Research Article ZnO Nanoparticles Treatment Induces ...downloads.hindawi.com/journals/bmri/2015/423287.pdf · Research Article ZnO Nanoparticles Treatment Induces Apoptosis by Increasing

Research ArticleZnO Nanoparticles Treatment Induces Apoptosis byIncreasing Intracellular ROS Levels in LTEP-a-2 Cells

Caixia Wang12 Xiaoke Hu1 Yan Gao3 and Yinglu Ji4

1Key Laboratory of Coastal Biology and Bioresource Utilization Yantai Institute of Coastal Zone ResearchChinese Academy of Sciences 17 Chunhui Road Laishan District Yantai 264003 China2University of Chinese Academy of Sciences Beijing 100049 China3National Oceanographic Center Qingdao 266071 China4College of Marine Life Science Ocean University of China Qingdao 266003 China

Correspondence should be addressed to Xiaoke Hu xkhuyicaccn

Received 22 May 2014 Accepted 7 November 2014

Academic Editor Jose Teixeira

Copyright copy 2015 Caixia Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Owing to thewide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life toxicological research onZnONPsis receiving increasing attention in these days In this study the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cellline LTEP-a-2was tested in vitro Log-phase cells were exposed to different levels of ZnONPs for hours followed by colorimetric cellviability assay using tetrazolium salt and cell survival rate assay using trypan blue dye Cell morphological changes were observed byGiemsa staining and light microscopy Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay Bothintracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method Resultsshowed that ZnONPs (ge001 120583gmL) significantly inhibited proliferation (119875 lt 005) and induced substantial apoptosis in LTEP-a-2cells after 4 h of exposure The intracellular ROS level rose up to 30ndash40 corresponding to significant depletion (approximately70ndash80) in GSH content in LTEP-a-2 cells (119875 lt 005) suggesting that ZnO NPs induced apoptosis mainly through increasedROS production This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells andprovides reference data for application of nanomaterials in the environment

1 Introduction

With rapid development of nanotechnology the applica-tion field and commercial manufacturing scale of syntheticnanomaterials and nanoparticles (hereinafter referred to asNPs) have undergone significant expansion worldwide Thissituation has increasingly aggravated the damage to ecologi-cal environment and human health mainly because variousNPs have diverse effects (small-scale surface quantum-sizeandor macroscopic quantum tunneling) [1] Research ofnanomaterial toxicology is presently at an early developmentstage Associated research has been conducted on carbonnanomaterials first and the test objective has been extendedfrom mouse [2] to aquatic organisms (largemouth bassDaphniamagnaTetrahymena thermophila and crucian carp)and human cells [3] Therefore the biological safety of NPs

has aroused great concerns by governments and academiccircles

Metal oxide nanomaterials such as zinc oxide (ZnO)NPs exhibit antibacterial anticorrosive antifungal and UV-filtering properties as well as certain cytotoxicity [1] Com-pared to titanium dioxide (TiO

2) NPs ZnO NPs exert

relatively strong toxic effects on human pulmonary epithelialcells and the toxicities of both kinds of metal oxide NPs arecontrolled by their physicochemical characteristics (eg sizeand crystal phase) [3] Regarding the underlying mechanismof toxicity TiO

2NPs promote the generation of intracellular

reactive oxygen species (ROS) bymodulating cellmetabolismwith light [4] whereas overproduction of ROS may damagethe antioxidant mechanism in macrophages [5] and causetoxic effects in brain microglia or other cells [6 7] SimilarlyZnO NPs may cause oxidative stress in macrophages and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 423287 9 pageshttpdxdoiorg1011552015423287

2 BioMed Research International

human cells resulting in lipid peroxidation cell membranedamage and ultimately cell death or apoptosis [8 9] Despiteprevious research achievements the toxicological mecha-nism of ZnO NPs has not been elucidated in certain speciesor cancer cells Exploring the exact mechanism of this novelnanomaterial is of great value for clinical trials of cancertreatment

In the present study we assessed the in vitro toxicity ofZnO NPs in a human pulmonary adenocarcinoma cell lineLTEP-a-2 Log-phase cells were exposed to different concen-trations of ZnO NPs for hours followed by in vitro tests ofcell viability survival rate morphological changes apoptosisand intracellular ROS and reduced glutathione (GSH) Theresults were analyzed to explore the toxicological mechanismof ZnO NPs in LTEP-a-2 cells further laying a foundationfor in-depth toxicological study and clinical trials of thisnanomaterial for cancer treatment

2 Materials and Methods

21 ZnO NPs All experiments were carried out on anultraclean bench to prevent interference of external factorsHighly purified (999) ZnO NPs were purchased fromSigma Aldrich (St Louis USA) Stock solutions of ZnONPs were prepared in Dulbeccorsquos modified Eaglersquos medium(DMEM) containing 50 120583gmL fetal bovine serum (FBS)To avoid particle aggregation the prepared solutions weresonicated three times (20 stime) prior to use [10 11] ZnONPs in DMEM were characterized in terms of morphologydiameter tendency of aggregation and intracellular distribu-tion using a scanning electron microscope (SEM Hitachi S-4800 Japan) Zeta potential analysis of ZnO NPs in DMEMwas performed by using dynamic light scattering (MalvernZetasizer ZS9 Worcestershire UK)

22 Cell Culture Human pulmonary adenocarcinoma cellsLTEP-a-2 were obtained from China Center for Type CultureCollection (Wuhan China) and maintained in DMEM cellculture medium (Gibco Grand Island NY USA) supple-mented with 10 FBS 100UmL penicillin and 100120583gmLstreptomycin (37∘C 5 CO

2) For each of the following tests

an aliquot of log-phase culture broth was taken and dilutedto obtain the density of 105-106 cellsmL

23 Cell Viability Assay The viability of LTEP-a-2 cellswas assayed by using the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT)method [12] Log-phasecells were harvested and thoroughly washed with phosphate-buffered saline (PBS) and then inoculated into 96-well plates(Nunc Roskilde Denmark) When the cell density reachedapproximately 5 times 104 cellswell different concentrations ofZnO NPs (0 control 001 025 05 10 and 15 120583gmL) wereadded into triplicate wells for 4 8 12 and 24 h of exposureAfter aspirated incubation a medium containing 20 120583L of5mgmL MTT was added and the culture was continuouslyincubated Four hours later blue formazan crystal appearedat the bottom of wells which was then dissolved with 150 120583Ldimethyl sulfoxide Cell viability was detected by measuring

the absorbance of cell culture broth at 490 nm using amicroplate reader (Thermo Varioskan Flash 3001 USA)

24 Trypan Blue Exclusion Test The lethality of ZnO NPson LTEP-a-2 cells was assessed by the trypan blue exclusiontest [13] Cells were seeded in 6-well plates with differentconcentrations of ZnO NPs (0 control 005 01 02 10and 50120583gmL) for 12 h of exposure in a humidified incu-bator (5 CO

2 37∘C) Thereafter cells were trypsinized

and resuspended in equal volumes of culture medium andtrypan Viable (unstained) and nonviable (blue-stained) cellswere counted using a haemocytometer to calculate the totalnumbers of living and dead cells

25 Morphological Assay LTEP-a-2 cells were cultured in6-well plates with different concentrations of ZnO NPs (0control 001 005 01 02 and 05 120583gmL) for 4 h of exposureand then fixed with methanol and dried The cells werestained for 20min with Giemsa staining solution rinsed indeionized water air-dried and examined under an opticalmicroscope (SH-60 Olympus Japan) equipped with a digitalcamera [14]The stained cells were examined in terms of sizeregularity of themargin andmorphological characteristics ofthe nucleus

26 Apoptosis Detection ZnO NPs-induced apoptosis after4 h of exposure was detected by acridine orangeethidiumbromide (AOEB) double staining Cells were stained with100 120583gmL AOEB (Sigma USA) for 2min followed byexamination using a fluorescence microscope (Leica DM5000B Leica Microsystems Germany) The detection crite-rion is that normal cells present uniformgreennuclei and lateapoptotic cells present orange to red nuclei with condensed orfragmented chromatin [15 16]

27 Caspase Activity Assay Caspase activity was assayedaccording to themethod ofVyas et al [17] Cells were culturedin 96-well plates with indicated concentrations of ZnO NPsfor 4 h and then harvested by centrifugation at 1000timesg for10min The activity of caspase-3 was detected by using acolorimetric assay kit (Nanjing Jiancheng BioengineeringInstitute Nanjing China) Cells were washed with PBSand resuspended in five volumes of lysis buffer (20mmolLHepes pH 79 20 Glycerol 200mmolL KCl 05mmolLEDTA 05 NP40 05mmolL DTT and 1 proteaseinhibitor cocktail) The content of protein was measured byusing the Bradfordmethod and the absorption of cell culturebroth at 405 nm was measured using a microplate reader(Infinite M200 Tecan Switzerland) [18] All treatments wereperformed in triplicate

28 Intracellular ROS Assay The intracellular ROS level wasmeasured by active oxygen detection [19ndash21] H2DCFDAwasdeacetylated intracellularly by using a nonspecific esteraseand then oxidized by cellular peroxides yielding a fluores-cent compound 27-dichlorofluorescein (DCF 120582EX120582EM =485 nm535 nm) Cells were treated with indicated concen-trations of ZnO NPs (0 control 001 005 05 10 and

BioMed Research International 3

(a)

0 1 2 3 4 5 6 7 8 9

0

5

10

15

20

25

30

35

40

ZnO

minus5

Num

ber o

f ZnO

par

ticle

s (

)

Size (d nm)

(b)

Nanoparticles Chemical composition

Source Diameter(nm)

Density (gmL)

pH Zeta potential(mV)(DI

water)

Average diameter in DLS

(nm)

ZnO Zn O Sigma Aldrich

lt100 lt3517 plusmn 01 7 plusmn 01 minus186

(c)

Figure 1 Major characteristics of ZnO nanoparticles used in this study (a) Scanning electronmicrograph (b) size distribution and (c) majorphysical properties

15 120583gmL) for 4 h and then washed with PBS and incubatedin 30 120583molL H

2DCFDA at 37∘C for 30min The content of

DCF was detected by using a microplate reader (VarioskanFlash 3001 Thermo USA) Each group was maintained withthe same number of cells in triplicate

29 Intracellular GSH Content Assay The intracellular GSHcontentwas determined by using amicroplate-readermethodwith a commercial kit (Nanjing Jiancheng BioengineeringInstitute Nanjing China) LTEP-a-2 cells were inoculatedinto 6-well plates at 106cellswell and then exposed to differentconcentrations of ZnO NPs (0 control 001 005 and025 120583gmL) for 4 h Cells were then harvested and washedwith PBS The content of GSH was assayed by measuring theabsorbance of cell extract at 412 nmusing amicroplate readercalculated according to a standard curve and normalizedby the protein concentration detected using the Bradfordmethod (Sangon Shanghai China) [22]

210 Statistical Analysis All experimental data are presentedas means plusmn standard error of the mean from at least threeindependent experiments Data comparison between treat-ments was accomplished by one-way analysis of variance andStudentrsquos 119905-test (119875 lt 005 considered statistically significant)

Statistical analysis was performed in SPSS160 (SPSS IncUSA) and Origin 60 (OriginLab Corp USA)

3 Results and Discussion

31 Characteristics of ZnO NPs A description of the mor-phology and physicochemical properties of ZnO NPs isregarded as a comparative study in the field of cytotoxicityresearch [23 24] In the present study SEM image showsthat the ZnO NPs in use are mainly anxiolytic shaped andare partially rhombic (Figure 1(a)) Mean grain diameter ofthe ZnO NPs is 30 plusmn 5 nm which matches the supplierrsquosdeclaration Zeta potential data indicate that the ZnO NPshave a positive surface charge minus186mV at pH 74 in DMEM(Figure 1(b)) which is inadequate to stabilize the suspensionof ZnO NPs via repulsive force and thus may cause NPsaggregation in DMEM The size distribution of ZnO NPs inDMEM as determined by dynamic light scattering showsgreat variations (Figure 1(c))

32 Cytotoxicity of ZnO NPs The cytotoxicity of ZnO NPsin LTEP-a-2 cells was tested by MTT assay using a protocoladopted from previously published reports and manufac-turerrsquos instructions [9 25 26] expressed as the percentage


0 001 025 05 1 15

00

01

02

03

04

05

06

07

08

Gro

wth

inhi

bitio

n (

of c

ontro

l)

Concentration of ZnO particles (120583gmL)

4h8h

minus01

lowast

lowastlowast lowastlowast

lowastlowast

(a)

0 001 025 05 1 15

0001020304050607080910

Gro

wth

inhi

bitio

n (

of c

ontro

l)


12h24h

minus01

lowastlowast lowast

(b)

Figure 2 Relative viability of LTEP-a-2 cells after 4ndash24 h of exposure to different concentrations of ZnO nanoparticles (0 120583gmL control)(a) 4 and 8 h and (b) 12 and 24 h lowast versus control 119875 lt 005 lowastlowast versus control 119875 lt 001 by Studentrsquos 119905-test

of cell mortality relative to the control treatment (Figure 2)After 4ndash24 h of exposure to ZnO NPs (001ndash15 120583gmL) cellviability declined substantially in a concentration- and time-dependent manner the declines were especially significantafter 8 h of exposure to ZnO NPs ge 025 120583gmL (119875 lt005) The number of cell deaths among all these doses hasbeen nearly 20 higher than the lower doses over the past24 h High cytotoxicity can be observed in cells treated withZnO NPs when compared to control group These resultsindicate that cell proliferationwas inhibited significantly withincreasing concentration of ZnO NPs

33 ZnO NPs Reduced Cell Survival Rate The dye exclusiontest was used to determine the number of viable cells presentin a cell suspension This method is based on the principlethat live cells possess intact cell membranes that excludecertain dyes such as trypan blue eosin or propidiumwhereas dead cells do not [27] In this test a cell suspensionwas simply mixed with dye and then visually examined todetermine whether cells take up or exclude dye A viablecell was identified with a clear cytoplasm and a nonviablecell with a blue cytoplasm Results showed that after 12 hof exposure to ZnO NPs (005ndash50 120583gmL) the survivalrate of LTEP-a-2 cells underwent substantial decreases in aconcentrate-dependent manner (Figure 3) In the presenceof low concentration of ZnO NPs (005 120583gmL) cell survivalrate remained above 60 showing a nearly 40 decreaserelative to the control treatment as the concentration of ZnONPs was increased to 01 120583gmL cell survival rate underwentanother 40 decrease down to approximately 20 onlyTogether these results confirm that the presence of ZnO NPssignificantly affected cell survival even at low concentrations(eg 005ndash01 120583gmL)

0 005 01 02 1 5

0

20

40

60

80

100

120

Cell

surv

ival

rate

()


Figure 3The survival rate of LTEP-a-2 cells detected by trypan blueexclusion test after 12 h of exposure to different concentrations ofZnO nanoparticles (0 120583gmL control)

34 ZnO NPs InducedMorphological Changes Giemsa stain-ing is commonly used for identifying morphological changesof monocytesmacrophages in cell preparation [28] In thepresent study Giesma staining was used to examine ZnONPs-induced morphological changes in LTEP-a-2 cells forfurther characterizing the cytotoxicity of this nanomaterialMicroscopic examinations revealed that Giemsa-stained con-trol cells (0120583gmL ZnO NPs) predominantly had roundregular cell margins and large nuclei (Figure 4) that is thecontrol cells were associated with rapid DNA synthesis andfast proliferation With increasing concentrations of ZnONPs (005 01 and 05 120583gmL) there were evident increases


0120583gmL 005 120583gmL

01 120583gmL 05 120583gmL

Figure 4 ZnO nanoparticles-induced morphological changes in LTEP-a-2 cells examined by Giemsa staining Note significant changes incells after 4 h of exposure to ZnO NPs (0120583gmL control)

in the number of pyknotic shrinking cells following a dose-dependent manner (Figure 4) the increasing morphologicalchanges in the presence of ZnO NPs coincided well withthe declines in cell survival rate (Figure 3) thus they wereassociated with proliferation inhibition andor cell death

35 ZnO NPs Induced Apoptosis AOEB staining is con-sidered an ideal method for distinguishing apoptotic cellsfrom necrotic ones [29 30] Here we use AOEB stainingto verify whether ZnO NPs inhibit proliferation of LTEP-a-2cells by inducing apoptosis or killing cells directly (necrosis)During morphologic examinations normal viable LTEP-a-2 control cells were stained green (0 120583gmL ZnO NPs)and the apoptotic cells exposed to ZnO NPs (005 01and 02 120583gmL) appeared as bright green arcs in an earlystage and with condensed yelloworange nuclei in the latestage (Figure 5(a)) Additionally the results of caspase-3activity assay showed that exposure of ZnO NPs inducedsignificant increases in caspase-3 activity compared to thecontrol treatment (119875 lt 005 for 001ndash005120583gmL ZnO NPs119875 lt 001 for 01ndash05120583gL ZnO NPs Figure 5(b)) As a keymediator caspase-3 plays a pivotal role in caspase-dependentapoptosis [31] Together these results confirm that exposureof ZnO NPs induced substantial apoptosis in LTEP-a-2 cellseven at low concentrations (eg 001120583gmL) thus inhibitingcell proliferation

36 ZnO NPs Increased Intracellular ROS Oxidative stressis considered one of the causative factors of apoptosis inpathogenesis and aggressiveness of most cancers [32] Amoderate rise in ROS level often induces cell proliferationwhereas excessive amounts of ROS induce apoptosis [33]To clarify the mechanism through which ZnO NPs induceapoptosis in LTEP-a-2 cells we determined the intracellularROS level by measuring the oxidation of nonfluorescentDCFH-DA to its highly fluorescent derivative DCF Resultsshowed that ZnO NPs stimulated ROS formation in cellsfollowing a concentration-dependent manner (Figure 6(a))Under a fluorescence microscope strong green fluorescencewas observed in LTEP-a-2 control cells whereas blue fluores-cence was observed in cells after exposure to ZnO NPs Withincreasing concentrations of ZnO NPs the blue fluorescencewas greatly strengthened accompanied by the appearanceof apoptosis vesicles (Figure 6(b)) Together these resultsdemonstrate that ZnO NPs induce apoptosis in LTEP-a-2cells through increased production of ROS consistent withprevious findings inmacrophages andhuman liver cells [8 9]as well as in vivo and in vitro tests of a wide range of NPsspecies [34 35]

37 ZnO NPs Decreased Intracellular GSH Content GSH isone of the most abundant intracellular antioxidant thiolswhich is involved in cell redox homeostasis and is central


(A) (B)

(C) (D)

(a)

0 001 005 01 02 05

117

118

119

120

121

122

123

124

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Casp

ase a

ctiv

ity (a

bsor

banc

e at4

05

nm)

Concentration of ZnO NPs (120583gmL)

(b)

Figure 5 ZnO nanoparticles-induced apoptosis in LTEP-a-2 cells after 4 h of exposure (a) Morphologic examination of LTEP-a-2 cells byAOEB fluorescence staining ((A) 0 120583gmL control (B) 005 120583gmL (C) 01 120583gmL and (D) 02 120583gmL) and (b) caspase-3 activity lowast versuscontrol 119875 lt 005 lowastlowast versus control 119875 lt 001 by Studentrsquos 119905-test

to defensive mechanisms against toxic agents and oxidant-mediated injury [36 37] In the present study the GSHcontent significantly declined in LTEP-a-2 cells exposedto ZnO NPs (001ndash025 120583gmL) for 4 h compared with thecontrol cells (119875 lt 005 Figure 7) The depletion of GSHcoincided with the enlarging tendency of intracellular ROSlevel (Figure 6) once again demonstrating that ZnO NPsdamaged the antioxidant mechanism of LTEP-a-2 cells

4 Conclusions

In recent decades nanomedicine has attracted considerableattention in the field of medicine [38ndash41] Despite theadvantages of nanotechnology appropriate securitymeasuresshould be taken to prevent potential hazardous effects ofNPs In the present study in vitro test results show that ZnONPs imposed distinct toxic effect on LTEP-a-2 cells and


Con

trol

0

200

400

600

800

1000

1200

1400

DCF

fluo

resc

ence

(RFU

)

2h4h

Concentration of ZnO NPs001120583

gm

L

005120583

gm

L

05120583

gm

L

15120583

gm

L

1120583

gm

L

(a)

01 120583gmL 05 120583gmL

001 120583gmL0120583gmL

(b)

Figure 6 Increased production of intracellular reactive oxygen species (ROS) in LTEP-a-2 cells after 4 h of exposure to ZnO nanoparticles(a) ROS level determined via spectrophotometry and (b) fluorescence intensity measured by microscopy

the declines in cell viability and survival rate coincided withspecific morphological changes and the occurrence of apop-tosis Further exploration of the toxicological mechanismrevealed that the increase in ROS coincided with depletionof GSH in apoptotic cells suggesting that oxidative stressmay be the primary toxicological mechanism of ZnO NPs in

LTEP-a-2 cells As a common mechanism for NPs-inducedcell oxidative damage increased ROS generation has beenconfirmed by in vivo and in vitro tests of a wide rangeof NPs species Oxidative stress often leads to cell deathby either apoptosis signaling pathways or necrosis signalingpathways depending on its extent of severity With increasing


0 001 005 025

5

10

15

20

25

30


The c

onte

nt o

f GSH

(120583m

g p

rote

in)

lowast

lowast lowast

Figure 7 Depletion of intracellular glutathione (GSH) in LTEP-a-2cells after 4 h of exposure to ZnO nanoparticles (0120583gmL control)GSH content determined by spectrophotometry lowast119875 lt 005 lowastlowast119875 lt001 versus control by Studentrsquos 119905-test

evidence for the toxicity of NPs it is important to plan outprecautionary measures and to prevent human exposures toNPs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding for this research was provided by the HundredTalents Program of Chinese Academy of Sciences and theYantai Municipal Science and Technology Project of Shan-dong Province China (Grant no 2012017)

References

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[2] C-W Lam J T James R McCluskey and R L HunterldquoPulmonary toxicity of single-wall carbon nanotubes in mice7 and 90 days after intractracheal instillationrdquo ToxicologicalSciences vol 77 no 1 pp 126ndash134 2004

[3] I-L Hsiao and Y-J Huang ldquoEffects of various physicochemicalcharacteristics on the toxicities of ZnO and TiO

2nanoparticles

toward human lung epithelial cellsrdquo Science of the Total Envi-ronment vol 409 no 7 pp 1219ndash1228 2011

[4] J Jiang G Oberdorster A Elder R Gelein P Mercer andP Biswas ldquoDoes nanoparticle activity depend upon size andcrystal phaserdquo Nanotoxicology vol 2 no 1 pp 33ndash42 2008

[5] D M Brown K Donaldson P J Borm et al ldquoCalcium andROS-mediated activation of transcription factors and TNF-120572cytokine gene expression in macrophages exposed to ultrafineparticlesrdquo American Journal of PhysiologymdashLung Cellular andMolecular Physiology vol 286 no 2 pp L344ndashL353 2004

[6] T C Long N Saleh R D Tilton G V Lowry and B VeronesildquoTitanium dioxide (P25) produces reactive oxygen species inimmortalized brain microglia (BV2) implications for nanopar-ticle neurotoxicityrdquo Environmental Science and Technology vol40 no 14 pp 4346ndash4352 2006

[7] A Nel T Xia LMadler andN Li ldquoToxic potential of materialsat the nanolevelrdquo Science vol 311 no 5761 pp 622ndash627 2006

[8] V Wilhelmi U Fischer H Weighardt et al ldquoZinc oxidenanoparticles induce necrosis and apoptosis in macrophages ina p47phox- and Nrf2-independent mannerrdquo PLoS ONE vol 8no 6 Article ID e65704 2013

[9] 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

[10] C Garcıa-Gomez M D Fernandez and M Babin ldquoEcotoxi-cological evaluation of sewage sludge contaminated with zincoxide nanoparticlesrdquo Archives of Environmental Contaminationand Toxicology vol 67 no 4 pp 494ndash506 2014

[11] C M Sayes K L Reed D B Warheit et al ldquoAssessing toxicityof fine and nanoparticles comparing in vitro measurements toin vivo pulmonarytoxicity profilesrdquo Toxicological Sciences vol97 no 1 pp 163ndash180 2007

[12] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[13] J Li C-Y Huang R-L Zheng K-R Cui and J-F Li ldquoHydro-gen peroxide induces apoptosis in human hepatoma cells andalters cell redox statusrdquo Cell Biology International vol 24 no 1pp 9ndash23 2000

[14] D S Kim S H Kim J H Song Y-T Chang S Y Hwang andT S Kim ldquoEnhancing effects of ceramide derivatives on 125-dihydroxyvitamin D

3-induced differentiation of human HL-60

leukemia cellsrdquo Life Sciences vol 81 no 25-26 pp 1638ndash16442007

[15] D L Pitrak H C Tsai K M Mullane S H Sutton andP Stevens ldquoAccelerated neutrophil apoptosis in the acquiredimmunodeficiency syndromerdquoThe Journal of Clinical Investiga-tion vol 98 no 12 pp 2714ndash2719 1996

[16] W Chengya J Eshleman J Lutterbaugh B Y J Willson and SMarkowitz ldquoSpontaneous apoptosis in human colon tumor celllines and the relation of wt p53 to apoptosisrdquo Chinese MedicalJournal vol 109 no 7 pp 537ndash541 1996

[17] K M Vyas R N Jadeja D Patel R V Devkar and VK Gupta ldquoA new pyrazolone based ternary Cu(II) complexsynthesis characterization crystal structure DNA bindingprotein binding and anti-cancer activity towards A549 humanlung carcinoma cells with a minimum cytotoxicity to non-cancerous cellsrdquo Polyhedron vol 65 pp 262ndash274 2013

[18] X Yuan B Zhang L Gan et al ldquoInvolvement of the mito-chondrion-dependent and the endoplasmic reticulum stress-signaling pathways in isoliquiritigenin-induced apoptosis ofHeLa cellrdquo Biomedical and Environmental Sciences vol 26 no4 pp 268ndash276 2013

[19] T L Vanden Hoek C Li Z Shao P T Schumacker and L BBecker ldquoSignificant levels of oxidants are generated by isolatedcardiomyocytes during ischemia prior to reperfusionrdquo Journalof Molecular and Cellular Cardiology vol 29 no 9 pp 2571ndash2583 1997

[20] J A Royall and H Ischiropoulos ldquoEvaluation of 2101584071015840-dichlo-rofluorescin and dihydrorhodamine 123 as fluorescent probes


for intracellular H2O2in cultured endothelial cellsrdquo Archives of

Biochemistry and Biophysics vol 302 no 2 pp 348ndash355 1993[21] B-G Park H-J Jung Y-W Cho H-W Lim and C-J Lim

ldquoPotentiation of antioxidative and anti-inflammatory propertiesof cultured wild ginseng root extract through probiotic fermen-tationrdquo Journal of Pharmacy and Pharmacology vol 65 no 3pp 457ndash464 2013

[22] C Chen X Jiang Y Hu and Z Zhang ldquoThe protective roleof resveratrol in the sodium arsenite-induced oxidative damagevia modulation of intracellular GSH homeostasisrdquo BiologicalTrace Element Research vol 155 no 1 pp 119ndash131 2013

[23] R J Vandebriel and W H de Jong ldquoA review of mammaliantoxicity of ZnO nanoparticlesrdquo Nanotechnology Science andApplications vol 2012 no 5 pp 61ndash71 2012

[24] H J Johnston G R Hutchison F M Christensen S PetersS Hankin and V Stone ldquoIdentification of the mechanismsthat drive the toxicity of TiO

2particulates the contribution of

physicochemical characteristicsrdquo Particle and Fibre Toxicologyvol 6 article 33 2009

[25] A Jaeger D G Weiss L Jonas and R Kriehuber ldquoOxidativestress-induced cytotoxic and genotoxic effects of nano-sizedtitanium dioxide particles in human HaCaT keratinocytesrdquoToxicology vol 296 no 1ndash3 pp 27ndash36 2012

[26] B C Heng X Zhao E C Tan et al ldquoEvaluation of the cytotoxicand inflammatory potential of differentially shaped zinc oxidenanoparticlesrdquo Archives of Toxicology vol 85 no 12 pp 1517ndash1528 2011

[27] W Strober ldquoTrypan blue exclusion test of cell viabilityrdquo CurrentProtocols in Immunology 2001

[28] W Strober ldquoWright-Giemsa andnonspecific esterase staining ofcellsrdquo inCurrent Protocols in Immunology appendix 3 appendix3D 2001

[29] J Monga S Pandit R S Chauhan C S Chauhan S SChauhan and M Sharma ldquoGrowth inhibition and apoptosisinduction by(+)-cyanidan-3-ol in hepatocellular carcinomardquoPLoS ONE vol 8 no 7 Article ID e68710 2013

[30] Y Wang Y S Xu L H Yin et al ldquoSynergistic anti-gliomaeffect of Hydroxygenkwanin and Apigenin in vitrordquo Chemico-Biological Interactions vol 206 no 2 pp 346ndash355 2013

[31] D Gao Z Xu P Qiao et al ldquoCadmium induces liver cellapoptosis through caspase-3A activation in purse red commoncarp (Cyprinus carpio)rdquo PLoS ONE vol 8 no 12 Article IDe83423 2013

[32] J F Curtin M Donovan and T G Cotter ldquoRegulationand measurement of oxidative stress in apoptosisrdquo Journal ofImmunological Methods vol 265 no 1-2 pp 49ndash72 2002

[33] T P Das S Suman and C Damodaran ldquoReactive oxygenspecies generation inhibits epithelial-mesenchymal transitionand promotes growth arrest in prostate cancer cellsrdquoMolecularCarcinogenesis vol 53 no 7 pp 537ndash547 2013

[34] M J Akhtar S Kumar H A Alhadlaq S A Alrokayan KM Abu-Salah and M Ahamed ldquoDose-dependent genotoxicityof copper oxide nanoparticles stimulated by reactive oxygenspecies in human lung epithelial cellsrdquoToxicology and IndustrialHealth In press

[35] K Donaldson V Stone A Seaton and W MacNee ldquoAmbientparticle inhalation and the cardiovascular system potentialmechanismsrdquo Environmental Health Perspectives vol 109 no 4pp 523ndash527 2001

[36] DM Townsend K D Tew andH Tapiero ldquoThe importance ofglutathione in human diseaserdquo Biomedicine and Pharmacother-apy vol 57 no 3ndash4 pp 145ndash155 2003

[37] S Catarzi F Favilli C Romagnoli et al ldquoOxidative state andIL-6 production in intestinal myofibroblasts of Crohnrsquos diseasepatientsrdquo Inflammatory Bowel Diseases vol 17 no 8 pp 1674ndash1684 2011

[38] M Ahamed M J Akhtar M Raja et al ldquoZnO nanorod-induced apoptosis in human alveolar adenocarcinoma cells viap53 survivin and baxbcl-2 pathways role of oxidative stressrdquoNanomedicine vol 7 no 6 pp 904ndash913 2011

[39] C Hanley J Layne A Punnoose et al ldquoPreferential killing ofcancer cells and activated human T cells using ZnO nanoparti-clesrdquo Nanotechnology vol 19 no 29 Article ID 295103 2008

[40] R Brayner R Ferrari-Iliou N Brivois S Djediat M FBenedetti and F Fievet ldquoToxicological impact studies based onEscherichia coli bacteria in ultrafineZnOnanoparticles colloidalmediumrdquo Nano Letters vol 6 no 4 pp 866ndash870 2006

[41] M Premanathan K Karthikeyan K Jeyasubramanian and GManivannan ldquoSelective toxicity of ZnO nanoparticles towardGram-positive bacteria and cancer cells by apoptosis throughlipid peroxidationrdquo Nanomedicine vol 7 no 2 pp 184ndash1922011

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human cells resulting in lipid peroxidation cell membranedamage and ultimately cell death or apoptosis [8 9] Despiteprevious research achievements the toxicological mecha-nism of ZnO NPs has not been elucidated in certain speciesor cancer cells Exploring the exact mechanism of this novelnanomaterial is of great value for clinical trials of cancertreatment

In the present study we assessed the in vitro toxicity ofZnO NPs in a human pulmonary adenocarcinoma cell lineLTEP-a-2 Log-phase cells were exposed to different concen-trations of ZnO NPs for hours followed by in vitro tests ofcell viability survival rate morphological changes apoptosisand intracellular ROS and reduced glutathione (GSH) Theresults were analyzed to explore the toxicological mechanismof ZnO NPs in LTEP-a-2 cells further laying a foundationfor in-depth toxicological study and clinical trials of thisnanomaterial for cancer treatment

2 Materials and Methods

21 ZnO NPs All experiments were carried out on anultraclean bench to prevent interference of external factorsHighly purified (999) ZnO NPs were purchased fromSigma Aldrich (St Louis USA) Stock solutions of ZnONPs were prepared in Dulbeccorsquos modified Eaglersquos medium(DMEM) containing 50 120583gmL fetal bovine serum (FBS)To avoid particle aggregation the prepared solutions weresonicated three times (20 stime) prior to use [10 11] ZnONPs in DMEM were characterized in terms of morphologydiameter tendency of aggregation and intracellular distribu-tion using a scanning electron microscope (SEM Hitachi S-4800 Japan) Zeta potential analysis of ZnO NPs in DMEMwas performed by using dynamic light scattering (MalvernZetasizer ZS9 Worcestershire UK)

22 Cell Culture Human pulmonary adenocarcinoma cellsLTEP-a-2 were obtained from China Center for Type CultureCollection (Wuhan China) and maintained in DMEM cellculture medium (Gibco Grand Island NY USA) supple-mented with 10 FBS 100UmL penicillin and 100120583gmLstreptomycin (37∘C 5 CO

2) For each of the following tests

an aliquot of log-phase culture broth was taken and dilutedto obtain the density of 105-106 cellsmL

23 Cell Viability Assay The viability of LTEP-a-2 cellswas assayed by using the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT)method [12] Log-phasecells were harvested and thoroughly washed with phosphate-buffered saline (PBS) and then inoculated into 96-well plates(Nunc Roskilde Denmark) When the cell density reachedapproximately 5 times 104 cellswell different concentrations ofZnO NPs (0 control 001 025 05 10 and 15 120583gmL) wereadded into triplicate wells for 4 8 12 and 24 h of exposureAfter aspirated incubation a medium containing 20 120583L of5mgmL MTT was added and the culture was continuouslyincubated Four hours later blue formazan crystal appearedat the bottom of wells which was then dissolved with 150 120583Ldimethyl sulfoxide Cell viability was detected by measuring

the absorbance of cell culture broth at 490 nm using amicroplate reader (Thermo Varioskan Flash 3001 USA)

24 Trypan Blue Exclusion Test The lethality of ZnO NPson LTEP-a-2 cells was assessed by the trypan blue exclusiontest [13] Cells were seeded in 6-well plates with differentconcentrations of ZnO NPs (0 control 005 01 02 10and 50120583gmL) for 12 h of exposure in a humidified incu-bator (5 CO

2 37∘C) Thereafter cells were trypsinized

and resuspended in equal volumes of culture medium andtrypan Viable (unstained) and nonviable (blue-stained) cellswere counted using a haemocytometer to calculate the totalnumbers of living and dead cells

25 Morphological Assay LTEP-a-2 cells were cultured in6-well plates with different concentrations of ZnO NPs (0control 001 005 01 02 and 05 120583gmL) for 4 h of exposureand then fixed with methanol and dried The cells werestained for 20min with Giemsa staining solution rinsed indeionized water air-dried and examined under an opticalmicroscope (SH-60 Olympus Japan) equipped with a digitalcamera [14]The stained cells were examined in terms of sizeregularity of themargin andmorphological characteristics ofthe nucleus

26 Apoptosis Detection ZnO NPs-induced apoptosis after4 h of exposure was detected by acridine orangeethidiumbromide (AOEB) double staining Cells were stained with100 120583gmL AOEB (Sigma USA) for 2min followed byexamination using a fluorescence microscope (Leica DM5000B Leica Microsystems Germany) The detection crite-rion is that normal cells present uniformgreennuclei and lateapoptotic cells present orange to red nuclei with condensed orfragmented chromatin [15 16]

27 Caspase Activity Assay Caspase activity was assayedaccording to themethod ofVyas et al [17] Cells were culturedin 96-well plates with indicated concentrations of ZnO NPsfor 4 h and then harvested by centrifugation at 1000timesg for10min The activity of caspase-3 was detected by using acolorimetric assay kit (Nanjing Jiancheng BioengineeringInstitute Nanjing China) Cells were washed with PBSand resuspended in five volumes of lysis buffer (20mmolLHepes pH 79 20 Glycerol 200mmolL KCl 05mmolLEDTA 05 NP40 05mmolL DTT and 1 proteaseinhibitor cocktail) The content of protein was measured byusing the Bradfordmethod and the absorption of cell culturebroth at 405 nm was measured using a microplate reader(Infinite M200 Tecan Switzerland) [18] All treatments wereperformed in triplicate

28 Intracellular ROS Assay The intracellular ROS level wasmeasured by active oxygen detection [19ndash21] H2DCFDAwasdeacetylated intracellularly by using a nonspecific esteraseand then oxidized by cellular peroxides yielding a fluores-cent compound 27-dichlorofluorescein (DCF 120582EX120582EM =485 nm535 nm) Cells were treated with indicated concen-trations of ZnO NPs (0 control 001 005 05 10 and


(a)

0 1 2 3 4 5 6 7 8 9

0

5

10

15

20

25

30

35

40

ZnO

minus5

Num

ber o

f ZnO

par

ticle

s (

)

Size (d nm)

(b)


Source Diameter(nm)

Density (gmL)


water)


(nm)



(c)












0 001 025 05 1 15

00

01

02

03

04

05

06

07

08

Gro

wth

inhi

bitio

n (

of c

ontro

l)


4h8h

minus01

lowast


lowastlowast

(a)

0 001 025 05 1 15

0001020304050607080910

Gro

wth

inhi

bitio

n (

of c

ontro

l)


12h24h

minus01

lowastlowast lowast

(b)




0 005 01 02 1 5

0

20

40

60

80

100

120

Cell

surv

ival

rate

()





0120583gmL 005 120583gmL

01 120583gmL 05 120583gmL







(A) (B)

(C) (D)

(a)

0 001 005 01 02 05

117

118

119

120

121

122

123

124

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Casp

ase a

ctiv

ity (a

bsor

banc

e at4

05

nm)


(b)



4 Conclusions



Con

trol

0

200

400

600

800

1000

1200

1400

DCF

fluo

resc

ence

(RFU

)

2h4h


gm

L

005120583

gm

L

05120583

gm

L

15120583

gm

L

1120583

gm

L

(a)

01 120583gmL 05 120583gmL

001 120583gmL0120583gmL

(b)





0 001 005 025

5

10

15

20

25

30


The c

onte

nt o

f GSH

(120583m

g p

rote

in)

lowast

lowast lowast





Acknowledgments


References




2nanoparticles






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

0 1 2 3 4 5 6 7 8 9

0

5

10

15

20

25

30

35

40

ZnO

minus5

Num

ber o

f ZnO

par

ticle

s (

)

Size (d nm)

(b)


Source Diameter(nm)

Density (gmL)


water)


(nm)



(c)












0 001 025 05 1 15

00

01

02

03

04

05

06

07

08

Gro

wth

inhi

bitio

n (

of c

ontro

l)


4h8h

minus01

lowast


lowastlowast

(a)

0 001 025 05 1 15

0001020304050607080910

Gro

wth

inhi

bitio

n (

of c

ontro

l)


12h24h

minus01

lowastlowast lowast

(b)




0 005 01 02 1 5

0

20

40

60

80

100

120

Cell

surv

ival

rate

()





0120583gmL 005 120583gmL

01 120583gmL 05 120583gmL







(A) (B)

(C) (D)

(a)

0 001 005 01 02 05

117

118

119

120

121

122

123

124

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Casp

ase a

ctiv

ity (a

bsor

banc

e at4

05

nm)


(b)



4 Conclusions



Con

trol

0

200

400

600

800

1000

1200

1400

DCF

fluo

resc

ence

(RFU

)

2h4h


gm

L

005120583

gm

L

05120583

gm

L

15120583

gm

L

1120583

gm

L

(a)

01 120583gmL 05 120583gmL

001 120583gmL0120583gmL

(b)





0 001 005 025

5

10

15

20

25

30


The c

onte

nt o

f GSH

(120583m

g p

rote

in)

lowast

lowast lowast





Acknowledgments


References




2nanoparticles






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 001 025 05 1 15

00

01

02

03

04

05

06

07

08

Gro

wth

inhi

bitio

n (

of c

ontro

l)


4h8h

minus01

lowast


lowastlowast

(a)

0 001 025 05 1 15

0001020304050607080910

Gro

wth

inhi

bitio

n (

of c

ontro

l)


12h24h

minus01

lowastlowast lowast

(b)




0 005 01 02 1 5

0

20

40

60

80

100

120

Cell

surv

ival

rate

()





0120583gmL 005 120583gmL

01 120583gmL 05 120583gmL







(A) (B)

(C) (D)

(a)

0 001 005 01 02 05

117

118

119

120

121

122

123

124

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Casp

ase a

ctiv

ity (a

bsor

banc

e at4

05

nm)


(b)



4 Conclusions



Con

trol

0

200

400

600

800

1000

1200

1400

DCF

fluo

resc

ence

(RFU

)

2h4h


gm

L

005120583

gm

L

05120583

gm

L

15120583

gm

L

1120583

gm

L

(a)

01 120583gmL 05 120583gmL

001 120583gmL0120583gmL

(b)





0 001 005 025

5

10

15

20

25

30


The c

onte

nt o

f GSH

(120583m

g p

rote

in)

lowast

lowast lowast





Acknowledgments


References




2nanoparticles






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0120583gmL 005 120583gmL

01 120583gmL 05 120583gmL







(A) (B)

(C) (D)

(a)

0 001 005 01 02 05

117

118

119

120

121

122

123

124

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Casp

ase a

ctiv

ity (a

bsor

banc

e at4

05

nm)


(b)



4 Conclusions



Con

trol

0

200

400

600

800

1000

1200

1400

DCF

fluo

resc

ence

(RFU

)

2h4h


gm

L

005120583

gm

L

05120583

gm

L

15120583

gm

L

1120583

gm

L

(a)

01 120583gmL 05 120583gmL

001 120583gmL0120583gmL

(b)





0 001 005 025

5

10

15

20

25

30


The c

onte

nt o

f GSH

(120583m

g p

rote

in)

lowast

lowast lowast





Acknowledgments


References




2nanoparticles






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(A) (B)

(C) (D)

(a)

0 001 005 01 02 05

117

118

119

120

121

122

123

124

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Casp

ase a

ctiv

ity (a

bsor

banc

e at4

05

nm)


(b)



4 Conclusions



Con

trol

0

200

400

600

800

1000

1200

1400

DCF

fluo

resc

ence

(RFU

)

2h4h


gm

L

005120583

gm

L

05120583

gm

L

15120583

gm

L

1120583

gm

L

(a)

01 120583gmL 05 120583gmL

001 120583gmL0120583gmL

(b)





0 001 005 025

5

10

15

20

25

30


The c

onte

nt o

f GSH

(120583m

g p

rote

in)

lowast

lowast lowast





Acknowledgments


References




2nanoparticles






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Con

trol

0

200

400

600

800

1000

1200

1400

DCF

fluo

resc

ence

(RFU

)

2h4h


gm

L

005120583

gm

L

05120583

gm

L

15120583

gm

L

1120583

gm

L

(a)

01 120583gmL 05 120583gmL

001 120583gmL0120583gmL

(b)





0 001 005 025

5

10

15

20

25

30


The c

onte

nt o

f GSH

(120583m

g p

rote

in)

lowast

lowast lowast





Acknowledgments


References




2nanoparticles






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 001 005 025

5

10

15

20

25

30


The c

onte

nt o

f GSH

(120583m

g p

rote

in)

lowast

lowast lowast





Acknowledgments


References




2nanoparticles






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article ZnO Nanoparticles Treatment Induces ...downloads.hindawi.com/journals/bmri/2015/423287.pdf · Research Article ZnO Nanoparticles Treatment Induces Apoptosis by Increasing