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In vitro cytotoxicity of silver nanoparticles and zinc oxide nanoparticles to human epithelial colorectal adenocarcinoma (Caco-2) cells
Mutation Research 769 (2014) 113–118
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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: 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 Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, China c Hubei University of Technology, Wuhan 430068, China b
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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 adenocarcinoma (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 dismutase (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 LD50 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 applications increase human and environmental exposure, thus the potential risk is related to their short-term and long-term toxicity [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 radicals 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. http://dx.doi.org/10.1016/j.mrfmmm.2014.08.001 0027-5107/© 2014 Elsevier B.V. All rights reserved.
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 applications in plastics, health, textiles, and paint industries [8]. However, enthusiasm for Ag NPs has been hampered by their cytotoxicity and genotoxicity [9]. Ag NPs may inhibit the segregation of chromosomes. Researchers have observed genotoxicity including DNA damages and chromosomal aberrations in human glioblastoma 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
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“nano-sized particle of Ag” as well as “ionic Ag+ ” contributed to the toxic effects of Ag NPs [10]. ZnO NPs belonging to one type of metal oxides are characterized by their photocatalytic and photo-oxidizing ability against chemical and biological species. The toxicological effects of ZnONPs attract increasing concerns as the field of nanotechnology progresses. Fin Dechsakulthorn indicated that human skin fibroblasts were sensitive to both ZnO NPs and TiO2 NPs through the MTS assay [11]. Mohd Javed Akhtar investigated the cytotoxicity of well-characterized ZnO NPs against three types of cancer cells (human hepatocellular carcinoma HepG2 , human lung adenocarcinoma A549, and human bronchial epithelial BEAS-2B) and two primary rat cells (astrocytes and hepatocytes), which showed that ZnO NPs selectively induced apoptosis in cancer cells, which was likely to be mediated by reactive oxygen species (ROS) via p53 pathway [12]. Barbara De Berardis assessed the cytotoxicity, oxidative stress, apoptosis and pro-inflammatory mediator release induced by ZnO NPs on human colon carcinoma LoVo cells. The experimental data showed that oxidative stress may be a key factor in inducing the cytotoxicity of ZnO NPs in colon carcinoma cells [13]. Maqusood Ahamed investigated the possible mechanisms of apoptosis induced by ZnO nanorods in human alveolar adenocarcinoma (A549) cells. The data demonstrated that ZnO nanorod induced apoptosis in A549 cells through ROS and oxidative stress via p53, survivin, bax/bcl-2 and caspase pathways [14]. Yi-Yun Kao concluded that exposure to ZnO NPs interfered with the homeostasis of cytosolic zinc concentration ([Zn2+ ]c ), and that elevated [Zn2+ ]c resulted in cell apoptosis [15]. Ma suggested a relatively high acute toxicity of ZnO NPs (in the low mg/L levels) to environmental species, and particle dissolution to ionic zinc and particle-induced generation of ROS representing the primary modes of action for ZnO NPs toxicity across all species tested [16]. Our studies have explored the influence of Ag NPs and ZnO NPs on the Caco-2 cell line. In summary, CCK-8 assay was used to evaluate cellular toxicity. ROS production, GSH detection and SOD detection were assessed in intracellular oxidative conditions. In this study, we reported that two types of nanoparticles (Ag NPs and ZnO NPs) exerted different cytotoxic effects. The potential application of Ag NPs and ZnO NPs as an antibacterial and an anticancer agent would provide new opportunities for this material in nano medicine. 2. Materials and methods 2.1. Samples Caco-2 cells (CBCAS, Shanghai, China) were cultured in DMEM medium (Gibco BRL, MD, USA), with fetal calf serum (10%), lglutamine (2.9 mg/mL), streptomycin (1 mg/mL), and penicillin (100 units/mL). The cells were cultured in a humidified incubator (at 37 ◦ C, 5% CO2 ). Culture media were changed every 2 days. Cells were passaged every 3–4 days. At 90% confluence, the cells were harvested using 0.25% trypsin and were subcultured into 50 cm2 flasks, 12-well plates, or 96-well plates according to the experiments. After the monolayers of cells were placed in 12 or 96-well plates, the cells were respectively treated with 10, 25, 50, 100, and 200 g/mL Ag NPs and ZnO NPs suspended in serum-free medium for 24 h. Ag NPs and ZnO NPs were purchased from Hangzhou Wan Jing New Limited. Ag NPs and ZnO NPs with anhydrous ethanol ultrasonic dispersion were characterized with transmission electron microscopy (TEM, JEOL Ltd., Tokyo, Japan).
ZnO NPs for 24 h in a humidified incubator (37 ◦ C, 5% CO2 ). Remove culture solution, and add 200 L mixture of (100 g/mL) acridine orange (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 the incubator, removed the supernatant and then observed the cells by a fluorescence microscope (Nikon Eclipse Ti, Japan). 2.3. Cell viability For proliferation assay, cells were seeded in 96-well plates at a density of 10,000 cells per well. Ag NPs and ZnO NPs in various concentrations (10, 25, 50, 100 and 200 g/mL) were added to each well. After being cultured in the incubator for 24 h, cells were measured by the CCK-8 (Beyotime Institute of Biotechnology, China). 2.4. Oxidative stress 2.4.1. SOD detection Cells 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 were harvested by scraping and washed twice with PBS. The part of supernatant was removed from the cell suspension by centrifuging for 5 min at 4500 rpm. After ultrasonic at 300 W for 2 min (ultrasonic 3 s Pause 2 s), the cell lysate was obtained. 20 L of prepared sample, 200 L of the SOD (Nanjing Jiancheng Bioengineering Institute, China) working solution, 20 L of dilution buffer, and 20 L of enzyme working solution was added to each well, and the mixture was mixed thoroughly. The plate was incubated at 37 for 20 min and the absorbance was read at 450 nm using a microplate reader. 2.4.2. GSH detection The 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 L were transferred to a fresh tube. The standard group was 25 M GSH (Nanjing Jiancheng Bioengineering Institute, China) dissolved in GSH buffer solution. The blank group was replaced by PBS. The absorbance was read to 405 nm using a microplate reader. Protein content was measured with the method of Bradford using BSA as the standard. 2.4.3. ROS assay ROS (Beyotime Institute of Biotechnology, China) was monitored 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 removed for 24 h, and the medium was renewed with DCF-DA dissolved in the medium for 30 min. After washing twice with the serum-free medium, the intensity of DCF-DA fluorescent was determined by using ELISA. 2.5. Statistical analysis The data were expressed as mean ± SD of three independent experiments and were subjected to statistical analysis by one-way analysis of variance. A value of p < 0.05 was considered significant SPSS 16.0 software was used for the statistical analysis [17]. 3. Results and discussion 3.1. Characterization of particles
2.2. Cell morphology Caco-2 cells were cultivated in a 12-well plate exposed to various concentrations (0, 25, 50, 100 and 200 g/mL) of Ag NPs and
The evaluation of nanomaterial was based on their size, shape, and distribution. Size and distribution of Ag NPs and ZnO NPs were assessed using a transmission election microscopy and Zetasizer
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Fig. 1. Microscopy characterizations of NPs. Transmission electron microscope (TEM) images of 90 nm. ZnO NPs (A) and Ag NPs (B). TEM scale bars: (A), 50 nm and (B), 200 nm.
instrumentation. Fig. 1 shows representative transmission election microscopy images of Ag NPs and ZnO NPs. The results showed the average particle diameters of ZnO NPs (A) and Ag NPs (B) were both around 90 nm for a nanosphere. Both nanoparticles under anhydrous ethanol as dispersants displayed uniform distributions, with some aggregated state. 3.2. Cell morphology The first and most readily noticeable effect of nanoparticles on the cell morphology after exposure of cells to toxic materials is the alteration in cell shape or morphology in a monolayer culture. Therefore, we examined the cell morphologic characteristics by fluorescent staining. AO was able to infiltrate into the viable cells, and the nuclei were stained a bright green color. Because of the integrity of the cell plasma membrane, EB was unable to infiltrate into the cells when the cells were alive or still in the early process
of apoptosis, while the dead cells had EB inside and the nuclei were stained a bright red color. Thus a viable cell (VN) had a uniform bright green nucleus and cytoplasm; an early apoptotic cell (VA), whose membranes were still intact and had a green nucleus, but a late apoptotic cells (NVA), whose chromatin condensation became visible in the form of bright orange areas of condensed chromatin in the nucleus; and a necrotic cell (NVN) had a uniform bright orange nucleus (Fig. 3). Therefore, with the help of AO/EB staining, different cells in the group could be differentiated clearly. Figs. 2 and 3 show various morphologies of Caco-2 cells stained with AO/EB. After 200, 50, and 10 g/mL ZnO-treated, the viable cells were remarkably decreased in comparison with the control cells (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). The results indicated the cell proliferation treated by 50 and 200 g/mL ZnO NPs was strongly inhibited. Besides, the cells cultivated with ZnO NPs showed that low dose of ZnO NPs had already damaged
Fig. 2. Morphology of Caco-2 cells after NPs exposure. Caco-2 morphology was observed with 200× magnification by optical microscope. Caco-2 cells were exposed for 24 h with ZnO NPs and Ag NPs: ZnO (200 g/mL) (A), ZnO (50 g/mL) (B), ZnO (10 g/mL) (C), Ag (200 g/mL) (D), Ag (10 g/mL) (E), control group (F).
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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/mL after 24 h incubation. There were sharp percentage reductions to 72.01%, 74.27%, 81.79%, 82.13% and 85.66% after ZnO NPs exposure at concentrations 10, 25, 50, 100, and 200 g/mL. ZnO NPs have dose-depended toxicity, and the LD50 of ZnO NPs in Caco-2 cells is 0.431 mg/L (Fig. 4). While Ag NPs caused a relatively slight depletion, the relative cell activity is 79.17%, 63.44%, 66.41%, 68.69%, and 63.25% after Ag NPs exposure at concentrations 10, 25, 50, 100, and 200 g/mL (Fig. 4). The results showed that ZnO NPs greatly affected cell proliferation than Ag NPs. The results might verify the subsequence of hormesis, namely, stimulatory effects caused by low levels of potentially toxic agents like feedback regulation. The CCK-8 assay revealed that the cells suffered more toxicity caused by ZnO NPs in comparison to Ag NPs. 3.4. Oxidative stress markers Fig. 3. Various morphologies of Caco-2 cells stained with AO/EB. Caco-2 cells were observed by AO/EB double staining after exposed 75 g/mL ZnO for 24 h (VN: viable cell; VA: early apoptotic cells; NVA: late apopotic cells and NVN: necrotic cells).
Caco-2 cells membrane (Fig. 2C). However, the cells cultivated with Ag NPs in the dose from 10 to 200 g/mL looked as the same as the control cells (Fig. 2D–F). In other words, no significant dead cells were observed in cells incubated with Ag NPs. It indicated that Ag NPs had very slight cell cytotoxicity compared to ZnO NPs. The microscopic studies demonstrated that cells at lower doses of ZnO NPs became abnormal in size, displaying cellular shrinkage, and acquisition of an irregular shape. When the concentration reached 50 g/mL, the ZnO NPs group showed significant depletion in the number of viable cells and a substantially increased number of early apoptotic cells, late apoptotic cells, and necrotic cells. Moreover, the Ag NPs (concentrations from 10 to 200 g/mL) group showed slight cytotoxicity. 3.3. Cell viability Viability assays are basic steps in toxicology that explain the cellular response to a toxicant. Also, they give information on the cell’s death, survival, and metabolic activities [18]. CCK-8, being nonradioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. Fig. 4 shows the cell activity variation of Caco2 cells which were exposed to 10, 25, 50, 100, and 200 g/mL of Ag NPs and ZnO NPs for 24 h. The abscissa was the concentration of nanoparticles, and the vertical coordinates were in cell activity.
Fig. 4. Cytotoxicity of NPs affects cell viability using the CCK-8 assay. Caco-2 cells were exposed on DMEM serum-free medium. With different concentrations of NPs for 24 h, results are expressed as the percent of cell activity compared to the control. The data are presented as the mean ± SE of at least three independent experiments. N = 5, Significance indicated by: *P < 0.05, **P < 0.01.
As not all disruptive effects result in membrane or metabolic function defects, more extensive cytotoxicity studies have attempted to determine the sub-lethal effects of NPs [19,20]. Evidence is accumulating that oxidative stress induced by NPs is a key route by which these NPs induce cell damage [21]. Oxidative stressinduced cell death utilizes supra physiological excesses of biologic induction of oxidants that results in a rapid and predictable killing of cells. Sensitive and optical detection of intracellular ROS generation can provide a valuable toxicity index value for a wide range of NPs as an early indicator for cellular responses [22]. Endocytosed NPs trigger an oxidative stress on cells by inducing the production of intracellular ROS, which is the very first event of cellular toxicity cascade reactions [23]. Under normal conditions, the mitochondria generate and release moderate levels of ROS into the cytosol that may function as signaling molecules for cell survival. However, when intracellular NPs induce to generate excessive amount of ROS beyond the limit of natural antioxidant defense systems like reductive GSH and antioxidant enzymes, cells start to lose normal functions with consequently causing cell death [24]. The chemical composition of NPs is a most decisive factor influencing ROS formation in lung epithelial cells [25]. ZnO NPs inducing significantly more oxidative damage than TiO2 , SiO2 , ZrO2 , and carbon black nanomaterials [26]. Fig. 5 showed the intracellular ROS, GSH, and SOD level of Caco2 cells which were exposed to 10, 25, 50, 100, and 200 g/mL of Ag NPs and ZnO NPs for 24 h. The abscissa was the concentration of nanoparticles, and the vertical coordinates were the intracellular ROS, GSH, and SOD level, respectively. The cells incubated without nanoparticles were the blank control. After 24 h exposure, Ag NPs did not significantly induce ROS at the concentration range of 0–200 g/mL in Caco-2 cells. As for ZnO NPs, at concentrations 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 intracellular ROS (Fig. 5A). According to the data, presumably the intense cytotoxicity of 200 g/mL ZnO NPs might have lead to a large number of cells death, so there were not enough cells to produce a great quantity ROS. Particle dissolution to ionic zinc and particle-induced generation of reactive oxygen species (ROS) represent the primary modes of action for ZnO NPs toxicity. GSH, an important endogenous antioxidant, has long been known to be involved in detoxifying reactions by protecting the thiol groups of enzymes and reacting with hydroxyl radicals, singlet oxygen, and hydrogen peroxide via GSH peroxidase (GPx) catalysis. The amount of GSH could reflect the antioxidant potential of an organelle [27]. In the present study, GSH is the most abundant non-protein thiol in cells participating in some processes,
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of NPs involved. This clearly demonstrated that ZnO NPs were more toxic than Ag NPs in Caco-2 cells. 4. Conclusions According to the AO/EB double staining and CCK-8 assay, the ZnO NPs cytotoxicity exhibited a dose-dependent effect. ROS, GSH, and SOD were considered as oxidative stress markers of cytotoxic mechanism. Exposed to ZnO NPs (10–200 g/mL), intracellular ROS and GSH level sharply increased, and SOD level was highly significantly decreased. Therefore, it can be concluded that ZnO NPs had the potency for the generation of ROS and eventual cytotoxicity. Ag NPs failed to induce ROS and SOD variation, while intracellular GSH level was obviously increased. The results demonstrated that ZnO NPs (10–200 g/mL) appeared high toxicity, and Ag NPs (10–200 g/mL) appeared no obvious toxicity in Caco-2 cells. Although it has been reported that Ag NPs have toxic effects to various cultured cells, the toxic effects at non-cytotoxic doses are still unknown. Besides, It was demonstrated that Ag NPs accelerated cell proliferation at low doses (<0.5 mg/L). The risk of any potentially toxic substance is not only a function of hazard but also a chance of exposure. It is useful to exploit the findings to engineer improved nanoparticles ultimately for use in consumer products. Although NPs cytotoxicity has been reported several times, but it is necessary to know that in vitro results can differ from what is found in vivo. Conflict of interest statement All authors of this research paper have directly participated in the planning, execution, or analysis of the study. All authors of this paper have read and approved the final version submitted. The contents of this manuscript have not been copyrighted or published previously. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. 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 with the control group. The data are presented as mean ± SE of at least three independent experiments. N = 4, significance indicated by: *P < 0.05, **P < 0.01.
including synthesis of DNA and proteins, regulation of enzyme activities, and inter and intracellular transportations [28]. According to Fig. 5B, compared with the control group, intracellular GSH induced by ZnO NPs (10–200 g/mL) highly significantly (p < 0.01) increased, and intracellular GSH level reached 212.68%, 223.52%, 285.39%, 209.65%, and 172.62%. The cells incubated at 50 g/mL ZnO NPs retained the most abundant GSH. However, GSH induced by ZnO NPs (10–50 g/mL) highly significantly (p < 0.01 vs. the control group) increased. The cells that were exposed to Ag NPs showed the slight depletion of GSH level in a dose-dependent manner. The cells exposed to ZnO NPs produced more GSH than Ag NPs. The results demonstrated that ZnO NPs showed significant toxicity to Caco-2 cells at 10–200 g/mL, but Ag NPs showed slight toxicity in the same concentrations. SOD is viewed as an antioxidant enzyme, which transforms superoxide anions (O2− ) into less reactive species-H2 O2 . The H2 O2 formed by SOD activity is decomposed to H2 O and O2 by glutathione peroxidase (GPx) in the presence of reduced GSH. According to Fig. 5C, for Caco-2 cells exposed to ZnO NPs, the SOD level was highly significant (p < 0.01) reduced at concentrations 10–200 g/mL for 24 h. While for Caco-2 cells exposed to Ag NPs, the SOD level had no significant variation. Although, the main mechanism of NPs cytotoxicity may differ, depending on the types
Acknowledgments This work was supported by Zhejiang Provincial Engineering Laboratory of Quality Controlling Technology and Instrumentation for Marine Food. We gratefully acknowledge financial support from Zhejiang Provincial Natural Science Foundation of China (LY14C200012) and Zhejiang Provincial Public Technology Application Research Project (2012C22052) and General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (201310120) and Hangzhou Science and Technology Development Project (20130432B66 and 20120232B72). References [1] L. Calzolai, D. Gilliland, F. Rossi, Measuring nanoparticles size distribution in food and consumer products: a review, Food Addit. Contam. A: Chem. Anal. Control Expo. Risk Assess. 29 (2012) 1183–1193. [2] C.M. Lopes, J.R. Fernandes, P. Martins-Lopes, Application of nanotechnology in the agro-food sector, Food Technol. Biotechnol. 51 (2013). [3] N. Lewinski, V. Colvin, R. Drezek, Cytotoxicity of nanoparticles, Small 4 (2008) 26–49. [4] R. Balansky, M. Longobardi, G. Ganchev, M. Iltcheva, N. Nedyalkov, P. Atanasov, R. Toshkova, S. De Flora, A. Izzotti, Transplacental clastogenic and epigenetic effects of gold nanoparticles in mice, Mutat. Res. (2013) . [5] O. Bondarenko, K. Juganson, A. Ivask, K. Kasemets, M. Mortimer, A. Kahru, Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review, Arch. Toxicol. 87 (2013) 1181–1200. [6] J.S. Kim, E. Kuk, K.N. Yu, J.-H. Kim, S.J. Park, H.J. Lee, S.H. Kim, Y.K. Park, Y.H. Park, C.-Y. Hwang, Antimicrobial effects of silver nanoparticles, Nanomed. Nanotechnol. Biol. Med. 3 (2007) 95–101.
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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: 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 Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, China c Hubei University of Technology, Wuhan 430068, China b
a r t i c l e
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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 adenocarcinoma (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 dismutase (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 LD50 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 applications increase human and environmental exposure, thus the potential risk is related to their short-term and long-term toxicity [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 radicals 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. http://dx.doi.org/10.1016/j.mrfmmm.2014.08.001 0027-5107/© 2014 Elsevier B.V. All rights reserved.
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 applications in plastics, health, textiles, and paint industries [8]. However, enthusiasm for Ag NPs has been hampered by their cytotoxicity and genotoxicity [9]. Ag NPs may inhibit the segregation of chromosomes. Researchers have observed genotoxicity including DNA damages and chromosomal aberrations in human glioblastoma 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
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“nano-sized particle of Ag” as well as “ionic Ag+ ” contributed to the toxic effects of Ag NPs [10]. ZnO NPs belonging to one type of metal oxides are characterized by their photocatalytic and photo-oxidizing ability against chemical and biological species. The toxicological effects of ZnONPs attract increasing concerns as the field of nanotechnology progresses. Fin Dechsakulthorn indicated that human skin fibroblasts were sensitive to both ZnO NPs and TiO2 NPs through the MTS assay [11]. Mohd Javed Akhtar investigated the cytotoxicity of well-characterized ZnO NPs against three types of cancer cells (human hepatocellular carcinoma HepG2 , human lung adenocarcinoma A549, and human bronchial epithelial BEAS-2B) and two primary rat cells (astrocytes and hepatocytes), which showed that ZnO NPs selectively induced apoptosis in cancer cells, which was likely to be mediated by reactive oxygen species (ROS) via p53 pathway [12]. Barbara De Berardis assessed the cytotoxicity, oxidative stress, apoptosis and pro-inflammatory mediator release induced by ZnO NPs on human colon carcinoma LoVo cells. The experimental data showed that oxidative stress may be a key factor in inducing the cytotoxicity of ZnO NPs in colon carcinoma cells [13]. Maqusood Ahamed investigated the possible mechanisms of apoptosis induced by ZnO nanorods in human alveolar adenocarcinoma (A549) cells. The data demonstrated that ZnO nanorod induced apoptosis in A549 cells through ROS and oxidative stress via p53, survivin, bax/bcl-2 and caspase pathways [14]. Yi-Yun Kao concluded that exposure to ZnO NPs interfered with the homeostasis of cytosolic zinc concentration ([Zn2+ ]c ), and that elevated [Zn2+ ]c resulted in cell apoptosis [15]. Ma suggested a relatively high acute toxicity of ZnO NPs (in the low mg/L levels) to environmental species, and particle dissolution to ionic zinc and particle-induced generation of ROS representing the primary modes of action for ZnO NPs toxicity across all species tested [16]. Our studies have explored the influence of Ag NPs and ZnO NPs on the Caco-2 cell line. In summary, CCK-8 assay was used to evaluate cellular toxicity. ROS production, GSH detection and SOD detection were assessed in intracellular oxidative conditions. In this study, we reported that two types of nanoparticles (Ag NPs and ZnO NPs) exerted different cytotoxic effects. The potential application of Ag NPs and ZnO NPs as an antibacterial and an anticancer agent would provide new opportunities for this material in nano medicine. 2. Materials and methods 2.1. Samples Caco-2 cells (CBCAS, Shanghai, China) were cultured in DMEM medium (Gibco BRL, MD, USA), with fetal calf serum (10%), lglutamine (2.9 mg/mL), streptomycin (1 mg/mL), and penicillin (100 units/mL). The cells were cultured in a humidified incubator (at 37 ◦ C, 5% CO2 ). Culture media were changed every 2 days. Cells were passaged every 3–4 days. At 90% confluence, the cells were harvested using 0.25% trypsin and were subcultured into 50 cm2 flasks, 12-well plates, or 96-well plates according to the experiments. After the monolayers of cells were placed in 12 or 96-well plates, the cells were respectively treated with 10, 25, 50, 100, and 200 g/mL Ag NPs and ZnO NPs suspended in serum-free medium for 24 h. Ag NPs and ZnO NPs were purchased from Hangzhou Wan Jing New Limited. Ag NPs and ZnO NPs with anhydrous ethanol ultrasonic dispersion were characterized with transmission electron microscopy (TEM, JEOL Ltd., Tokyo, Japan).
ZnO NPs for 24 h in a humidified incubator (37 ◦ C, 5% CO2 ). Remove culture solution, and add 200 L mixture of (100 g/mL) acridine orange (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 the incubator, removed the supernatant and then observed the cells by a fluorescence microscope (Nikon Eclipse Ti, Japan). 2.3. Cell viability For proliferation assay, cells were seeded in 96-well plates at a density of 10,000 cells per well. Ag NPs and ZnO NPs in various concentrations (10, 25, 50, 100 and 200 g/mL) were added to each well. After being cultured in the incubator for 24 h, cells were measured by the CCK-8 (Beyotime Institute of Biotechnology, China). 2.4. Oxidative stress 2.4.1. SOD detection Cells 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 were harvested by scraping and washed twice with PBS. The part of supernatant was removed from the cell suspension by centrifuging for 5 min at 4500 rpm. After ultrasonic at 300 W for 2 min (ultrasonic 3 s Pause 2 s), the cell lysate was obtained. 20 L of prepared sample, 200 L of the SOD (Nanjing Jiancheng Bioengineering Institute, China) working solution, 20 L of dilution buffer, and 20 L of enzyme working solution was added to each well, and the mixture was mixed thoroughly. The plate was incubated at 37 for 20 min and the absorbance was read at 450 nm using a microplate reader. 2.4.2. GSH detection The 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 L were transferred to a fresh tube. The standard group was 25 M GSH (Nanjing Jiancheng Bioengineering Institute, China) dissolved in GSH buffer solution. The blank group was replaced by PBS. The absorbance was read to 405 nm using a microplate reader. Protein content was measured with the method of Bradford using BSA as the standard. 2.4.3. ROS assay ROS (Beyotime Institute of Biotechnology, China) was monitored 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 removed for 24 h, and the medium was renewed with DCF-DA dissolved in the medium for 30 min. After washing twice with the serum-free medium, the intensity of DCF-DA fluorescent was determined by using ELISA. 2.5. Statistical analysis The data were expressed as mean ± SD of three independent experiments and were subjected to statistical analysis by one-way analysis of variance. A value of p < 0.05 was considered significant SPSS 16.0 software was used for the statistical analysis [17]. 3. Results and discussion 3.1. Characterization of particles
2.2. Cell morphology Caco-2 cells were cultivated in a 12-well plate exposed to various concentrations (0, 25, 50, 100 and 200 g/mL) of Ag NPs and
The evaluation of nanomaterial was based on their size, shape, and distribution. Size and distribution of Ag NPs and ZnO NPs were assessed using a transmission election microscopy and Zetasizer
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Fig. 1. Microscopy characterizations of NPs. Transmission electron microscope (TEM) images of 90 nm. ZnO NPs (A) and Ag NPs (B). TEM scale bars: (A), 50 nm and (B), 200 nm.
instrumentation. Fig. 1 shows representative transmission election microscopy images of Ag NPs and ZnO NPs. The results showed the average particle diameters of ZnO NPs (A) and Ag NPs (B) were both around 90 nm for a nanosphere. Both nanoparticles under anhydrous ethanol as dispersants displayed uniform distributions, with some aggregated state. 3.2. Cell morphology The first and most readily noticeable effect of nanoparticles on the cell morphology after exposure of cells to toxic materials is the alteration in cell shape or morphology in a monolayer culture. Therefore, we examined the cell morphologic characteristics by fluorescent staining. AO was able to infiltrate into the viable cells, and the nuclei were stained a bright green color. Because of the integrity of the cell plasma membrane, EB was unable to infiltrate into the cells when the cells were alive or still in the early process
of apoptosis, while the dead cells had EB inside and the nuclei were stained a bright red color. Thus a viable cell (VN) had a uniform bright green nucleus and cytoplasm; an early apoptotic cell (VA), whose membranes were still intact and had a green nucleus, but a late apoptotic cells (NVA), whose chromatin condensation became visible in the form of bright orange areas of condensed chromatin in the nucleus; and a necrotic cell (NVN) had a uniform bright orange nucleus (Fig. 3). Therefore, with the help of AO/EB staining, different cells in the group could be differentiated clearly. Figs. 2 and 3 show various morphologies of Caco-2 cells stained with AO/EB. After 200, 50, and 10 g/mL ZnO-treated, the viable cells were remarkably decreased in comparison with the control cells (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). The results indicated the cell proliferation treated by 50 and 200 g/mL ZnO NPs was strongly inhibited. Besides, the cells cultivated with ZnO NPs showed that low dose of ZnO NPs had already damaged
Fig. 2. Morphology of Caco-2 cells after NPs exposure. Caco-2 morphology was observed with 200× magnification by optical microscope. Caco-2 cells were exposed for 24 h with ZnO NPs and Ag NPs: ZnO (200 g/mL) (A), ZnO (50 g/mL) (B), ZnO (10 g/mL) (C), Ag (200 g/mL) (D), Ag (10 g/mL) (E), control group (F).
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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/mL after 24 h incubation. There were sharp percentage reductions to 72.01%, 74.27%, 81.79%, 82.13% and 85.66% after ZnO NPs exposure at concentrations 10, 25, 50, 100, and 200 g/mL. ZnO NPs have dose-depended toxicity, and the LD50 of ZnO NPs in Caco-2 cells is 0.431 mg/L (Fig. 4). While Ag NPs caused a relatively slight depletion, the relative cell activity is 79.17%, 63.44%, 66.41%, 68.69%, and 63.25% after Ag NPs exposure at concentrations 10, 25, 50, 100, and 200 g/mL (Fig. 4). The results showed that ZnO NPs greatly affected cell proliferation than Ag NPs. The results might verify the subsequence of hormesis, namely, stimulatory effects caused by low levels of potentially toxic agents like feedback regulation. The CCK-8 assay revealed that the cells suffered more toxicity caused by ZnO NPs in comparison to Ag NPs. 3.4. Oxidative stress markers Fig. 3. Various morphologies of Caco-2 cells stained with AO/EB. Caco-2 cells were observed by AO/EB double staining after exposed 75 g/mL ZnO for 24 h (VN: viable cell; VA: early apoptotic cells; NVA: late apopotic cells and NVN: necrotic cells).
Caco-2 cells membrane (Fig. 2C). However, the cells cultivated with Ag NPs in the dose from 10 to 200 g/mL looked as the same as the control cells (Fig. 2D–F). In other words, no significant dead cells were observed in cells incubated with Ag NPs. It indicated that Ag NPs had very slight cell cytotoxicity compared to ZnO NPs. The microscopic studies demonstrated that cells at lower doses of ZnO NPs became abnormal in size, displaying cellular shrinkage, and acquisition of an irregular shape. When the concentration reached 50 g/mL, the ZnO NPs group showed significant depletion in the number of viable cells and a substantially increased number of early apoptotic cells, late apoptotic cells, and necrotic cells. Moreover, the Ag NPs (concentrations from 10 to 200 g/mL) group showed slight cytotoxicity. 3.3. Cell viability Viability assays are basic steps in toxicology that explain the cellular response to a toxicant. Also, they give information on the cell’s death, survival, and metabolic activities [18]. CCK-8, being nonradioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. Fig. 4 shows the cell activity variation of Caco2 cells which were exposed to 10, 25, 50, 100, and 200 g/mL of Ag NPs and ZnO NPs for 24 h. The abscissa was the concentration of nanoparticles, and the vertical coordinates were in cell activity.
Fig. 4. Cytotoxicity of NPs affects cell viability using the CCK-8 assay. Caco-2 cells were exposed on DMEM serum-free medium. With different concentrations of NPs for 24 h, results are expressed as the percent of cell activity compared to the control. The data are presented as the mean ± SE of at least three independent experiments. N = 5, Significance indicated by: *P < 0.05, **P < 0.01.
As not all disruptive effects result in membrane or metabolic function defects, more extensive cytotoxicity studies have attempted to determine the sub-lethal effects of NPs [19,20]. Evidence is accumulating that oxidative stress induced by NPs is a key route by which these NPs induce cell damage [21]. Oxidative stressinduced cell death utilizes supra physiological excesses of biologic induction of oxidants that results in a rapid and predictable killing of cells. Sensitive and optical detection of intracellular ROS generation can provide a valuable toxicity index value for a wide range of NPs as an early indicator for cellular responses [22]. Endocytosed NPs trigger an oxidative stress on cells by inducing the production of intracellular ROS, which is the very first event of cellular toxicity cascade reactions [23]. Under normal conditions, the mitochondria generate and release moderate levels of ROS into the cytosol that may function as signaling molecules for cell survival. However, when intracellular NPs induce to generate excessive amount of ROS beyond the limit of natural antioxidant defense systems like reductive GSH and antioxidant enzymes, cells start to lose normal functions with consequently causing cell death [24]. The chemical composition of NPs is a most decisive factor influencing ROS formation in lung epithelial cells [25]. ZnO NPs inducing significantly more oxidative damage than TiO2 , SiO2 , ZrO2 , and carbon black nanomaterials [26]. Fig. 5 showed the intracellular ROS, GSH, and SOD level of Caco2 cells which were exposed to 10, 25, 50, 100, and 200 g/mL of Ag NPs and ZnO NPs for 24 h. The abscissa was the concentration of nanoparticles, and the vertical coordinates were the intracellular ROS, GSH, and SOD level, respectively. The cells incubated without nanoparticles were the blank control. After 24 h exposure, Ag NPs did not significantly induce ROS at the concentration range of 0–200 g/mL in Caco-2 cells. As for ZnO NPs, at concentrations 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 intracellular ROS (Fig. 5A). According to the data, presumably the intense cytotoxicity of 200 g/mL ZnO NPs might have lead to a large number of cells death, so there were not enough cells to produce a great quantity ROS. Particle dissolution to ionic zinc and particle-induced generation of reactive oxygen species (ROS) represent the primary modes of action for ZnO NPs toxicity. GSH, an important endogenous antioxidant, has long been known to be involved in detoxifying reactions by protecting the thiol groups of enzymes and reacting with hydroxyl radicals, singlet oxygen, and hydrogen peroxide via GSH peroxidase (GPx) catalysis. The amount of GSH could reflect the antioxidant potential of an organelle [27]. In the present study, GSH is the most abundant non-protein thiol in cells participating in some processes,
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of NPs involved. This clearly demonstrated that ZnO NPs were more toxic than Ag NPs in Caco-2 cells. 4. Conclusions According to the AO/EB double staining and CCK-8 assay, the ZnO NPs cytotoxicity exhibited a dose-dependent effect. ROS, GSH, and SOD were considered as oxidative stress markers of cytotoxic mechanism. Exposed to ZnO NPs (10–200 g/mL), intracellular ROS and GSH level sharply increased, and SOD level was highly significantly decreased. Therefore, it can be concluded that ZnO NPs had the potency for the generation of ROS and eventual cytotoxicity. Ag NPs failed to induce ROS and SOD variation, while intracellular GSH level was obviously increased. The results demonstrated that ZnO NPs (10–200 g/mL) appeared high toxicity, and Ag NPs (10–200 g/mL) appeared no obvious toxicity in Caco-2 cells. Although it has been reported that Ag NPs have toxic effects to various cultured cells, the toxic effects at non-cytotoxic doses are still unknown. Besides, It was demonstrated that Ag NPs accelerated cell proliferation at low doses (<0.5 mg/L). The risk of any potentially toxic substance is not only a function of hazard but also a chance of exposure. It is useful to exploit the findings to engineer improved nanoparticles ultimately for use in consumer products. Although NPs cytotoxicity has been reported several times, but it is necessary to know that in vitro results can differ from what is found in vivo. Conflict of interest statement All authors of this research paper have directly participated in the planning, execution, or analysis of the study. All authors of this paper have read and approved the final version submitted. The contents of this manuscript have not been copyrighted or published previously. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. 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 with the control group. The data are presented as mean ± SE of at least three independent experiments. N = 4, significance indicated by: *P < 0.05, **P < 0.01.
including synthesis of DNA and proteins, regulation of enzyme activities, and inter and intracellular transportations [28]. According to Fig. 5B, compared with the control group, intracellular GSH induced by ZnO NPs (10–200 g/mL) highly significantly (p < 0.01) increased, and intracellular GSH level reached 212.68%, 223.52%, 285.39%, 209.65%, and 172.62%. The cells incubated at 50 g/mL ZnO NPs retained the most abundant GSH. However, GSH induced by ZnO NPs (10–50 g/mL) highly significantly (p < 0.01 vs. the control group) increased. The cells that were exposed to Ag NPs showed the slight depletion of GSH level in a dose-dependent manner. The cells exposed to ZnO NPs produced more GSH than Ag NPs. The results demonstrated that ZnO NPs showed significant toxicity to Caco-2 cells at 10–200 g/mL, but Ag NPs showed slight toxicity in the same concentrations. SOD is viewed as an antioxidant enzyme, which transforms superoxide anions (O2− ) into less reactive species-H2 O2 . The H2 O2 formed by SOD activity is decomposed to H2 O and O2 by glutathione peroxidase (GPx) in the presence of reduced GSH. According to Fig. 5C, for Caco-2 cells exposed to ZnO NPs, the SOD level was highly significant (p < 0.01) reduced at concentrations 10–200 g/mL for 24 h. While for Caco-2 cells exposed to Ag NPs, the SOD level had no significant variation. Although, the main mechanism of NPs cytotoxicity may differ, depending on the types
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