g/mL, with the highest maximum mutation rate
to approximatelyabove 30 and 50𝜇g/mL, respectively, with measuredas1.63±0.15and1.63±0.21,respectively.
fewerdeadcellsobservedaftertreatmentwithalkalizedNPs, These results show that alkalized ZnO NPs have muta-
indicating that alkalized ZnO NPs are less cytotoxic than genic capacity surpassing that of pristine NPs at certain
pristineZnONPs. concentrations. This is inconsistent with the previously
6 JournalofNanomaterials
Control 10𝜇g/ml 15𝜇g/ml
e
n
Pristi
50𝜇m 50𝜇m 50𝜇m
Control 10𝜇g/ml 20𝜇g/ml
ys
a
d
0
2
50𝜇m 50𝜇m 50𝜇m
Control 20𝜇g/ml 30𝜇g/ml 40𝜇g/ml
ys
a
d
0
6
50𝜇m 50𝜇m 50𝜇m 50𝜇m
Control 20𝜇g/ml 40𝜇g/ml 50𝜇g/ml 60𝜇g/ml
ys
a
d
0
2
1
50𝜇m 50𝜇m 50𝜇m 50𝜇m 50𝜇m
Figure2:MorphologychangesinAL cellsafterexposuretopristineoralkalizednanoparticles(NPs)for72hinsupplementalHam’sF12
medium.AL cellmorphologywasobservedwithanopticalmicroscopeat20xmagnificationforcontrolgroupsand10xmagnificationfor
NP-treatedgroups.
observedtrendoflowerZnONPcytotoxicitywithincreased NPs alkalized for 20 days (15𝜇g/mL), NPs alkalized for 60
transformationtimeanddeservesfurtherstudy. days(30𝜇g/mL),andNPsalkalizedfor120days(60𝜇g/mL)
remainedat95.16±4.11%,77.79±1.78%,75.63±3.26%,and
3.4. Contribution of Released Zn2+ to the Toxicity of Pristine 64.44±1.78%,respectively.Althoughtherewasalkalization
2+
and Alkalized ZnO NPs. To determine the role of Zn in time-dependentincreaseincytotoxicityattributabletotreat-
ZnO NP toxicity during the natural alkalization process, mentwiththeZnONP supernatant,cellviabilityremained
2+
we used a dose approaching the LC100 that still exhibited above60%.TheseresultsindicatethatZn didnotcontribute
mutagenicity. After high-speed centrifugation and mem- significantlytothecytotoxiceffectsofZnONPs.Incontrast,
brane filtration, ZnO NP supernatants were dispersed in themutagenicityofpristineandalkalizedZnONPswasmore
2+
Milli-Qwaterataconcentrationof50mg/mL;ICP-OESwas dependent on the concentration of Zn in the ZnO NP
used to determine the amount of Zn2+ released from the supernatant. Specifically, the mutation rate in cells treated
NPsduringthealkalizationprocess.AsshowninTable1,the with supernatant from pristine NPs was 1.21 ± 0.22, which
concentrationofdissolvedZn2+intheZnONPsuspensions was approximately half of that observed with the ZnO NP
increasedwiththealkalizationtime,andthemaximumcon- suspension(2.32±0.21)(𝑝 < 0.05)(Figure5(b)),indicating
centrationofZn2+after120dayswas28.17𝜇g/mL.Ourresults thatthemutagenicityofpristineNPsisattributabletoboth
2+
showthatZnONPdissolutionisslightlyenhancedbyalka- the particles and Zn released into the supernatant. When
2+
lization, accompanied by transformation of the crystalline the transformation time was extended to 20 days, the Zn
phase. After quantitative determination of the dissolved released into the supernatantplayed a moresignificant role
Zn2+ concentration,AL cellsweretreatedwiththeZnONP in the mutagenic effects of the ZnO NPs. The mutagenic
2+
supernatants. As shown in Figure5(a), the viability of cells frequency of Zn released into the supernatant from ZnO
treated with the supernatants of pristine NPs (15𝜇g/mL), NPsalkalizedfor60and120dayswas1.27±0.21 and1.54±
JournalofNanomaterials 7
∗
100 2.4
∗
p<0.05 ants 2.0
urvival fraction (%) 468000 −hange of CD59mut5per 10survivors 011...826 ∗∗ ∗∗∗∗ ∗∗∗∗
S d c
20 ol
F 0.4
0 0.0
Control 5 10 15 20 30 40 50 60 Pristine 20 days 60 days 120 days
ZnO NPs (g/ml) ZnO NPs
Pristine 60 days Control 20g/ml
20 days 120 days 5g/ml 30g/ml
10g/ml 40g/ml
Figure 3: Survival fractions of AL cells after pristine or alkalized 15g/ml 50g/ml
nanoparticle(NP)exposure.NaturallyalkalizedZnOnanoparticles
(NPs)inducedlowercytotoxicitythanthatofpristineNPsinALcells Figure4:MutationfrequenciesinALcellsafterpristineoralkalized
growninsupplementalHam’sF12medium.Resultsareexpressedas nanoparticle(NP)exposure.Therelativeratioofincreasedmutation
thepercentofcellactivitycomparedtothecontrolusingdifferent frequenciesisexpressedasafoldincreaseoverthebackgroundafter
NPconcentrationsfor72h.Thedataarepresentedasthemeans ± treatment with different NP concentrations for 72h. The data are
SDofatleastthreeindependentexperiments.Thevaluesrepresented presentedasmeans±SDofatleastthreeindependentexperiments.
byadashedlinearesignificant(𝑝 < 0.05)whencomparedtothe Significanceisindicatedby∗𝑝<0.05.
survivalfractionofthecontrolgroup.
0.22, respectively, which is similar to the values measured of transformed NPs affect their fate, transport, and toxicity
for alkalized ZnO NP suspensions (1.48 ± 0.06 and 1.42 ± [10]. Studies have shown that NPs undergo a series of
0.01) (Figure5(b)). These data indicate that Zn2+ is solely chemicalconversions,includingchemicaladsorptionoftoxic
responsibleforthemutagenicpotentialofZnONPsalkalized metalanions,complexationoforganicmolecules,andsubse-
for120days(Figure5(b)). quentdissolution[41–43].Inaddition,NPaging(weathering)
affects dissolution and reactivity [13, 44, 45]. Previously,
3.5. High-Frequency Exposure to Air Led to a More Intense we reported that ZnO NPs undergo a dramatic physico-
Transformation and Variations in ZnO NP Toxicity. The chemical transformation with aging [22]. Here, we confirm
above results were based on pristine NPs as well as air- a similar transformation process and the neoformation of
exposed alkalized NPs. To determine the key factors influ- Zn5(CO3)2(OH)6 (Figure1). Further, we clarify that the
encingthealkalizationtransformationprocess,weobserved transformationis a natural alkalization (without exogenous
changes in toxicity and cell morphology in AL cells after alkaline substances added) process. We suggest that the
treatmentwithZnONPsundergoingthealkalizationprocess alkalizationisattributabletotheexposureofsuspendedZnO
for 60 days in the absence of air. As shown in Figure6, NPstoairratherthanjuststorageinultrapurewater.
we observed dramatic differences in cells treated with air- Studies have reported similar transformations of ZnO
exposedalkalizedZnONPs;however,therewerenoobvious NPstoZnCO3andZn5(CO3)2(OH)6;however,thereislittle
changes in morphology between cells treated with pristine data regarding the corresponding toxicity [19–21]. Further-
NPs and those that underwent the alkalization process in more,thepresenceofadditionalchemicals,suchassulfides
theabsenceofair(Figures6(a)and6(c)).Forcellsexposed andphosphates,mayresultinadditionalchemicalreactions
to ZnO NPs alkalized for 60 days in the absence of air, andfacilitateNPtransformation[15,16].Therehasbeenlittle
phenotype(Figures6(d)and6(f))andcellviability(Figures researchonthenaturaltransformationprocessofNPs;Zhang
6(g) and 6(i)) were almost the same as those of pristine etal.(2016)[23]recentlyanalyzedthephysicochemicaltrans-
ZnO NPs at the same dose. These results illustrate that, formation of ZnO NPs during the aging process as well as
in the absence of air, ZnO NPs are less likely to undergo toxicityingreenalgae,inwhichnaturalNPtransformations
alkalization. Subsequently, few changes were observed for wereobservedatroomtemperatureovertime(0–210days);
ZnONPcytotoxicity. however,theauthorsdidnotmentiondetailsofhowtheaging
processwasmodeled.Inourpresentwork,weconfirmedthe
4.Discussion chemical transformation (natural alkalization) of ZnO NPs
2+
intoZn5(CO3)2(OH)6,Zn(OH)2,andZn (Figure1),aswell
Nanoparticles are highly reactive and dynamic in natural aschangesinthechemicalpropertiesthatcouldresultinvari-
systems,anditisimportanttounderstandhowtheproperties ations in ZnO NP cytotoxicity and mutagenicity, especially
8 JournalofNanomaterials
2.8
∗ ∗
100
2.4 ∗
∗ ∗ nts
a
vival fraction (%) 468000 ∗ −nge of CD59mut5er 10survivors 112...260
ur hap 0.8
S 20 d c
ol
F 0.4
0
0.0
Control Pristine 20 days 60 days 120 days Control Pristine 20 days 60 days 120 days
ZnO NPs ZnO NPs
Suspension Suspension
Supernatant Supernatant
(a) (b)
2+
Figure5:ThecontributionofZn inthesupernatantfractiontocellsurvival.(a)ThemutationpotentialofpristineandalkalizedZnO
nanoparticles (NPs) at a concentration approaching the lethal concentration (LC100, pristine NPs: 15𝜇g/mL; NPs alkalized for 20 days:
15𝜇g/mL;NPsalkalizedfor60days:30𝜇g/mL;andNPsalkalizedfor120day:60𝜇g/mL)for72hisshown.(b)Afterhigh-speedcentrifugation
andmembranefiltration,ALcellsweretreatedwiththeNPsupernatantsatconcentrationsapproachingtheLC100for72h.TheNPsuspension
withoutcentrifugationwasusedasacontrol.Bars:±SD.∗indicates𝑝<0.05.
themutagenicpotentialofZnONPsgivenattheLC100.ZnO and exhibited a higher frequency of mutations than that
NP aggregation occurring during the alkalization process, of surviving cells treated with a lower dose (Figure4). In
and the hydrodynamic particle size of the NPs in water other words, transformed ZnO NPs with low cytotoxicity
suspensionsincreasedfrom324.4±81nm(pristine)to633± caused genetic damage and mutations in surviving cells.
97nm (NPs alkalized for 120 days) (Table1). After dilution These results suggest that the adverse effects attributable
in cell culture medium, the extent of aggregation declined to transformed NPs could be more serious than those of
obviously. We also observed that, with the extension of pristine NPs. Therefore, it is very important to select more
alkalizationtime,thedegreeofNPagglomerationincreased, effectiveandappropriatedetectionsystemsandmethodsfor
whiletheircytotoxicitygraduallyweakened(Figures2and3), evaluatingNPsafety.
whichcouldpartlyexplainthedifferenceintoxicityobserved Dissolution is an important process that affects surface
between pristine and alkalized ZnO NPs. Furthermore, we properties, toxicity, and NP persistence. This is particularly
found that high-frequency exposure to air is necessary for trueforNPsmadefromGroupBsoftmetalcations,suchas
variations in ZnO NP alkalization and toxicity to occur Ag,Zn,andCu[10].Theroleofdissolutionandundissolved
(Figure6).ThisislikelyattributabletotheconversionofZnO particles in ZnO NP toxicity has been studied in vitro and
NPsintoZn5(CO3)2(OH)6,whichrequirestheparticipation invivo[26,47,48].However,noconsistentconclusionshave
ofCO2. been reached concerning the primary contributor to ZnO
Unlike cytotoxicity, which is a lethal effect of toxic
NP toxicity. It is well accepted that ZnO NPs dissolve and
substances,genotoxicityreflectsthemutageniceffectsoftoxic 2+
release Zn ions, which reduces persistence but increases
and exogenous substances to DNA and RNA, as well as 2+
toxicity[10].Songetal.[24]reportedthatdissolvedZn is
deleteriousgeneticchangesinoffspring.MutationstoDNA,
theprimarycontributortocelldeathinmousemacrophage
which are associated with cancer, may occur if the damage
Ana-1 cells. In contrast, microbial growth is inhibited by
is not repaired in time [46]; therefore, determining the
ZnO NP suspensions rather than suspension supernatants,
mutagenicityofZnONPsisimportant.Previously,wefound
thatagedZnONPshavesignificantprotuberance(𝑝<0.05), indicatingthatthetoxiceffectsareprimarilyattributableto
2+
whichagreeswithZnONPresearchinbacteria[19,25].Based ZnONPsratherthandissolvedZn [34].Theseconflicting
on the results of the CD59 gene loci mutation assay in AL observations may be attributable to the origin of ZnO NPs
cells, we determined that dying or severely damaged cells transformed in the aqueous environment, stability of the
wereabletorecoverfromthetemporarygrowthlagafterZnO stocksuspensions,exposuremethod,and/orthenatureofthe
NPs treatment; therefore, we focused on the mutagenicity experimental models. A fundamental understanding of the
of (nearly) lethal doses of ZnO NPs. After treatment with scopeandextentofthecontributionfrombothdissolvedand
ZnO NPs at a dose approaching the LC100, some cells near undissolvedparticulatestoNPtoxicitycanprovideessential
death were able to recover from the temporary growth lag information regarding the potential risks to ecosystems
JournalofNanomaterials 9
0.2
휇m
(a) (b) (c)
50
휇m
(d) (e) (f)
100 100 100
∗
∗ ∗
%) 80 %) 80 %) 80
Survival fraction ( 246000 Survival fraction ( 246000 Survival fraction ( 246000
∗ ∗
0 0 0
Control 5 10 15 Control 5 10 15 Control 5 10 15
(g/ml) (g/ml) (g/ml)
Pristine NPs 60-day NPs with air exposure 60-day NPs without air exposure
(g) (h) (i)
Figure6:MorphologychangesandvariationsinthesurvivalfractionofALcellstreatedwithZnONPswithhigh/low-frequencyexposureto
air.Lowresolutiontransmissionelectronmicroscopy(TEM)imagesof(a)pristineNPs,(b)NPsalkalizedfor60dayswithairexposure,and
(c)NPsalkalizedfor60dayswithoutairexposure.ImagesofALcellstreatedwith(d)pristine,(e)NPsalkalizedfor60dayswithairexposure,
and(f)NPsalkalizedfor60dayswithoutairexposureat15𝜇g/mLfor72h.SurvivalfractionsofALcellstreatedwith(g)pristine,(h)NPs
alkalizedfor60dayswithairexposure,and(i)NPsalkalizedfor60dayswithoutairexposurefor72h.Thedataarepresentedasmeans±SD
ofatleastthreeindependentexperiments.Significanceisindicatedby∗𝑝<0.05.
and humans. In the present study, the role that dissolved major contributor to toxicity from these results. Although
2+ 2+
Zn in ZnO NP supernatant plays in overall toxicity was numerous studies have discussed the role of Zn ions in
investigated. We focused on the toxicity of alkalized ZnO ZnO NP toxicity [24, 34, 49], there are no relevant studies
NP concentrations approaching the LC100. As shown in regarding the positive mutagenicity results. It is possible
2+
Figure5(a), the presence of Zn could not fully explain thatthemechanismsunderlyingZnONPcytotoxicitydiffer
viability of AL cells exposed to a dose of alkalized ZnO fromthoseunderlyingZnONPmutagenicity.Previously,we
NPsapproachingtheLC100.However,mutationfrequencies revealedthat,duringtheagingprocess,Zn2+ ionsandsolid
inducedbyalkalizedZnONPs,especiallythosealkalizedfor particles contribute differently to the mutagenicity of ZnO
2+
120days,couldstemfromtheprimarycontributionofZn NPs[22].Theseobservationsindicatethatthemechanismsof
2+
(Figure5(b)). We could not determine whether Zn is a ZnONPtoxicityarecomplicatedandmorestudiesfocusing
10 JournalofNanomaterials
onmutagenicityandNPtransformationsintheenvironment (2015M82023 and 2016M600477), by Scientific Research
areneeded. of BSKY (XJ201506) from Anhui Medical University, and
Inaddition,itshouldbenotedthatthisstudyonlyexam- by CPSF-CAS Joint Foundation for Excellent Postdoctoral
ined the transformed products using several fundamental Fellows(2015LH0017).
and routine qualitative methods (TEM, DLS, FTIR/Raman,
and XRD spectroscopy). The use of more accurate and References
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by grants from China Postdoctoral Science Foundation “Sulfidation mechanism for zinc oxide nanoparticles and the