Research ArticleCerium Oxide Nanoparticles Induced Toxicity in Human LungCells Role of ROS Mediated DNA Damage and Apoptosis

Sandeep Mittal12 and Alok K Pandey12

1 Academy of Scientific and Innovative Research (AcSIR) New Delhi 110025 India2Nanomaterial Toxicology Group CSIR-Indian Institute of Toxicology Research (CSIR-IITR) PO Box 80 Mahatma Gandhi MargLucknow Uttar Pradesh 226001 India

Correspondence should be addressed to Alok K Pandey pandeyalok2006gmailcom

Received 27 February 2014 Revised 22 April 2014 Accepted 12 May 2014 Published 1 June 2014

Academic Editor Haseeb Ahmad Khan

Copyright copy 2014 S Mittal and A K Pandey This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cerium oxide nanoparticles (CeO2NPs) have promising industrial and biomedical applications In spite of their applications the

toxicity of these NPs in biologicalphysiological environment is a major concern Present study aimed to understand the molecularmechanism underlying the toxicity of CeO

2NPs on lung adenocarcinoma (A549) cells After internalization CeO

2NPs caused

significant cytotoxicity and morphological changes in A549 cells Further the cell death was found to be apoptotic as shown byloss in mitochondrial membrane potential and increase in annexin-V positive cells and confirmed by immunoblot analysis of BAXBCl-2 Cyt C AIF caspase-3 and caspase-9 A significant increase in oxidative DNA damage was found which was confirmedby phosphorylation of p53 gene and presence of cleaved poly ADP ribose polymerase (PARP) This damage could be attributedto increased production of reactive oxygen species (ROS) with concomitant decrease in antioxidant ldquoglutathione (GSH)rdquo levelDNA damage and cell death were attenuated by the application of ROS and apoptosis inhibitors N-acetyl-L- cysteine (NAC) andZ-DEVD-fmk respectively Our study concludes that ROS mediated DNA damage and cell cycle arrest play a major role in CeO

2

NPs induced apoptotic cell death in A549 cells Apart from beneficial applications these NPs also impart potential harmful effectswhich should be properly evaluated prior to their use

1 Introduction

Over the past few years there has been rapid increase in theuse of different nanomaterials owing to their unique physic-ochemical and bioreactive properties Different metal oxidenanoparticles (NPs) have potentially been used in industriesincluding sunscreens food paints textile electronics sportsand biomedical application and imaging [1 2] It is estimatedthat the value of engineered nanoparticles (ENPs) marketwould increase up to 20ndash30 billion dollars by 2015 [3] Thishas raised concerns over the unforeseen adverse harmfulhealth effects caused due to interactions with the livingsystems

Amongst rare earth elements cerium oxide (CeO2) NPs

are widely used in a variety of applications such as glassceramic polishing agent television tubes solar cells fuel cellsultraviolet absorbents and gas sensors [4ndash7] Besides theseindustrial applications various biomedical applications of

CeO2NPs such as protection against radiation induced dam-

age and retinal neurodegeneration and anti-inflammatoryand antioxidant activity have also been explored [8ndash11]Recently the use of CeO

2NPs as a diesel fuel additive to

reduce the ignition temperature of carbonaceous dieselexhaust particle (DEP) and subsequently to reduce the emis-sion of particulate matter from diesel engines has beenexplored [12] Although this addition enhances the ability ofdiesel engines it leads to direct emission of CeO

2NPs in the

environment Health Effect Institute (HEI) has also reportedthat the CeO

2NPs emission will reach up to 22 million

pounds annually in European Union after this addition [12]Thus commercially used CeO

2NPs are released into the

environment and their evaluation in the living system isworthwhile and relevant for society and human welfare [13]Human exposure to the nanoparticles is possible both fromworkplace (occupational) and environmental release through

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 891934 14 pageshttpdxdoiorg1011552014891934

2 BioMed Research International

inhalation and ingestion as major routes Since CeO2NPs

are poorly absorbed by the intestine inhalation appears tobe the major route of exposure It should also be noted thatcomplete respiratory system acts as repository for depositionof different sizes of NPs Although several studies havebeen performed to evaluate the adverse effect of CeO

2NPs

on environment and human health they are not providingproper conclusionCeO

2NPshave been reported to act as cel-

lular antioxidants with colocalizations inside mitochondrialysosomes endoplasmic reticulum nucleus and cytoplasmusing keratinocytemodel systems [14 15] However the studylacked genotoxicity assessment of these particles Contrary tothis previous studies have reported that CeO

2NPs generate

oxidative stress and may induce apoptosis in human lungepithelial cells [16 17] Therefore CeO

2NPs may portend

cytotoxic and genotoxic effects upon cellular internalizationIt has been reported that the CeO

2NPs of various sizes

showed significant toxic effects on E coli and human cellsrespectively due to adsorption of NPs and oxidative stress[18 19] Along this reports have also been postulated thatCeO2NPs of smaller sizes do not cause any adverse effect

but can protect cells from harmful effects of radiation andoxidative stress although this protectionwas cell type specific[20 21] In vivo studies have resulted that CeO

2NPs exposure

via inhalation and intratracheal instillation route can induceacute pulmonary and systemic toxicity in rat and mice dueto proinflammatory responses [22 23] Besides these CeO

2

NPs have been shown to protect cells from reactive oxygenspecies due to their inherent antioxidant properties [15 24]this protective effect was thought to be due to the presenceof dual oxidation state of CeO

2NPs or the pH value of cell

compartment where the nanoparticles internalizeWith thesecontrasting results the toxicity of CeO

2NPs remains elusive

and specific toxicity endpoints relevant to human healthneeds to be addressed It was therefore prudent to conduct asystematic study to understand the comprehensivemolecularmechanism of toxicity of CeO

2NPs as well as to see the role

of DNA damage and halt of cell cycle in cell deathCharacterization ofNPs is an essential step before toxicity

assessment as properties of NPs vary significantly with shapeand size It is also essential to characterize their agglomera-tionaggregation tendency in the culture media for accuratetoxicity assessment In vitro cell based assays are rapid andallow reliable toxicity fingerprints for NPs In the presentstudy an attempt was made to (1) characterize CeO

2NPs to

assess their behavior in culture medium (2) evaluate cyto-toxic and oxidative stress potential (3) estimate their DNAdamaging potential and subsequent cell cycle arrest and (4)evaluate apoptotic index

2 Materials and Methods

21 Chemicals Cerium (IV) oxide nanopowder (purity9995 lt25 nm particle size BET) propidium iodide 27-dichlorofluorescein diacetate (DCFDA) dye 551015840 661015840-tetra-chloro-1110158403101584031015840-tetraethylbenzimidazolecarbocyanine iodide(JC-1) dye low melting point agarose (LMA) ethidiumbromide (EtBr) Triton X-100 and ethyl methanesulfonate(EMS) were purchased from Sigma Chemical Co Ltd (StLouis MO USA) Normal melting agarose (NMA) and

ethylenediaminetetraacetic acid (EDTA) disodium salt werepurchased from Himedia Pvt Ltd (Mumbai India) For-mamidopyrimidine DNA glycosylase (Fpg) enzyme wasobtained from Trevigen Inc (USA) Hydrogen peroxide(H2O2) was purchased from Ranbaxy Fine Chemical Ltd

(New Delhi India) Phosphate buffered saline (Ca+2 Mg+2free PBS) Dulbeccorsquos modified eagle medium nutrient mix-ture F-12 (Ham) (1 1) powder (DMEM F-12) trypsin-EDTAfoetal bovine serum (FBS) trypan blue and antibiotic andantimycotic solution (10000UmL penicillin 10mgmLstreptomycin 25120583gmL amphotericin-B) were purchasedfrom Life Technologies Pvt Ltd (New Delhi India) Allother chemicals were obtained locally and were of analyticalreagent grade Cell culture plastic wares were obtained fromThermo Scientific Nunc (Rochester New York)

22 Characterization of CeO2NPs CeO

2NPs were charac-

terized using transmission electron microscopy (TEM) anddynamic light scattering (DLS) technique

221 Transmission Electron Microscopy (TEM) TEM anal-ysis was carried out for the assessment of primary particlesize and morphology of CeO

2NPs Samples were prepared

by suspending NPs in Milli-Q water at a concentration of50 120583gmL A drop of suspension was added to the formvar-coated copper grid and allowed to air dry prior to measure-ment TEMmeasurements were performed at an acceleratingvoltage of 80 kV on a Tecnai G2 spirit (FEI Netherlands)instrument

222 Dynamic Light Scattering (DLS) CeO2NPs were

suspended in Milli-Q water and culture medium (supple-mented with 10 FBS) separately at a final concentration of150 120583gmL and subjected to probe sonication for 10min at 30watt for 2min pulse on and 1min pulse off cycles Size andzeta potential of CeO

2NPs (100 120583gmL) were analyzed using

dynamic light scattering and phase analysis light scatteringtechnique in a Zetasizer Nano-ZS equipped with 40mW633 nm laser (model ZEN 3600 Malvern Instruments LtdMalvern UK)

23 Cell Based Assays Thehuman lung alveolar basal epithe-lial cell line (A549) was obtained from National Centrefor Cell Science (NCCS) Pune India and maintained inDMEMF-12 medium (1 1) supplemented with 10 foetalbovine serum 1 antibiotic-antimycotic solution at 37∘C ina humidified environment of 5 CO

2 Cells were cultured in

96-well 12-well and 6-well culture plates and 75 cm2 cultureflask for different experiments Cells at a confluency of 80were used for all the experiments

Stock suspension (150 120583gmL) of CeO2NPs was prepared

and diluted to varying concentrations (1120583gmLndash100120583gmL)as described above Cells were exposed to these concentra-tions for a specified time schedule (3 h 6 h 24 h and 48 h) asper each experimental design

231 Internalization of CeO2NPs Flow cytometric analysis

was performed for the assessment of CeO2NPs internaliza-

tion in A549 cells according to the method of Suzuki et al

BioMed Research International 3

[25] using light scattering principlesThe analysis is based onthe principle that increase in the intensity of side scattered(SSC) light with constant intensity of forward scattered (FSC)light in cells reveals increased granularity of cells correlatedto cellular uptake of NPs In brief 1 times 105 cellsmLwell wereseeded in 12-well culture plate and allowed to attach thesurface for 22 h Cells were exposed to varying concentra-tions of CeO

2NPs (1 120583gmLndash100120583gmL) for 24 h and 48 h

Treatment was removed and cells were harvested using 025trypsin-EDTA and resuspended in 500120583L of 1x PBS Analysiswas made using flow cytometer equipped with 488 nm laser(FACS Canto II and FACS Diva software (version 612) BDBiosciences San Jose CA USA)

232 Cytotoxicity Assessment Cytotoxicity potential ofCeO2NPs was assessed using propidium iodide (PI) staining

and trypan blue dye exclusion assay for quantification ofdead cells

Propidium Iodide (PI) Staining Assay Live-dead assessmentof A549 cells exposed to CeO

2NPs was carried out by flow

cytometry based propidium iodide (PI) dye uptake assayCells (1 times 105 cellsmLwell) were seeded in 12-well cul-

ture plate and after 22 h exposed to varying concentrations(1 120583gmLndash100 120583gmL) of CeO

2NPs for 24 h and 48 h After

completion treated cells were harvested and resuspendedin 100 120583L PBS and incubated with propidium iodide dye(stock 5mgmL working 2 120583L100 120583L) for 10min at roomtemperature This suspension was diluted by adding 400 120583LPBS and red fluorescence emitted fromPI was collected usingBD FACS Canto II flow cytometer (BD Biosciences San JoseCA USA) coupled with 650 plusmn 13 nm band pass filter Theproportions of live or dead cells were analyzed using FACSDiva software (version 612) (BD Biosciences San Jose CAUSA)

Trypan Blue Dye Exclusion Assay Viability of A549 cellsexposed to CeO

2NPs was determined by trypan blue dye

exclusion assay Briefly 5 times 104 cellsmLwell were seeded andexposed to different concentrations (1120583gmLndash100120583gmL) for24 h and 48 h Exposed cells were harvested washed withPBS and mixed with equal volume (10120583L cells + 10 120583L dye)of 025 trypan blue dye solution for 5min Ten microlitresfrom this solution were used to count the viable cells by usinga Countess automated cell counter (Invitrogen UK) Loss inviability was expressed as percent dead cells

24 Measurement of Oxidative Stress Markers

241 Determination of Intracellular Reactive Oxygen Species(ROS) Generation Generation of intracellular reactive oxy-gen species in A549 cells exposed to CeO

2NPs was mea-

sured using 27-dichlorofluorescein diacetate (DCFDA) dyeaccording to the method of Wang and Joseph [26] Briefly 1times 104 cells100 120583Lwell were seeded in 96-well black bottomplate and incubated for 22 h before the exposure Cells wereexposed to increasing concentrations (1120583gmLndash100 120583gmL)of CeO

2NPs for 3 6 and 24 h in the presence and absence

of N-acetyl-L-cysteine (NAC) at a concentration of 10mM

Following the exposure cells werewashedwith PBS and incu-bated with DCFDA dye (20120583M in PBS) for 30min at 37∘CFurther the dye solution was replaced by 200 120583L of PBSand fluorescence was read at 485 nm excitation and 528 nmemission wavelength in multiwell plate reader (SYNERGY-HT Biotek USA) using KC-4 software The results wereexpressed as ROS generation compared to control Todetect the auto fluorescence and interference of NPs withDCFDA dye a cell free experiment in presence and absenceof CeO

2NPs was also conducted in parallel to the treatment

experiment

242 Measurement of Glutathione (GSH) Level A549 cellstreated with CeO

2NPs were collected and assessed for the

changes in the level of cellular GSH according to the methodof Ellman [27]

Briefly cells were cultured in 75 cm2 flask and exposedto 1 120583gmLndash100120583gmL concentrations of NPs for 3 h and 6 hAfter the treatment cells from control and treated groupswere lysed in cell lysis buffer (20mM Tris-HCl (pH 75)150mM sodium chloride (NaCl) 1mM Na

2EDTA 1 Tri-

ton X-100 and 25mM sodium pyrophosphate) Followingcentrifugation the cell extract was maintained on ice untilassayed for the cellular GSH A mixture of 01mL cell extractand 09mL of 5 tri-chloro acetic acid (TCA) was cen-trifuged (2300timesg for 15min at 4∘C) Further 05mL of thesupernatant was added to 15mL of 001 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) and the reaction was monitoredat 412 nm The amount of GSH was expressed in terms ofumolmg protein

25 Determination of Mitochondrial Membrane Potentialand Apoptosis Determination of mitochondrial membranepotential (MMP)was done using lipophilic cationic 551015840 661015840-tetrachloro-1 110158403101584031015840-tetraethylbenzimidazolecarbocyanineiodide (JC-1) dye This dye has dual fluorescence nature andmitochondrial membrane permeability This dye passivelyenters the mitochondria and forms aggregate which givesthe red fluorescence When the potential of mitochondriacollapse this dye can no longer accumulate in the mitochon-dria and remains in cytoplasm in form of monomer whichgives the green fluorescence

For the MMP analysis 1 times 105 cellsmLwell cultured in12-well plate and exposed to 1 120583gmLndash100120583gmLofCeO

2NPs

for 24 h were harvested and washed with PBS and incubatedwith 10 120583M JC-1 dye in culture medium for 15min at 37∘CCells were again washed with PBS and resuspended in 400 120583Lof PBSThe cells were analyzed for red and green fluorescencein a BD FACS Canto II flow cytometer (BD Biosciences SanJose CA USA) coupled with 485 nm excitation and 590 nmemission filters

Apoptosis analysis in CeO2NPs exposed A549 cells was

carried out using the Annexin V-FITC apoptosis detectionkit (BD Biosciences San Jose CA USA) according to themanufacturerrsquos protocol Briefly cells (1 times 105 cellsmLwell)exposed to different concentrations (1120583gmLndash100120583gmL) ofCeO2NPs in presence and absence of Z-DEVD-fmk (a

Caspase-3 inhibitor) at a concentration of 60 120583M for 24 hwere harvested washed with PBS resuspended in 100 120583L of

4 BioMed Research International

binding buffer containing 5 120583L of annexin V and propidiumiodide (PI) and incubated for 10min at room temperaturein dark After incubation samples were diluted by adding400 120583L of binding buffer and analyzed using BD FACSCanto II flow cytometer equipped with FACS Diva softwareversion 612 (BDBiosciences San Jose CAUSA)The resultswere expressed as Annexin V-FITC+ and PIminus cells identifiedas apoptotic cells Annexin V-FITC+ and PI+ cells as lateapoptotic cells Annexin V-FITCminus and PI+ cells as necroticcells and Annexin V-FITCminus and PIminus as healthy cells

Along this acridine orange staining of A549 cellstreated with different concentrations of CeO

2NPs was also

carried out for the assessment of apoptosis Briefly 1 times105 cellsmLwell were seeded in 12-well plate exposed to1 120583gmLndash100 120583gmL concentrations of NPs for 24 h har-vested and cytocentrifuged at 1000 rpm for 10min Cellswere air dried fixed in 90 chilled methanol for 10min andstained with 10 120583gmL solution of acridine orange dye for5minThe visualization of cells and scoring of the slides weredone at 400x magnification using fluorescent microscope(DMLB Leica Germany) coupled with CCD camera

26 Assessment of DNA Damage Induction by CeO2NPs

The DNA damaging potential of CeO2NPs was determined

by standard alkaline Comet assay and fpg-modified Cometassay

261 Standard Alkaline Comet Assay The induction of DNAdamage by CeO

2NPs was assessed by using alkaline Comet

assay according to the method of Singh et al [28] In brief1 times 105 cellsmLwell in 12-well culture plate was exposedto increasing concentrations (1120583gmLndash100120583gmL) of CeO

2

NPs for 6 h Following the exposure cells were harvestedand resuspended in 100 120583L PBS Comet slides were preparedaccording to the method of Bajpayee et al [29] and keptin lysis solution at 4∘C overnight Duplicate slides for eachconcentration were prepared The slides were subjected toDNAunwinding electrophoresis and neutralization processSlides were stained with 20 120583gmL of ethidium bromidesolution and kept in a humidified slide chamber until scoringThe scoring of the slides was done at 400x magnifica-tion using fluorescent microscope (DMLB Leica Germany)coupled with CCD camera The analysis was done usingimage analysis software (KOMET 50 Kinetic Imaging UK)attached with microscope Images from 50 Comet cells (25cells from each replicate slide) were analyzed and the Cometparameters that is Tail DNA and Olive tail moment weremeasured in cells according to the defined protocol [30]

262 Fpg-Modified Comet Assay Fpg-modified Comet assaywas done according to the method of Collins [31] to identifythe oxidative stress mediated DNA damage involving theinduction of oxidized bases Briefly up to the lysing the pro-cess was performed the same as with standard alkaline Cometassay After lysing slides were washed with enzyme buffer(40mMHEPES 01M KCl 05mM EDTA pH 8 02mgmLbovine serum albumin BSA) for three times and subse-quently incubated with 30 120583L of fpg enzyme (1 3000 dilutionin enzyme buffer) for 30min at 37∘C Further the slides

were subjected to unwinding electrophoresis staining andimaging the same as with standard alkaline Comet assay

27 Cell Cycle Analysis It is well known that presence of cellsin subG1 phase of cell cycle correlates theDNA fragmentationwith apoptosis [32] Effect of CeO

2NPs on cell cycle progres-

sion of A549 cells was assessed using Flow cytometry Briefly2 times 105 cellsmLwell in 6-well culture plates were exposedto different concentrations (1120583gmLndash100 120583gmL) of NPs for24 h After exposure cells were harvested washed with PBSand fixed with 70 ice cold ethanol overnight at minus20∘C Cellswere centrifuged lysed for 30min at 4∘C (using 02 TritonX 100) and treated with 10mgmL RNase for 30min at 37∘CSamples were stained with 1mgmL solution of propidiumiodide dye for 30min at 4∘C and analyzed using BD FACSCanto II flow cytometer equipped with FACS Diva softwareversion 612 (BD Biosciences San Jose CA USA) Resultswere expressed as percentage of cells in each phase of cellcycle

28 Western Blotting For analysis of different proteinsinvolved in CeO

2NPs induced toxicity 2 times 105 cellsmLwell

grown in 6-well culture plate were exposed with NPs concen-trations (1 120583gmLndash100120583gmL) for 24 h After completion ofexposure cells were washed three times with PBS and proteinwas extracted from cells for electrophoresis The amountof protein was estimated using Bradford method [33] andresolved by SDS-PAGE and transferred to polyvinylidenefluoride (PVDF)membrane For the detection of specific pro-teins PVDF membrane was incubated with anti-BAX anti-BCl-2 anti-caspase-3 anti-caspase-9 anti-cytochrome Canti-PARP anti-p53 and anti-120573-actin after blocking in 3BSA solution Detection of protein bands was carried outusing chemiluminiscence and densitometric analysis wasdone using Quantity One Quantitation Software version 431(Bio-Rad USA)

29 Statistical Analysis All the assays were repeated at leastthree times and the data are presented as mean plusmn standarderror In all experiments CeO

2NPs treated samples were

compared with their respective controls and the mean dif-ference was calculated using one way analysis of variance(ANOVA) Along this mean difference between two groupswas assessed using the two-tailed Studentrsquos 119905-test and119875 lt 005was considered as statistically significant in all the experi-ments

3 Results

31 Characterization of Cerium Oxide Nanoparticles TEManalysis was done for the assessment of primary particle sizeand morphology CeO

2NPs were cuboidal in shape and size

observed from TEM was approximately in the range from8 nm to 20 nmalthough some agglomerationwas also present(Figure 1)

CeO2NPs were also characterized using dynamic light

scattering technique in Milli-Q water as well as DMEM F-12 culture medium In Milli-Q water they tend to form

BioMed Research International 5

Table 1 Characterization of CeO2 NPs by dynamic light scattering

S number Medium Hydrodynamic size (dnm) Polydispersity index (PDI) Zeta potential (mV)1 Milli-Q water 5768 0862 minus1192 Culture medium (DMEM F-12 with 10 FBS) 1774 0248 minus137

Figure 1 Characterization of CeO2NPs by TEM TEM analysis

revealed that CeO2NPs were cuboidal in shape with size range from

sim8 nm to 20 nm with some agglomeration

agglomerate with average diameter 5768 plusmn 045 nm InDMEM F-12 medium the average size and zeta potential ofCeO2NPs was shown to be 1774 plusmn 023 nm and minus137 plusmn

025mV respectively with polydispersity index (PDI) 0248(Table 1)

32 Cellular Internalization of Cerium Oxide NanoparticlesThere was a statistically significant (119875 lt 005) concentrationand time dependent internalization of nanoparticles as evi-dent by increase in the SSC intensity of treated cells Resultshowed that 372 838 and 1533 increase in intensityof SSC at 25120583gmL 50 120583gmL and 100 120583gmL respectivelyafter 24 h was found which increased to 432 1034 and1943 after 48 h (Figure 2)

33 Morphological Analysis Morphological analysis of A549cells after NPs exposure exhibited that there was an obviouschange in cell morphology after 24 h exposure (Figure 3)Cells lost their morphology and started to become round inshape at 25120583gmL concentration These changes markedlyincreased with increasing concentrations and at 100 120583gmLmany of the cells detached and formed clumps with irregularshape Cell density was also reduced at higher dose

34 Cytotoxicity Assessment To determine the cytotoxic-ity A549 cells were incubated with varying concentrations(1 120583gmLndash100 120583gmL) of CeO

2NPs for 24 h and 48 h Pro-

pidium iodide (PI) uptake method was utilized to assess theviability in terms of cell death Cell death was increased ina concentration and time dependent manner following theexposure of nanoparticles (Figure 4(a))

0

5

10

15

20

25

Control 1 10 25 50 100

SSC

inte

nsity

()

Concentration of CeO2 NPs (120583gmL)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

24h48h

Figure 2 Internalization of CeO2NPs in A549 cells after 24 h

and 48 h exposure Cells were harvested and analyzed using flowcytometry Increase in SSC intensity which correlates the increasedgranularity of cells was used as a marker for uptake of NPs Valuesrepresent mean plusmn SEM of three independent experiments (lowast119875 lt005 lowastlowast119875 lt 001 compared with control)

There was a statistically significant (119875 lt 001) increasein cell death (725 937 1235 and 1407 in 10 120583gmL25 gmL 50 120583gmL and 100 120583gmL exposed cells resp) after24 h exposure which increased to 962 1169 1534 and1898 respectively after 48 h exposure Similar results werealso obtained by trypan blue assay in which viability of cellswas decreased with increasing concentrations of NPs (Figure4(b))

35 Determination of ROS and GSH Amount CeO2NPs

showed a significant (119875 lt 001) concentration and timedependent increase in production of ROS in terms of increasein DCF fluorescence intensity (Figure 5(a)) DCF fluores-cence intensity increased to 171 200 and 259 after 3 hand 240 266 and 286 after 6 h exposure of 25 120583gmL50 120583gmL and 100 120583gmL respectively as compared to con-trol However ROS generation decreased (115 118 and109 at 25 120583gmL 50 120583gmL and 100 120583gmL) after 24 hexposure This ROS was completely reduced in the presenceof N-acetyl-L-cysteine (NAC) as shown in Figure 5(b)

With the increase in ROS production cellular GSHsignificantly (119875 lt 005) depleted after 3 h and 6 h at 25 120583gmL50 120583gmL and 100 120583gmL exposure as compared to control(Figure 5(c))

36 Mitochondrial Membrane Potential (MMP) Analysis andApoptosis Cells treated with CeO

2NPs showed a significant

(119875 lt 005) concentration dependent decrease in mitochon-drial membrane polarization as evident by JC-1 dye (Figure6(a)) There was 727 1676 and 1851 decrease inMMP at 25 120583gmL 50120583gmL and 100 120583gmL exposurerespectively as compared to control

6 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

Figure 3 Morphological changes in A549 cells exposed to CeO2NPs for 24 h Cells were exposed washed and examined under phase

contrast microscope (a) control cells (b) 1 120583gmL (c) 10120583gmL (d) 25 120583gmL (e) 50 120583gmL and (f) 100 120583gmL concentration exposed cells(magnification times20)

Both the decrease in MMP and accumulation of ROSare hallmarks of mitochondria mediated apoptosis [34] Asevident in Figure 6(b) the amount of apoptotic cells alsoincreased in concentration dependent manner There wasincrease in apoptotic cells from 279 (control) to 679(25 120583gmL) 737 (50 120583gmL) and 892 (100 120583gmL) How-ever in the presence of Z-DEVD-fmk this death was com-pletely attenuated which clearly indicated that the death wascaspase dependent

37 Acridine Orange Staining Determination of apoptosisinduction in A549 cells by CeO

2NPs was also assessed by

acridine orange staining Cells with chromatin condensation

nuclear fragmentation and apoptotic bodies were found inthe cells exposed to CeO

2NPs as compared to control (Figure

7)

38 DNA Damage by CeO2NPs Cells exposed to 25 120583gmL

50 120583gmL and 100 120583gmL concentration of CeO2NPs for 6 h

exhibited a significant (119875 lt 005) induction in DNA damagecompared to control cells as evident by Comet parametersOlive tail moment and Tail DNA in standard alkalineComet assay (Table 2) Moreover the values for Tail DNAand Olive tail moment in fpg-modified Comet assay weresignificantly (119875 lt 005) higher than the standard alkalineComet assayThis result suggests the involvement of oxidativestress towards the DNA damage

BioMed Research International 7

0

5

10

15

20

25

30

35D

ead

cells

()

lowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

5

10

15

20

25

30

35

Dea

d ce

lls (

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

24h48h

lowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 4 Cytotoxicity assessment of CeO2NPs in A549 cells by (a) propidium iodide (PI) uptake method and (b) trypan blue dye exclusion

assay after 24 h and 48 h exposure Cells were stainedwith PI and subjected to flow cytometer whereas in trypan blue assay cells were subjectedto automatic cell counter The results were expressed as mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001 comparedwith control)

0

50

100

150

200

250

300

350

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

24h3h6h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

25

50

75

100

125

150

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

Control 1 10 25 50 100

3h6h

Concentration of CeO2 NPs (120583gmL)+ NAC (10mM)

(b)

000005010015020025030035040045

Glu

tath

ione

cont

ent

lowastlowast

lowastlowastlowastlowast

lowastlowast

3h6h

(120583m

olm

g of

pro

tein

)

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(c)

Figure 5 Effect of CeO2NPs on (a b) ROS production and (c) cellular level of glutathione (GSH) in A549 cells (a b) Cells were exposed

and incubated with H2DCFDA (c) After exposure cells were collected lysed and incubated with reaction mixture (TCA DTNB) Both

measurements were done usingmicroplate reader and data represents mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001compared to control)

8 BioMed Research International

Table 2 DNA damage induction by CeO2 NPs as evident by alkaline and fpg modified Comet assay in A549 cells after 6 h exposure

Groups OTM (arbitrary unit) Tail DNA ()Fpg (minus) Fpg (+) Fpg (minus) Fpg (+)

Control 089 plusmn 006 113 plusmn 009 632 plusmn 023 696 plusmn 012

CeO2 NPs (1 120583gmL) 119 plusmn 012lowast

168 plusmn 014lowast

776 plusmn 038 1081 plusmn 028lowastlowast

CeO2 NPs (10120583gmL) 155 plusmn 007lowastlowast

194 plusmn 015lowastlowast

901 plusmn 022lowast

1182 plusmn 024lowastlowast

CeO2 NPs (25120583gmL) 173 plusmn 008lowastlowast

228 plusmn 015lowastlowast

1030 plusmn 067lowastlowast

1345 plusmn 018lowastlowast

CeO2 NPs (50120583gmL) 189 plusmn 007lowastlowast

270 plusmn 014lowastlowast

1315 plusmn 061lowastlowast

1563 plusmn 026lowastlowast

CeO2 NPs (100120583gmL) 213 plusmn 007lowastlowast

376 plusmn 016lowastlowast

1639 plusmn 065lowastlowast

1804 plusmn 027lowastlowast

Positive control-H2O2 (25 120583M) 439 plusmn 011lowastlowast

633 plusmn 014lowastlowast

2367 plusmn 158lowastlowast

2928 plusmn 036lowastlowast

Comet slides were prepared according to defined protocol and 50 Comet cells were scored using fluorescence microscope Results from three independentexperiments in form of mean plusmn SEM were reported (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control 119875 lt 005 when compared with respective valuesin standard Comet assay)

02468

101214161820

MM

P lo

ss (

)

lowastlowastlowastlowast

lowastlowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

02468

1012141618

Apoptotic cells (absence of Z-DEVD-fmk)Apoptotic cells (presence of Z-DEVd-fmk)

Apop

totic

cells

()

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 6 Mitochondrial membrane potential alteration and apoptosis induction by CeO2NPs in A549 cells after 24 h exposure Cells were

exposed to 1 120583gmLndash100 120583gmL concentration of NPs harvested and stainedwith JC-1 dye forMMP (Figure 6(a)) and annexinV-FITCPI forapoptosis (Figure 6(b)) Cells were subjected to flow cytometry and values from three independent experiments (mean plusmn SEM) were reported(lowast119875 lt 005 lowastlowast119875 lt 001 compared to control)

39 Cell Cycle Analysis The effect of NPs on A549 Cell cycleprogression was assessed by flow cytometry In response toDNA damage relevant checkpoints can arrest the cell cycle ata certain stage Data showed that there was a significant (119875 lt005) dose dependent increase in cells present in subG1 phaseof cell cycle in comparison to control while the percentageof cells in G2 phase of cell cycle declined progressivelyThe amount of cells in Sub G1 phase increased from 07(control) to 295 345 and 51 at 25 120583gmL 50120583gmLand 100 120583gmL concentration respectively (Figure 8)

310 Western Blot Analysis Western blot analysis data exhib-ited that there was a significant (119875 lt 005) increase in expres-sion of proapoptotic protein Bax (41 fold 45 fold 51 fold)tumor suppressor protein p53 (18-fold 24-fold and 27-fold) and PARP (25-fold 31-fold and 33-fold) withdecreased expression level of Bcl-2 (066-fold 049-fold and023-fold) as compared to control at 25 120583gmL 50120583gmLand 100 120583gmL concentrations respectively Furthermoresignificant (119875 lt 005) increase in expression level of cyto-chrome C (33-fold 36-fold and 41-fold) Apaf-1 (20-fold26-fold and 29-fold) caspase-3 (22-fold 27-fold 29-fold)

and caspase-9 (14-fold 19-fold and 2-fold) were also foundwhich confirms the mitochondrial mediated apoptotic celldeath induction by CeO

2NPs in A549 cell (Figure 9)

4 Discussion

Due to the conflicting information of cerium oxide nanopar-ticles effects over human and environment the present studywas carried out to precisely define their final fate in a cellMoreover the major regulator behind the CeO

2NPs induced

cell death if any was also explored Our study systematicallyconcludes that CeO

2NPs get internalized in cells in a sig-

nificant manner and lead to mitochondria mediated cas-pase dependent cell death We also showed that CeO

2NPs

produced increased amount of ROS which contributed toextensive DNA damage and perturbation in cell cycle withincreasing apoptotic cells in subG1 phase Further in thepresence of specific inhibitor for ROS and apoptosis bothprocesses were completely attenuated proving that the causeof CeO

2toxicity is ROS mediated DNA damage leading to

apoptosis

BioMed Research International 9

(a) (b)

(c) (d)

(e) (f)Figure 7 Determination of apoptotic cells by acridine orange staining of A549 cells exposed to CeO

2NPs for 24 h exposure Cells were

exposed washed and stained with acridine orange dye (a) Control cells (b c) treated cells showing nuclear fragmentation (d e) chromatincondensation and (f) apoptotic bodies

0

10

20

30

40

50

60

SubG1 phaseG1 phase

S phaseG2 phase

Cel

ls (

)

lowastlowast lowastlowast lowastlowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

Figure 8 Cell cycle analysis of A549 cells exposed to CeO2NPs for

24 h exposure Cells were exposed fixed and stained with propid-ium iodide dye before subjecting to flow cytometric analysis Valuesfrom three independent experiments (mean plusmn SEM) were reportedwhere lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control

The characterization of NPs is essential for the toxicitystudies as it has been shown that the size shape surface reac-tivity solubility and degree of aggregation impart for theirdifferential response in biological system [35]Hence the par-ticle morphology average size and agglomeration status ofCeO2were examined by TEMWe observed that the particles

were in the range of 8 nm to 20 nm in size and cuboidal inshape although there was some agglomeration also presentwhich range in the size of sim40 nm Also to mimic the realexposure scenario the CeO

2NPs were characterized in the

Milli-Q water as well as in culture medium by DLS Weobserved that the average hydrodynamic size of the particle inMilli-Qwaterwas 5768 nmplusmn 045 nmand in culturemedium1774 nm plusmn 023 nm The difference in the average hydrody-namic size of NPs observed in different solvent could beattributed to their aggregation state In the culturemedia NPswere more stable andmonodispersed due to the formation ofprotein nanoparticles corona (Table 1)

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

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AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

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Medicinal ChemistryInternational Journal of

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AddictionJournal of

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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

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ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Pharmaceutics

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MEDIATORSINFLAMMATION

of

2 BioMed Research International

inhalation and ingestion as major routes Since CeO2NPs

are poorly absorbed by the intestine inhalation appears tobe the major route of exposure It should also be noted thatcomplete respiratory system acts as repository for depositionof different sizes of NPs Although several studies havebeen performed to evaluate the adverse effect of CeO

2NPs

on environment and human health they are not providingproper conclusionCeO

2NPshave been reported to act as cel-

lular antioxidants with colocalizations inside mitochondrialysosomes endoplasmic reticulum nucleus and cytoplasmusing keratinocytemodel systems [14 15] However the studylacked genotoxicity assessment of these particles Contrary tothis previous studies have reported that CeO

2NPs generate

oxidative stress and may induce apoptosis in human lungepithelial cells [16 17] Therefore CeO

2NPs may portend

cytotoxic and genotoxic effects upon cellular internalizationIt has been reported that the CeO

2NPs of various sizes

showed significant toxic effects on E coli and human cellsrespectively due to adsorption of NPs and oxidative stress[18 19] Along this reports have also been postulated thatCeO2NPs of smaller sizes do not cause any adverse effect

but can protect cells from harmful effects of radiation andoxidative stress although this protectionwas cell type specific[20 21] In vivo studies have resulted that CeO

2NPs exposure

via inhalation and intratracheal instillation route can induceacute pulmonary and systemic toxicity in rat and mice dueto proinflammatory responses [22 23] Besides these CeO

2

NPs have been shown to protect cells from reactive oxygenspecies due to their inherent antioxidant properties [15 24]this protective effect was thought to be due to the presenceof dual oxidation state of CeO

2NPs or the pH value of cell

compartment where the nanoparticles internalizeWith thesecontrasting results the toxicity of CeO

2NPs remains elusive

and specific toxicity endpoints relevant to human healthneeds to be addressed It was therefore prudent to conduct asystematic study to understand the comprehensivemolecularmechanism of toxicity of CeO

2NPs as well as to see the role

of DNA damage and halt of cell cycle in cell deathCharacterization ofNPs is an essential step before toxicity

assessment as properties of NPs vary significantly with shapeand size It is also essential to characterize their agglomera-tionaggregation tendency in the culture media for accuratetoxicity assessment In vitro cell based assays are rapid andallow reliable toxicity fingerprints for NPs In the presentstudy an attempt was made to (1) characterize CeO

2NPs to

assess their behavior in culture medium (2) evaluate cyto-toxic and oxidative stress potential (3) estimate their DNAdamaging potential and subsequent cell cycle arrest and (4)evaluate apoptotic index

2 Materials and Methods

21 Chemicals Cerium (IV) oxide nanopowder (purity9995 lt25 nm particle size BET) propidium iodide 27-dichlorofluorescein diacetate (DCFDA) dye 551015840 661015840-tetra-chloro-1110158403101584031015840-tetraethylbenzimidazolecarbocyanine iodide(JC-1) dye low melting point agarose (LMA) ethidiumbromide (EtBr) Triton X-100 and ethyl methanesulfonate(EMS) were purchased from Sigma Chemical Co Ltd (StLouis MO USA) Normal melting agarose (NMA) and

ethylenediaminetetraacetic acid (EDTA) disodium salt werepurchased from Himedia Pvt Ltd (Mumbai India) For-mamidopyrimidine DNA glycosylase (Fpg) enzyme wasobtained from Trevigen Inc (USA) Hydrogen peroxide(H2O2) was purchased from Ranbaxy Fine Chemical Ltd

(New Delhi India) Phosphate buffered saline (Ca+2 Mg+2free PBS) Dulbeccorsquos modified eagle medium nutrient mix-ture F-12 (Ham) (1 1) powder (DMEM F-12) trypsin-EDTAfoetal bovine serum (FBS) trypan blue and antibiotic andantimycotic solution (10000UmL penicillin 10mgmLstreptomycin 25120583gmL amphotericin-B) were purchasedfrom Life Technologies Pvt Ltd (New Delhi India) Allother chemicals were obtained locally and were of analyticalreagent grade Cell culture plastic wares were obtained fromThermo Scientific Nunc (Rochester New York)

22 Characterization of CeO2NPs CeO

2NPs were charac-

terized using transmission electron microscopy (TEM) anddynamic light scattering (DLS) technique

221 Transmission Electron Microscopy (TEM) TEM anal-ysis was carried out for the assessment of primary particlesize and morphology of CeO

2NPs Samples were prepared

by suspending NPs in Milli-Q water at a concentration of50 120583gmL A drop of suspension was added to the formvar-coated copper grid and allowed to air dry prior to measure-ment TEMmeasurements were performed at an acceleratingvoltage of 80 kV on a Tecnai G2 spirit (FEI Netherlands)instrument

222 Dynamic Light Scattering (DLS) CeO2NPs were

suspended in Milli-Q water and culture medium (supple-mented with 10 FBS) separately at a final concentration of150 120583gmL and subjected to probe sonication for 10min at 30watt for 2min pulse on and 1min pulse off cycles Size andzeta potential of CeO

2NPs (100 120583gmL) were analyzed using

dynamic light scattering and phase analysis light scatteringtechnique in a Zetasizer Nano-ZS equipped with 40mW633 nm laser (model ZEN 3600 Malvern Instruments LtdMalvern UK)

23 Cell Based Assays Thehuman lung alveolar basal epithe-lial cell line (A549) was obtained from National Centrefor Cell Science (NCCS) Pune India and maintained inDMEMF-12 medium (1 1) supplemented with 10 foetalbovine serum 1 antibiotic-antimycotic solution at 37∘C ina humidified environment of 5 CO

2 Cells were cultured in

96-well 12-well and 6-well culture plates and 75 cm2 cultureflask for different experiments Cells at a confluency of 80were used for all the experiments

Stock suspension (150 120583gmL) of CeO2NPs was prepared

and diluted to varying concentrations (1120583gmLndash100120583gmL)as described above Cells were exposed to these concentra-tions for a specified time schedule (3 h 6 h 24 h and 48 h) asper each experimental design

231 Internalization of CeO2NPs Flow cytometric analysis

was performed for the assessment of CeO2NPs internaliza-

tion in A549 cells according to the method of Suzuki et al

BioMed Research International 3

[25] using light scattering principlesThe analysis is based onthe principle that increase in the intensity of side scattered(SSC) light with constant intensity of forward scattered (FSC)light in cells reveals increased granularity of cells correlatedto cellular uptake of NPs In brief 1 times 105 cellsmLwell wereseeded in 12-well culture plate and allowed to attach thesurface for 22 h Cells were exposed to varying concentra-tions of CeO

2NPs (1 120583gmLndash100120583gmL) for 24 h and 48 h

Treatment was removed and cells were harvested using 025trypsin-EDTA and resuspended in 500120583L of 1x PBS Analysiswas made using flow cytometer equipped with 488 nm laser(FACS Canto II and FACS Diva software (version 612) BDBiosciences San Jose CA USA)

232 Cytotoxicity Assessment Cytotoxicity potential ofCeO2NPs was assessed using propidium iodide (PI) staining

and trypan blue dye exclusion assay for quantification ofdead cells

Propidium Iodide (PI) Staining Assay Live-dead assessmentof A549 cells exposed to CeO

2NPs was carried out by flow

cytometry based propidium iodide (PI) dye uptake assayCells (1 times 105 cellsmLwell) were seeded in 12-well cul-

ture plate and after 22 h exposed to varying concentrations(1 120583gmLndash100 120583gmL) of CeO

2NPs for 24 h and 48 h After

completion treated cells were harvested and resuspendedin 100 120583L PBS and incubated with propidium iodide dye(stock 5mgmL working 2 120583L100 120583L) for 10min at roomtemperature This suspension was diluted by adding 400 120583LPBS and red fluorescence emitted fromPI was collected usingBD FACS Canto II flow cytometer (BD Biosciences San JoseCA USA) coupled with 650 plusmn 13 nm band pass filter Theproportions of live or dead cells were analyzed using FACSDiva software (version 612) (BD Biosciences San Jose CAUSA)

Trypan Blue Dye Exclusion Assay Viability of A549 cellsexposed to CeO

2NPs was determined by trypan blue dye

exclusion assay Briefly 5 times 104 cellsmLwell were seeded andexposed to different concentrations (1120583gmLndash100120583gmL) for24 h and 48 h Exposed cells were harvested washed withPBS and mixed with equal volume (10120583L cells + 10 120583L dye)of 025 trypan blue dye solution for 5min Ten microlitresfrom this solution were used to count the viable cells by usinga Countess automated cell counter (Invitrogen UK) Loss inviability was expressed as percent dead cells

24 Measurement of Oxidative Stress Markers

241 Determination of Intracellular Reactive Oxygen Species(ROS) Generation Generation of intracellular reactive oxy-gen species in A549 cells exposed to CeO

2NPs was mea-

sured using 27-dichlorofluorescein diacetate (DCFDA) dyeaccording to the method of Wang and Joseph [26] Briefly 1times 104 cells100 120583Lwell were seeded in 96-well black bottomplate and incubated for 22 h before the exposure Cells wereexposed to increasing concentrations (1120583gmLndash100 120583gmL)of CeO

2NPs for 3 6 and 24 h in the presence and absence

of N-acetyl-L-cysteine (NAC) at a concentration of 10mM

Following the exposure cells werewashedwith PBS and incu-bated with DCFDA dye (20120583M in PBS) for 30min at 37∘CFurther the dye solution was replaced by 200 120583L of PBSand fluorescence was read at 485 nm excitation and 528 nmemission wavelength in multiwell plate reader (SYNERGY-HT Biotek USA) using KC-4 software The results wereexpressed as ROS generation compared to control Todetect the auto fluorescence and interference of NPs withDCFDA dye a cell free experiment in presence and absenceof CeO

2NPs was also conducted in parallel to the treatment

experiment

242 Measurement of Glutathione (GSH) Level A549 cellstreated with CeO

2NPs were collected and assessed for the

changes in the level of cellular GSH according to the methodof Ellman [27]

Briefly cells were cultured in 75 cm2 flask and exposedto 1 120583gmLndash100120583gmL concentrations of NPs for 3 h and 6 hAfter the treatment cells from control and treated groupswere lysed in cell lysis buffer (20mM Tris-HCl (pH 75)150mM sodium chloride (NaCl) 1mM Na

2EDTA 1 Tri-

ton X-100 and 25mM sodium pyrophosphate) Followingcentrifugation the cell extract was maintained on ice untilassayed for the cellular GSH A mixture of 01mL cell extractand 09mL of 5 tri-chloro acetic acid (TCA) was cen-trifuged (2300timesg for 15min at 4∘C) Further 05mL of thesupernatant was added to 15mL of 001 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) and the reaction was monitoredat 412 nm The amount of GSH was expressed in terms ofumolmg protein

25 Determination of Mitochondrial Membrane Potentialand Apoptosis Determination of mitochondrial membranepotential (MMP)was done using lipophilic cationic 551015840 661015840-tetrachloro-1 110158403101584031015840-tetraethylbenzimidazolecarbocyanineiodide (JC-1) dye This dye has dual fluorescence nature andmitochondrial membrane permeability This dye passivelyenters the mitochondria and forms aggregate which givesthe red fluorescence When the potential of mitochondriacollapse this dye can no longer accumulate in the mitochon-dria and remains in cytoplasm in form of monomer whichgives the green fluorescence

For the MMP analysis 1 times 105 cellsmLwell cultured in12-well plate and exposed to 1 120583gmLndash100120583gmLofCeO

2NPs

for 24 h were harvested and washed with PBS and incubatedwith 10 120583M JC-1 dye in culture medium for 15min at 37∘CCells were again washed with PBS and resuspended in 400 120583Lof PBSThe cells were analyzed for red and green fluorescencein a BD FACS Canto II flow cytometer (BD Biosciences SanJose CA USA) coupled with 485 nm excitation and 590 nmemission filters

Apoptosis analysis in CeO2NPs exposed A549 cells was

carried out using the Annexin V-FITC apoptosis detectionkit (BD Biosciences San Jose CA USA) according to themanufacturerrsquos protocol Briefly cells (1 times 105 cellsmLwell)exposed to different concentrations (1120583gmLndash100120583gmL) ofCeO2NPs in presence and absence of Z-DEVD-fmk (a

Caspase-3 inhibitor) at a concentration of 60 120583M for 24 hwere harvested washed with PBS resuspended in 100 120583L of

4 BioMed Research International

binding buffer containing 5 120583L of annexin V and propidiumiodide (PI) and incubated for 10min at room temperaturein dark After incubation samples were diluted by adding400 120583L of binding buffer and analyzed using BD FACSCanto II flow cytometer equipped with FACS Diva softwareversion 612 (BDBiosciences San Jose CAUSA)The resultswere expressed as Annexin V-FITC+ and PIminus cells identifiedas apoptotic cells Annexin V-FITC+ and PI+ cells as lateapoptotic cells Annexin V-FITCminus and PI+ cells as necroticcells and Annexin V-FITCminus and PIminus as healthy cells

Along this acridine orange staining of A549 cellstreated with different concentrations of CeO

2NPs was also

carried out for the assessment of apoptosis Briefly 1 times105 cellsmLwell were seeded in 12-well plate exposed to1 120583gmLndash100 120583gmL concentrations of NPs for 24 h har-vested and cytocentrifuged at 1000 rpm for 10min Cellswere air dried fixed in 90 chilled methanol for 10min andstained with 10 120583gmL solution of acridine orange dye for5minThe visualization of cells and scoring of the slides weredone at 400x magnification using fluorescent microscope(DMLB Leica Germany) coupled with CCD camera

26 Assessment of DNA Damage Induction by CeO2NPs

The DNA damaging potential of CeO2NPs was determined

by standard alkaline Comet assay and fpg-modified Cometassay

261 Standard Alkaline Comet Assay The induction of DNAdamage by CeO

2NPs was assessed by using alkaline Comet

assay according to the method of Singh et al [28] In brief1 times 105 cellsmLwell in 12-well culture plate was exposedto increasing concentrations (1120583gmLndash100120583gmL) of CeO

2

NPs for 6 h Following the exposure cells were harvestedand resuspended in 100 120583L PBS Comet slides were preparedaccording to the method of Bajpayee et al [29] and keptin lysis solution at 4∘C overnight Duplicate slides for eachconcentration were prepared The slides were subjected toDNAunwinding electrophoresis and neutralization processSlides were stained with 20 120583gmL of ethidium bromidesolution and kept in a humidified slide chamber until scoringThe scoring of the slides was done at 400x magnifica-tion using fluorescent microscope (DMLB Leica Germany)coupled with CCD camera The analysis was done usingimage analysis software (KOMET 50 Kinetic Imaging UK)attached with microscope Images from 50 Comet cells (25cells from each replicate slide) were analyzed and the Cometparameters that is Tail DNA and Olive tail moment weremeasured in cells according to the defined protocol [30]

262 Fpg-Modified Comet Assay Fpg-modified Comet assaywas done according to the method of Collins [31] to identifythe oxidative stress mediated DNA damage involving theinduction of oxidized bases Briefly up to the lysing the pro-cess was performed the same as with standard alkaline Cometassay After lysing slides were washed with enzyme buffer(40mMHEPES 01M KCl 05mM EDTA pH 8 02mgmLbovine serum albumin BSA) for three times and subse-quently incubated with 30 120583L of fpg enzyme (1 3000 dilutionin enzyme buffer) for 30min at 37∘C Further the slides

were subjected to unwinding electrophoresis staining andimaging the same as with standard alkaline Comet assay

27 Cell Cycle Analysis It is well known that presence of cellsin subG1 phase of cell cycle correlates theDNA fragmentationwith apoptosis [32] Effect of CeO

2NPs on cell cycle progres-

sion of A549 cells was assessed using Flow cytometry Briefly2 times 105 cellsmLwell in 6-well culture plates were exposedto different concentrations (1120583gmLndash100 120583gmL) of NPs for24 h After exposure cells were harvested washed with PBSand fixed with 70 ice cold ethanol overnight at minus20∘C Cellswere centrifuged lysed for 30min at 4∘C (using 02 TritonX 100) and treated with 10mgmL RNase for 30min at 37∘CSamples were stained with 1mgmL solution of propidiumiodide dye for 30min at 4∘C and analyzed using BD FACSCanto II flow cytometer equipped with FACS Diva softwareversion 612 (BD Biosciences San Jose CA USA) Resultswere expressed as percentage of cells in each phase of cellcycle

28 Western Blotting For analysis of different proteinsinvolved in CeO

2NPs induced toxicity 2 times 105 cellsmLwell

grown in 6-well culture plate were exposed with NPs concen-trations (1 120583gmLndash100120583gmL) for 24 h After completion ofexposure cells were washed three times with PBS and proteinwas extracted from cells for electrophoresis The amountof protein was estimated using Bradford method [33] andresolved by SDS-PAGE and transferred to polyvinylidenefluoride (PVDF)membrane For the detection of specific pro-teins PVDF membrane was incubated with anti-BAX anti-BCl-2 anti-caspase-3 anti-caspase-9 anti-cytochrome Canti-PARP anti-p53 and anti-120573-actin after blocking in 3BSA solution Detection of protein bands was carried outusing chemiluminiscence and densitometric analysis wasdone using Quantity One Quantitation Software version 431(Bio-Rad USA)

29 Statistical Analysis All the assays were repeated at leastthree times and the data are presented as mean plusmn standarderror In all experiments CeO

2NPs treated samples were

compared with their respective controls and the mean dif-ference was calculated using one way analysis of variance(ANOVA) Along this mean difference between two groupswas assessed using the two-tailed Studentrsquos 119905-test and119875 lt 005was considered as statistically significant in all the experi-ments

3 Results

31 Characterization of Cerium Oxide Nanoparticles TEManalysis was done for the assessment of primary particle sizeand morphology CeO

2NPs were cuboidal in shape and size

observed from TEM was approximately in the range from8 nm to 20 nmalthough some agglomerationwas also present(Figure 1)

CeO2NPs were also characterized using dynamic light

scattering technique in Milli-Q water as well as DMEM F-12 culture medium In Milli-Q water they tend to form

BioMed Research International 5

Table 1 Characterization of CeO2 NPs by dynamic light scattering

S number Medium Hydrodynamic size (dnm) Polydispersity index (PDI) Zeta potential (mV)1 Milli-Q water 5768 0862 minus1192 Culture medium (DMEM F-12 with 10 FBS) 1774 0248 minus137

Figure 1 Characterization of CeO2NPs by TEM TEM analysis

revealed that CeO2NPs were cuboidal in shape with size range from

sim8 nm to 20 nm with some agglomeration

agglomerate with average diameter 5768 plusmn 045 nm InDMEM F-12 medium the average size and zeta potential ofCeO2NPs was shown to be 1774 plusmn 023 nm and minus137 plusmn

025mV respectively with polydispersity index (PDI) 0248(Table 1)

32 Cellular Internalization of Cerium Oxide NanoparticlesThere was a statistically significant (119875 lt 005) concentrationand time dependent internalization of nanoparticles as evi-dent by increase in the SSC intensity of treated cells Resultshowed that 372 838 and 1533 increase in intensityof SSC at 25120583gmL 50 120583gmL and 100 120583gmL respectivelyafter 24 h was found which increased to 432 1034 and1943 after 48 h (Figure 2)

33 Morphological Analysis Morphological analysis of A549cells after NPs exposure exhibited that there was an obviouschange in cell morphology after 24 h exposure (Figure 3)Cells lost their morphology and started to become round inshape at 25120583gmL concentration These changes markedlyincreased with increasing concentrations and at 100 120583gmLmany of the cells detached and formed clumps with irregularshape Cell density was also reduced at higher dose

34 Cytotoxicity Assessment To determine the cytotoxic-ity A549 cells were incubated with varying concentrations(1 120583gmLndash100 120583gmL) of CeO

2NPs for 24 h and 48 h Pro-

pidium iodide (PI) uptake method was utilized to assess theviability in terms of cell death Cell death was increased ina concentration and time dependent manner following theexposure of nanoparticles (Figure 4(a))

0

5

10

15

20

25

Control 1 10 25 50 100

SSC

inte

nsity

()

Concentration of CeO2 NPs (120583gmL)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

24h48h

Figure 2 Internalization of CeO2NPs in A549 cells after 24 h

and 48 h exposure Cells were harvested and analyzed using flowcytometry Increase in SSC intensity which correlates the increasedgranularity of cells was used as a marker for uptake of NPs Valuesrepresent mean plusmn SEM of three independent experiments (lowast119875 lt005 lowastlowast119875 lt 001 compared with control)

There was a statistically significant (119875 lt 001) increasein cell death (725 937 1235 and 1407 in 10 120583gmL25 gmL 50 120583gmL and 100 120583gmL exposed cells resp) after24 h exposure which increased to 962 1169 1534 and1898 respectively after 48 h exposure Similar results werealso obtained by trypan blue assay in which viability of cellswas decreased with increasing concentrations of NPs (Figure4(b))

35 Determination of ROS and GSH Amount CeO2NPs

showed a significant (119875 lt 001) concentration and timedependent increase in production of ROS in terms of increasein DCF fluorescence intensity (Figure 5(a)) DCF fluores-cence intensity increased to 171 200 and 259 after 3 hand 240 266 and 286 after 6 h exposure of 25 120583gmL50 120583gmL and 100 120583gmL respectively as compared to con-trol However ROS generation decreased (115 118 and109 at 25 120583gmL 50 120583gmL and 100 120583gmL) after 24 hexposure This ROS was completely reduced in the presenceof N-acetyl-L-cysteine (NAC) as shown in Figure 5(b)

With the increase in ROS production cellular GSHsignificantly (119875 lt 005) depleted after 3 h and 6 h at 25 120583gmL50 120583gmL and 100 120583gmL exposure as compared to control(Figure 5(c))

36 Mitochondrial Membrane Potential (MMP) Analysis andApoptosis Cells treated with CeO

2NPs showed a significant

(119875 lt 005) concentration dependent decrease in mitochon-drial membrane polarization as evident by JC-1 dye (Figure6(a)) There was 727 1676 and 1851 decrease inMMP at 25 120583gmL 50120583gmL and 100 120583gmL exposurerespectively as compared to control

6 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

Figure 3 Morphological changes in A549 cells exposed to CeO2NPs for 24 h Cells were exposed washed and examined under phase

contrast microscope (a) control cells (b) 1 120583gmL (c) 10120583gmL (d) 25 120583gmL (e) 50 120583gmL and (f) 100 120583gmL concentration exposed cells(magnification times20)

Both the decrease in MMP and accumulation of ROSare hallmarks of mitochondria mediated apoptosis [34] Asevident in Figure 6(b) the amount of apoptotic cells alsoincreased in concentration dependent manner There wasincrease in apoptotic cells from 279 (control) to 679(25 120583gmL) 737 (50 120583gmL) and 892 (100 120583gmL) How-ever in the presence of Z-DEVD-fmk this death was com-pletely attenuated which clearly indicated that the death wascaspase dependent

37 Acridine Orange Staining Determination of apoptosisinduction in A549 cells by CeO

2NPs was also assessed by

acridine orange staining Cells with chromatin condensation

nuclear fragmentation and apoptotic bodies were found inthe cells exposed to CeO

2NPs as compared to control (Figure

7)

38 DNA Damage by CeO2NPs Cells exposed to 25 120583gmL

50 120583gmL and 100 120583gmL concentration of CeO2NPs for 6 h

exhibited a significant (119875 lt 005) induction in DNA damagecompared to control cells as evident by Comet parametersOlive tail moment and Tail DNA in standard alkalineComet assay (Table 2) Moreover the values for Tail DNAand Olive tail moment in fpg-modified Comet assay weresignificantly (119875 lt 005) higher than the standard alkalineComet assayThis result suggests the involvement of oxidativestress towards the DNA damage

BioMed Research International 7

0

5

10

15

20

25

30

35D

ead

cells

()

lowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

5

10

15

20

25

30

35

Dea

d ce

lls (

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

24h48h

lowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 4 Cytotoxicity assessment of CeO2NPs in A549 cells by (a) propidium iodide (PI) uptake method and (b) trypan blue dye exclusion

assay after 24 h and 48 h exposure Cells were stainedwith PI and subjected to flow cytometer whereas in trypan blue assay cells were subjectedto automatic cell counter The results were expressed as mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001 comparedwith control)

0

50

100

150

200

250

300

350

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

24h3h6h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

25

50

75

100

125

150

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

Control 1 10 25 50 100

3h6h

Concentration of CeO2 NPs (120583gmL)+ NAC (10mM)

(b)

000005010015020025030035040045

Glu

tath

ione

cont

ent

lowastlowast

lowastlowastlowastlowast

lowastlowast

3h6h

(120583m

olm

g of

pro

tein

)

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(c)

Figure 5 Effect of CeO2NPs on (a b) ROS production and (c) cellular level of glutathione (GSH) in A549 cells (a b) Cells were exposed

and incubated with H2DCFDA (c) After exposure cells were collected lysed and incubated with reaction mixture (TCA DTNB) Both

measurements were done usingmicroplate reader and data represents mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001compared to control)

8 BioMed Research International

Table 2 DNA damage induction by CeO2 NPs as evident by alkaline and fpg modified Comet assay in A549 cells after 6 h exposure

Groups OTM (arbitrary unit) Tail DNA ()Fpg (minus) Fpg (+) Fpg (minus) Fpg (+)

Control 089 plusmn 006 113 plusmn 009 632 plusmn 023 696 plusmn 012

CeO2 NPs (1 120583gmL) 119 plusmn 012lowast

168 plusmn 014lowast

776 plusmn 038 1081 plusmn 028lowastlowast

CeO2 NPs (10120583gmL) 155 plusmn 007lowastlowast

194 plusmn 015lowastlowast

901 plusmn 022lowast

1182 plusmn 024lowastlowast

CeO2 NPs (25120583gmL) 173 plusmn 008lowastlowast

228 plusmn 015lowastlowast

1030 plusmn 067lowastlowast

1345 plusmn 018lowastlowast

CeO2 NPs (50120583gmL) 189 plusmn 007lowastlowast

270 plusmn 014lowastlowast

1315 plusmn 061lowastlowast

1563 plusmn 026lowastlowast

CeO2 NPs (100120583gmL) 213 plusmn 007lowastlowast

376 plusmn 016lowastlowast

1639 plusmn 065lowastlowast

1804 plusmn 027lowastlowast

Positive control-H2O2 (25 120583M) 439 plusmn 011lowastlowast

633 plusmn 014lowastlowast

2367 plusmn 158lowastlowast

2928 plusmn 036lowastlowast

Comet slides were prepared according to defined protocol and 50 Comet cells were scored using fluorescence microscope Results from three independentexperiments in form of mean plusmn SEM were reported (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control 119875 lt 005 when compared with respective valuesin standard Comet assay)

02468

101214161820

MM

P lo

ss (

)

lowastlowastlowastlowast

lowastlowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

02468

1012141618

Apoptotic cells (absence of Z-DEVD-fmk)Apoptotic cells (presence of Z-DEVd-fmk)

Apop

totic

cells

()

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 6 Mitochondrial membrane potential alteration and apoptosis induction by CeO2NPs in A549 cells after 24 h exposure Cells were

exposed to 1 120583gmLndash100 120583gmL concentration of NPs harvested and stainedwith JC-1 dye forMMP (Figure 6(a)) and annexinV-FITCPI forapoptosis (Figure 6(b)) Cells were subjected to flow cytometry and values from three independent experiments (mean plusmn SEM) were reported(lowast119875 lt 005 lowastlowast119875 lt 001 compared to control)

39 Cell Cycle Analysis The effect of NPs on A549 Cell cycleprogression was assessed by flow cytometry In response toDNA damage relevant checkpoints can arrest the cell cycle ata certain stage Data showed that there was a significant (119875 lt005) dose dependent increase in cells present in subG1 phaseof cell cycle in comparison to control while the percentageof cells in G2 phase of cell cycle declined progressivelyThe amount of cells in Sub G1 phase increased from 07(control) to 295 345 and 51 at 25 120583gmL 50120583gmLand 100 120583gmL concentration respectively (Figure 8)

310 Western Blot Analysis Western blot analysis data exhib-ited that there was a significant (119875 lt 005) increase in expres-sion of proapoptotic protein Bax (41 fold 45 fold 51 fold)tumor suppressor protein p53 (18-fold 24-fold and 27-fold) and PARP (25-fold 31-fold and 33-fold) withdecreased expression level of Bcl-2 (066-fold 049-fold and023-fold) as compared to control at 25 120583gmL 50120583gmLand 100 120583gmL concentrations respectively Furthermoresignificant (119875 lt 005) increase in expression level of cyto-chrome C (33-fold 36-fold and 41-fold) Apaf-1 (20-fold26-fold and 29-fold) caspase-3 (22-fold 27-fold 29-fold)

and caspase-9 (14-fold 19-fold and 2-fold) were also foundwhich confirms the mitochondrial mediated apoptotic celldeath induction by CeO

2NPs in A549 cell (Figure 9)

4 Discussion

Due to the conflicting information of cerium oxide nanopar-ticles effects over human and environment the present studywas carried out to precisely define their final fate in a cellMoreover the major regulator behind the CeO

2NPs induced

cell death if any was also explored Our study systematicallyconcludes that CeO

2NPs get internalized in cells in a sig-

nificant manner and lead to mitochondria mediated cas-pase dependent cell death We also showed that CeO

2NPs

produced increased amount of ROS which contributed toextensive DNA damage and perturbation in cell cycle withincreasing apoptotic cells in subG1 phase Further in thepresence of specific inhibitor for ROS and apoptosis bothprocesses were completely attenuated proving that the causeof CeO

2toxicity is ROS mediated DNA damage leading to

apoptosis

BioMed Research International 9

(a) (b)

(c) (d)

(e) (f)Figure 7 Determination of apoptotic cells by acridine orange staining of A549 cells exposed to CeO

2NPs for 24 h exposure Cells were

exposed washed and stained with acridine orange dye (a) Control cells (b c) treated cells showing nuclear fragmentation (d e) chromatincondensation and (f) apoptotic bodies

0

10

20

30

40

50

60

SubG1 phaseG1 phase

S phaseG2 phase

Cel

ls (

)

lowastlowast lowastlowast lowastlowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

Figure 8 Cell cycle analysis of A549 cells exposed to CeO2NPs for

24 h exposure Cells were exposed fixed and stained with propid-ium iodide dye before subjecting to flow cytometric analysis Valuesfrom three independent experiments (mean plusmn SEM) were reportedwhere lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control

The characterization of NPs is essential for the toxicitystudies as it has been shown that the size shape surface reac-tivity solubility and degree of aggregation impart for theirdifferential response in biological system [35]Hence the par-ticle morphology average size and agglomeration status ofCeO2were examined by TEMWe observed that the particles

were in the range of 8 nm to 20 nm in size and cuboidal inshape although there was some agglomeration also presentwhich range in the size of sim40 nm Also to mimic the realexposure scenario the CeO

2NPs were characterized in the

Milli-Q water as well as in culture medium by DLS Weobserved that the average hydrodynamic size of the particle inMilli-Qwaterwas 5768 nmplusmn 045 nmand in culturemedium1774 nm plusmn 023 nm The difference in the average hydrody-namic size of NPs observed in different solvent could beattributed to their aggregation state In the culturemedia NPswere more stable andmonodispersed due to the formation ofprotein nanoparticles corona (Table 1)

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

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AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Pharmaceutics

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MEDIATORSINFLAMMATION

of

BioMed Research International 3

[25] using light scattering principlesThe analysis is based onthe principle that increase in the intensity of side scattered(SSC) light with constant intensity of forward scattered (FSC)light in cells reveals increased granularity of cells correlatedto cellular uptake of NPs In brief 1 times 105 cellsmLwell wereseeded in 12-well culture plate and allowed to attach thesurface for 22 h Cells were exposed to varying concentra-tions of CeO

2NPs (1 120583gmLndash100120583gmL) for 24 h and 48 h

Treatment was removed and cells were harvested using 025trypsin-EDTA and resuspended in 500120583L of 1x PBS Analysiswas made using flow cytometer equipped with 488 nm laser(FACS Canto II and FACS Diva software (version 612) BDBiosciences San Jose CA USA)

232 Cytotoxicity Assessment Cytotoxicity potential ofCeO2NPs was assessed using propidium iodide (PI) staining

and trypan blue dye exclusion assay for quantification ofdead cells

Propidium Iodide (PI) Staining Assay Live-dead assessmentof A549 cells exposed to CeO

2NPs was carried out by flow

cytometry based propidium iodide (PI) dye uptake assayCells (1 times 105 cellsmLwell) were seeded in 12-well cul-

ture plate and after 22 h exposed to varying concentrations(1 120583gmLndash100 120583gmL) of CeO

2NPs for 24 h and 48 h After

completion treated cells were harvested and resuspendedin 100 120583L PBS and incubated with propidium iodide dye(stock 5mgmL working 2 120583L100 120583L) for 10min at roomtemperature This suspension was diluted by adding 400 120583LPBS and red fluorescence emitted fromPI was collected usingBD FACS Canto II flow cytometer (BD Biosciences San JoseCA USA) coupled with 650 plusmn 13 nm band pass filter Theproportions of live or dead cells were analyzed using FACSDiva software (version 612) (BD Biosciences San Jose CAUSA)

Trypan Blue Dye Exclusion Assay Viability of A549 cellsexposed to CeO

2NPs was determined by trypan blue dye

exclusion assay Briefly 5 times 104 cellsmLwell were seeded andexposed to different concentrations (1120583gmLndash100120583gmL) for24 h and 48 h Exposed cells were harvested washed withPBS and mixed with equal volume (10120583L cells + 10 120583L dye)of 025 trypan blue dye solution for 5min Ten microlitresfrom this solution were used to count the viable cells by usinga Countess automated cell counter (Invitrogen UK) Loss inviability was expressed as percent dead cells

24 Measurement of Oxidative Stress Markers

241 Determination of Intracellular Reactive Oxygen Species(ROS) Generation Generation of intracellular reactive oxy-gen species in A549 cells exposed to CeO

2NPs was mea-

sured using 27-dichlorofluorescein diacetate (DCFDA) dyeaccording to the method of Wang and Joseph [26] Briefly 1times 104 cells100 120583Lwell were seeded in 96-well black bottomplate and incubated for 22 h before the exposure Cells wereexposed to increasing concentrations (1120583gmLndash100 120583gmL)of CeO

2NPs for 3 6 and 24 h in the presence and absence

of N-acetyl-L-cysteine (NAC) at a concentration of 10mM

Following the exposure cells werewashedwith PBS and incu-bated with DCFDA dye (20120583M in PBS) for 30min at 37∘CFurther the dye solution was replaced by 200 120583L of PBSand fluorescence was read at 485 nm excitation and 528 nmemission wavelength in multiwell plate reader (SYNERGY-HT Biotek USA) using KC-4 software The results wereexpressed as ROS generation compared to control Todetect the auto fluorescence and interference of NPs withDCFDA dye a cell free experiment in presence and absenceof CeO

2NPs was also conducted in parallel to the treatment

experiment

242 Measurement of Glutathione (GSH) Level A549 cellstreated with CeO

2NPs were collected and assessed for the

changes in the level of cellular GSH according to the methodof Ellman [27]

Briefly cells were cultured in 75 cm2 flask and exposedto 1 120583gmLndash100120583gmL concentrations of NPs for 3 h and 6 hAfter the treatment cells from control and treated groupswere lysed in cell lysis buffer (20mM Tris-HCl (pH 75)150mM sodium chloride (NaCl) 1mM Na

2EDTA 1 Tri-

ton X-100 and 25mM sodium pyrophosphate) Followingcentrifugation the cell extract was maintained on ice untilassayed for the cellular GSH A mixture of 01mL cell extractand 09mL of 5 tri-chloro acetic acid (TCA) was cen-trifuged (2300timesg for 15min at 4∘C) Further 05mL of thesupernatant was added to 15mL of 001 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) and the reaction was monitoredat 412 nm The amount of GSH was expressed in terms ofumolmg protein

25 Determination of Mitochondrial Membrane Potentialand Apoptosis Determination of mitochondrial membranepotential (MMP)was done using lipophilic cationic 551015840 661015840-tetrachloro-1 110158403101584031015840-tetraethylbenzimidazolecarbocyanineiodide (JC-1) dye This dye has dual fluorescence nature andmitochondrial membrane permeability This dye passivelyenters the mitochondria and forms aggregate which givesthe red fluorescence When the potential of mitochondriacollapse this dye can no longer accumulate in the mitochon-dria and remains in cytoplasm in form of monomer whichgives the green fluorescence

For the MMP analysis 1 times 105 cellsmLwell cultured in12-well plate and exposed to 1 120583gmLndash100120583gmLofCeO

2NPs

for 24 h were harvested and washed with PBS and incubatedwith 10 120583M JC-1 dye in culture medium for 15min at 37∘CCells were again washed with PBS and resuspended in 400 120583Lof PBSThe cells were analyzed for red and green fluorescencein a BD FACS Canto II flow cytometer (BD Biosciences SanJose CA USA) coupled with 485 nm excitation and 590 nmemission filters

Apoptosis analysis in CeO2NPs exposed A549 cells was

carried out using the Annexin V-FITC apoptosis detectionkit (BD Biosciences San Jose CA USA) according to themanufacturerrsquos protocol Briefly cells (1 times 105 cellsmLwell)exposed to different concentrations (1120583gmLndash100120583gmL) ofCeO2NPs in presence and absence of Z-DEVD-fmk (a

Caspase-3 inhibitor) at a concentration of 60 120583M for 24 hwere harvested washed with PBS resuspended in 100 120583L of

4 BioMed Research International

binding buffer containing 5 120583L of annexin V and propidiumiodide (PI) and incubated for 10min at room temperaturein dark After incubation samples were diluted by adding400 120583L of binding buffer and analyzed using BD FACSCanto II flow cytometer equipped with FACS Diva softwareversion 612 (BDBiosciences San Jose CAUSA)The resultswere expressed as Annexin V-FITC+ and PIminus cells identifiedas apoptotic cells Annexin V-FITC+ and PI+ cells as lateapoptotic cells Annexin V-FITCminus and PI+ cells as necroticcells and Annexin V-FITCminus and PIminus as healthy cells

Along this acridine orange staining of A549 cellstreated with different concentrations of CeO

2NPs was also

carried out for the assessment of apoptosis Briefly 1 times105 cellsmLwell were seeded in 12-well plate exposed to1 120583gmLndash100 120583gmL concentrations of NPs for 24 h har-vested and cytocentrifuged at 1000 rpm for 10min Cellswere air dried fixed in 90 chilled methanol for 10min andstained with 10 120583gmL solution of acridine orange dye for5minThe visualization of cells and scoring of the slides weredone at 400x magnification using fluorescent microscope(DMLB Leica Germany) coupled with CCD camera

26 Assessment of DNA Damage Induction by CeO2NPs

The DNA damaging potential of CeO2NPs was determined

by standard alkaline Comet assay and fpg-modified Cometassay

261 Standard Alkaline Comet Assay The induction of DNAdamage by CeO

2NPs was assessed by using alkaline Comet

assay according to the method of Singh et al [28] In brief1 times 105 cellsmLwell in 12-well culture plate was exposedto increasing concentrations (1120583gmLndash100120583gmL) of CeO

2

NPs for 6 h Following the exposure cells were harvestedand resuspended in 100 120583L PBS Comet slides were preparedaccording to the method of Bajpayee et al [29] and keptin lysis solution at 4∘C overnight Duplicate slides for eachconcentration were prepared The slides were subjected toDNAunwinding electrophoresis and neutralization processSlides were stained with 20 120583gmL of ethidium bromidesolution and kept in a humidified slide chamber until scoringThe scoring of the slides was done at 400x magnifica-tion using fluorescent microscope (DMLB Leica Germany)coupled with CCD camera The analysis was done usingimage analysis software (KOMET 50 Kinetic Imaging UK)attached with microscope Images from 50 Comet cells (25cells from each replicate slide) were analyzed and the Cometparameters that is Tail DNA and Olive tail moment weremeasured in cells according to the defined protocol [30]

262 Fpg-Modified Comet Assay Fpg-modified Comet assaywas done according to the method of Collins [31] to identifythe oxidative stress mediated DNA damage involving theinduction of oxidized bases Briefly up to the lysing the pro-cess was performed the same as with standard alkaline Cometassay After lysing slides were washed with enzyme buffer(40mMHEPES 01M KCl 05mM EDTA pH 8 02mgmLbovine serum albumin BSA) for three times and subse-quently incubated with 30 120583L of fpg enzyme (1 3000 dilutionin enzyme buffer) for 30min at 37∘C Further the slides

were subjected to unwinding electrophoresis staining andimaging the same as with standard alkaline Comet assay

27 Cell Cycle Analysis It is well known that presence of cellsin subG1 phase of cell cycle correlates theDNA fragmentationwith apoptosis [32] Effect of CeO

2NPs on cell cycle progres-

sion of A549 cells was assessed using Flow cytometry Briefly2 times 105 cellsmLwell in 6-well culture plates were exposedto different concentrations (1120583gmLndash100 120583gmL) of NPs for24 h After exposure cells were harvested washed with PBSand fixed with 70 ice cold ethanol overnight at minus20∘C Cellswere centrifuged lysed for 30min at 4∘C (using 02 TritonX 100) and treated with 10mgmL RNase for 30min at 37∘CSamples were stained with 1mgmL solution of propidiumiodide dye for 30min at 4∘C and analyzed using BD FACSCanto II flow cytometer equipped with FACS Diva softwareversion 612 (BD Biosciences San Jose CA USA) Resultswere expressed as percentage of cells in each phase of cellcycle

28 Western Blotting For analysis of different proteinsinvolved in CeO

2NPs induced toxicity 2 times 105 cellsmLwell

grown in 6-well culture plate were exposed with NPs concen-trations (1 120583gmLndash100120583gmL) for 24 h After completion ofexposure cells were washed three times with PBS and proteinwas extracted from cells for electrophoresis The amountof protein was estimated using Bradford method [33] andresolved by SDS-PAGE and transferred to polyvinylidenefluoride (PVDF)membrane For the detection of specific pro-teins PVDF membrane was incubated with anti-BAX anti-BCl-2 anti-caspase-3 anti-caspase-9 anti-cytochrome Canti-PARP anti-p53 and anti-120573-actin after blocking in 3BSA solution Detection of protein bands was carried outusing chemiluminiscence and densitometric analysis wasdone using Quantity One Quantitation Software version 431(Bio-Rad USA)

29 Statistical Analysis All the assays were repeated at leastthree times and the data are presented as mean plusmn standarderror In all experiments CeO

2NPs treated samples were

compared with their respective controls and the mean dif-ference was calculated using one way analysis of variance(ANOVA) Along this mean difference between two groupswas assessed using the two-tailed Studentrsquos 119905-test and119875 lt 005was considered as statistically significant in all the experi-ments

3 Results

31 Characterization of Cerium Oxide Nanoparticles TEManalysis was done for the assessment of primary particle sizeand morphology CeO

2NPs were cuboidal in shape and size

observed from TEM was approximately in the range from8 nm to 20 nmalthough some agglomerationwas also present(Figure 1)

CeO2NPs were also characterized using dynamic light

scattering technique in Milli-Q water as well as DMEM F-12 culture medium In Milli-Q water they tend to form

BioMed Research International 5

Table 1 Characterization of CeO2 NPs by dynamic light scattering

S number Medium Hydrodynamic size (dnm) Polydispersity index (PDI) Zeta potential (mV)1 Milli-Q water 5768 0862 minus1192 Culture medium (DMEM F-12 with 10 FBS) 1774 0248 minus137

Figure 1 Characterization of CeO2NPs by TEM TEM analysis

revealed that CeO2NPs were cuboidal in shape with size range from

sim8 nm to 20 nm with some agglomeration

agglomerate with average diameter 5768 plusmn 045 nm InDMEM F-12 medium the average size and zeta potential ofCeO2NPs was shown to be 1774 plusmn 023 nm and minus137 plusmn

025mV respectively with polydispersity index (PDI) 0248(Table 1)

32 Cellular Internalization of Cerium Oxide NanoparticlesThere was a statistically significant (119875 lt 005) concentrationand time dependent internalization of nanoparticles as evi-dent by increase in the SSC intensity of treated cells Resultshowed that 372 838 and 1533 increase in intensityof SSC at 25120583gmL 50 120583gmL and 100 120583gmL respectivelyafter 24 h was found which increased to 432 1034 and1943 after 48 h (Figure 2)

33 Morphological Analysis Morphological analysis of A549cells after NPs exposure exhibited that there was an obviouschange in cell morphology after 24 h exposure (Figure 3)Cells lost their morphology and started to become round inshape at 25120583gmL concentration These changes markedlyincreased with increasing concentrations and at 100 120583gmLmany of the cells detached and formed clumps with irregularshape Cell density was also reduced at higher dose

34 Cytotoxicity Assessment To determine the cytotoxic-ity A549 cells were incubated with varying concentrations(1 120583gmLndash100 120583gmL) of CeO

2NPs for 24 h and 48 h Pro-

pidium iodide (PI) uptake method was utilized to assess theviability in terms of cell death Cell death was increased ina concentration and time dependent manner following theexposure of nanoparticles (Figure 4(a))

0

5

10

15

20

25

Control 1 10 25 50 100

SSC

inte

nsity

()

Concentration of CeO2 NPs (120583gmL)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

24h48h

Figure 2 Internalization of CeO2NPs in A549 cells after 24 h

and 48 h exposure Cells were harvested and analyzed using flowcytometry Increase in SSC intensity which correlates the increasedgranularity of cells was used as a marker for uptake of NPs Valuesrepresent mean plusmn SEM of three independent experiments (lowast119875 lt005 lowastlowast119875 lt 001 compared with control)

There was a statistically significant (119875 lt 001) increasein cell death (725 937 1235 and 1407 in 10 120583gmL25 gmL 50 120583gmL and 100 120583gmL exposed cells resp) after24 h exposure which increased to 962 1169 1534 and1898 respectively after 48 h exposure Similar results werealso obtained by trypan blue assay in which viability of cellswas decreased with increasing concentrations of NPs (Figure4(b))

35 Determination of ROS and GSH Amount CeO2NPs

showed a significant (119875 lt 001) concentration and timedependent increase in production of ROS in terms of increasein DCF fluorescence intensity (Figure 5(a)) DCF fluores-cence intensity increased to 171 200 and 259 after 3 hand 240 266 and 286 after 6 h exposure of 25 120583gmL50 120583gmL and 100 120583gmL respectively as compared to con-trol However ROS generation decreased (115 118 and109 at 25 120583gmL 50 120583gmL and 100 120583gmL) after 24 hexposure This ROS was completely reduced in the presenceof N-acetyl-L-cysteine (NAC) as shown in Figure 5(b)

With the increase in ROS production cellular GSHsignificantly (119875 lt 005) depleted after 3 h and 6 h at 25 120583gmL50 120583gmL and 100 120583gmL exposure as compared to control(Figure 5(c))

36 Mitochondrial Membrane Potential (MMP) Analysis andApoptosis Cells treated with CeO

2NPs showed a significant

(119875 lt 005) concentration dependent decrease in mitochon-drial membrane polarization as evident by JC-1 dye (Figure6(a)) There was 727 1676 and 1851 decrease inMMP at 25 120583gmL 50120583gmL and 100 120583gmL exposurerespectively as compared to control

6 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

Figure 3 Morphological changes in A549 cells exposed to CeO2NPs for 24 h Cells were exposed washed and examined under phase

contrast microscope (a) control cells (b) 1 120583gmL (c) 10120583gmL (d) 25 120583gmL (e) 50 120583gmL and (f) 100 120583gmL concentration exposed cells(magnification times20)

Both the decrease in MMP and accumulation of ROSare hallmarks of mitochondria mediated apoptosis [34] Asevident in Figure 6(b) the amount of apoptotic cells alsoincreased in concentration dependent manner There wasincrease in apoptotic cells from 279 (control) to 679(25 120583gmL) 737 (50 120583gmL) and 892 (100 120583gmL) How-ever in the presence of Z-DEVD-fmk this death was com-pletely attenuated which clearly indicated that the death wascaspase dependent

37 Acridine Orange Staining Determination of apoptosisinduction in A549 cells by CeO

2NPs was also assessed by

acridine orange staining Cells with chromatin condensation

nuclear fragmentation and apoptotic bodies were found inthe cells exposed to CeO

2NPs as compared to control (Figure

7)

38 DNA Damage by CeO2NPs Cells exposed to 25 120583gmL

50 120583gmL and 100 120583gmL concentration of CeO2NPs for 6 h

exhibited a significant (119875 lt 005) induction in DNA damagecompared to control cells as evident by Comet parametersOlive tail moment and Tail DNA in standard alkalineComet assay (Table 2) Moreover the values for Tail DNAand Olive tail moment in fpg-modified Comet assay weresignificantly (119875 lt 005) higher than the standard alkalineComet assayThis result suggests the involvement of oxidativestress towards the DNA damage

BioMed Research International 7

0

5

10

15

20

25

30

35D

ead

cells

()

lowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

5

10

15

20

25

30

35

Dea

d ce

lls (

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

24h48h

lowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 4 Cytotoxicity assessment of CeO2NPs in A549 cells by (a) propidium iodide (PI) uptake method and (b) trypan blue dye exclusion

assay after 24 h and 48 h exposure Cells were stainedwith PI and subjected to flow cytometer whereas in trypan blue assay cells were subjectedto automatic cell counter The results were expressed as mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001 comparedwith control)

0

50

100

150

200

250

300

350

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

24h3h6h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

25

50

75

100

125

150

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

Control 1 10 25 50 100

3h6h

Concentration of CeO2 NPs (120583gmL)+ NAC (10mM)

(b)

000005010015020025030035040045

Glu

tath

ione

cont

ent

lowastlowast

lowastlowastlowastlowast

lowastlowast

3h6h

(120583m

olm

g of

pro

tein

)

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(c)

Figure 5 Effect of CeO2NPs on (a b) ROS production and (c) cellular level of glutathione (GSH) in A549 cells (a b) Cells were exposed

and incubated with H2DCFDA (c) After exposure cells were collected lysed and incubated with reaction mixture (TCA DTNB) Both

measurements were done usingmicroplate reader and data represents mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001compared to control)

8 BioMed Research International

Table 2 DNA damage induction by CeO2 NPs as evident by alkaline and fpg modified Comet assay in A549 cells after 6 h exposure

Groups OTM (arbitrary unit) Tail DNA ()Fpg (minus) Fpg (+) Fpg (minus) Fpg (+)

Control 089 plusmn 006 113 plusmn 009 632 plusmn 023 696 plusmn 012

CeO2 NPs (1 120583gmL) 119 plusmn 012lowast

168 plusmn 014lowast

776 plusmn 038 1081 plusmn 028lowastlowast

CeO2 NPs (10120583gmL) 155 plusmn 007lowastlowast

194 plusmn 015lowastlowast

901 plusmn 022lowast

1182 plusmn 024lowastlowast

CeO2 NPs (25120583gmL) 173 plusmn 008lowastlowast

228 plusmn 015lowastlowast

1030 plusmn 067lowastlowast

1345 plusmn 018lowastlowast

CeO2 NPs (50120583gmL) 189 plusmn 007lowastlowast

270 plusmn 014lowastlowast

1315 plusmn 061lowastlowast

1563 plusmn 026lowastlowast

CeO2 NPs (100120583gmL) 213 plusmn 007lowastlowast

376 plusmn 016lowastlowast

1639 plusmn 065lowastlowast

1804 plusmn 027lowastlowast

Positive control-H2O2 (25 120583M) 439 plusmn 011lowastlowast

633 plusmn 014lowastlowast

2367 plusmn 158lowastlowast

2928 plusmn 036lowastlowast

Comet slides were prepared according to defined protocol and 50 Comet cells were scored using fluorescence microscope Results from three independentexperiments in form of mean plusmn SEM were reported (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control 119875 lt 005 when compared with respective valuesin standard Comet assay)

02468

101214161820

MM

P lo

ss (

)

lowastlowastlowastlowast

lowastlowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

02468

1012141618

Apoptotic cells (absence of Z-DEVD-fmk)Apoptotic cells (presence of Z-DEVd-fmk)

Apop

totic

cells

()

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 6 Mitochondrial membrane potential alteration and apoptosis induction by CeO2NPs in A549 cells after 24 h exposure Cells were

exposed to 1 120583gmLndash100 120583gmL concentration of NPs harvested and stainedwith JC-1 dye forMMP (Figure 6(a)) and annexinV-FITCPI forapoptosis (Figure 6(b)) Cells were subjected to flow cytometry and values from three independent experiments (mean plusmn SEM) were reported(lowast119875 lt 005 lowastlowast119875 lt 001 compared to control)

39 Cell Cycle Analysis The effect of NPs on A549 Cell cycleprogression was assessed by flow cytometry In response toDNA damage relevant checkpoints can arrest the cell cycle ata certain stage Data showed that there was a significant (119875 lt005) dose dependent increase in cells present in subG1 phaseof cell cycle in comparison to control while the percentageof cells in G2 phase of cell cycle declined progressivelyThe amount of cells in Sub G1 phase increased from 07(control) to 295 345 and 51 at 25 120583gmL 50120583gmLand 100 120583gmL concentration respectively (Figure 8)

310 Western Blot Analysis Western blot analysis data exhib-ited that there was a significant (119875 lt 005) increase in expres-sion of proapoptotic protein Bax (41 fold 45 fold 51 fold)tumor suppressor protein p53 (18-fold 24-fold and 27-fold) and PARP (25-fold 31-fold and 33-fold) withdecreased expression level of Bcl-2 (066-fold 049-fold and023-fold) as compared to control at 25 120583gmL 50120583gmLand 100 120583gmL concentrations respectively Furthermoresignificant (119875 lt 005) increase in expression level of cyto-chrome C (33-fold 36-fold and 41-fold) Apaf-1 (20-fold26-fold and 29-fold) caspase-3 (22-fold 27-fold 29-fold)

and caspase-9 (14-fold 19-fold and 2-fold) were also foundwhich confirms the mitochondrial mediated apoptotic celldeath induction by CeO

2NPs in A549 cell (Figure 9)

4 Discussion

Due to the conflicting information of cerium oxide nanopar-ticles effects over human and environment the present studywas carried out to precisely define their final fate in a cellMoreover the major regulator behind the CeO

2NPs induced

cell death if any was also explored Our study systematicallyconcludes that CeO

2NPs get internalized in cells in a sig-

nificant manner and lead to mitochondria mediated cas-pase dependent cell death We also showed that CeO

2NPs

produced increased amount of ROS which contributed toextensive DNA damage and perturbation in cell cycle withincreasing apoptotic cells in subG1 phase Further in thepresence of specific inhibitor for ROS and apoptosis bothprocesses were completely attenuated proving that the causeof CeO

2toxicity is ROS mediated DNA damage leading to

apoptosis

BioMed Research International 9

(a) (b)

(c) (d)

(e) (f)Figure 7 Determination of apoptotic cells by acridine orange staining of A549 cells exposed to CeO

2NPs for 24 h exposure Cells were

exposed washed and stained with acridine orange dye (a) Control cells (b c) treated cells showing nuclear fragmentation (d e) chromatincondensation and (f) apoptotic bodies

0

10

20

30

40

50

60

SubG1 phaseG1 phase

S phaseG2 phase

Cel

ls (

)

lowastlowast lowastlowast lowastlowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

Figure 8 Cell cycle analysis of A549 cells exposed to CeO2NPs for

24 h exposure Cells were exposed fixed and stained with propid-ium iodide dye before subjecting to flow cytometric analysis Valuesfrom three independent experiments (mean plusmn SEM) were reportedwhere lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control

The characterization of NPs is essential for the toxicitystudies as it has been shown that the size shape surface reac-tivity solubility and degree of aggregation impart for theirdifferential response in biological system [35]Hence the par-ticle morphology average size and agglomeration status ofCeO2were examined by TEMWe observed that the particles

were in the range of 8 nm to 20 nm in size and cuboidal inshape although there was some agglomeration also presentwhich range in the size of sim40 nm Also to mimic the realexposure scenario the CeO

2NPs were characterized in the

Milli-Q water as well as in culture medium by DLS Weobserved that the average hydrodynamic size of the particle inMilli-Qwaterwas 5768 nmplusmn 045 nmand in culturemedium1774 nm plusmn 023 nm The difference in the average hydrody-namic size of NPs observed in different solvent could beattributed to their aggregation state In the culturemedia NPswere more stable andmonodispersed due to the formation ofprotein nanoparticles corona (Table 1)

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

4 BioMed Research International

binding buffer containing 5 120583L of annexin V and propidiumiodide (PI) and incubated for 10min at room temperaturein dark After incubation samples were diluted by adding400 120583L of binding buffer and analyzed using BD FACSCanto II flow cytometer equipped with FACS Diva softwareversion 612 (BDBiosciences San Jose CAUSA)The resultswere expressed as Annexin V-FITC+ and PIminus cells identifiedas apoptotic cells Annexin V-FITC+ and PI+ cells as lateapoptotic cells Annexin V-FITCminus and PI+ cells as necroticcells and Annexin V-FITCminus and PIminus as healthy cells

Along this acridine orange staining of A549 cellstreated with different concentrations of CeO

2NPs was also

carried out for the assessment of apoptosis Briefly 1 times105 cellsmLwell were seeded in 12-well plate exposed to1 120583gmLndash100 120583gmL concentrations of NPs for 24 h har-vested and cytocentrifuged at 1000 rpm for 10min Cellswere air dried fixed in 90 chilled methanol for 10min andstained with 10 120583gmL solution of acridine orange dye for5minThe visualization of cells and scoring of the slides weredone at 400x magnification using fluorescent microscope(DMLB Leica Germany) coupled with CCD camera

26 Assessment of DNA Damage Induction by CeO2NPs

The DNA damaging potential of CeO2NPs was determined

by standard alkaline Comet assay and fpg-modified Cometassay

261 Standard Alkaline Comet Assay The induction of DNAdamage by CeO

2NPs was assessed by using alkaline Comet

assay according to the method of Singh et al [28] In brief1 times 105 cellsmLwell in 12-well culture plate was exposedto increasing concentrations (1120583gmLndash100120583gmL) of CeO

2

NPs for 6 h Following the exposure cells were harvestedand resuspended in 100 120583L PBS Comet slides were preparedaccording to the method of Bajpayee et al [29] and keptin lysis solution at 4∘C overnight Duplicate slides for eachconcentration were prepared The slides were subjected toDNAunwinding electrophoresis and neutralization processSlides were stained with 20 120583gmL of ethidium bromidesolution and kept in a humidified slide chamber until scoringThe scoring of the slides was done at 400x magnifica-tion using fluorescent microscope (DMLB Leica Germany)coupled with CCD camera The analysis was done usingimage analysis software (KOMET 50 Kinetic Imaging UK)attached with microscope Images from 50 Comet cells (25cells from each replicate slide) were analyzed and the Cometparameters that is Tail DNA and Olive tail moment weremeasured in cells according to the defined protocol [30]

262 Fpg-Modified Comet Assay Fpg-modified Comet assaywas done according to the method of Collins [31] to identifythe oxidative stress mediated DNA damage involving theinduction of oxidized bases Briefly up to the lysing the pro-cess was performed the same as with standard alkaline Cometassay After lysing slides were washed with enzyme buffer(40mMHEPES 01M KCl 05mM EDTA pH 8 02mgmLbovine serum albumin BSA) for three times and subse-quently incubated with 30 120583L of fpg enzyme (1 3000 dilutionin enzyme buffer) for 30min at 37∘C Further the slides

were subjected to unwinding electrophoresis staining andimaging the same as with standard alkaline Comet assay

27 Cell Cycle Analysis It is well known that presence of cellsin subG1 phase of cell cycle correlates theDNA fragmentationwith apoptosis [32] Effect of CeO

2NPs on cell cycle progres-

sion of A549 cells was assessed using Flow cytometry Briefly2 times 105 cellsmLwell in 6-well culture plates were exposedto different concentrations (1120583gmLndash100 120583gmL) of NPs for24 h After exposure cells were harvested washed with PBSand fixed with 70 ice cold ethanol overnight at minus20∘C Cellswere centrifuged lysed for 30min at 4∘C (using 02 TritonX 100) and treated with 10mgmL RNase for 30min at 37∘CSamples were stained with 1mgmL solution of propidiumiodide dye for 30min at 4∘C and analyzed using BD FACSCanto II flow cytometer equipped with FACS Diva softwareversion 612 (BD Biosciences San Jose CA USA) Resultswere expressed as percentage of cells in each phase of cellcycle

28 Western Blotting For analysis of different proteinsinvolved in CeO

2NPs induced toxicity 2 times 105 cellsmLwell

grown in 6-well culture plate were exposed with NPs concen-trations (1 120583gmLndash100120583gmL) for 24 h After completion ofexposure cells were washed three times with PBS and proteinwas extracted from cells for electrophoresis The amountof protein was estimated using Bradford method [33] andresolved by SDS-PAGE and transferred to polyvinylidenefluoride (PVDF)membrane For the detection of specific pro-teins PVDF membrane was incubated with anti-BAX anti-BCl-2 anti-caspase-3 anti-caspase-9 anti-cytochrome Canti-PARP anti-p53 and anti-120573-actin after blocking in 3BSA solution Detection of protein bands was carried outusing chemiluminiscence and densitometric analysis wasdone using Quantity One Quantitation Software version 431(Bio-Rad USA)

29 Statistical Analysis All the assays were repeated at leastthree times and the data are presented as mean plusmn standarderror In all experiments CeO

2NPs treated samples were

compared with their respective controls and the mean dif-ference was calculated using one way analysis of variance(ANOVA) Along this mean difference between two groupswas assessed using the two-tailed Studentrsquos 119905-test and119875 lt 005was considered as statistically significant in all the experi-ments

3 Results

31 Characterization of Cerium Oxide Nanoparticles TEManalysis was done for the assessment of primary particle sizeand morphology CeO

2NPs were cuboidal in shape and size

observed from TEM was approximately in the range from8 nm to 20 nmalthough some agglomerationwas also present(Figure 1)

CeO2NPs were also characterized using dynamic light

scattering technique in Milli-Q water as well as DMEM F-12 culture medium In Milli-Q water they tend to form

BioMed Research International 5

Table 1 Characterization of CeO2 NPs by dynamic light scattering

S number Medium Hydrodynamic size (dnm) Polydispersity index (PDI) Zeta potential (mV)1 Milli-Q water 5768 0862 minus1192 Culture medium (DMEM F-12 with 10 FBS) 1774 0248 minus137

Figure 1 Characterization of CeO2NPs by TEM TEM analysis

revealed that CeO2NPs were cuboidal in shape with size range from

sim8 nm to 20 nm with some agglomeration

agglomerate with average diameter 5768 plusmn 045 nm InDMEM F-12 medium the average size and zeta potential ofCeO2NPs was shown to be 1774 plusmn 023 nm and minus137 plusmn

025mV respectively with polydispersity index (PDI) 0248(Table 1)

32 Cellular Internalization of Cerium Oxide NanoparticlesThere was a statistically significant (119875 lt 005) concentrationand time dependent internalization of nanoparticles as evi-dent by increase in the SSC intensity of treated cells Resultshowed that 372 838 and 1533 increase in intensityof SSC at 25120583gmL 50 120583gmL and 100 120583gmL respectivelyafter 24 h was found which increased to 432 1034 and1943 after 48 h (Figure 2)

33 Morphological Analysis Morphological analysis of A549cells after NPs exposure exhibited that there was an obviouschange in cell morphology after 24 h exposure (Figure 3)Cells lost their morphology and started to become round inshape at 25120583gmL concentration These changes markedlyincreased with increasing concentrations and at 100 120583gmLmany of the cells detached and formed clumps with irregularshape Cell density was also reduced at higher dose

34 Cytotoxicity Assessment To determine the cytotoxic-ity A549 cells were incubated with varying concentrations(1 120583gmLndash100 120583gmL) of CeO

2NPs for 24 h and 48 h Pro-

pidium iodide (PI) uptake method was utilized to assess theviability in terms of cell death Cell death was increased ina concentration and time dependent manner following theexposure of nanoparticles (Figure 4(a))

0

5

10

15

20

25

Control 1 10 25 50 100

SSC

inte

nsity

()

Concentration of CeO2 NPs (120583gmL)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

24h48h

Figure 2 Internalization of CeO2NPs in A549 cells after 24 h

and 48 h exposure Cells were harvested and analyzed using flowcytometry Increase in SSC intensity which correlates the increasedgranularity of cells was used as a marker for uptake of NPs Valuesrepresent mean plusmn SEM of three independent experiments (lowast119875 lt005 lowastlowast119875 lt 001 compared with control)

There was a statistically significant (119875 lt 001) increasein cell death (725 937 1235 and 1407 in 10 120583gmL25 gmL 50 120583gmL and 100 120583gmL exposed cells resp) after24 h exposure which increased to 962 1169 1534 and1898 respectively after 48 h exposure Similar results werealso obtained by trypan blue assay in which viability of cellswas decreased with increasing concentrations of NPs (Figure4(b))

35 Determination of ROS and GSH Amount CeO2NPs

showed a significant (119875 lt 001) concentration and timedependent increase in production of ROS in terms of increasein DCF fluorescence intensity (Figure 5(a)) DCF fluores-cence intensity increased to 171 200 and 259 after 3 hand 240 266 and 286 after 6 h exposure of 25 120583gmL50 120583gmL and 100 120583gmL respectively as compared to con-trol However ROS generation decreased (115 118 and109 at 25 120583gmL 50 120583gmL and 100 120583gmL) after 24 hexposure This ROS was completely reduced in the presenceof N-acetyl-L-cysteine (NAC) as shown in Figure 5(b)

With the increase in ROS production cellular GSHsignificantly (119875 lt 005) depleted after 3 h and 6 h at 25 120583gmL50 120583gmL and 100 120583gmL exposure as compared to control(Figure 5(c))

36 Mitochondrial Membrane Potential (MMP) Analysis andApoptosis Cells treated with CeO

2NPs showed a significant

(119875 lt 005) concentration dependent decrease in mitochon-drial membrane polarization as evident by JC-1 dye (Figure6(a)) There was 727 1676 and 1851 decrease inMMP at 25 120583gmL 50120583gmL and 100 120583gmL exposurerespectively as compared to control

6 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

Figure 3 Morphological changes in A549 cells exposed to CeO2NPs for 24 h Cells were exposed washed and examined under phase

contrast microscope (a) control cells (b) 1 120583gmL (c) 10120583gmL (d) 25 120583gmL (e) 50 120583gmL and (f) 100 120583gmL concentration exposed cells(magnification times20)

Both the decrease in MMP and accumulation of ROSare hallmarks of mitochondria mediated apoptosis [34] Asevident in Figure 6(b) the amount of apoptotic cells alsoincreased in concentration dependent manner There wasincrease in apoptotic cells from 279 (control) to 679(25 120583gmL) 737 (50 120583gmL) and 892 (100 120583gmL) How-ever in the presence of Z-DEVD-fmk this death was com-pletely attenuated which clearly indicated that the death wascaspase dependent

37 Acridine Orange Staining Determination of apoptosisinduction in A549 cells by CeO

2NPs was also assessed by

acridine orange staining Cells with chromatin condensation

nuclear fragmentation and apoptotic bodies were found inthe cells exposed to CeO

2NPs as compared to control (Figure

7)

38 DNA Damage by CeO2NPs Cells exposed to 25 120583gmL

50 120583gmL and 100 120583gmL concentration of CeO2NPs for 6 h

exhibited a significant (119875 lt 005) induction in DNA damagecompared to control cells as evident by Comet parametersOlive tail moment and Tail DNA in standard alkalineComet assay (Table 2) Moreover the values for Tail DNAand Olive tail moment in fpg-modified Comet assay weresignificantly (119875 lt 005) higher than the standard alkalineComet assayThis result suggests the involvement of oxidativestress towards the DNA damage

BioMed Research International 7

0

5

10

15

20

25

30

35D

ead

cells

()

lowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

5

10

15

20

25

30

35

Dea

d ce

lls (

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

24h48h

lowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 4 Cytotoxicity assessment of CeO2NPs in A549 cells by (a) propidium iodide (PI) uptake method and (b) trypan blue dye exclusion

assay after 24 h and 48 h exposure Cells were stainedwith PI and subjected to flow cytometer whereas in trypan blue assay cells were subjectedto automatic cell counter The results were expressed as mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001 comparedwith control)

0

50

100

150

200

250

300

350

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

24h3h6h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

25

50

75

100

125

150

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

Control 1 10 25 50 100

3h6h

Concentration of CeO2 NPs (120583gmL)+ NAC (10mM)

(b)

000005010015020025030035040045

Glu

tath

ione

cont

ent

lowastlowast

lowastlowastlowastlowast

lowastlowast

3h6h

(120583m

olm

g of

pro

tein

)

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(c)

Figure 5 Effect of CeO2NPs on (a b) ROS production and (c) cellular level of glutathione (GSH) in A549 cells (a b) Cells were exposed

and incubated with H2DCFDA (c) After exposure cells were collected lysed and incubated with reaction mixture (TCA DTNB) Both

measurements were done usingmicroplate reader and data represents mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001compared to control)

8 BioMed Research International

Table 2 DNA damage induction by CeO2 NPs as evident by alkaline and fpg modified Comet assay in A549 cells after 6 h exposure

Groups OTM (arbitrary unit) Tail DNA ()Fpg (minus) Fpg (+) Fpg (minus) Fpg (+)

Control 089 plusmn 006 113 plusmn 009 632 plusmn 023 696 plusmn 012

CeO2 NPs (1 120583gmL) 119 plusmn 012lowast

168 plusmn 014lowast

776 plusmn 038 1081 plusmn 028lowastlowast

CeO2 NPs (10120583gmL) 155 plusmn 007lowastlowast

194 plusmn 015lowastlowast

901 plusmn 022lowast

1182 plusmn 024lowastlowast

CeO2 NPs (25120583gmL) 173 plusmn 008lowastlowast

228 plusmn 015lowastlowast

1030 plusmn 067lowastlowast

1345 plusmn 018lowastlowast

CeO2 NPs (50120583gmL) 189 plusmn 007lowastlowast

270 plusmn 014lowastlowast

1315 plusmn 061lowastlowast

1563 plusmn 026lowastlowast

CeO2 NPs (100120583gmL) 213 plusmn 007lowastlowast

376 plusmn 016lowastlowast

1639 plusmn 065lowastlowast

1804 plusmn 027lowastlowast

Positive control-H2O2 (25 120583M) 439 plusmn 011lowastlowast

633 plusmn 014lowastlowast

2367 plusmn 158lowastlowast

2928 plusmn 036lowastlowast

Comet slides were prepared according to defined protocol and 50 Comet cells were scored using fluorescence microscope Results from three independentexperiments in form of mean plusmn SEM were reported (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control 119875 lt 005 when compared with respective valuesin standard Comet assay)

02468

101214161820

MM

P lo

ss (

)

lowastlowastlowastlowast

lowastlowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

02468

1012141618

Apoptotic cells (absence of Z-DEVD-fmk)Apoptotic cells (presence of Z-DEVd-fmk)

Apop

totic

cells

()

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 6 Mitochondrial membrane potential alteration and apoptosis induction by CeO2NPs in A549 cells after 24 h exposure Cells were

exposed to 1 120583gmLndash100 120583gmL concentration of NPs harvested and stainedwith JC-1 dye forMMP (Figure 6(a)) and annexinV-FITCPI forapoptosis (Figure 6(b)) Cells were subjected to flow cytometry and values from three independent experiments (mean plusmn SEM) were reported(lowast119875 lt 005 lowastlowast119875 lt 001 compared to control)

39 Cell Cycle Analysis The effect of NPs on A549 Cell cycleprogression was assessed by flow cytometry In response toDNA damage relevant checkpoints can arrest the cell cycle ata certain stage Data showed that there was a significant (119875 lt005) dose dependent increase in cells present in subG1 phaseof cell cycle in comparison to control while the percentageof cells in G2 phase of cell cycle declined progressivelyThe amount of cells in Sub G1 phase increased from 07(control) to 295 345 and 51 at 25 120583gmL 50120583gmLand 100 120583gmL concentration respectively (Figure 8)

310 Western Blot Analysis Western blot analysis data exhib-ited that there was a significant (119875 lt 005) increase in expres-sion of proapoptotic protein Bax (41 fold 45 fold 51 fold)tumor suppressor protein p53 (18-fold 24-fold and 27-fold) and PARP (25-fold 31-fold and 33-fold) withdecreased expression level of Bcl-2 (066-fold 049-fold and023-fold) as compared to control at 25 120583gmL 50120583gmLand 100 120583gmL concentrations respectively Furthermoresignificant (119875 lt 005) increase in expression level of cyto-chrome C (33-fold 36-fold and 41-fold) Apaf-1 (20-fold26-fold and 29-fold) caspase-3 (22-fold 27-fold 29-fold)

and caspase-9 (14-fold 19-fold and 2-fold) were also foundwhich confirms the mitochondrial mediated apoptotic celldeath induction by CeO

2NPs in A549 cell (Figure 9)

4 Discussion

Due to the conflicting information of cerium oxide nanopar-ticles effects over human and environment the present studywas carried out to precisely define their final fate in a cellMoreover the major regulator behind the CeO

2NPs induced

cell death if any was also explored Our study systematicallyconcludes that CeO

2NPs get internalized in cells in a sig-

nificant manner and lead to mitochondria mediated cas-pase dependent cell death We also showed that CeO

2NPs

produced increased amount of ROS which contributed toextensive DNA damage and perturbation in cell cycle withincreasing apoptotic cells in subG1 phase Further in thepresence of specific inhibitor for ROS and apoptosis bothprocesses were completely attenuated proving that the causeof CeO

2toxicity is ROS mediated DNA damage leading to

apoptosis

BioMed Research International 9

(a) (b)

(c) (d)

(e) (f)Figure 7 Determination of apoptotic cells by acridine orange staining of A549 cells exposed to CeO

2NPs for 24 h exposure Cells were

exposed washed and stained with acridine orange dye (a) Control cells (b c) treated cells showing nuclear fragmentation (d e) chromatincondensation and (f) apoptotic bodies

0

10

20

30

40

50

60

SubG1 phaseG1 phase

S phaseG2 phase

Cel

ls (

)

lowastlowast lowastlowast lowastlowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

Figure 8 Cell cycle analysis of A549 cells exposed to CeO2NPs for

24 h exposure Cells were exposed fixed and stained with propid-ium iodide dye before subjecting to flow cytometric analysis Valuesfrom three independent experiments (mean plusmn SEM) were reportedwhere lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control

The characterization of NPs is essential for the toxicitystudies as it has been shown that the size shape surface reac-tivity solubility and degree of aggregation impart for theirdifferential response in biological system [35]Hence the par-ticle morphology average size and agglomeration status ofCeO2were examined by TEMWe observed that the particles

were in the range of 8 nm to 20 nm in size and cuboidal inshape although there was some agglomeration also presentwhich range in the size of sim40 nm Also to mimic the realexposure scenario the CeO

2NPs were characterized in the

Milli-Q water as well as in culture medium by DLS Weobserved that the average hydrodynamic size of the particle inMilli-Qwaterwas 5768 nmplusmn 045 nmand in culturemedium1774 nm plusmn 023 nm The difference in the average hydrody-namic size of NPs observed in different solvent could beattributed to their aggregation state In the culturemedia NPswere more stable andmonodispersed due to the formation ofprotein nanoparticles corona (Table 1)

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

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BioMed Research International 5

Table 1 Characterization of CeO2 NPs by dynamic light scattering

S number Medium Hydrodynamic size (dnm) Polydispersity index (PDI) Zeta potential (mV)1 Milli-Q water 5768 0862 minus1192 Culture medium (DMEM F-12 with 10 FBS) 1774 0248 minus137

Figure 1 Characterization of CeO2NPs by TEM TEM analysis

revealed that CeO2NPs were cuboidal in shape with size range from

sim8 nm to 20 nm with some agglomeration

agglomerate with average diameter 5768 plusmn 045 nm InDMEM F-12 medium the average size and zeta potential ofCeO2NPs was shown to be 1774 plusmn 023 nm and minus137 plusmn

025mV respectively with polydispersity index (PDI) 0248(Table 1)

32 Cellular Internalization of Cerium Oxide NanoparticlesThere was a statistically significant (119875 lt 005) concentrationand time dependent internalization of nanoparticles as evi-dent by increase in the SSC intensity of treated cells Resultshowed that 372 838 and 1533 increase in intensityof SSC at 25120583gmL 50 120583gmL and 100 120583gmL respectivelyafter 24 h was found which increased to 432 1034 and1943 after 48 h (Figure 2)

33 Morphological Analysis Morphological analysis of A549cells after NPs exposure exhibited that there was an obviouschange in cell morphology after 24 h exposure (Figure 3)Cells lost their morphology and started to become round inshape at 25120583gmL concentration These changes markedlyincreased with increasing concentrations and at 100 120583gmLmany of the cells detached and formed clumps with irregularshape Cell density was also reduced at higher dose

34 Cytotoxicity Assessment To determine the cytotoxic-ity A549 cells were incubated with varying concentrations(1 120583gmLndash100 120583gmL) of CeO

2NPs for 24 h and 48 h Pro-

pidium iodide (PI) uptake method was utilized to assess theviability in terms of cell death Cell death was increased ina concentration and time dependent manner following theexposure of nanoparticles (Figure 4(a))

0

5

10

15

20

25

Control 1 10 25 50 100

SSC

inte

nsity

()

Concentration of CeO2 NPs (120583gmL)

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

24h48h

Figure 2 Internalization of CeO2NPs in A549 cells after 24 h

and 48 h exposure Cells were harvested and analyzed using flowcytometry Increase in SSC intensity which correlates the increasedgranularity of cells was used as a marker for uptake of NPs Valuesrepresent mean plusmn SEM of three independent experiments (lowast119875 lt005 lowastlowast119875 lt 001 compared with control)

There was a statistically significant (119875 lt 001) increasein cell death (725 937 1235 and 1407 in 10 120583gmL25 gmL 50 120583gmL and 100 120583gmL exposed cells resp) after24 h exposure which increased to 962 1169 1534 and1898 respectively after 48 h exposure Similar results werealso obtained by trypan blue assay in which viability of cellswas decreased with increasing concentrations of NPs (Figure4(b))

35 Determination of ROS and GSH Amount CeO2NPs

showed a significant (119875 lt 001) concentration and timedependent increase in production of ROS in terms of increasein DCF fluorescence intensity (Figure 5(a)) DCF fluores-cence intensity increased to 171 200 and 259 after 3 hand 240 266 and 286 after 6 h exposure of 25 120583gmL50 120583gmL and 100 120583gmL respectively as compared to con-trol However ROS generation decreased (115 118 and109 at 25 120583gmL 50 120583gmL and 100 120583gmL) after 24 hexposure This ROS was completely reduced in the presenceof N-acetyl-L-cysteine (NAC) as shown in Figure 5(b)

With the increase in ROS production cellular GSHsignificantly (119875 lt 005) depleted after 3 h and 6 h at 25 120583gmL50 120583gmL and 100 120583gmL exposure as compared to control(Figure 5(c))

36 Mitochondrial Membrane Potential (MMP) Analysis andApoptosis Cells treated with CeO

2NPs showed a significant

(119875 lt 005) concentration dependent decrease in mitochon-drial membrane polarization as evident by JC-1 dye (Figure6(a)) There was 727 1676 and 1851 decrease inMMP at 25 120583gmL 50120583gmL and 100 120583gmL exposurerespectively as compared to control

6 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

Figure 3 Morphological changes in A549 cells exposed to CeO2NPs for 24 h Cells were exposed washed and examined under phase

contrast microscope (a) control cells (b) 1 120583gmL (c) 10120583gmL (d) 25 120583gmL (e) 50 120583gmL and (f) 100 120583gmL concentration exposed cells(magnification times20)

Both the decrease in MMP and accumulation of ROSare hallmarks of mitochondria mediated apoptosis [34] Asevident in Figure 6(b) the amount of apoptotic cells alsoincreased in concentration dependent manner There wasincrease in apoptotic cells from 279 (control) to 679(25 120583gmL) 737 (50 120583gmL) and 892 (100 120583gmL) How-ever in the presence of Z-DEVD-fmk this death was com-pletely attenuated which clearly indicated that the death wascaspase dependent

37 Acridine Orange Staining Determination of apoptosisinduction in A549 cells by CeO

2NPs was also assessed by

acridine orange staining Cells with chromatin condensation

nuclear fragmentation and apoptotic bodies were found inthe cells exposed to CeO

2NPs as compared to control (Figure

7)

38 DNA Damage by CeO2NPs Cells exposed to 25 120583gmL

50 120583gmL and 100 120583gmL concentration of CeO2NPs for 6 h

exhibited a significant (119875 lt 005) induction in DNA damagecompared to control cells as evident by Comet parametersOlive tail moment and Tail DNA in standard alkalineComet assay (Table 2) Moreover the values for Tail DNAand Olive tail moment in fpg-modified Comet assay weresignificantly (119875 lt 005) higher than the standard alkalineComet assayThis result suggests the involvement of oxidativestress towards the DNA damage

BioMed Research International 7

0

5

10

15

20

25

30

35D

ead

cells

()

lowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

5

10

15

20

25

30

35

Dea

d ce

lls (

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

24h48h

lowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 4 Cytotoxicity assessment of CeO2NPs in A549 cells by (a) propidium iodide (PI) uptake method and (b) trypan blue dye exclusion

assay after 24 h and 48 h exposure Cells were stainedwith PI and subjected to flow cytometer whereas in trypan blue assay cells were subjectedto automatic cell counter The results were expressed as mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001 comparedwith control)

0

50

100

150

200

250

300

350

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

24h3h6h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

25

50

75

100

125

150

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

Control 1 10 25 50 100

3h6h

Concentration of CeO2 NPs (120583gmL)+ NAC (10mM)

(b)

000005010015020025030035040045

Glu

tath

ione

cont

ent

lowastlowast

lowastlowastlowastlowast

lowastlowast

3h6h

(120583m

olm

g of

pro

tein

)

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(c)

Figure 5 Effect of CeO2NPs on (a b) ROS production and (c) cellular level of glutathione (GSH) in A549 cells (a b) Cells were exposed

and incubated with H2DCFDA (c) After exposure cells were collected lysed and incubated with reaction mixture (TCA DTNB) Both

measurements were done usingmicroplate reader and data represents mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001compared to control)

8 BioMed Research International

Table 2 DNA damage induction by CeO2 NPs as evident by alkaline and fpg modified Comet assay in A549 cells after 6 h exposure

Groups OTM (arbitrary unit) Tail DNA ()Fpg (minus) Fpg (+) Fpg (minus) Fpg (+)

Control 089 plusmn 006 113 plusmn 009 632 plusmn 023 696 plusmn 012

CeO2 NPs (1 120583gmL) 119 plusmn 012lowast

168 plusmn 014lowast

776 plusmn 038 1081 plusmn 028lowastlowast

CeO2 NPs (10120583gmL) 155 plusmn 007lowastlowast

194 plusmn 015lowastlowast

901 plusmn 022lowast

1182 plusmn 024lowastlowast

CeO2 NPs (25120583gmL) 173 plusmn 008lowastlowast

228 plusmn 015lowastlowast

1030 plusmn 067lowastlowast

1345 plusmn 018lowastlowast

CeO2 NPs (50120583gmL) 189 plusmn 007lowastlowast

270 plusmn 014lowastlowast

1315 plusmn 061lowastlowast

1563 plusmn 026lowastlowast

CeO2 NPs (100120583gmL) 213 plusmn 007lowastlowast

376 plusmn 016lowastlowast

1639 plusmn 065lowastlowast

1804 plusmn 027lowastlowast

Positive control-H2O2 (25 120583M) 439 plusmn 011lowastlowast

633 plusmn 014lowastlowast

2367 plusmn 158lowastlowast

2928 plusmn 036lowastlowast

Comet slides were prepared according to defined protocol and 50 Comet cells were scored using fluorescence microscope Results from three independentexperiments in form of mean plusmn SEM were reported (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control 119875 lt 005 when compared with respective valuesin standard Comet assay)

02468

101214161820

MM

P lo

ss (

)

lowastlowastlowastlowast

lowastlowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

02468

1012141618

Apoptotic cells (absence of Z-DEVD-fmk)Apoptotic cells (presence of Z-DEVd-fmk)

Apop

totic

cells

()

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 6 Mitochondrial membrane potential alteration and apoptosis induction by CeO2NPs in A549 cells after 24 h exposure Cells were

exposed to 1 120583gmLndash100 120583gmL concentration of NPs harvested and stainedwith JC-1 dye forMMP (Figure 6(a)) and annexinV-FITCPI forapoptosis (Figure 6(b)) Cells were subjected to flow cytometry and values from three independent experiments (mean plusmn SEM) were reported(lowast119875 lt 005 lowastlowast119875 lt 001 compared to control)

39 Cell Cycle Analysis The effect of NPs on A549 Cell cycleprogression was assessed by flow cytometry In response toDNA damage relevant checkpoints can arrest the cell cycle ata certain stage Data showed that there was a significant (119875 lt005) dose dependent increase in cells present in subG1 phaseof cell cycle in comparison to control while the percentageof cells in G2 phase of cell cycle declined progressivelyThe amount of cells in Sub G1 phase increased from 07(control) to 295 345 and 51 at 25 120583gmL 50120583gmLand 100 120583gmL concentration respectively (Figure 8)

310 Western Blot Analysis Western blot analysis data exhib-ited that there was a significant (119875 lt 005) increase in expres-sion of proapoptotic protein Bax (41 fold 45 fold 51 fold)tumor suppressor protein p53 (18-fold 24-fold and 27-fold) and PARP (25-fold 31-fold and 33-fold) withdecreased expression level of Bcl-2 (066-fold 049-fold and023-fold) as compared to control at 25 120583gmL 50120583gmLand 100 120583gmL concentrations respectively Furthermoresignificant (119875 lt 005) increase in expression level of cyto-chrome C (33-fold 36-fold and 41-fold) Apaf-1 (20-fold26-fold and 29-fold) caspase-3 (22-fold 27-fold 29-fold)

and caspase-9 (14-fold 19-fold and 2-fold) were also foundwhich confirms the mitochondrial mediated apoptotic celldeath induction by CeO

2NPs in A549 cell (Figure 9)

4 Discussion

Due to the conflicting information of cerium oxide nanopar-ticles effects over human and environment the present studywas carried out to precisely define their final fate in a cellMoreover the major regulator behind the CeO

2NPs induced

cell death if any was also explored Our study systematicallyconcludes that CeO

2NPs get internalized in cells in a sig-

nificant manner and lead to mitochondria mediated cas-pase dependent cell death We also showed that CeO

2NPs

produced increased amount of ROS which contributed toextensive DNA damage and perturbation in cell cycle withincreasing apoptotic cells in subG1 phase Further in thepresence of specific inhibitor for ROS and apoptosis bothprocesses were completely attenuated proving that the causeof CeO

2toxicity is ROS mediated DNA damage leading to

apoptosis

BioMed Research International 9

(a) (b)

(c) (d)

(e) (f)Figure 7 Determination of apoptotic cells by acridine orange staining of A549 cells exposed to CeO

2NPs for 24 h exposure Cells were

exposed washed and stained with acridine orange dye (a) Control cells (b c) treated cells showing nuclear fragmentation (d e) chromatincondensation and (f) apoptotic bodies

0

10

20

30

40

50

60

SubG1 phaseG1 phase

S phaseG2 phase

Cel

ls (

)

lowastlowast lowastlowast lowastlowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

Figure 8 Cell cycle analysis of A549 cells exposed to CeO2NPs for

24 h exposure Cells were exposed fixed and stained with propid-ium iodide dye before subjecting to flow cytometric analysis Valuesfrom three independent experiments (mean plusmn SEM) were reportedwhere lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control

The characterization of NPs is essential for the toxicitystudies as it has been shown that the size shape surface reac-tivity solubility and degree of aggregation impart for theirdifferential response in biological system [35]Hence the par-ticle morphology average size and agglomeration status ofCeO2were examined by TEMWe observed that the particles

were in the range of 8 nm to 20 nm in size and cuboidal inshape although there was some agglomeration also presentwhich range in the size of sim40 nm Also to mimic the realexposure scenario the CeO

2NPs were characterized in the

Milli-Q water as well as in culture medium by DLS Weobserved that the average hydrodynamic size of the particle inMilli-Qwaterwas 5768 nmplusmn 045 nmand in culturemedium1774 nm plusmn 023 nm The difference in the average hydrody-namic size of NPs observed in different solvent could beattributed to their aggregation state In the culturemedia NPswere more stable andmonodispersed due to the formation ofprotein nanoparticles corona (Table 1)

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

6 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

Figure 3 Morphological changes in A549 cells exposed to CeO2NPs for 24 h Cells were exposed washed and examined under phase

contrast microscope (a) control cells (b) 1 120583gmL (c) 10120583gmL (d) 25 120583gmL (e) 50 120583gmL and (f) 100 120583gmL concentration exposed cells(magnification times20)

Both the decrease in MMP and accumulation of ROSare hallmarks of mitochondria mediated apoptosis [34] Asevident in Figure 6(b) the amount of apoptotic cells alsoincreased in concentration dependent manner There wasincrease in apoptotic cells from 279 (control) to 679(25 120583gmL) 737 (50 120583gmL) and 892 (100 120583gmL) How-ever in the presence of Z-DEVD-fmk this death was com-pletely attenuated which clearly indicated that the death wascaspase dependent

37 Acridine Orange Staining Determination of apoptosisinduction in A549 cells by CeO

2NPs was also assessed by

acridine orange staining Cells with chromatin condensation

nuclear fragmentation and apoptotic bodies were found inthe cells exposed to CeO

2NPs as compared to control (Figure

7)

38 DNA Damage by CeO2NPs Cells exposed to 25 120583gmL

50 120583gmL and 100 120583gmL concentration of CeO2NPs for 6 h

exhibited a significant (119875 lt 005) induction in DNA damagecompared to control cells as evident by Comet parametersOlive tail moment and Tail DNA in standard alkalineComet assay (Table 2) Moreover the values for Tail DNAand Olive tail moment in fpg-modified Comet assay weresignificantly (119875 lt 005) higher than the standard alkalineComet assayThis result suggests the involvement of oxidativestress towards the DNA damage

BioMed Research International 7

0

5

10

15

20

25

30

35D

ead

cells

()

lowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

5

10

15

20

25

30

35

Dea

d ce

lls (

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

24h48h

lowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 4 Cytotoxicity assessment of CeO2NPs in A549 cells by (a) propidium iodide (PI) uptake method and (b) trypan blue dye exclusion

assay after 24 h and 48 h exposure Cells were stainedwith PI and subjected to flow cytometer whereas in trypan blue assay cells were subjectedto automatic cell counter The results were expressed as mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001 comparedwith control)

0

50

100

150

200

250

300

350

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

24h3h6h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

25

50

75

100

125

150

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

Control 1 10 25 50 100

3h6h

Concentration of CeO2 NPs (120583gmL)+ NAC (10mM)

(b)

000005010015020025030035040045

Glu

tath

ione

cont

ent

lowastlowast

lowastlowastlowastlowast

lowastlowast

3h6h

(120583m

olm

g of

pro

tein

)

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(c)

Figure 5 Effect of CeO2NPs on (a b) ROS production and (c) cellular level of glutathione (GSH) in A549 cells (a b) Cells were exposed

and incubated with H2DCFDA (c) After exposure cells were collected lysed and incubated with reaction mixture (TCA DTNB) Both

measurements were done usingmicroplate reader and data represents mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001compared to control)

8 BioMed Research International

Table 2 DNA damage induction by CeO2 NPs as evident by alkaline and fpg modified Comet assay in A549 cells after 6 h exposure

Groups OTM (arbitrary unit) Tail DNA ()Fpg (minus) Fpg (+) Fpg (minus) Fpg (+)

Control 089 plusmn 006 113 plusmn 009 632 plusmn 023 696 plusmn 012

CeO2 NPs (1 120583gmL) 119 plusmn 012lowast

168 plusmn 014lowast

776 plusmn 038 1081 plusmn 028lowastlowast

CeO2 NPs (10120583gmL) 155 plusmn 007lowastlowast

194 plusmn 015lowastlowast

901 plusmn 022lowast

1182 plusmn 024lowastlowast

CeO2 NPs (25120583gmL) 173 plusmn 008lowastlowast

228 plusmn 015lowastlowast

1030 plusmn 067lowastlowast

1345 plusmn 018lowastlowast

CeO2 NPs (50120583gmL) 189 plusmn 007lowastlowast

270 plusmn 014lowastlowast

1315 plusmn 061lowastlowast

1563 plusmn 026lowastlowast

CeO2 NPs (100120583gmL) 213 plusmn 007lowastlowast

376 plusmn 016lowastlowast

1639 plusmn 065lowastlowast

1804 plusmn 027lowastlowast

Positive control-H2O2 (25 120583M) 439 plusmn 011lowastlowast

633 plusmn 014lowastlowast

2367 plusmn 158lowastlowast

2928 plusmn 036lowastlowast

Comet slides were prepared according to defined protocol and 50 Comet cells were scored using fluorescence microscope Results from three independentexperiments in form of mean plusmn SEM were reported (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control 119875 lt 005 when compared with respective valuesin standard Comet assay)

02468

101214161820

MM

P lo

ss (

)

lowastlowastlowastlowast

lowastlowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

02468

1012141618

Apoptotic cells (absence of Z-DEVD-fmk)Apoptotic cells (presence of Z-DEVd-fmk)

Apop

totic

cells

()

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 6 Mitochondrial membrane potential alteration and apoptosis induction by CeO2NPs in A549 cells after 24 h exposure Cells were

exposed to 1 120583gmLndash100 120583gmL concentration of NPs harvested and stainedwith JC-1 dye forMMP (Figure 6(a)) and annexinV-FITCPI forapoptosis (Figure 6(b)) Cells were subjected to flow cytometry and values from three independent experiments (mean plusmn SEM) were reported(lowast119875 lt 005 lowastlowast119875 lt 001 compared to control)

39 Cell Cycle Analysis The effect of NPs on A549 Cell cycleprogression was assessed by flow cytometry In response toDNA damage relevant checkpoints can arrest the cell cycle ata certain stage Data showed that there was a significant (119875 lt005) dose dependent increase in cells present in subG1 phaseof cell cycle in comparison to control while the percentageof cells in G2 phase of cell cycle declined progressivelyThe amount of cells in Sub G1 phase increased from 07(control) to 295 345 and 51 at 25 120583gmL 50120583gmLand 100 120583gmL concentration respectively (Figure 8)

310 Western Blot Analysis Western blot analysis data exhib-ited that there was a significant (119875 lt 005) increase in expres-sion of proapoptotic protein Bax (41 fold 45 fold 51 fold)tumor suppressor protein p53 (18-fold 24-fold and 27-fold) and PARP (25-fold 31-fold and 33-fold) withdecreased expression level of Bcl-2 (066-fold 049-fold and023-fold) as compared to control at 25 120583gmL 50120583gmLand 100 120583gmL concentrations respectively Furthermoresignificant (119875 lt 005) increase in expression level of cyto-chrome C (33-fold 36-fold and 41-fold) Apaf-1 (20-fold26-fold and 29-fold) caspase-3 (22-fold 27-fold 29-fold)

and caspase-9 (14-fold 19-fold and 2-fold) were also foundwhich confirms the mitochondrial mediated apoptotic celldeath induction by CeO

2NPs in A549 cell (Figure 9)

4 Discussion

Due to the conflicting information of cerium oxide nanopar-ticles effects over human and environment the present studywas carried out to precisely define their final fate in a cellMoreover the major regulator behind the CeO

2NPs induced

cell death if any was also explored Our study systematicallyconcludes that CeO

2NPs get internalized in cells in a sig-

nificant manner and lead to mitochondria mediated cas-pase dependent cell death We also showed that CeO

2NPs

produced increased amount of ROS which contributed toextensive DNA damage and perturbation in cell cycle withincreasing apoptotic cells in subG1 phase Further in thepresence of specific inhibitor for ROS and apoptosis bothprocesses were completely attenuated proving that the causeof CeO

2toxicity is ROS mediated DNA damage leading to

apoptosis

BioMed Research International 9

(a) (b)

(c) (d)

(e) (f)Figure 7 Determination of apoptotic cells by acridine orange staining of A549 cells exposed to CeO

2NPs for 24 h exposure Cells were

exposed washed and stained with acridine orange dye (a) Control cells (b c) treated cells showing nuclear fragmentation (d e) chromatincondensation and (f) apoptotic bodies

0

10

20

30

40

50

60

SubG1 phaseG1 phase

S phaseG2 phase

Cel

ls (

)

lowastlowast lowastlowast lowastlowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

Figure 8 Cell cycle analysis of A549 cells exposed to CeO2NPs for

24 h exposure Cells were exposed fixed and stained with propid-ium iodide dye before subjecting to flow cytometric analysis Valuesfrom three independent experiments (mean plusmn SEM) were reportedwhere lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control

The characterization of NPs is essential for the toxicitystudies as it has been shown that the size shape surface reac-tivity solubility and degree of aggregation impart for theirdifferential response in biological system [35]Hence the par-ticle morphology average size and agglomeration status ofCeO2were examined by TEMWe observed that the particles

were in the range of 8 nm to 20 nm in size and cuboidal inshape although there was some agglomeration also presentwhich range in the size of sim40 nm Also to mimic the realexposure scenario the CeO

2NPs were characterized in the

Milli-Q water as well as in culture medium by DLS Weobserved that the average hydrodynamic size of the particle inMilli-Qwaterwas 5768 nmplusmn 045 nmand in culturemedium1774 nm plusmn 023 nm The difference in the average hydrody-namic size of NPs observed in different solvent could beattributed to their aggregation state In the culturemedia NPswere more stable andmonodispersed due to the formation ofprotein nanoparticles corona (Table 1)

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 7

0

5

10

15

20

25

30

35D

ead

cells

()

lowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

5

10

15

20

25

30

35

Dea

d ce

lls (

)

lowastlowast

lowastlowastlowastlowast

lowastlowast

24h48h

lowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 4 Cytotoxicity assessment of CeO2NPs in A549 cells by (a) propidium iodide (PI) uptake method and (b) trypan blue dye exclusion

assay after 24 h and 48 h exposure Cells were stainedwith PI and subjected to flow cytometer whereas in trypan blue assay cells were subjectedto automatic cell counter The results were expressed as mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001 comparedwith control)

0

50

100

150

200

250

300

350

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

24h3h6h

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

0

25

50

75

100

125

150

ROS

gene

ratio

n (

)(c

ompa

red

to co

ntro

l)

Control 1 10 25 50 100

3h6h

Concentration of CeO2 NPs (120583gmL)+ NAC (10mM)

(b)

000005010015020025030035040045

Glu

tath

ione

cont

ent

lowastlowast

lowastlowastlowastlowast

lowastlowast

3h6h

(120583m

olm

g of

pro

tein

)

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(c)

Figure 5 Effect of CeO2NPs on (a b) ROS production and (c) cellular level of glutathione (GSH) in A549 cells (a b) Cells were exposed

and incubated with H2DCFDA (c) After exposure cells were collected lysed and incubated with reaction mixture (TCA DTNB) Both

measurements were done usingmicroplate reader and data represents mean plusmn SEM of three independent experiments (lowast119875 lt 005 lowastlowast119875 lt 001compared to control)

8 BioMed Research International

Table 2 DNA damage induction by CeO2 NPs as evident by alkaline and fpg modified Comet assay in A549 cells after 6 h exposure

Groups OTM (arbitrary unit) Tail DNA ()Fpg (minus) Fpg (+) Fpg (minus) Fpg (+)

Control 089 plusmn 006 113 plusmn 009 632 plusmn 023 696 plusmn 012

CeO2 NPs (1 120583gmL) 119 plusmn 012lowast

168 plusmn 014lowast

776 plusmn 038 1081 plusmn 028lowastlowast

CeO2 NPs (10120583gmL) 155 plusmn 007lowastlowast

194 plusmn 015lowastlowast

901 plusmn 022lowast

1182 plusmn 024lowastlowast

CeO2 NPs (25120583gmL) 173 plusmn 008lowastlowast

228 plusmn 015lowastlowast

1030 plusmn 067lowastlowast

1345 plusmn 018lowastlowast

CeO2 NPs (50120583gmL) 189 plusmn 007lowastlowast

270 plusmn 014lowastlowast

1315 plusmn 061lowastlowast

1563 plusmn 026lowastlowast

CeO2 NPs (100120583gmL) 213 plusmn 007lowastlowast

376 plusmn 016lowastlowast

1639 plusmn 065lowastlowast

1804 plusmn 027lowastlowast

Positive control-H2O2 (25 120583M) 439 plusmn 011lowastlowast

633 plusmn 014lowastlowast

2367 plusmn 158lowastlowast

2928 plusmn 036lowastlowast

Comet slides were prepared according to defined protocol and 50 Comet cells were scored using fluorescence microscope Results from three independentexperiments in form of mean plusmn SEM were reported (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control 119875 lt 005 when compared with respective valuesin standard Comet assay)

02468

101214161820

MM

P lo

ss (

)

lowastlowastlowastlowast

lowastlowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

02468

1012141618

Apoptotic cells (absence of Z-DEVD-fmk)Apoptotic cells (presence of Z-DEVd-fmk)

Apop

totic

cells

()

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 6 Mitochondrial membrane potential alteration and apoptosis induction by CeO2NPs in A549 cells after 24 h exposure Cells were

exposed to 1 120583gmLndash100 120583gmL concentration of NPs harvested and stainedwith JC-1 dye forMMP (Figure 6(a)) and annexinV-FITCPI forapoptosis (Figure 6(b)) Cells were subjected to flow cytometry and values from three independent experiments (mean plusmn SEM) were reported(lowast119875 lt 005 lowastlowast119875 lt 001 compared to control)

39 Cell Cycle Analysis The effect of NPs on A549 Cell cycleprogression was assessed by flow cytometry In response toDNA damage relevant checkpoints can arrest the cell cycle ata certain stage Data showed that there was a significant (119875 lt005) dose dependent increase in cells present in subG1 phaseof cell cycle in comparison to control while the percentageof cells in G2 phase of cell cycle declined progressivelyThe amount of cells in Sub G1 phase increased from 07(control) to 295 345 and 51 at 25 120583gmL 50120583gmLand 100 120583gmL concentration respectively (Figure 8)

310 Western Blot Analysis Western blot analysis data exhib-ited that there was a significant (119875 lt 005) increase in expres-sion of proapoptotic protein Bax (41 fold 45 fold 51 fold)tumor suppressor protein p53 (18-fold 24-fold and 27-fold) and PARP (25-fold 31-fold and 33-fold) withdecreased expression level of Bcl-2 (066-fold 049-fold and023-fold) as compared to control at 25 120583gmL 50120583gmLand 100 120583gmL concentrations respectively Furthermoresignificant (119875 lt 005) increase in expression level of cyto-chrome C (33-fold 36-fold and 41-fold) Apaf-1 (20-fold26-fold and 29-fold) caspase-3 (22-fold 27-fold 29-fold)

and caspase-9 (14-fold 19-fold and 2-fold) were also foundwhich confirms the mitochondrial mediated apoptotic celldeath induction by CeO

2NPs in A549 cell (Figure 9)

4 Discussion

Due to the conflicting information of cerium oxide nanopar-ticles effects over human and environment the present studywas carried out to precisely define their final fate in a cellMoreover the major regulator behind the CeO

2NPs induced

cell death if any was also explored Our study systematicallyconcludes that CeO

2NPs get internalized in cells in a sig-

nificant manner and lead to mitochondria mediated cas-pase dependent cell death We also showed that CeO

2NPs

produced increased amount of ROS which contributed toextensive DNA damage and perturbation in cell cycle withincreasing apoptotic cells in subG1 phase Further in thepresence of specific inhibitor for ROS and apoptosis bothprocesses were completely attenuated proving that the causeof CeO

2toxicity is ROS mediated DNA damage leading to

apoptosis

BioMed Research International 9

(a) (b)

(c) (d)

(e) (f)Figure 7 Determination of apoptotic cells by acridine orange staining of A549 cells exposed to CeO

2NPs for 24 h exposure Cells were

exposed washed and stained with acridine orange dye (a) Control cells (b c) treated cells showing nuclear fragmentation (d e) chromatincondensation and (f) apoptotic bodies

0

10

20

30

40

50

60

SubG1 phaseG1 phase

S phaseG2 phase

Cel

ls (

)

lowastlowast lowastlowast lowastlowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

Figure 8 Cell cycle analysis of A549 cells exposed to CeO2NPs for

24 h exposure Cells were exposed fixed and stained with propid-ium iodide dye before subjecting to flow cytometric analysis Valuesfrom three independent experiments (mean plusmn SEM) were reportedwhere lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control

The characterization of NPs is essential for the toxicitystudies as it has been shown that the size shape surface reac-tivity solubility and degree of aggregation impart for theirdifferential response in biological system [35]Hence the par-ticle morphology average size and agglomeration status ofCeO2were examined by TEMWe observed that the particles

were in the range of 8 nm to 20 nm in size and cuboidal inshape although there was some agglomeration also presentwhich range in the size of sim40 nm Also to mimic the realexposure scenario the CeO

2NPs were characterized in the

Milli-Q water as well as in culture medium by DLS Weobserved that the average hydrodynamic size of the particle inMilli-Qwaterwas 5768 nmplusmn 045 nmand in culturemedium1774 nm plusmn 023 nm The difference in the average hydrody-namic size of NPs observed in different solvent could beattributed to their aggregation state In the culturemedia NPswere more stable andmonodispersed due to the formation ofprotein nanoparticles corona (Table 1)

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

8 BioMed Research International

Table 2 DNA damage induction by CeO2 NPs as evident by alkaline and fpg modified Comet assay in A549 cells after 6 h exposure

Groups OTM (arbitrary unit) Tail DNA ()Fpg (minus) Fpg (+) Fpg (minus) Fpg (+)

Control 089 plusmn 006 113 plusmn 009 632 plusmn 023 696 plusmn 012

CeO2 NPs (1 120583gmL) 119 plusmn 012lowast

168 plusmn 014lowast

776 plusmn 038 1081 plusmn 028lowastlowast

CeO2 NPs (10120583gmL) 155 plusmn 007lowastlowast

194 plusmn 015lowastlowast

901 plusmn 022lowast

1182 plusmn 024lowastlowast

CeO2 NPs (25120583gmL) 173 plusmn 008lowastlowast

228 plusmn 015lowastlowast

1030 plusmn 067lowastlowast

1345 plusmn 018lowastlowast

CeO2 NPs (50120583gmL) 189 plusmn 007lowastlowast

270 plusmn 014lowastlowast

1315 plusmn 061lowastlowast

1563 plusmn 026lowastlowast

CeO2 NPs (100120583gmL) 213 plusmn 007lowastlowast

376 plusmn 016lowastlowast

1639 plusmn 065lowastlowast

1804 plusmn 027lowastlowast

Positive control-H2O2 (25 120583M) 439 plusmn 011lowastlowast

633 plusmn 014lowastlowast

2367 plusmn 158lowastlowast

2928 plusmn 036lowastlowast

Comet slides were prepared according to defined protocol and 50 Comet cells were scored using fluorescence microscope Results from three independentexperiments in form of mean plusmn SEM were reported (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control 119875 lt 005 when compared with respective valuesin standard Comet assay)

02468

101214161820

MM

P lo

ss (

)

lowastlowastlowastlowast

lowastlowast

lowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(a)

02468

1012141618

Apoptotic cells (absence of Z-DEVD-fmk)Apoptotic cells (presence of Z-DEVd-fmk)

Apop

totic

cells

()

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

(b)

Figure 6 Mitochondrial membrane potential alteration and apoptosis induction by CeO2NPs in A549 cells after 24 h exposure Cells were

exposed to 1 120583gmLndash100 120583gmL concentration of NPs harvested and stainedwith JC-1 dye forMMP (Figure 6(a)) and annexinV-FITCPI forapoptosis (Figure 6(b)) Cells were subjected to flow cytometry and values from three independent experiments (mean plusmn SEM) were reported(lowast119875 lt 005 lowastlowast119875 lt 001 compared to control)

39 Cell Cycle Analysis The effect of NPs on A549 Cell cycleprogression was assessed by flow cytometry In response toDNA damage relevant checkpoints can arrest the cell cycle ata certain stage Data showed that there was a significant (119875 lt005) dose dependent increase in cells present in subG1 phaseof cell cycle in comparison to control while the percentageof cells in G2 phase of cell cycle declined progressivelyThe amount of cells in Sub G1 phase increased from 07(control) to 295 345 and 51 at 25 120583gmL 50120583gmLand 100 120583gmL concentration respectively (Figure 8)

310 Western Blot Analysis Western blot analysis data exhib-ited that there was a significant (119875 lt 005) increase in expres-sion of proapoptotic protein Bax (41 fold 45 fold 51 fold)tumor suppressor protein p53 (18-fold 24-fold and 27-fold) and PARP (25-fold 31-fold and 33-fold) withdecreased expression level of Bcl-2 (066-fold 049-fold and023-fold) as compared to control at 25 120583gmL 50120583gmLand 100 120583gmL concentrations respectively Furthermoresignificant (119875 lt 005) increase in expression level of cyto-chrome C (33-fold 36-fold and 41-fold) Apaf-1 (20-fold26-fold and 29-fold) caspase-3 (22-fold 27-fold 29-fold)

and caspase-9 (14-fold 19-fold and 2-fold) were also foundwhich confirms the mitochondrial mediated apoptotic celldeath induction by CeO

2NPs in A549 cell (Figure 9)

4 Discussion

Due to the conflicting information of cerium oxide nanopar-ticles effects over human and environment the present studywas carried out to precisely define their final fate in a cellMoreover the major regulator behind the CeO

2NPs induced

cell death if any was also explored Our study systematicallyconcludes that CeO

2NPs get internalized in cells in a sig-

nificant manner and lead to mitochondria mediated cas-pase dependent cell death We also showed that CeO

2NPs

produced increased amount of ROS which contributed toextensive DNA damage and perturbation in cell cycle withincreasing apoptotic cells in subG1 phase Further in thepresence of specific inhibitor for ROS and apoptosis bothprocesses were completely attenuated proving that the causeof CeO

2toxicity is ROS mediated DNA damage leading to

apoptosis

BioMed Research International 9

(a) (b)

(c) (d)

(e) (f)Figure 7 Determination of apoptotic cells by acridine orange staining of A549 cells exposed to CeO

2NPs for 24 h exposure Cells were

exposed washed and stained with acridine orange dye (a) Control cells (b c) treated cells showing nuclear fragmentation (d e) chromatincondensation and (f) apoptotic bodies

0

10

20

30

40

50

60

SubG1 phaseG1 phase

S phaseG2 phase

Cel

ls (

)

lowastlowast lowastlowast lowastlowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

Figure 8 Cell cycle analysis of A549 cells exposed to CeO2NPs for

24 h exposure Cells were exposed fixed and stained with propid-ium iodide dye before subjecting to flow cytometric analysis Valuesfrom three independent experiments (mean plusmn SEM) were reportedwhere lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control

The characterization of NPs is essential for the toxicitystudies as it has been shown that the size shape surface reac-tivity solubility and degree of aggregation impart for theirdifferential response in biological system [35]Hence the par-ticle morphology average size and agglomeration status ofCeO2were examined by TEMWe observed that the particles

were in the range of 8 nm to 20 nm in size and cuboidal inshape although there was some agglomeration also presentwhich range in the size of sim40 nm Also to mimic the realexposure scenario the CeO

2NPs were characterized in the

Milli-Q water as well as in culture medium by DLS Weobserved that the average hydrodynamic size of the particle inMilli-Qwaterwas 5768 nmplusmn 045 nmand in culturemedium1774 nm plusmn 023 nm The difference in the average hydrody-namic size of NPs observed in different solvent could beattributed to their aggregation state In the culturemedia NPswere more stable andmonodispersed due to the formation ofprotein nanoparticles corona (Table 1)

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 9

(a) (b)

(c) (d)

(e) (f)Figure 7 Determination of apoptotic cells by acridine orange staining of A549 cells exposed to CeO

2NPs for 24 h exposure Cells were

exposed washed and stained with acridine orange dye (a) Control cells (b c) treated cells showing nuclear fragmentation (d e) chromatincondensation and (f) apoptotic bodies

0

10

20

30

40

50

60

SubG1 phaseG1 phase

S phaseG2 phase

Cel

ls (

)

lowastlowast lowastlowast lowastlowastlowast

Control 1 10 25 50 100Concentration of CeO2 NPs (120583gmL)

Figure 8 Cell cycle analysis of A549 cells exposed to CeO2NPs for

24 h exposure Cells were exposed fixed and stained with propid-ium iodide dye before subjecting to flow cytometric analysis Valuesfrom three independent experiments (mean plusmn SEM) were reportedwhere lowast119875 lt 005 and lowastlowast119875 lt 001 compared to control

The characterization of NPs is essential for the toxicitystudies as it has been shown that the size shape surface reac-tivity solubility and degree of aggregation impart for theirdifferential response in biological system [35]Hence the par-ticle morphology average size and agglomeration status ofCeO2were examined by TEMWe observed that the particles

were in the range of 8 nm to 20 nm in size and cuboidal inshape although there was some agglomeration also presentwhich range in the size of sim40 nm Also to mimic the realexposure scenario the CeO

2NPs were characterized in the

Milli-Q water as well as in culture medium by DLS Weobserved that the average hydrodynamic size of the particle inMilli-Qwaterwas 5768 nmplusmn 045 nmand in culturemedium1774 nm plusmn 023 nm The difference in the average hydrody-namic size of NPs observed in different solvent could beattributed to their aggregation state In the culturemedia NPswere more stable andmonodispersed due to the formation ofprotein nanoparticles corona (Table 1)

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

10 BioMed Research International

Bcl-2

Cyto C

Bax

0

1

2

3

4

5

6

Bax Bcl-2 Cyt C

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

Caspase-3

Caspase-3

Caspase-9

Caspase-9

Apaf1

Apaf1

Fold

chan

ge re

lativ

e to

cont

rol

Uncleaved PARP

Cleaved PARP

p53

Phospho p53

0

1

2

3

4

5

6

PARP Cleaved PARP

Fold

chan

ge re

lativ

e to

cont

rol

0

1

2

3

4

5

6

p53 Pp53

Fold

chan

ge re

lativ

e to

cont

rol

(Cci) (Ccii)

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowastlowastlowastlowast

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL10120583gmL 25120583gmL50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

Control 1120583gmL 10120583gmL 25120583gmL 50120583gmL 100120583gmL

120573-Actin 120573-Actin

120573-Actin

23kD

26kD

42kD

15kD

(A) (B)

(C)

(Aa) (Bb)

lowastlowast

lowast

lowastlowast

120 kD

17kD

35kD

42kD

120 kD

25kD

53kD

55kD

42kD

lowastlowast

Figure 9 Dose dependent expression level analysis (A B and C) of various apoptotic proteins in A549 cells after 24 h exposure of CeO2

NPs Cells exposed to indicated concentration protein lysate were collected and assayed by western blotting 120573-actin was used as an internalcontrol All blots (A B and C) and respective Bar graph (Aa Bb and Cc) values (mean plusmn SEM) are representative of three independentexperiments (lowast119875 lt 005 lowastlowast119875 lt 001 compared to respective control)

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 11

For toxicity studies of the NPs it is important to assessNPs uptake and correlate it with the cellular response In thepresent study the internalization of the CeO

2NPs in A549

cells were determined by the flow cytometry using the pro-tocol described by our earlier studies [36 37] We observeda concentration and time dependent significant (119875 lt 005)increase in the side scattered intensity of the treated A549cells as compared to control (Figure 2) Additionally phasecontrast images of A549 cells treated with CeO

2NPs showed

their accumulation in cytoplasm The uptake potential ofCeO2NPs in various human cell lines as well as in mouse

macrophage cells has also been reported previously [38 39]which correlates with our findings Also we observed somemorphological changes in the CeO

2NPs treated cells such as

at concentration 25120583gmL few cells become spherical whileat concentration 100 120583gmL the number of the spherical cellsincreased and many of them were detached from the surfaceof culture plate (Figure 3) Earlier studies have also shownthe internalization of CeO

2NPs in human keratinocytes

cells human bronchial epithelial cells cardiac progenitorcells human monocytes and so forth using TEM and flowcytometry [40ndash42]

Various metal oxide nanoparticles have shown to inducecell death after internalization [37 43 44] We assessed thecytotoxicity potential of CeO

2NPs using propidium iodide

(PI) uptake method and trypan blue dye exclusion assay PIdye selectively enters the cells with compromised membraneintegrity and binds to the DNA which causes increase inthe fluorescence intensity of PI dye whereas in trypan bluedye exclusion assay live cell excludes the dye and dead cellsstained with blue color Our data suggested that followingthe exposure of CeO

2NPs there was a significant (119875 lt

005) concentration and time dependent cytotoxicity in A549cells at 25 120583gmL 50 120583gmL and 100 120583gmL concentrationsat 24 h and 48 h in both the assays These results were inaccordance with the earlier studies which have reported thetoxic behavior of CeO

2NPs in various cell lines [17 41] Cell

death induction by various metal oxide NPs are due to theirdissolution in culture medium which causes release of ionsThese ions contributed to the cell death [43 45 46] But CeO

2

NPs did not dissolve in medium [47] so that their toxicityis due to NPs and not by ceric ions although data are alsoavailable which show that CeO

2NPs exposure did not induce

cell death in lung cells and phagocytic cells [46]NPs mediated cell death is supposed to be due to the

production of ROS leading to oxidative stress caused bynanoparticlesrsquo small size and large surface area to volumeratio [48 49] Our result showed that CeO

2NPs are capable

to produce ROS in a concentration and time dependentmanner up to 6 h (Figure 5(a)) But level of ROS was foundto be decreased at 24 h The possible reasons may be eitherdue to the increase in amount of cell death or generationof ROS stabilized after a certain time period However theamount of ROS was also decreased in the presence of NACa ROS inhibitor as shown in Figure 5(b) Further with theincrease in amount of ROS level of GSH was decreased inpresent study It is well known that excess production of ROSleads to decreased level of antioxidant in cultured cells Thuscombined data showed that CeO

2NPs are capable to produce

ROS which is due to decrease in level of cellular antioxidantswhich are correlated with previous studies [16] CeO

2NPs

have also been reported to show antioxidant behavior [15]which has been attributed to the presence of mixed valencestates of CeO

2NPs on their surface [9 50] However the

exact mechanism of oxidantantioxidant behavior of CeO2

NPs remains elusiveAny imbalance between ROS level and antioxidant level

may lead to genomic instability So we assessed the effect ofCeO2NPs induced ROS on genomic DNA by alkaline Comet

assay and fpg modified Comet assay It was found that theamount of DNA damage was increased in concentration andtime dependent manner as suggested by Comet assay param-eters namely Olive tail moment and Tail DNA (Table 2)Theamount of DNA damage was higher in fpg modified Cometassay in comparison to alkaline Comet assay proving theoxidative stress inducedDNAdamage In earlier studies it hasbeen reported that CeO

2NPs did not induce damage to DNA

and chromatin in human cells [51] and have antioxidativeeffect over trichloroethane (TCEtn) exposure [52] which arein contrary to our results But in these studies cell lineswere ofdifferent origins and in the earlier study [52] effect of ceriumoxide alone has not been shown

Excess production of ROS with concomitant decrease inGSH level also leads to mitochondrial membrane permeabil-ity and induction of apoptotic cell death in cultured cells [53ndash55] MMP is important to maintain the potential differenceacross the mitochondrial membrane Loss in MMP is anearly event during apoptosis in cultured cells which results inaccumulation of ROS and redistribution of apoptotic factorsacross the mitochondria [56] In present study we assessedthe MMP loss by the use of JC-1 dye which has dual colorproperties In healthy cells this dye enters the mitochondriaand forms aggregate which gives red color but when thepotentials of mitochondria collapse this dye remains in cyto-plasm and gives green color Thus increase in red to greenfluorescence ratio was used as a marker of MMP loss Ourresults showed that there was a significant (119875 lt 005) con-centration dependent decrease in mitochondrial membranepotential Increased production of ROS with progressivedecrease inMMPhas been proposed asmain event during theapoptosis [34] Here we assessed the apoptosis induction byCeO2NPs using annexinV-FITCPIWe found that therewas

a concentration dependent increase in apoptotic cells whichwere reversed in the presence of Z-DEVD-fmk Howevernecrotic and late apoptotic cells were not found Moreoverin cell cycle analysis we found the significant increase inamount of cell present in sub G1 phase as compared tocontrol

Further to understand the molecular mechanism behindthe apoptotic cell death induced by CeO

2NPs in A549 cells

Western blot analysis was carried out after 24 h exposureOur results showed that there was a concentration dependentincrease in p53 level of treated cells as compared to controlcells (Figure 9(C)) Expression of p53 proteins in mammalsis tightly regulated and serves to protect the organism fromDNA-damaging stimuli In case of extensive DNA damagep53 activates and leads to cell cycle arrest or apoptosis[57] It has also been shown that activation of p53 leads to

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

12 BioMed Research International

upregulation of proapoptotic member of Bcl-2 family suchas Bax and Bak which translocates to mitochondria andsuppresses the activity of antiapoptotic member of Bcl-2family [58] This modulation in BaxBcl-2 ratio leads topermeabilization of mitochondria and release of apoptoticproteins such as cytochromeCwhich binds to cytosolic Apaf-1 [59] This binding results in oligomerization of Apaf-1 andthis complex further binds to caspase-9 to form apoptosomewhich cleaves and activates the downstream caspases (3 6and 7) to initiate mitochondrial mediated apoptosis [60]In addition to this our data also exhibited that there wasa concentration dependent increase in Bax cytochrome Ccaspase-9 and Apaf-1 with progressive decrease in Bcl-2protein expression which suggest the induction of apoptoticcell death pathway in A549 cells exposed to CeO

2NPs

(Figures 9(A) and 9(B)) Consequently there was an increasein expression level of caspase-3 along with PARP a down-stream substrate of activated caspase-3 which confirms theinvolvement of mitochondria in apoptotic cell death

5 Conclusion

Present study showed that cerium oxide NPs get internalizedin A549 cells which cause morphological alterations and tox-icity in a concentration and time dependent manner TheseNPs increase the production of ROS which leads to decreasein antioxidant level of cells and apoptotic cell death Alongwith this ROS production also causes damage to DNA andhalts cell cycle progression which contributed to increasedamount of cell death Immunoblot analysis exhibited that theapoptotic death was mediated through mitochondria whichinvolves the activation of Bcl-2 family mediator includingactivation of PARP Thus extensive DNA damage mediatedmolecular perturbations leads to apoptotic cell death in CeO

2

NPs exposed A549 cells

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding from CSIRNew Delhi for the EMPOWER scheme (OLP-06) and net-work project NanoSHE (BSC-0112) Sandeep Mittal thanksUniversity Grant Commission (UGC) New Delhi for theaward of Junior Research Fellowship Authors are also thank-ful to Dr L K S Chauhan for his help in TEM imaging

References

[1] G J Nohynek J Lademann C Ribaud and M S RobertsldquoGrey Goo on the skin Nanotechnology cosmetic and sun-screen safetyrdquo Critical Reviews in Toxicology vol 37 no 3 pp251ndash277 2007

[2] M Gorensek and P Recelj ldquoNanosilver functionalized cottonfabricrdquo Textile Research Journal vol 77 no 3 pp 138ndash141 2007

[3] AMcWilliamsNanotechnology A ReliasticMarket AssessmentBCC research Wellesley Mass USA 2010

[4] A Corma P Atienzar H Garcıa and J Y Chane-Ching ldquoHier-archically mesostructured doped CeO

2with potential for solar-

cell userdquo Nature Materials vol 3 no 6 pp 394ndash397 2004[5] F Gao Q Lu and S Komarneni ldquoFast synthesis of cerium oxide

nanoparticles and nanorodsrdquo Journal of Nanoscience and Nano-technology vol 6 no 12 pp 3812ndash3819 2006

[6] N IzuW Shin I Matsubara andNMurayama ldquoDevelopmentof resistive oxygen sensors based on cerium oxide thick filmrdquoJournal of Electroceramics vol 13 no 1ndash3 pp 703ndash706 2004

[7] S B Khan M Faisal M M Rahman and A Jamal ldquoExplora-tion of CeO

2nanoparticles as a chemi-sensor and photo-cata-

lyst for environmental applicationsrdquo Science of the Total Envi-ronment vol 409 no 15 pp 2987ndash2992 2011

[8] J Chen S Patil S Seal and J F McGinnis ldquoRare earthnanoparticles prevent retinal degeneration induced by intracel-lular peroxidesrdquo Nature Nanotechnology vol 1 no 2 pp 142ndash150 2006

[9] M Das S Patil N Bhargava et al ldquoAuto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neuronsrdquoBiomaterials vol 28 no 10 pp 1918ndash1925 2007

[10] J Niu A Azfer L M Rogers X Wang and P E KolattukudyldquoCardioprotective effects of cerium oxide nanoparticles in atransgenic murine model of cardiomyopathyrdquo CardiovascularResearch vol 73 no 3 pp 549ndash559 2007

[11] R W Tarnuzzer J Colon S Patil and S Seal ldquoVacancy engi-neered ceria nanostructures for protection from radiation-induced cellular damagerdquo Nano Letters vol 5 no 12 pp 2573ndash2577 2005

[12] Health Effect Institute (HEI) ldquoEvaluation of human health riskfromCerium added to diesel fuelrdquoCommunication vol 9 2001

[13] Organisation for economic cooperation and development(OECD) List of Manufactured Nanomaterials and List of EndPoints for Phase One of the OECD Testing Programme 2008

[14] S Singh A Kumar A Karakoti S Seal andW T Self ldquoUnveil-ing the mechanism of uptake and sub-cellular distribution ofcerium oxide nanoparticlesrdquo Molecular BioSystems vol 6 no10 pp 1813ndash1820 2010

[15] S Chen Y Hou G Cheng C Zhang S Wang and J ZhangldquoCerium oxide nanoparticles protect endothelial cells fromapoptosis induced by oxidative stressrdquo Biological Trace ElementResearch vol 154 no 1 pp 156ndash166 2013

[16] W Lin YWHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[17] E J Park J Choi Y-K Park and K Park ldquoOxidative stressinduced by cerium oxide nanoparticles in cultured BEAS-2Bcellsrdquo Toxicology vol 245 no 1-2 pp 90ndash100 2008

[18] G Cheng W Guo L Han et al ldquoCerium oxide nanoparticlesinduce cytotoxicity in human hepatoma SMMC-7721 cells viaoxidative stress and the activation of MAPK signaling path-waysrdquo Toxicology in Vitro vol 27 no 3 pp 1082ndash1088 2013

[19] K Babin F Antoine D M Goncalves and D Girard ldquoTiO2

CeO2and ZnO nanoparticles and modulation of the degran-

ulation process in human neutrophilsrdquo Toxicology Letters vol221 no 1 pp 57ndash63 2013

[20] G Ciofani G G Genchi B Mazzolai and V Mattoli ldquoTran-scriptional profile of genes involved in oxidative stress and anti-oxidant defense in PC12 cells following treatment with ceriumoxide nanoparticlesrdquo Biochimica et Biophysica Acta vol 1840no 1 pp 495ndash506 2013

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 13

[21] A Clark A Zhu K Sun and H R Petty ldquoCerium oxide andplatinum nanoparticles protect cells from oxidant-mediatedapoptosisrdquo Journal of Nanoparticle Research vol 13 no 10 pp5547ndash5555 2011

[22] J YMa R RMercer M Barger et al ldquoInduction of pulmonaryfibrosis by cerium oxide nanoparticlesrdquo Toxicology and AppliedPharmacology vol 262 no 3 pp 255ndash264 2012

[23] S Aalapati S Ganapathy S Manapuram G Anumolu and BM Prakya ldquoToxicity and bio-accumulation of inhaled ceriumoxide nanoparticles in CD1 micerdquo Nanotoxicology vol 8 no 7pp 786ndash798 2014

[24] S M Hirst A Karakoti S Singh et al ldquoBio-distribution and invivo antioxidant effects of cerium oxide nanoparticles in micerdquoEnvironmental Toxicology vol 28 no 2 pp 107ndash118 2013

[25] H Suzuki T Toyooka andY Ibuki ldquoSimple and easymethod toevaluate uptake potential of nanoparticles in mammalian cellsusing a flow cytometric light scatter analysisrdquo EnvironmentalScience and Technology vol 41 no 8 pp 3018ndash3024 2007

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[29] M Bajpayee A K Pandey D Parmar N Mathur P K SethandADhawan ldquoComet assay responses in human lymphocytesare not influenced by the menstrual cycle a study in healthyIndian femalesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 565 no 2 pp 163ndash172 2005

[30] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquoEnvironmentalMolecularMutagenesis vol 35 no 3 pp206ndash221 2000

[31] A R Collins ldquoThe comet assay for DNA damage and repairprinciples applications and limitationsrdquo Applied Biochemistryand BiotechnologymdashPart BMolecular Biotechnology vol 26 no3 pp 249ndash261 2004

[32] H Tuschl and C E Schwab ldquoFlow cytometric methods usedas screening tests for basal toxicity of chemicalsrdquo Toxicology inVitro vol 18 no 4 pp 483ndash491 2004

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[35] A Asati S Santra C Kaittanis and J M Perez ldquoSurface-charge-dependent cell localization and cytotoxicity of ceriumoxide nanoparticlesrdquo ACS Nano vol 4 no 9 pp 5321ndash53312010

[36] A Kumar A K Pandey S S Singh R Shanker andADhawanldquoA flow cytometric method to assess nanoparticle uptake inbacteriardquo Cytometry Part A vol 79 no 9 pp 707ndash712 2011

[37] R K Shukla A Kumar D Gurbani A K Pandey S Singh andADhawan ldquoTiO

2nanoparticles induce oxidativeDNAdamage

and apoptosis in human liver cellsrdquoNanotoxicology vol 7 no 1pp 48ndash60 2013

[38] S R S Ting J M Whitelock R Tomic et al ldquoCellular uptakeand activity of heparin functionalised cerium oxide nano-particles in monocytesrdquo Biomaterials vol 34 no 17 pp 4377ndash4386 2013

[39] M S LordM JungW Y Teoh et al ldquoCellular uptake and reac-tive oxygen species modulation of cerium oxide nanoparticlesin humanmonocyte cell line U937rdquo Biomaterials vol 33 no 31pp 7915ndash7924 2012

[40] H J Eom and J Choi ldquoOxidative stress of CeO2nanoparticles

via p38-Nrf-2 signaling pathway in human bronchial epithelialcell Beas-2Brdquo Toxicology Letters vol 187 no 2 pp 77ndash83 2009

[41] S Hussain F Al-Nsour A B Rice et al ldquoCerium dioxidenanoparticles induce apoptosis and autophagy in human peri-pheral blood monocytesrdquo ACS Nano vol 6 no 7 pp 5820ndash5829 2012

[42] F Pagliari CMandoli G Forte et al ldquoCeriumoxide nanoparti-cles protect cardiac progenitor cells from oxidative stressrdquo ACSNano vol 6 no 5 pp 3767ndash3775 2012

[43] M Horie K Nishio K Fujita et al ldquoUltrafine NiO particlesinduce cytotoxicity in vitro by cellular uptake and subsequentNi(II) releaserdquo Chemical Research in Toxicology vol 22 no 8pp 1415ndash1426 2009

[44] V Sharma D Anderson and A Dhawan ldquoZinc oxide nanopar-ticles induce oxidative DNA damage and ROS-triggered mito-chondria mediated apoptosis in human liver cells (HepG2)rdquoApoptosis vol 17 no 8 pp 852ndash870 2012

[45] N M Franklin N J Rogers S C Apte G E Batley G EGadd and P S Casey ldquoComparative toxicity of nanopartic-ulate ZnO bulk ZnO and ZnCl2 to a freshwater microalga(Pseudokirchneriella subcapitata) the importance of particlesolubilityrdquoEnvironmental Science andTechnology vol 41 no 24pp 8484ndash8490 2007

[46] T XiaMKovochichM Liong et al ldquoComparison of themech-anism of toxicity of zinc oxide and cerium oxide nanoparticlesbased on dissolution and oxidative stress propertiesrdquoACSNanovol 2 no 10 pp 2121ndash2134 2008

[47] T J Brunner P Wick P Manser et al ldquoIn vitro cytotoxicityof oxide nanoparticles comparison to asbestos silica and theeffect of particle solubilityrdquo Environmental Science and Technol-ogy vol 40 no 14 pp 4374ndash4381 2006

[48] Y Higuchi ldquoGlutathione depletion-induced chromosomalDNA fragmentation associated with apoptosis and necrosisrdquoJournal of Cellular andMolecularMedicine vol 8 no 4 pp 455ndash464 2004

[49] G Oberdorster E Oberdorster and J Oberdorster ldquoNan-otoxicology an emerging discipline evolving from studies ofultrafine particlesrdquo Environmental Health Perspectives vol 113no 7 pp 823ndash839 2005

[50] D Schubert R Dargusch J Raitano and S-W Chan ldquoCeriumand yttrium oxide nanoparticles are neuroprotectiverdquo Biochem-ical and Biophysical Research Communications vol 342 no 1pp 86ndash91 2006

[51] B K Pierscionek Y Li A A Yasseen L M Colhoun R ASchachar and W Chen ldquoNanoceria have no genotoxic effecton human lens epithelial cellsrdquo Nanotechnology vol 21 no 3Article ID 035102 2010

[52] K T Rim S J Kim S W Song and J S Park ldquoEffect ofcerium oxide nanoparticles to inflammation and oxidativeDNAdamages inH9c2 cellsrdquoMolecular andCellular Toxicologyvol 8 no 3 pp 271ndash280 2012

[53] P Jezek and L Hlavata ldquoMitochondria in homeostasis of reac-tive oxygen species in cell tissues and organismrdquo International

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

14 BioMed Research International

Journal of Biochemistry andCell Biology vol 37 no 12 pp 2478ndash2503 2005

[54] P Kaur M Aschner and T Syversen ldquoGlutathione modulationinfluences methyl mercury induced neurotoxicity in primarycell cultures of neurons and astrocytesrdquo NeuroToxicology vol27 no 4 pp 492ndash500 2006

[55] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[56] J E Chipuk L Bouchier-Hayes and D R Green ldquoMitochon-drial outer membrane permeabilization during apoptosis theinnocent bystander scenariordquo Cell Death and Differentiationvol 13 no 8 pp 1396ndash1402 2006

[57] K C Lee W L P Goh M Xu et al ldquoDetection of the p53response in zebrafish embryos using new monoclonal antibod-iesrdquo Oncogene vol 27 no 5 pp 629ndash640 2008

[58] D C S Huang and A Strasser ldquoBH3-only proteinsmdashessentialinitiators of apoptotic cell deathrdquo Cell vol 103 no 6 pp 839ndash842 2000

[59] S Cory and J M Adams ldquoThe BCL2 family regulators of thecellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[60] D Green and G Kroemer ldquoThe central executioners of apop-tosis caspases or mitochondriardquo Trends in Cell Biology vol 8no 7 pp 267ndash271 1998

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of