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Toxicology Letters 227 (2014) 29–40

Contents lists available at ScienceDirect

Toxicology Letters

j our na l ho me page: www.elsev ier .com/ locate / tox le t

inc oxide nanoparticles induce apoptosis by enhancement ofutophagy via PI3K/Akt/mTOR inhibition

uchi Roya,b, Sunil Kumar Singhc, L.K.S. Chauhand, Mukul Dasa,b, Anurag Tripathia,∗∗,remendra D. Dwivedia,b,∗

Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), M.G. Marg, Post Box No. 80, Lucknow 226001, IndiaAcademy of Scientific and Innovative Research (AcSIR), New Delhi, IndiaDivision of Parasitology, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, IndiaElectron Microscopy Facility, CSIR-Indian Institute of Toxicology Research, M.G. Marg. Post Box No. 80, Lucknow 226001, India

i g h l i g h t s

ZNPs induced ROS generationwhereas depleted antioxidantenzymes.ZNPs were found concentratedwithin autophagosomes andlysosomes.ZNPs followed extrinsic andintrinsic pathways of apoptosisin macrophages.Autophagy involved PI3K/pAKTpMTOR cascade in ZNPsinduced macrophages.NAC pre-treatment and LC3B-siRNAsuppressed apoptosis and autophagy.

g r a p h i c a l a b s t r a c t

Schematic representation of the possible way of internalization of ZnO NPs into the macrophages and itsafter-effects.

r t i c l e i n f o

rticle history:eceived 16 January 2014eceived in revised form 25 February 2014ccepted 26 February 2014vailable online 12 March 2014

eywords:inc oxide nanoparticlesxidative stress

a b s t r a c t

Zinc oxide nanoparticles (ZnO NPs) induced macrophage cell death and its mechanism remains to besolved. Herein, we report that ZnO NPs induced ROS generation by depleting antioxidant enzymes,increasing lipid peroxidation and protein carbonyl contents in macrophages. The oxidative stress wasinduced by the inhibition of Nrf2 transcription factor release. ZnO NPs also activated the cleavage ofapoptosis markers like caspases 3, 8 and 9. �H2Ax activation and cleavage of poly (ADP-ribose) poly-merase (PARP) that are known indicators of genotoxicity were found to be activated by following p53,p21/waf1 signaling. ZnO NPs increased the number of autophagosomes and autophagy marker proteinssuch as microtubule-associated protein 1 light chain 3-isoform II (MAP-LC3-II) and Beclin 1 after 0.5–24 h

acrophagesutophagypoptosis

of treatment. Phosphorylated Akt, PI3K and mTOR were significantly decreased on ZnO NPs exposure.Moreover, the apoptotic and autophagic cell death could be inhibited on blocking of ROS generation byN-acetylcysteine (NAC) which demonstrated the critical role of ROS in both types of cell death. In addi-tion, inhibition of LC3-II by siRNA-dependent knockdown attenuated the cleavage of caspase 3. This studydemonstrates autophagy supp

∗ Corresponding author at: Food, Drug and Chemical Toxicology Division, CSIR-Indian Inel.: +91 522 2620107/2620106/2616191; fax: +91 522 2628227.∗∗ Corresponding author at: Food, Drug and Chemical Toxicology Division, CSIR-Indian In

E-mail addresses: anurag1706@gmail.com (A. Tripathi), pddwivedi@yahoo.com, pddw

ttp://dx.doi.org/10.1016/j.toxlet.2014.02.024378-4274/© 2014 Elsevier Ireland Ltd. All rights reserved.

orts apoptosis on ZnO NPs exposure.

© 2014 Elsevier Ireland Ltd. All rights reserved.

stitute of Toxicology Research, P.O. Box No. 80, M.G. Marg, Lucknow 226 001, India.

stitute of Toxicology Research, P.O. Box No. 80, M.G. Marg, Lucknow 226001, India.ivedi@iitr.res.in (P.D. Dwivedi).

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. Introduction

Zinc oxide nanoparticles (ZnO NPs) are widely used in manyelds, such as rubber manufacture, cosmetics, pigments, food addi-ives, medicine, chemical fiber and electronics industries. Becausef the traditional concept that zinc oxide is non-toxic, the toxicolog-cal studies on ZnO NPs are far behind the speed of their application.n recent years, with the phenomenal increase in uses of ZnO NPs,ssociated health risks with its use is of concern.

Several studies have reported that the immune organs are theain sites for the deposition of nanoparticles after systemic expo-

ure. It is highly probable that macrophages carry nanoparticleso the different organs, as their accumulation has been found inhe phagolysosomes of tissue residing macrophages (Sadauskast al., 2007; Adlersberg and Singer, 1973). Accumulation of ZnOPs occurred in the liver, spleen, lungs, kidney and heart after

ingle intraperitoneal administration (Li et al., 2012; Wang et al.,008). Nanoparticles can travel from various entry routes suchs dermal, oral and respiratory tract to other parts of the body.acrophages unlike other cells are capable of efficient uptake of

articles by phagocytosis and are present in many tissues as resi-ent macrophages such as alveolar macrophages and Langerhansells etc. ZnO NPs are widely added to the feed as additives. Dingt al. (2007) showed that the oral exposure to the low dosage ofnO NPs in mice increased the phagocytic activity of mouse peri-oneal macrophages. However, exposure to high doses of ZnO NPsnduced the edema and degeneration of hepatocytes, inflammationf the pancreas and damage to the stomach and spleen (Wang et al.,008).

There are several reports on the effect of different shapes andizes on the toxicity of ZnO NPs (Lin et al., 2009; Deng et al., 2009;uan et al., 2010). Previously, we had reported that ZnO NPs can

nduce reactive oxygen species (ROS) generation and provoke mod-lation in immune responses (Roy et al., 2011, 2014). Most often,he harmful effects of ROS may be manifested by DNA damage,xidations of poly-unsaturated fatty acids in lipids and oxidationsf amino acids in proteins (Limbach et al., 2007). Although, intra-ellular ROS had a significant correlation with cell viability buthe link between ZnO NPs-induced cell death and oxidative stresss not well established in macrophages. Thus, in this study, weelected mouse primary peritoneal macrophages and investigatedor oxidative damage to DNA, lipids and proteins of macrophagesn ZnO NPs exposure. Further, we tried to find the evidences ofutophagosome formation and its possible association with celleath. Autophagy is a key mechanism in various physiopathologicalrocesses including cell death and survival (Mizushima et al., 2008;ubinsztein, 2006). Several reports have shown that autophagy notnly enhances caspase-dependent cell death, but also required fort (Espert et al., 2006). In contrast, it has been shown that autophagylays an important role in promoting cell survival against apo-tosis. Nanoparticles can induce dysfunction or overstimulationf autophagy pathway that may be further involved in their selfellular consumption and cytotoxicity. Defects in the autophagyathway have been linked to a number of pathologies in humansuch as chronic infection, muscular disorders, and cancer and neu-odegenerative disease (Ravikumar et al., 2010).

Collectively, the aforesaid reports demonstrate that exposureo ZnO NPs affect macrophages and T cells mediated immuneeactions. However, it is currently unclear whether cell survivalechanisms are influenced by ZnO NPs in macrophages or not.

n light of the available evidences showing the impacts of ZnOPs on the functionality of immune cells, the objective of the

resent study was to investigate the effect of ZnO NPs on autophagynd apoptosis cell death mechanisms as well as the possiblessociation between them in macrophages exposed with ZnOPs.

ers 227 (2014) 29–40

2. Materials and methods

2.1. Cell culture

Inbred strains of female Balb/c mice (8–10 weeks old) were sacrificed accord-ing to the guidelines for the care and use of the Animal Ethical Committee of IndianInstitute of Toxicology Research, Lucknow, India. This study was carried out after theapproval of the Institutional Ethics Committee (No. of approval-IITR/IAEC/47/11).Peritoneal exudate was collected from the peritoneal cavity of mice by injectingchilled RPMI 1640 medium and added to 96-well cell culture flat bottom plate.After 3 h of incubation in a CO2 incubator (5% CO2) at 37 ◦C, the non-adherent cellswere removed by vigorous washing (three times) with warm RPMI 1640 medium.Furthermore, adhered cells were incubated overnight in RPMI 1640 medium sup-plemented with heat-inactivated 10% FBS, penicillin (100 U/ml) and streptomycin(100 U/ml) at 37 ◦C in the humid air containing 5% CO2 to form macrophage mono-layers. More than 95% of the adherent cell populations were macrophages asdetermined by morphology and nonspecific esterase staining.

2.2. Transmission electron microscopy (TEM)

ZnO NPs powder was purchased from Sigma, USA and characterized for size andzeta-potential by transmission electron microscope (TEM) and dynamic light scat-tering (DLS). TEM result showed ZnO NPs were of ∼50 nm and DLS size showed sizedistribution with an average diameter of 278.8 nm in culture media supplementedwith 10% FBS. The zeta potential of ZnO NPs was −11.5 mV in complete mediumas shown in our previous study (Roy et al., 2014). Macrophages (2 × 106 cells/well)were plated in 12-well cell culture plate. After overnight incubation, the cells wereexposed to ZnO NPs (2.5 �g/ml) for 24 h. After exposure, the medium was aspiratedand the cells were washed twice with 1× PBS. Cells were then centrifuged and thepellet was fixed with 2.5% glutaraldehyde and 2% paraformaldehyde. After washingwith 0.1 M sodium cacodylate buffer the pellet was post fixed in 1% osmium tetraox-ide for 3 h. The fixed pellet was washed and dehydrated through increasing grades(30–100%) of acetone. The sample was infiltrated with Araldite® resin overnight atroom temperature and finally embedded in pure resin. The blocks were incubated at60 ◦C for 72 h. After incubation, ultrathin sections were prepared using Reichert-Jungultra microtome (Vienna, Austria). The sections were stained with uranyl acetate andReynold’s lead citrate. To avoid confusions of any staining artifacts the grids wereexamined before and after staining under TEM (JEM-2100, JEOL Ltd., Tokyo, Japan)at 60 or 80 kV.

2.3. Assessment of necrosis and apoptosis

Apoptosis in macrophages was measured by using the annexin-V/propidiumiodide (PI) apoptosis detection kit (BD Biosciences, San Diego, USA). Macrophageswere incubated with 2.5 �g/ml of ZnO NPs for 0.5, 3, 6, 12, 24 h and 10 mM N-acetylcysteine (NAC) pretreatment followed by ZnO NPs for 24 h. After exposure,cells were trypsinized and centrifuged at 1000 rpm; the cell pellet was washed withPBS once and re-suspended in 100 �l of binding buffer, then incubated with 2 �lannexin-V-FITC for 10 min, which was followed by staining with 1 �g/ml PI. Then,the samples were diluted by adding 400 �l binding and at least 10,000 cells werecounted for each sample. The cell population of interest was gated on the basis ofthe forward and side-scatter properties. Vertical and horizontal lines were designedon the basis of autofluorescence of untreated control cells. Then different cell pop-ulations were identified, where FITC negative and PI negative were designated asviable cells; FITC positive and PI negative as early apoptotic cells; FITC positive andPI positive as late apoptotic cells or necrotic cells; and FITC negative and PI posi-tive as necrotic cells. The data analysis was performed using BD FACS Diva software(Becton Dickinson, USA).

To analyze apoptotic cells by Hoechst labeling, both untreated and treated cells(1, 2.5, 5 or 10 �g/ml of ZnO NPs) were fixed with 4% formaldehyde followed bystaining with Hoechst 33342 and then observed by confocal microscopy.

2.4. ROS measurement

In all 4× 105 cells/well were seeded into 96-well (black, flat bottom) cell cul-ture plate. Cells were incubated with 2.5 �g/ml of ZnO NPs for different time-points(0.5, 3, 6, 12 and 24 h). For determination of specific free radical role in ZnO NPs-induced cell death, different specific scavengers against various free radicals suchas 20 mM mannitol and 20 mM ethanol for hydroxyl free radicals (Shen et al.,1997; Suthanthiran et al., 1984), 10 mM dimethyl formamide (DMF) for superox-ide free radicals (Song and Yen, 2002) and 40 �M tocopherol for singlet oxygen[1O2] species (Berton et al., 1998) were added to macrophages for 0.5 h prior to2.5 �g/ml of ZnO NPs exposure for 3 h. After incubation, the medium was takenout and cells were washed twice with 1× PBS. Thereafter, culture medium contain-

ing 2,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dye (20 �M) was addedto each well. The plate was incubated for 0.5 h at 37 ◦C and the medium containingDCFDA was discarded. PBS (200 �l) was then added to each well and DCF fluores-cence was recorded in a fluorimeter at excitation and emission wavelengths of 485and 528 nm, respectively.

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.5. Oxidative stress markers

To study the time dependent effect of ZnO NPs on various oxidative stressarkers, macrophages were treated with 2.5 �g/ml of ZnO NPs for 3, 6, 12 and

4 h. Reduced glutathione (GSH) content was assayed in the cells homogenateccording to the method of Ellman (Ellman, 1959). Lipid peroxidation (LPO) inhe cell homogenate was determined by estimating the formation of malondialde-yde (MDA) (Utley et al., 1967). Protein carbonyl content was measured in theomogenate according to the method of Levine et al. (Levine et al., 1990). Cata-

ase activity was determined by the method of Sinha (Sinha, 1972). Glutathioneeductase (GR), Glutathione peroxidase (GPx) and Glutathione S transferase (GST)ctivities were assayed according to the method of Moron (Moron et al., 1979).

.6. Detection of changes in mitochondrial membrane potential (MMP)

Mitochondrial membrane permeability was determined using the mitochon-rial staining dye, Rhodamine 123. When the mitochondrial membrane potentialollapses, the dye can no longer accumulate within the mitochondria and fluorescesreen. Macrophages were treated with 2.5 �g/ml of ZnO NPs (0.5, 3, 6, 12 and 24 h)nd 10 mM of NAC + 2.5 �g/ml of ZnO NPs treated groups in black bottom 96-welllate and were rinsed with PBS twice, stained with 0.3 �M Rhodamine-123 for 0.5 ht 37 ◦C after their incubations. Cells were rinsed with PBS twice, resuspended in00 �l PBS, and instantly assessed by fluorimeter at 488 nm of excitation and emis-ion of 535 nm to quantify the population of mitochondria with green fluorescence.

.7. Determination of autophagosome formation

FITC tagged-LC3 antibody was used for detecting the formation of autophago-ome by using Cyto-ID® autophagy detection kit (Enzo Life Sciences, Switzerland)s per the manufacturers’ protocol. Rapamycin (500 nM), an autophagy inducernd a known positive control of autophagy was added to cells for 16–18 h. Theucleus was stained with Hoechst 33342 stain. After the completion of staining, cellsere washed twice with 1× assay buffer provided in the kit. Images of autophagic

ells were taken by using a confocal microscope (TCS-SPE Confocal microscope,annheim, Germany) and fluorescence (autophagy flux) of total autophagic cells

er group was read at FITC filter (Excitation 480 nm, Emission 530 nm) by using auorimeter.

.8. Real Time PCR (RT-PCR) of Atgs genes

RT-PCR analyses of Atg genes (Atg5, Atg10 and Atg12) in macrophages werearried out using gene-specific primers in control, 2.5 �g/ml of ZnO NPs (0.5,, 6, 12 and 24 h) and NAC + ZnO NPs treated groups. The oligonucleotiderimers used were Atg5: forward – primer-5′-AAGTCTGTCCTTCCGCAGTC-3′ , reverse

primer-5′-TGAAGAAAGTTATCTGGGTAGCTCA-3′; Atg10: forward – primer-5′-CCAGTGTGCTCACATGTCT-3′ , reverse – primer-5′-TCGTCACTTCAGAATCATCCA-′; Atg12: forward – primer-5′-CATTGACTTCATCAAAAAGTTCCTT-3′ , reverse –rimer-5′-GGCAAAGGACTGATTCACATAA-3′ and GAPDH: forward – primer-5′-TCACCACCATGGAGAAGGC-3′ , reverse – primer-5′-GGCATGGACTGTGGTCATGA-3′ .otal RNA from cells were isolated with RNAzol®RT (Molecular Research Center,nc., Cincinnati, OH, USA). cDNA from different groups were prepared by using Highapacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA)ccording to the manufacturer’s instructions. Quantitative RT-PCR was carried outsing a SYBER Green PCR master mix (Thermo Scientific, Waltham, MA, USA) accord-

ng to the manufacturers’ instructions. Amplification of housekeeping gene GAPDHas taken as an endogenous control. Normalized values were used for plotting the

ar graphs.

.9. Microtubule-associated protein 1 light chain 3-isoform II (MAP-LC3-II)-siRNAransfection

Mouse MAP-LC3-II-siRNA was purchased from Santa Cruz Biotechnology. TheiRNA was transfected into macrophages by using the transfection reagent (Santaruz Biotechnology, USA) and further treated with ZnO NPs for 24 h. The influence ofutophagy on apoptosis was seen by blocking LC3 and then the cleavage of caspase

and LC-3 were identified.

.10. Total cell lysate preparation

Macrophages were treated with 2.5 �g/ml of ZnO NPs (0.5, 3, 6, 12 and 24 h)nd NAC + ZnO NPs. Following treatment to the cells, ice-cold lysis buffer (50 mMris–HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5%

P-40, 1% Triton X-100, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, pH.4) was added to the groups. Plates were placed over ice for 0.5 h and the lysateas collected in a microfuge tube. The lysates were cleared by centrifugation at

4,000 × g for 15 min at 4 ◦C and the supernatants (total cell lysate) were eithersed immediately or stored at −80 ◦C.

ers 227 (2014) 29–40 31

2.11. Western blotting

A forty microgram of protein was resolved in 10% SDS-PAGE and then elec-troblotted onto nitrocellulose membranes and incubated with different antibodiesi.e. anti-H-Ras, anti-PI3K (BD Biosciences, USA) Beclin 1 (Abcam, USA); anti-nuclearfactor-erythroid 2 (Nrf2), anti-p-mTOR, anti-�-H2AX (Ser139) (Santa Cruz, USA);anti-LC3 (Biogenx, USA); anti-p-Akt, anti-poly (ADP-ribose) polymerase (PARP),anti-p21, anti-p53, anti-Bax, anti-Bcl2, anti-cytochrome-c, anti-caspase 8, anti-caspase 9, anti-caspase 3 (Cell Signaling Technologies, Boston, MA). Immunoblotswere detected with horseradish peroxidase conjugated anti-mouse or anti-rabbitIgG using chemiluminescence kit and visualized by Versa Doc Imaging System (Bio-rad, CA, USA). To quantify equal loading, the membrane was reprobed with eitheranti-�-actin or anti-�-tubulin antibody. The density of each band was estimatedby the Alfa Imager software. The �-actin was taken as an endogenous control andits respective densitometry values were used for normalizing different proteinsexpressions. Normalized values were indicated on the respective bands.

2.12. Statistical analysis

Data was expressed as mean ± SE and analyzed by Prism5.0 software usingone-way and two-way ANOVA followed by Bonferroni intergroup comparison test.Correlation analysis was performed by determining the Pearson’s correlation coef-ficient (two-tailed) with a confidence level of 95% using SPSS software. p < 0.05 wasconsidered as significant.

3. Results

3.1. Cell death induction by ZnO NPs

Cells were exposed to a series of concentrations of ZnO NPs (1,2.5, 5 and 10 �g/ml) and cell death was analyzed by annexin/PI andHoechst staining. Percentage of apoptotic cells death was calculatedby adding the percentage of early apoptotic gated cells (annexin-V+cells) and late apoptotic gated cells (annexin-V+/PI+). A signifi-cant increase (↑13.95%) in the apoptotic cells was observed with2.5 �g/ml exposure to ZnO NPs (Fig. 1a and b) whereas with 1, 5and 10 �g/ml of ZnO NPs, the apoptotic cell death was 8.5, 7.35 and7.15% observed respectively. In addition to this, a dose-dependentincrease (1, 2.5, 5 and 10 �g/ml: 12.5, 13.6, 21.4 and 23.3% necroticcells) in the percent of necrotic cells (PI+ stained) as well as nuclearalteration with chromatin condensation was observed (Fig. 1c).

3.2. ROS generation by ZnO NPs

Various nanoparticles are known inducer of ROS mediated celldeath in several cell types. To probe ROS generation in macrophagesafter ZnO NPs exposure, H2DCFDA was used. After 3 h of exposureto 2.5 �g/ml of ZnO NPs, nearly two fold increase in ROS genera-tion was observed over untreated control (Correlation co-efficient,r = −0.145) (Fig. 1d). With the increase of concentration (1, 2.5, 5 and10 �g/ml), it was found that ROS was significantly accumulated.

Apoptotic cell death and ROS generation were significantlyobserved at the dose of 2.5 �g/ml. Therefore, further experimentswere performed on this concentration.

3.3. The effect of scavengers of ROS on ZnO NPs-induced ROS

ROS generation was enhanced at 2.5 �g/ml of ZnO NPs expo-sure therefore; determination of specific free radical role in ZnONPs-induced cell death was identified on the same dose by usingdifferent specific scavengers such as mannitol, ethanol, DMF andtocopherol. We found that mannitol, ethanol and DMF provideda nearly complete reduction in ROS level which was induced byZnO NPs (Fig. 1e) whereas tocopherol resulted in non-significantreduction in ROS.

3.4. Effect of ZnO NPs on oxidative stress markers in macrophages

Effect of ZnO NPs on oxidative stress markers in macrophagescan be seen by a significant increase in LPO (2–4 fold) and protein

32 R. Roy et al. / Toxicology Letters 227 (2014) 29–40

Fig. 1. Cell death mechanism and ROS generation. Macrophages were exposed to ZnO NPs (1, 2.5, 5 and 10 �g/ml for 24 h) and stained with (a) annexin V-FITC/PI beforeanalyzed by flow cytometer. (b) Percent of total apoptotic cells represents the mean ± S.E.M. of five replicates. (c) Hoechst staining images of macrophages. Nuclear alterationand chromatin condensation are visible in ZnO NPs treated cells. (d) ROS generation on ZnO NPs treatment. Cells were treated with 2.5, 5 and 10 �g/ml ZnO NPs for 0.5,1, 3, 6 and 12 h before incubation with 20 �M H2DCFDA and measured by fluorimeter. Percent increase in ROS production in treated cells was compared with untreatedcells. Each reported value represents the mean ± S.E.M. of five replicates (*p < 0.05, compared with untreated control). (e) Effects of antioxidants (indicated in bar diagram asTocopherol: T; Mannitol: M; Ethanol: E; and DMF: D) on ROS induced by ZnO NPs. Antioxidants were added 0.5 h prior to ZnO NPs exposure and ROS was measured after24 h. Data in terms of percentage are presented as the mean ± SE of five values. (*p < 0.05, indicated significant compared with untreated control).

R. Roy et al. / Toxicology Letters 227 (2014) 29–40 33

Fig. 2. Effect of ZnO NPs on oxidative stress markers in macrophages. Time-dependent effect of ZnO NPs on (a) LPO, (b) protein carbonyl, (c) GSH content, and activities of(d) GR, (e) GPx, (f) catalase and (g) GST of macrophages exposed for 3, 6, 12 and 24 h. Each value represents the mean ± SE of five values. *p < 0.05, significant with respect tocontrol group.

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arbonyl (∼2 fold) along with a significant decrease in GSH con-ent (one-third fold of control) on ZnO NPs exposure (Fig. 2a–c),hile GPx and GST were significantly decreased and the activities

f catalase and GR were gradually inhibited or reached to null at4 h (Fig. 2d–g).

.5. Effect of ZnO NPs on the oxidative stress mediated apoptoticignaling

Since Nrf2, a transcription factor plays a significant role in coun-eracting stress by inducing the expression of many other geneshat are involved in antioxidant responses. We studied the effectf ZnO NPs on Nrf2 levels. As shown in Fig. 3a, ZnO NPs applica-ion resulted in enhancement of Nrf2 levels after 0.5 h but levelsontinued to decrease substantially after 3 h. Effect of ZnO NPsn the expression of p53 and p21/waf1 proteins in macrophagesas also observed. Expression of these proteins was significantly

nhanced after treatment of ZnO NPs for 0.5–12 h as comparedo control. Further, ZnO NPs application for 0.5–24 h resulted inignificant over expression of Bax along with reduction of Bcl2,hich was associated with an increase in cytochrome c level in

he cytoplasm (Fig. 3b). Caspase 8 cleavage was enhanced after 3 hf exposure of ZnO NPs to macrophages (Fig. 3c) while the expres-ions of cleaved caspase 9 and caspase 3 were enhanced maximallyt 24 (Fig. 3d). Pretreatment of NAC reduced the cleavage of cas-ase 3, caspase 9 and caspase 8. Activation of caspase 3 resulted

n PARP cleavage and �H2Ax activation that was observed in aime-dependent manner after ZnO NPs application to macrophagesFig. 3e) and these were also reduced on ROS inhibition by usingAC.

.6. Antioxidant revert ROS induced cell death

In general, ROS induces autophagy and apoptosis therefore, weere interested to find effect of ROS on macrophages cell death.e pre-treated the macrophages with NAC, a well known inhibitor

f ROS before ZnO NPs exposure and checked the percentage ofpoptotic cells. Apoptotic cell death was significantly reduced from4% to 5.4% and all over cell death decreased from 28% to 15% onreatment of NAC (Fig. 4a). This clearly suggested that cell deathas induced as a result of ROS generation by ZnO NPs exposure.

Further, we performed experiment related to cell death whichs loss of mitochondrial membrane potential (MMP). Remarkableoss of MMP was observed after exposure of ZnO NPs in a time-ependent manner (Fig. 4b). On pretreatment of NAC, MMP losstopped and it was found comparable to control.

.7. Internalization of ZnO NPs shown by TEM

Electron micrographs of macrophages exposed to ZnO NPshowed a significant cellular uptake of ZnO NPs (Fig. 5a). Multipleembrane foldings and plasma membrane (pseudopodial) exten-

ions were fused together to engulf the ZnO NPs as indicated byhe arrows. The nanoparticles detectable in the cytoplasmic regionf macrophages showed accumulation of ZnO NPs within micron-ized vesicles resembling late endosomes and lysosomes, which areypical features of autophagosomes. In case of control, the mem-ranous boundary was not at all folded and no vesicle was foundhere.

.8. Autophagy induced by ZnO NPs

Next we examined whether ZnO NPs could induce autophagyr not. We incubated macrophages with ZnO NPs and foundutophagosome accumulation (FITC+ LC3+ cells), a hallmark ofutophagy after 0.5 h (Fig. 5b and c). Moreover we also assessed

ers 227 (2014) 29–40

the expression of three other well known markers of autophagynamely Atg5, Atg10 and Atg12 genes in ZnO NPs treated cells andobserved significant induction in their expression as compared tocontrol when quantified by real time analysis (Fig. 6a). Also, at pro-tein expression level, autophagy was confirmed by the increase inLC3-II and Beclin 1 levels in macrophages treated with ZnO NPs(Fig. 6b). Taken together, these results showed that ZnO NPs clearlyinduced autophagy in macrophages.

Further, the role of ROS in autophagy was determined by inhibit-ing ROS with NAC. We observed that NAC pre-treatment to ZnO NPsexposure leads to inhibition of Atg5, 10 and 12 mRNA expressions,LC3-II formation and Beclin 1 expression.

3.9. PI3K/AKT/mTOR signaling pathway activation by ZnO NPs forautophagy

To assess the role of PI3K and mTOR in ZnO NPs inducedautophagy, we analyzed the phosphorylation levels of mTORand PI3K by using western blotting and found that ZnO NPsreduced their expression in a time dependent manner (Fig. 6c).On treatment with ZnO NPs, level of phosphorylated Akt wassignificantly decreased. The effect of ZnO NPs on mTOR inhibi-tion started 6 h after exposure. These results confirmed that ZnONPs induced autophagy via PI3K/Akt/mTOR classical pathway inmacrophages.

3.10. Influence of autophagy in apoptosis

Role of autophagy in induction of apoptosis remains unclear,and can have a protective or deleterious effect. Here, we examinedthe role of LC3 in the regulation of ZnO NPs-induced apoptosis andcell death. In LC3B-siRNA infected macrophages ZnO NPs displayeddecreased formation of LC3-II in comparison to control (Fig. 6d).In LC3B-siRNA-treated macrophages, ZnO NPs treatment inducedlower levels of apoptosis, as evidenced by the decreased cleavageof caspase-3.

4. Discussion

There are few reports concerning the cytotoxic effect of ZnO NPson the immune cells. Previous studies showed that ZnO NPs expo-sure resulted in the induction of inflammatory responses and thesewere internalized in macrophages via caveolae mediated pathway(Roy et al., 2013, 2014). ZnO NPs have the potential to generate ROSunder in vitro conditions that could be correlated to their potentialto induce cellular inflammation under in vivo situations (Landsiedelet al., 2010; Ayres et al., 2008). Various studies have implicatedintracellular oxidative stress as the cause of toxicity induced by ZnONPs (Kim et al., 2010; Meyer et al., 2011; Yazdi et al., 2010). ZnONPs induced ROS generation at 3 h could be due to sudden burdenor exposure of NPs to metabolize these invaders or initiate the sig-naling required for balancing the intracellular homeostasis. Cellshave adaptive and dynamic programs to create balance betweengeneration and removal of ROS, hence for maintaining this bal-ance Nrf2 transcription factor has been identified as the masterregulator (Kensler et al., 2007). ZnO NPs suppressed Nrf2 that maycause significant depletion of antioxidant content and inhibitionof anti-oxidant enzymes activity along with enhanced lipid perox-idation and protein carbonyls formation. It was found that DMF,ethanol and mannitol decreased the ROS generation which implythat superoxide ions and hydroxyl radicals were induced by ZnONPs.

Exposure of ZnO NPs (2.5 �g/ml) caused significant enhance-ment in ROS generation at 3 h indicated that ROS generationfollowing ZnO NPs exposure is an early event resulting in alter-ations of oxidative stress markers after 6 h (anti-oxidant and

R. Roy et al. / Toxicology Letters 227 (2014) 29–40 35

Fig. 3. ZnO NPs induced oxidative stress, genotoxicity and apoptotic markers in macrophages. (a) Nrf2, p53 and p21/waf1 levels in 2.5 �g/ml ZnO NPs treated macrophagesfor 0.5, 3, 6, 12 and 24 h. (b) Bax, Bcl2, and cytochome c levels in cytosolic fraction of macrophages. (c) Caspases 3, 8, and 9 cleavages in ZnO NPs (0.5, 3, 6, 12 and 24 h) andNAC + ZnO NPs treated macrophages. (d) �H2Ax activation and cleavage of PARP in ZnO NPs (0.5, 3, 6, 12 and 24 h) and NAC + ZnO NPs treated macrophages were analyzedby immunoblotting in whole cell lysates of macrophages.

36 R. Roy et al. / Toxicology Letters 227 (2014) 29–40

Fig. 4. Effect of antioxidant on ZnO NPs-induced apoptosis. (a) Cells were treated with NAC + ZnO NPs for 24 h before staining with annexin V-FITC/PI and analyzed by flowc respecg phageN

ateeZSel

ytometer. Data represent the mean ± SE of five values. *p < 0.05, significant with

roup. (b) Effect of ZnO NPs on mitochondrial membrane potential (MMP) in macroPs, stained with Rhodamine-123 and subjected to fluorimeter.

nti-oxidant enzymes decreased while damage to lipid and pro-ein increased). Our results were supported by a report wherearly ROS generation had later effect on oxidative enzymes (Kumart al., 2011). Depletion in GR, catalase, GST and GPx activities after

nO NPs exposure emphasized the occurrence of oxidative stress.everal studies suggested that enhanced free radicals may causextensive damage to macromolecules of cell, i.e., DNA, protein andipid, thus altering their structure and function (Das et al., 2005;

t to the control group. #p < 0.05, significant with respect to ZnO NPs-only treateds. Cells were incubated with 2.5 �g/ml ZnO NPs for 0.5, 3, 6, 12, 24 h and NAC + ZnO

Martin and Barrett, 2002; Gerloff et al., 2011). ZnO NPs exposurefor 6–24 h caused significant DNA damage by enhancing cleavageof PARP following p53/p21 pathway and increasing expression of�H2Ax, suggesting its genotoxic and mutagenic potential. This find-

ing was supported by the others studies, where ZnO NPs wereconsidered to be genotoxic (Sharma et al., 2009, 2011). The toxicconsequences might be due to decrease in mitochondrial mem-brane potential and presence of ZnO NPs in the nucleus. Cell death

R. Roy et al. / Toxicology Letters 227 (2014) 29–40 37

Fig. 5. ZnO NPs trigger autophagy. (a) TEM images of autophagosomes and cellular structures in macrophages treated with ZnO NPs. Black arrows point to ZNP clusters.Autophagosome formations in ZNP treated cells as indicated by red arrows. High magnification view of a large autolysosome containing clusters of ZnO NPs and cellulardebris. Nucleus of the treated cells also contain large numbers of dense ZnO NPs. (b) Detection of autophagic vacuoles in macrophages after different time points (control, 0.5,3, 6, 12 and 24 h) treated cells and Rapamycin (positive control) treated cells. (c) Autophagic flux in macrophages on ZnO NPs exposure measured by staining with LC3-FITCantibody and assessed by fluorimeter.

38 R. Roy et al. / Toxicology Letters 227 (2014) 29–40

Fig. 6. Quantitative mRNA levels of autophagy related genes. (a) Time-dependent upregulation of Atg10, Atg12 and Atg5 mRNA expression after ZnO NPs treatment (2.5 �g/mlfor 0.5, 3, 6, 12, 24 h and NAC + ZnO NPs for 24 h). (b) Autophagic marker proteins (LC3-II and Beclin 1) in lysates of treated macrophages (2.5 �g/ml for 0.5, 3, 6, 12, 24 hand NAC + ZnO NPs for 24 h) assayed by western blotting. �-Actin was used as the loading control. All blots shown are representative of three independent experiments. (c)Effect of ZnO NPs on Ras-PI3K-mTOR-Akt signaling pathway. Cells were treated with ZnO NPs (2.5 �g/ml for 0.5, 3, 6, 12 and 24 h) and protein lysates were assayed for theexpression of Ras-PI3K-pmTOR-Akt. (d) Effect of LC3-II-siRNA on LC3-II and cleavage of caspase 3 expressions in control, siRNA treated control, ZnO NPs treated and siRNAtransfected followed by ZnO NPs treated groups.

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R. Roy et al. / Toxicolog

ue to mitochondrial collapse was well supported by couple ofrevious studies (Scherz-Shouval et al., 2007; Azad et al., 2009).

Up-regulation of pro-apoptotic protein Bax, down-regulationf Bcl-2 and release of cytochrome c indicated the occurrence ofpoptosis due to ZnO NPs. Cytochrome c release in the cytoplasmctivates apoptosis protease activating factor 1 (Apaf 1), whicheads to the activation of caspase 3, the execution caspase. Stud-es of Cecconi (1999) and Hsiao and Huang (2011) corroboratedur findings. Subsequently, ZnO NPs activated caspase 3 cleavedARP proteins (DNA repair enzyme in the cell and among the firstarget of executioner caspases). Cleavage of caspases 3, 8 and 9 indi-ated that ZnO NPs exposure followed both extrinsic and intrinsicathways of apoptosis to ensure survival during stress.

However, another cell death mechanism, autophagy alsoontributes to the abnormal ROS accumulation by selectively pro-oting the degradation of major enzymatic ROS scavengers which

orroborate our results (Yu et al., 2006). Autophagy involves the deovo formation of small double-membrane-bound vesicles calledutophagosomes within the cytoplasm that sequester cytosoliconstituents. ZnO NPs-induced autophagosomes were observed inhe cytoplasm of macrophages. These NPs induced the transcriptevels of Atgs (Atg10, Atg12 and Atg5) that are required for thenitiation of autophagy. Atg10 is essential to form Atg12 conjuga-ion with Atg5 and this conjugate is necessary to form autophagicesicles. Further, these NPs induced Atgs catalyzed the formationf microtubule-associated protein 1 light chain 3 (MAP-LC3-I) toAP-LC3-II and release of (Atg6) Beclin 1. Both of these proteins are

ey activators and constituents of the autophagosome membraneormation (Geng and Klionsky, 2008; He and Klionsky, 2009). Theseesults were in accordance with the activation of caspase-8, sug-esting a regulatory role of LC3-II in extrinsic apoptosis activationChen et al., 2010).

Our results suggested that oxidative stress induced by ROSormation may lead to apoptosis, but whether these ROS weressential for autophagosome formation or not? To address thisuestion, we tested the effect of NAC, a general antioxidant, onpoptosis and autophagosomes formation after ZnO NPs exposure.ddition of NAC to the growth medium before ZnO NPs exposure

nhibited the apoptotic cell death, reduced the formation of LC3-IInd abolished the formation of Beclin 1. Antioxidants are knowno abolish the formation of autophagosomes and the consequentegradation of autophagy-related proteins (Scherz-Shouval et al.,007).

To further understand the mechanism of autophagy induced bynO NPs, PI3K/mTOR/AKT pathway was explored that plays a crit-cal role in the early stages of autophagosome formation (Petiott al., 2000; Tassa et al., 2003). Initial activation of PI3K under stressondition may lead to rise in free radicals in the vicinity of mito-hondria. Further, a significant reduction in p-Akt and p-mTORxpressions, downstream of classical PI3k/Akt/mTOR signalingathway favored autophagy. To scrutinize the regulatory role ofC3-II over apoptosis, cleavage of caspase 3 was observed in LC3-IIilenced macrophages. Inhibition of LC3-II reduced the cleavage ofaspase 3. He and Klionsky (2009) reported that siRNA-dependentnockdown of LC3-II conferred protection against epithelial cellpoptosis. Thus, it seems that ROS production may be the rea-on of cell death and autophagy supported apoptosis on ZnO NPsxposure.

. Conclusions

ZnO NPs exposure induced oxidative stress in macrophagesnitiated autophagy as well as apoptosis simultaneously. Forma-ion of autophagosomes may be a cellular defense mechanismgainst oxidative stress. Moreover, autophagy induced by ZnO NPs

ers 227 (2014) 29–40 39

followed PI3K/mTOR/Akt signaling cascade. Disruption in mecha-nism of cell death may hamper immune functions that may lead toserious diseases.

Conflict of interest

None of the authors have any conflict of interest to disclose.

Transparency document

The Transparency document associated with this article can befound in the online version.

Acknowledgements

The authors wish to thank Dr. K.C. Gupta, Director, CSIR-IITR forhis support. Ruchi Roy is thankful to the University Grants Commis-sion (UGC), New Delhi for the award of Senior Research Fellowshipand conveys her gratitude to Academy of Scientific and InnovativeResearch (AcSIR), New Delhi. Acknowledgment to TCS-SPE Confocalmicroscopy facility of CSIR-IITR, Lucknow, India. Financial sup-port from CSIR network project Nano-SHE (Grant no.- BSC-0112)is acknowledged. This is CSIR-IITR manuscript no. 3203.

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