in vitro cytotoxicity of silver nanoparticles and zinc oxide nanoparticles to human epithelial...

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Mutation Research 769 (2014) 113–118 Contents lists available at ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis j ourna l h om epage: www.elsevier.com/l ocate/molmut Comm unit y ad dress: www.elsevier.com/locate/mutres In vitro cytotoxicity of silver nanoparticles and zinc oxide nanoparticles to human epithelial colorectal adenocarcinoma (Caco-2) cells Yijuan Song a,1 , Rongfa Guan a,,1 , Fei Lyu b , Tianshu Kang a , Yihang Wu a , Xiaoqiang Chen c a Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018, China b Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, China c Hubei University of Technology, Wuhan 430068, China a r t i c l e i n f o Article history: Received 20 November 2013 Received in revised form 27 July 2014 Accepted 4 August 2014 Available online 12 August 2014 Keywords: Zinc oxide nanoparticles Silver nanoparticles Cytotoxicity Human epithelial colorectal adenocarcinoma cells a b s t r a c t With the increasing applications of silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) in foods and cosmetics, the concerns about the potential toxicities to human have been raised. The aims of this study are to observe the cytotoxicity of Ag NPs and ZnO NPs to human epithelial colorectal adeno- carcinoma (Caco-2) cells in vitro, and to discover the toxicity mechanism of nanoparticles on Caco-2 cells. Caco-2 cells were exposed to 10, 25, 50, 100, 200 g/mL of Ag NPs and ZnO NPs (90 nm). AO/EB double staining was used to characterize the morphology of the treated cells. The cell counting kit-8 (CCK-8) assay was used to detect the proliferation of the cells. Reactive oxygen species (ROS), superoxide dismu- tase (SOD) and glutathione (GSH) assay were used to explore the oxidative damage of Caco-2 cells. The results showed that Ag NPs and ZnO NPs (0–200 g/mL) had highly significant effect on the Caco-2 cells activity. ZnO NPs exerted higher cytotoxicity than Ag NPs in the same concentration range. ZnO NPs have dose-depended toxicity. The LD 50 of ZnO NPs in Caco-2 cells is 0.431 mg/L. Significant depletion of SOD level, variation in GSH level and release of ROS in cells treated by ZnO NPs were observed, which suggests that cytotoxicity of ZnO NPs in intestine cells might be mediated through cellular oxidative stress. While Caco-2 cells treated with Ag NPs at all experimental concentrations showed no cellular oxidative damage. Moreover, the cells’ antioxidant capacity increased, and reached the highest level when the concentration of Ag NPs was 50 g/mL. Therefore, it can be concluded that Ag NPs are safer antibacterial material in food packaging materials than ZnO NPs. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Nanoparticles are already used in several consumer products including food packages, food containers, food additives, water purification systems and so on [1,2]. However, these broad appli- cations increase human and environmental exposure, thus the potential risk is related to their short-term and long-term toxic- ity [3,4]. The nanoparticles may also penetrate the cell and affect cellular respiration through inactivating the essential enzymes by forming complications with the catalytic sulfur of thiol groups in cysteine residues and through the production of toxic radi- cals such as superoxide, hydrogen peroxide, and hydroxyl ions. Corresponding author at: Xueyuan Road 258, Hangzhou 310018, China. Tel.: +86 571 87676187; fax: +86 571 86914449. E-mail addresses: [email protected], [email protected] (R. Guan). 1 These authors contributed equally to this work. Owing to their wide industrial and commercial applications, Ag NPs and ZnO NPs attracted more attentions. Data analysis showed that the majority of articles concerned the applications of Ag NPs (7699 papers, 59%), followed by ZnO NPs (4640 papers, 36%) from 1989 to 2013 [5]. There is an increasing concern related to the biological impacts of the use of Ag NPs and ZnO NPs on a large scale, and the possible risks to the environment and health. Ag NPs show outstanding antibacterial properties [6,7]. Many investigations have focused on their bacterial effects and applica- tions in plastics, health, textiles, and paint industries [8]. However, enthusiasm for Ag NPs has been hampered by their cytotoxic- ity and genotoxicity [9]. Ag NPs may inhibit the segregation of chromosomes. Researchers have observed genotoxicity including DNA damages and chromosomal aberrations in human glioblas- toma cells treated with Ag NPs. Koji Kawata investigated toxic effects of Ag NPs to human hepatoma derived cell line HepG2 that were exposed to Ag NPs at low doses. It was concluded that both http://dx.doi.org/10.1016/j.mrfmmm.2014.08.001 0027-5107/© 2014 Elsevier B.V. All rights reserved.

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Mutation Research 769 (2014) 113–118

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

j ourna l h om epage: www.elsev ier .com/ l ocate /molmutComm uni t y ad dress : www.elsev ier .com/ locate /mutres

n vitro cytotoxicity of silver nanoparticles and zinc oxideanoparticles to human epithelial colorectal adenocarcinomaCaco-2) cells

ijuan Songa,1, Rongfa Guana,∗,1, Fei Lyub, Tianshu Kanga, Yihang Wua, Xiaoqiang Chenc

Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018, ChinaDepartment of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, ChinaHubei University of Technology, Wuhan 430068, China

r t i c l e i n f o

rticle history:eceived 20 November 2013eceived in revised form 27 July 2014ccepted 4 August 2014vailable online 12 August 2014

eywords:inc oxide nanoparticlesilver nanoparticlesytotoxicityuman epithelial colorectaldenocarcinoma cells

a b s t r a c t

With the increasing applications of silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs)in foods and cosmetics, the concerns about the potential toxicities to human have been raised. The aimsof this study are to observe the cytotoxicity of Ag NPs and ZnO NPs to human epithelial colorectal adeno-carcinoma (Caco-2) cells in vitro, and to discover the toxicity mechanism of nanoparticles on Caco-2 cells.Caco-2 cells were exposed to 10, 25, 50, 100, 200 �g/mL of Ag NPs and ZnO NPs (90 nm). AO/EB doublestaining was used to characterize the morphology of the treated cells. The cell counting kit-8 (CCK-8)assay was used to detect the proliferation of the cells. Reactive oxygen species (ROS), superoxide dismu-tase (SOD) and glutathione (GSH) assay were used to explore the oxidative damage of Caco-2 cells. Theresults showed that Ag NPs and ZnO NPs (0–200 �g/mL) had highly significant effect on the Caco-2 cellsactivity. ZnO NPs exerted higher cytotoxicity than Ag NPs in the same concentration range. ZnO NPs havedose-depended toxicity. The LD50 of ZnO NPs in Caco-2 cells is 0.431 mg/L. Significant depletion of SODlevel, variation in GSH level and release of ROS in cells treated by ZnO NPs were observed, which suggests

that cytotoxicity of ZnO NPs in intestine cells might be mediated through cellular oxidative stress. WhileCaco-2 cells treated with Ag NPs at all experimental concentrations showed no cellular oxidative damage.Moreover, the cells’ antioxidant capacity increased, and reached the highest level when the concentrationof Ag NPs was 50 �g/mL. Therefore, it can be concluded that Ag NPs are safer antibacterial material infood packaging materials than ZnO NPs.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Nanoparticles are already used in several consumer productsncluding food packages, food containers, food additives, waterurification systems and so on [1,2]. However, these broad appli-ations increase human and environmental exposure, thus theotential risk is related to their short-term and long-term toxic-

ty [3,4]. The nanoparticles may also penetrate the cell and affectellular respiration through inactivating the essential enzymes by

orming complications with the catalytic sulfur of thiol groupsn cysteine residues and through the production of toxic radi-als such as superoxide, hydrogen peroxide, and hydroxyl ions.

∗ Corresponding author at: Xueyuan Road 258, Hangzhou 310018, China.el.: +86 571 87676187; fax: +86 571 86914449.

E-mail addresses: [email protected], [email protected] (R. Guan).1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.mrfmmm.2014.08.001027-5107/© 2014 Elsevier B.V. All rights reserved.

Owing to their wide industrial and commercial applications, AgNPs and ZnO NPs attracted more attentions. Data analysis showedthat the majority of articles concerned the applications of AgNPs (7699 papers, 59%), followed by ZnO NPs (4640 papers, 36%)from 1989 to 2013 [5]. There is an increasing concern relatedto the biological impacts of the use of Ag NPs and ZnO NPson a large scale, and the possible risks to the environment andhealth.

Ag NPs show outstanding antibacterial properties [6,7]. Manyinvestigations have focused on their bacterial effects and applica-tions in plastics, health, textiles, and paint industries [8]. However,enthusiasm for Ag NPs has been hampered by their cytotoxic-ity and genotoxicity [9]. Ag NPs may inhibit the segregation ofchromosomes. Researchers have observed genotoxicity including

DNA damages and chromosomal aberrations in human glioblas-toma cells treated with Ag NPs. Koji Kawata investigated toxiceffects of Ag NPs to human hepatoma derived cell line HepG2 thatwere exposed to Ag NPs at low doses. It was concluded that both

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nano-sized particle of Ag” as well as “ionic Ag+” contributed to theoxic effects of Ag NPs [10].

ZnO NPs belonging to one type of metal oxides are character-zed by their photocatalytic and photo-oxidizing ability againsthemical and biological species. The toxicological effects of ZnO-Ps attract increasing concerns as the field of nanotechnologyrogresses. Fin Dechsakulthorn indicated that human skin fibro-lasts were sensitive to both ZnO NPs and TiO2 NPs through theTS assay [11]. Mohd Javed Akhtar investigated the cytotoxic-

ty of well-characterized ZnO NPs against three types of cancerells (human hepatocellular carcinoma HepG2, human lung ade-ocarcinoma A549, and human bronchial epithelial BEAS-2B) andwo primary rat cells (astrocytes and hepatocytes), which showedhat ZnO NPs selectively induced apoptosis in cancer cells, whichas likely to be mediated by reactive oxygen species (ROS) via53 pathway [12]. Barbara De Berardis assessed the cytotoxicity,xidative stress, apoptosis and pro-inflammatory mediator releasenduced by ZnO NPs on human colon carcinoma LoVo cells. Thexperimental data showed that oxidative stress may be a key fac-or in inducing the cytotoxicity of ZnO NPs in colon carcinomaells [13]. Maqusood Ahamed investigated the possible mecha-isms of apoptosis induced by ZnO nanorods in human alveolardenocarcinoma (A549) cells. The data demonstrated that ZnOanorod induced apoptosis in A549 cells through ROS and oxida-ive stress via p53, survivin, bax/bcl-2 and caspase pathways [14].i-Yun Kao concluded that exposure to ZnO NPs interfered withhe homeostasis of cytosolic zinc concentration ([Zn2+]c), and thatlevated [Zn2+]c resulted in cell apoptosis [15]. Ma suggested aelatively high acute toxicity of ZnO NPs (in the low mg/L lev-ls) to environmental species, and particle dissolution to ionic zincnd particle-induced generation of ROS representing the primaryodes of action for ZnO NPs toxicity across all species tested [16].Our studies have explored the influence of Ag NPs and ZnO

Ps on the Caco-2 cell line. In summary, CCK-8 assay was used tovaluate cellular toxicity. ROS production, GSH detection and SODetection were assessed in intracellular oxidative conditions. In thistudy, we reported that two types of nanoparticles (Ag NPs andnO NPs) exerted different cytotoxic effects. The potential applica-ion of Ag NPs and ZnO NPs as an antibacterial and an anticancergent would provide new opportunities for this material in nanoedicine.

. Materials and methods

.1. Samples

Caco-2 cells (CBCAS, Shanghai, China) were cultured in DMEMedium (Gibco BRL, MD, USA), with fetal calf serum (10%), l-

lutamine (2.9 mg/mL), streptomycin (1 mg/mL), and penicillin100 units/mL). The cells were cultured in a humidified incubatorat 37 ◦C, 5% CO2). Culture media were changed every 2 days. Cellsere passaged every 3–4 days. At 90% confluence, the cells werearvested using 0.25% trypsin and were subcultured into 50 cm2

asks, 12-well plates, or 96-well plates according to the experi-ents.After the monolayers of cells were placed in 12 or 96-well

lates, the cells were respectively treated with 10, 25, 50, 100, and00 �g/mL Ag NPs and ZnO NPs suspended in serum-free mediumor 24 h. Ag NPs and ZnO NPs were purchased from Hangzhou Waning New Limited. Ag NPs and ZnO NPs with anhydrous ethanolltrasonic dispersion were characterized with transmission elec-ron microscopy (TEM, JEOL Ltd., Tokyo, Japan).

.2. Cell morphology

Caco-2 cells were cultivated in a 12-well plate exposed to var-ous concentrations (0, 25, 50, 100 and 200 �g/mL) of Ag NPs and

ch 769 (2014) 113–118

ZnO NPs for 24 h in a humidified incubator (37 ◦C, 5% CO2). Removeculture solution, and add 200 �L mixture of (100 �g/mL) acridineorange (AO) and (100 �g/mL) ethidium bromide (EB) (Sigma USA)with the 1:1 AO to EB, and incubated the plate for 3 min in theincubator, removed the supernatant and then observed the cells bya fluorescence microscope (Nikon Eclipse Ti, Japan).

2.3. Cell viability

For proliferation assay, cells were seeded in 96-well plates ata density of 10,000 cells per well. Ag NPs and ZnO NPs in vari-ous concentrations (10, 25, 50, 100 and 200 �g/mL) were addedto each well. After being cultured in the incubator for 24 h, cellswere measured by the CCK-8 (Beyotime Institute of Biotechnology,China).

2.4. Oxidative stress

2.4.1. SOD detectionCells were cultured in 50 cm2 culture flasks and exposed to Ag

NPs and ZnO NPs (10–200 �g/mL) for 24 h. Then the cells wereharvested by scraping and washed twice with PBS. The part ofsupernatant was removed from the cell suspension by centrifugingfor 5 min at 4500 rpm. After ultrasonic at 300 W for 2 min (ultra-sonic 3 s Pause 2 s), the cell lysate was obtained. 20 �L of preparedsample, 200 �L of the SOD (Nanjing Jiancheng Bioengineering Insti-tute, China) working solution, 20 �L of dilution buffer, and 20 �L ofenzyme working solution was added to each well, and the mixturewas mixed thoroughly. The plate was incubated at 37 for 20 minand the absorbance was read at 450 nm using a microplate reader.

2.4.2. GSH detectionThe cell pellet was sonicated at 300 W (amplitude 100%, pulse

5 s/10 s, 2 min) to obtain the cell lysate. A cell suspension of 600 �L,reaction buffer solution of 600 �L, and substrate solution of 150 �Lwere transferred to a fresh tube. The standard group was 25 �MGSH (Nanjing Jiancheng Bioengineering Institute, China) dissolvedin GSH buffer solution. The blank group was replaced by PBS. Theabsorbance was read to 405 nm using a microplate reader. Proteincontent was measured with the method of Bradford using BSA asthe standard.

2.4.3. ROS assayROS (Beyotime Institute of Biotechnology, China) was moni-

tored by measurement of hydrogen peroxide generation. In brief,cells were seeded (20,000 cells per well) in the 96-well plates.Then, the serum-free medium with nanoparticles was removedfor 24 h, and the medium was renewed with DCF-DA dissolved inthe medium for 30 min. After washing twice with the serum-freemedium, the intensity of DCF-DA fluorescent was determined byusing ELISA.

2.5. Statistical analysis

The data were expressed as mean ± SD of three independentexperiments and were subjected to statistical analysis by one-wayanalysis of variance. A value of p < 0.05 was considered significantSPSS 16.0 software was used for the statistical analysis [17].

3. Results and discussion

3.1. Characterization of particles

The evaluation of nanomaterial was based on their size, shape,and distribution. Size and distribution of Ag NPs and ZnO NPs wereassessed using a transmission election microscopy and Zetasizer

Y. Song et al. / Mutation Research 769 (2014) 113–118 115

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ig. 1. Microscopy characterizations of NPs. Transmission electron microscope (T00 nm.

nstrumentation. Fig. 1 shows representative transmission electionicroscopy images of Ag NPs and ZnO NPs. The results showed the

verage particle diameters of ZnO NPs (A) and Ag NPs (B) were bothround 90 nm for a nanosphere. Both nanoparticles under anhy-rous ethanol as dispersants displayed uniform distributions, withome aggregated state.

.2. Cell morphology

The first and most readily noticeable effect of nanoparticles onhe cell morphology after exposure of cells to toxic materials ishe alteration in cell shape or morphology in a monolayer culture.herefore, we examined the cell morphologic characteristics by flu-

rescent staining. AO was able to infiltrate into the viable cells,nd the nuclei were stained a bright green color. Because of thentegrity of the cell plasma membrane, EB was unable to infiltratento the cells when the cells were alive or still in the early process

ig. 2. Morphology of Caco-2 cells after NPs exposure. Caco-2 morphology was observed

ith ZnO NPs and Ag NPs: ZnO (200 �g/mL) (A), ZnO (50 �g/mL) (B), ZnO (10 �g/mL) (C),

ages of 90 nm. ZnO NPs (A) and Ag NPs (B). TEM scale bars: (A), 50 nm and (B),

of apoptosis, while the dead cells had EB inside and the nuclei werestained a bright red color. Thus a viable cell (VN) had a uniformbright green nucleus and cytoplasm; an early apoptotic cell (VA),whose membranes were still intact and had a green nucleus, but alate apoptotic cells (NVA), whose chromatin condensation becamevisible in the form of bright orange areas of condensed chromatin inthe nucleus; and a necrotic cell (NVN) had a uniform bright orangenucleus (Fig. 3). Therefore, with the help of AO/EB staining, differentcells in the group could be differentiated clearly.

Figs. 2 and 3 show various morphologies of Caco-2 cells stainedwith AO/EB. After 200, 50, and 10 �g/mL ZnO-treated, the viablecells were remarkably decreased in comparison with the controlcells (Fig. 2A–C and F), and there were lots of NVA and NVN, and all

the cells shrank and became irregular in shape (Fig. 2A and B). Theresults indicated the cell proliferation treated by 50 and 200 �g/mLZnO NPs was strongly inhibited. Besides, the cells cultivated withZnO NPs showed that low dose of ZnO NPs had already damaged

with 200× magnification by optical microscope. Caco-2 cells were exposed for 24 h Ag (200 �g/mL) (D), Ag (10 �g/mL) (E), control group (F).

116 Y. Song et al. / Mutation Resear

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ig. 3. Various morphologies of Caco-2 cells stained with AO/EB. Caco-2 cells werebserved by AO/EB double staining after exposed 75 �g/mL ZnO for 24 h (VN: viableell; VA: early apoptotic cells; NVA: late apopotic cells and NVN: necrotic cells).

aco-2 cells membrane (Fig. 2C). However, the cells cultivated withg NPs in the dose from 10 to 200 �g/mL looked as the same as

he control cells (Fig. 2D–F). In other words, no significant deadells were observed in cells incubated with Ag NPs. It indicated thatg NPs had very slight cell cytotoxicity compared to ZnO NPs. Theicroscopic studies demonstrated that cells at lower doses of ZnOPs became abnormal in size, displaying cellular shrinkage, andcquisition of an irregular shape. When the concentration reached0 �g/mL, the ZnO NPs group showed significant depletion in theumber of viable cells and a substantially increased number of earlypoptotic cells, late apoptotic cells, and necrotic cells. Moreover, theg NPs (concentrations from 10 to 200 �g/mL) group showed slightytotoxicity.

.3. Cell viability

Viability assays are basic steps in toxicology that explain theellular response to a toxicant. Also, they give information on theell’s death, survival, and metabolic activities [18]. CCK-8, beingonradioactive, allows sensitive colorimetric assays for the deter-ination of the number of viable cells in cell proliferation and

ytotoxicity assays. Fig. 4 shows the cell activity variation of Caco- cells which were exposed to 10, 25, 50, 100, and 200 �g/mL ofg NPs and ZnO NPs for 24 h. The abscissa was the concentrationf nanoparticles, and the vertical coordinates were in cell activity.

ig. 4. Cytotoxicity of NPs affects cell viability using the CCK-8 assay. Caco-2 cellsere exposed on DMEM serum-free medium. With different concentrations of NPs

or 24 h, results are expressed as the percent of cell activity compared to the control.he data are presented as the mean ± SE of at least three independent experiments.

= 5, Significance indicated by: *P < 0.05, **P < 0.01.

ch 769 (2014) 113–118

The cells incubated without nanoparticles were the blank control.As shown in Fig. 4, Ag NPs and ZnO NPs highly significantly (p < 0.01)inhibited cell proliferation of Caco-2 cells from 0 to 200 �g/mLafter 24 h incubation. There were sharp percentage reductions to72.01%, 74.27%, 81.79%, 82.13% and 85.66% after ZnO NPs exposureat concentrations 10, 25, 50, 100, and 200 �g/mL. ZnO NPs havedose-depended toxicity, and the LD50 of ZnO NPs in Caco-2 cells is0.431 mg/L (Fig. 4). While Ag NPs caused a relatively slight deple-tion, the relative cell activity is 79.17%, 63.44%, 66.41%, 68.69%, and63.25% after Ag NPs exposure at concentrations 10, 25, 50, 100,and 200 �g/mL (Fig. 4). The results showed that ZnO NPs greatlyaffected cell proliferation than Ag NPs. The results might verify thesubsequence of hormesis, namely, stimulatory effects caused bylow levels of potentially toxic agents like feedback regulation. TheCCK-8 assay revealed that the cells suffered more toxicity causedby ZnO NPs in comparison to Ag NPs.

3.4. Oxidative stress markers

As not all disruptive effects result in membrane or metabolicfunction defects, more extensive cytotoxicity studies haveattempted to determine the sub-lethal effects of NPs [19,20]. Evi-dence is accumulating that oxidative stress induced by NPs is a keyroute by which these NPs induce cell damage [21]. Oxidative stress-induced cell death utilizes supra physiological excesses of biologicinduction of oxidants that results in a rapid and predictable killingof cells. Sensitive and optical detection of intracellular ROS genera-tion can provide a valuable toxicity index value for a wide range ofNPs as an early indicator for cellular responses [22]. EndocytosedNPs trigger an oxidative stress on cells by inducing the productionof intracellular ROS, which is the very first event of cellular toxicitycascade reactions [23]. Under normal conditions, the mitochon-dria generate and release moderate levels of ROS into the cytosolthat may function as signaling molecules for cell survival. How-ever, when intracellular NPs induce to generate excessive amountof ROS beyond the limit of natural antioxidant defense systems likereductive GSH and antioxidant enzymes, cells start to lose normalfunctions with consequently causing cell death [24]. The chemicalcomposition of NPs is a most decisive factor influencing ROS for-mation in lung epithelial cells [25]. ZnO NPs inducing significantlymore oxidative damage than TiO2, SiO2, ZrO2, and carbon blacknanomaterials [26].

Fig. 5 showed the intracellular ROS, GSH, and SOD level of Caco-2 cells which were exposed to 10, 25, 50, 100, and 200 �g/mL ofAg NPs and ZnO NPs for 24 h. The abscissa was the concentrationof nanoparticles, and the vertical coordinates were the intracel-lular ROS, GSH, and SOD level, respectively. The cells incubatedwithout nanoparticles were the blank control. After 24 h expo-sure, Ag NPs did not significantly induce ROS at the concentrationrange of 0–200 �g/mL in Caco-2 cells. As for ZnO NPs, at concen-trations 10, 25, 50, 100, and 200 �g/mL, the fold of ROS levels(relative to control) was 2.28, 2.47, 2.43, 2.42, and 1.61. ZnO NPs(10–100 �g/mL) resulted in a significant increase in intracellularROS (Fig. 5A). According to the data, presumably the intense cyto-toxicity of 200 �g/mL ZnO NPs might have lead to a large numberof cells death, so there were not enough cells to produce a greatquantity ROS. Particle dissolution to ionic zinc and particle-inducedgeneration of reactive oxygen species (ROS) represent the primarymodes of action for ZnO NPs toxicity.

GSH, an important endogenous antioxidant, has long beenknown to be involved in detoxifying reactions by protecting thethiol groups of enzymes and reacting with hydroxyl radicals, singlet

oxygen, and hydrogen peroxide via GSH peroxidase (GPx) catal-ysis. The amount of GSH could reflect the antioxidant potentialof an organelle [27]. In the present study, GSH is the most abun-dant non-protein thiol in cells participating in some processes,

Y. Song et al. / Mutation Resear

Fig. 5. Effects of ZnO and Ag NPs on oxidative stress. Effects of NPs on ROS (A), GSH(B), SOD (C) levels after 24 h exposure. Data were expressed as the comparison withte

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test organisms and mammalian cells in vitro: a critical review, Arch. Toxicol.

he control group. The data are presented as mean ± SE of at least three independentxperiments. N = 4, significance indicated by: *P < 0.05, **P < 0.01.

ncluding synthesis of DNA and proteins, regulation of enzymectivities, and inter and intracellular transportations [28]. Accord-ng to Fig. 5B, compared with the control group, intracellular GSHnduced by ZnO NPs (10–200 �g/mL) highly significantly (p < 0.01)ncreased, and intracellular GSH level reached 212.68%, 223.52%,85.39%, 209.65%, and 172.62%. The cells incubated at 50 �g/mLnO NPs retained the most abundant GSH. However, GSH inducedy ZnO NPs (10–50 �g/mL) highly significantly (p < 0.01 vs. the con-rol group) increased. The cells that were exposed to Ag NPs showedhe slight depletion of GSH level in a dose-dependent manner. Theells exposed to ZnO NPs produced more GSH than Ag NPs. Theesults demonstrated that ZnO NPs showed significant toxicity toaco-2 cells at 10–200 �g/mL, but Ag NPs showed slight toxicity inhe same concentrations.

SOD is viewed as an antioxidant enzyme, which transformsuperoxide anions (O2−) into less reactive species-H2O2. The2O2 formed by SOD activity is decomposed to H2O and O2 bylutathione peroxidase (GPx) in the presence of reduced GSH.ccording to Fig. 5C, for Caco-2 cells exposed to ZnO NPs, theOD level was highly significant (p < 0.01) reduced at concentra-

ions 10–200 �g/mL for 24 h. While for Caco-2 cells exposed to AgPs, the SOD level had no significant variation. Although, the mainechanism of NPs cytotoxicity may differ, depending on the types

ch 769 (2014) 113–118 117

of NPs involved. This clearly demonstrated that ZnO NPs were moretoxic than Ag NPs in Caco-2 cells.

4. Conclusions

According to the AO/EB double staining and CCK-8 assay, theZnO NPs cytotoxicity exhibited a dose-dependent effect. ROS, GSH,and SOD were considered as oxidative stress markers of cytotoxicmechanism. Exposed to ZnO NPs (10–200 �g/mL), intracellular ROSand GSH level sharply increased, and SOD level was highly sig-nificantly decreased. Therefore, it can be concluded that ZnO NPshad the potency for the generation of ROS and eventual cyto-toxicity. Ag NPs failed to induce ROS and SOD variation, whileintracellular GSH level was obviously increased. The results demon-strated that ZnO NPs (10–200 �g/mL) appeared high toxicity, andAg NPs (10–200 �g/mL) appeared no obvious toxicity in Caco-2cells. Although it has been reported that Ag NPs have toxic effectsto various cultured cells, the toxic effects at non-cytotoxic dosesare still unknown. Besides, It was demonstrated that Ag NPs accel-erated cell proliferation at low doses (<0.5 mg/L). The risk of anypotentially toxic substance is not only a function of hazard but alsoa chance of exposure. It is useful to exploit the findings to engineerimproved nanoparticles ultimately for use in consumer products.Although NPs cytotoxicity has been reported several times, but itis necessary to know that in vitro results can differ from what isfound in vivo.

Conflict of interest statement

All authors of this research paper have directly participated inthe planning, execution, or analysis of the study. All authors of thispaper have read and approved the final version submitted. Thecontents of this manuscript have not been copyrighted or publishedpreviously. We declare that we do not have any commercial or asso-ciative interest that represents a conflict of interest in connectionwith the work submitted.

Acknowledgments

This work was supported by Zhejiang Provincial EngineeringLaboratory of Quality Controlling Technology and Instrumenta-tion for Marine Food. We gratefully acknowledge financial supportfrom Zhejiang Provincial Natural Science Foundation of China(LY14C200012) and Zhejiang Provincial Public Technology Appli-cation Research Project (2012C22052) and General Administrationof Quality Supervision, Inspection and Quarantine of the People’sRepublic of China (201310120) and Hangzhou Science and Tech-nology Development Project (20130432B66 and 20120232B72).

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