- Email: [email protected]
Cytotoxic effect and apoptosis induction by silver nanoparticles in HeLa cells
Biochemical and Biophysical Research Communications 390 (2009) 733–737
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Cytotoxic effect and apoptosis induction by silver nanoparticles in HeLa cells Nobuhiko Miura a,*, Yasushi Shinohara b a b
Division of Health Effects Research, National Institute of Occupational Safety and Health (JNIOSH), 6-21-1 Nagao, Tama-ku, Kawasaki 214-8585, Japan Division of Work Environment Research, National Institute of Occupational Safety and Health (JNIOSH), 6-21-1 Nagao, Tama-ku, Kawasaki 214-8585, Japan
a r t i c l e
i n f o
Article history: Received 5 October 2009 Available online 21 October 2009 Keywords: Silver nanoparticles Silver nitrate Apoptosis Metallothionein Heme oxygenase
a b s t r a c t Nanosilver has well-known antibacterial properties, and is widely used in daily life as various medical and general products. In comparison with silver ion, there is serious lacking of information concerning the biological effects of nanoAg. In this study, we observed the cytotoxic effect of nanoAg in HeLa cells. The nanoAg-induced cytotoxicity was lower than that of AgNO3, used as a silver ion source. Apoptosis evaluated by flowcytometric analysis was associated with this cell death. Further, the expressions of ho-1 and mt-2A, well-known oxidative stress-related genes, were up-regulated by nanoAg treatment. Our results showed that nanoAg possesses the potential for cytotoxicity, therefore, in the case of exposure at high concentrations, we should consider to protect from nanoAg-induced toxicity. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Nanosilver (nanoAg) is one of the most commonly used nanomaterials because of its strong antibacterial properties. For example, nanoAg is used for medical purpose by coating or embedding for wound dressings, surgical instruments, implants and bone prostheses [1–3]. NanoAg coatings are also used on various textiles for manufacture of clothing and socks [4,5]. Further, nanoAg is marketed as deodorants, room sprays, water cleaners, laundry detergents and wall paints. Therefore daily exposure to nanoAg is occurred to these consumers, in addition to workers who labor at manufacturing factory of these products. The inhibitory effect of ionic silver, well known highly toxic to bacteria, is due to several biological events such as its adsorption to the negatively charged bacterial cell wall, generating reactive oxygen species (ROS) and de-activating cellular enzymes [6–9]. In comparison, possible mechanisms by which nanoAg inhibit bacterial growth include nanoAg attachment to cell membranes, changes of membrane permeability and intracellular ROS accumulation [10–12]. Despite the widespread use of nanoAg products, relatively few studies have been undertaken to determine the biological effects of nanoAg exposure to mammalian cells. In this paper, we evaluated the toxicity of nanoAg and examined influences on the
Abbreviations: ho-1, heme oxygenase-1; mt-2A, metallothionein-2A; HSP70, heat shock protein 70 kDa; NanoAg, silver nanoparticles; EDS, energy dispersive spectrometer. * Corresponding author. Fax: +81 44 865 6124. E-mail address: [email protected] (N. Miura). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.039
expression levels of several stress-response genes by nanoAg in cultured human cells. Materials and methods Materials. NanoAg solution was purchased from Mitsuboshi Belting Ltd. (Kobe, Japan); this solution was supplied as 10% (w/ v) in pure water. Silver nitrate (AgNO3) was purchased from nacalai tesque (Tokyo, Japan). Annexin V fluorescein conjugate (ApoScreen Annexin V) was purchased from SouthernBiotech (Birmingham, USA). Other reagents were purchased from Wako Pure Chemical (Tokyo, Japan) unless otherwise stated. Transmission electron microscopy (TEM). The sizes and aggregational states of nanoAg were examined using a TEM (JEM-2100 equipped with JED-2300T energy dispersive spectrometer (EDS) system, JEOL, Tokyo). NanoAg stock solution (10%, 200 ll) was sonicated in water-bath using Bioruptor (Diagenode, Belgium) with a maximum power of cycling of ON (5 s)–OFF (10 s) for 10 cycles, and diluted to 120 lg Ag/ml with ultra pure water (UPW). The samples were deposited on carbon-coated nickel grids and were air dried overnight before TEM analysis. Cell culture and cell survival rate. HeLa S3 cells were maintained in Eagle’s minimum essential medium (Nissui pharmaceutical) supplemented with 10% calf serum and non-essential amino acids. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in air. For estimation of the cell viability against nanoAg and AgNO3, cells were seeded at 1 104 cells/90 ll in 96-well plates and precultured for 3 days. Before treatment, nanoAg solution was sonicated as mentioned above. NanoAg or AgNO3 were diluted to appropriate concentration with UPW, and then treated to
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HeLa cells (10 ll, n = 3). The cell survival rates were estimated after 24 h incubation with Ag reagents using AlamarBlue reagent (Invtrogen, Carlsbad, USA) as previously described [13]. All experiments were performed at least on three separate occasions. Apoptosis analysis. HeLa S3 cells were seeded at 1 105 cells/ 650 ll in 12-well plates and cultured for 24 h. Cells were then treated with various concentrations of nanoAg, AgNO3, or H2O2 (6.5 ll, n = 3) and incubated for 3 h. Cells were washed with 1 ml of PBS twice and scraped. The cell pellet was resuspended in binding buffer (10 mM Hepes (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA) and stained with ApoScreen Annexin V (FITC-conjugated) for 15 min followed by staining with propidium iodide (PI; final 1 lg/ml). Apoptosis was analyzed by flow cytometry using Epics XL-MCL instrument and Epics XL EXPO32 analysis software (Beckman Coulter, Japan). Expression levels of stress genes. HeLa S3 cells were seeded at 1 105 cells/650 ll in 12-well plates and cultured for 3 days. Cells were then treated with various concentrations of nanoAg, AgNO3, or CdSO4 (6.5 ll, n = 3) and incubated for 4 h. Total RNA from HeLa cells was isolated by MagNA Pure LC instrument (Roche Diagnostics, Germany) using MagNA Pure LC RNA isolation kit III (Roche) according to the manufacture’s instructions. Concentrations of total RNA were estimated by measuring absorbance at 260 nm using NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, USA). The oligo (dT)-based first strand cDNA was synthesized from 1 lg of total RNA using Transcriptor first strand cDNA synthesis kit (Roche) according to the manufacture’s instructions. The expression levels of stress genes were estimated by real-time PCR using LightCycler 480 system (Roche). The sequences of primer sets used were: heme oxygenase-1 (ho-1) sense, 50 -CAGGCAGAGAATGCTGAGTTC-30 and antisense, 50 -GCTTCACATAGCGCTGCA-30 ; metallothionein-2A (mt-2A) sense, 50 -AGTCCCAGCGAACCC-30 and antisense, 50 -ATTATCATTCACATTATTTCATAG-30 ; heat shock protein 70 (hsp70) sense 50 -AGCTGGAGCAGGTGTGTAAC-30 and antisense, 50 -CTCCTGACCT
CAAGTGATCC-30 . PCR reactions were performed using LightCycler 480 SYBR green I master kit (Roche) according to manufacture’s instructions. Statistical analysis. All experiments were done in triplicate, and all of data were presented as mean ± standard deviation (SD). Statistical significance was determined by Student’s t-test. Results NanoAg induced cytotoxicity The data sheet from Mitsuboshi Belting Ltd. indicates that the properties of nanoAg are: mean diameter sizes of 5–10 nm; included protectant (patented material) to suppress ionizing of Ag; supplied as 10% (w/v) in pure water; free of Pb, Cd, halogen, and sulfide. In our observation, nanoAg was diluted to 120 lg Ag/ml (a maximum final concentration for cell viability assay) in UPW, then sonicated and deposited on carbon-coated nickel grids (see Materials and methods). TEM observation showed that the mean diameter of nanoAg particles was in the comparatively uniform nanosize range (2–5 nm) (Fig. 1A). NanoAg particles were well dispersed in both UPW (Fig. 1A and C) and the cell-free culture medium whereas some particles seemed to be caught in some polymeric masses existed in cell-free culture medium (Fig. 1B). These nanoAg particles were identified from their EDS spectra (Fig. 1D). Using this nanoAg dispersion, we assessed the effect of nanoAg on cell viability. HeLa cells were incubated with nanoAg or AgNO3 for 24 h and viable cells were measured by fluorometric method (see Materials and methods). NanoAg showed cytotoxicity at concentrations of 80 lg Ag/ml or higher (Fig. 2). AgNO3 showed stronger cytotoxicity compared to that of nanoAg: cell viability reduction was observed at concentrations of 12 lg Ag/ml. The IC50 values of nanoAg and AgNO3 were about 92 and 17 lg Ag/
Fig. 1. Well-dispersion of nanoAg in UPW and cell-free culture medium. NanoAg stock solution (10%) was sonicated and diluted to 120 lg Ag/ml with UPW. The samples were deposited on carbon-coated nickel grids, air dried overnight before TEM analysis. Images of nanoAg in UPW (A,C) and cell-free culture medium (B) were indicated. Arrow in (B) indicates some polymeric masses existed in cell-free culture medium. (D) EDS spectra of nanoAg particle in UPW (upper) and background area of carbon-coated support film (lower). Si is a contaminant from carbon evapolator at the time of grid preparation.
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120
nanoAg
100
Cell viability (%)
Cell viability (%)
120
*
80 60 40
*
20
*
AgNO3
100 80
*
60 40 20
0
* * * * *
0 0
20
40
60
80 100 120
0
Concentration (µg Ag/ml)
8
16
24
32
40
Concentration (µg Ag/ml)
Overlay Cell viability (%)
120
nanoAg
100 80 60
AgNO3
40 20 0
0
20
40
60
80
100 120
Concentration (µg Ag/ml) Fig. 2. Comparison of cytotoxicic effect by nanoAg and AgNO3 treatment. HeLa cells were precultured for 3 days, and then incubated with various concentrations (lg Ag/ml) of nanoAg or AgNO3 for 24 h. Cell viability was estimated by AlamarBlue reagents. Each value indicates mean ± SD. Lower ‘‘Overlay” panel: overlaid upper panels to compare the cell viabilities of both reagents. *Significantly different from control (p < 0.01).
Cells / total cells (%)
(A) Total apoptosis
(B) Early apoptosis
*
100
*
*
100 80
80
60
60
*
40 20
*
*
40
*
20
*
*
*
#
*
* 0
0 C None
30 60 90 120 8 nanoAg (µ µg Ag/ml)
16 27
C
32
30 60 90 120 8
None
AgNO3 (µg Ag/ml)
nanoAg (µg Ag/ml)
16 27
32
AgNO3 (µg Ag/ml)
Cells / total cells (%)
(C) Late apoptosis 100
*
*
80
*
60 40
*
20
#
#
*
0 C None
30 60 90 120 8 nanoAg (µg Ag/ml)
16 27
32
AgNO3 (µg Ag/ml)
Fig. 3. Induction of apoptosis by nanoAg treatment. HeLa cells were precultured overnight, and then incubated with various concentrations (lg Ag/ml) of nanoAg or AgNO3 for 3 h. Cells were washed with PBS, resuspended in binding buffer containing 2.5 mM CaCl2 followed by staining with FITC-conjugated Annexin V and PI. The distribution of apoptotic cells was analyzed by flow cytometry. *,#Significantly different from control (p < 0.01 and p < 0.05, respectively).
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ml, respectively (Fig. 2). These results demonstrate that nanoAg possesses cytotoxicity though it is not stronger than AgNO3.
binding, oxidative stress and protein homeostasis. As typical genes of these biological responses, we selected mt-2A, ho-1 and hsp70 genes for this analysis. NanoAg remarkably induced mt-2A and ho-1 gene expressions. However, nanoAg gave little or no effect on hsp70 gene expression (Fig. 4A). Similar induction pattern was recognized by AgNO3 treatment (Fig. 4B), though induction ratios were higher in AgNO3. In the case of CdSO4, a strong inducer of these genes, hsp70 gene expression was markedly induced in addition to mt-2A and ho-1 gene (Fig. 4C). Therefore, nanoAg seems to possess similar biological effects or mechanisms to AgNO3.
NanoAg triggered apoptosis To further analyze the feature of cell death induced by nanoAg, we performed apoptosis analysis using flow cytometry. Cells were double-stained with Annexin V-FITC and propidium iodide at 3 h after the silver treatment. As shown in Fig. 3A, nanoAg induced apoptosis in a dose dependent manner. Although the ratio of early apoptosis to total apoptosis was frequent at lower doses (<60 lg Ag/ml: Fig. 3B), the late-apoptosis ratio became remarkable at higher dose (120 lg Ag/ml: Fig. 3C); each ratio of early- and lateapoptosis to total apoptosis was summarized under Fig. 3B and C, respectively. The apoptosis induction by AgNO3 was more strongly than by nanoAg as well as the result of cytotoxicity (Fig. 3). These results indicated that apoptosis took part in the mechanism of cell death by nanoAg.
Discussion Our results showed that nanoAg possessed cytotoxic effect to human cells. The IC50 value of nanoAg was higher than that of AgNO3 (about 5.4-fold different; Fig. 2). Therefore, although nanoAg does not have strong cytotoxicity like AgNO3, nanoAg seem to show clear cytotoxicity when exposing it to cells in higher concentrations. Both nanoAg and AgNO3 induced apoptosis (total-, early- and late-apoptosis) in a dose dependent manner, and the strength of apoptosis induction by AgNO3 was strong compared to nanoAg as well as cytotoxicity. The apoptotic effect of nanoAg was reported using mouse NIH3T 3 cells [14]. Further, apoptosis-related genes
NanoAg induced stress-response genes The biological effect of nanoAg was estimated by gene expressions as an index. It is well known that heavy metals such as cadmium (Cd) induced several stress-response genes related for metal
nanoAg Induction (fold)
(A)
mt-2A gene
ho-1 gene
hsp70 gene
50
600
60
40
500
50
400
40
300
30
200
20
30
*
20 10
*
** *
* 0 **
0
(B)
50
AgNO3 Induction (fold)
0
40
20
40
60
*
80
100
*
30
* *
(C)
50
CdSO4 Induction (fold)
40
8
12
100
50
500
*
*
*
*
0
0
4
* *
*
8
12
16
0
50
100
150
200
*
0
4
60
80
100
8
12
16
*
50
*
40
*
30
200
20
100
10
*
* *
*
0
0
0
*
60
20 10
*
300
10
40
30
*
400
20
20
40
*
200
600
*
0
500
16
**
30
80
60
0 4
60
600
100
0 0
40
300
*
10 0
20
400
20 10
0
*
*
100
0
50
100
150
200
0
50
100
150
200
Fig. 4. Up-regulation of mt-2A and ho-1 genes by nanoAg treatment. HeLa cells were precultured for 3 days, and then incubated with various concentrations of nanoAg or AgNO3 (lg Ag/ml) or CdSO4 (lM) for 4 h. Total RNA (1 lg) was reverse transcribed to cDNA in the presence of oligo (dT) primers. The expression levels of stress genes (mt-2A, ho-1 and hsp70) were estimated by real-time PCR using LightCycler 480 system. Values were indicated as fold-induction to control. *Significantly different from control (p < 0.01).
N. Miura, Y. Shinohara / Biochemical and Biophysical Research Communications 390 (2009) 733–737
were altered in the liver of nanoAg-fed mice [15]. Therefore, apoptosis takes part in the mechanisms of the cell death by nanoAg. It has long been known that apoptosis occurs in response to various biological stresses including oxidative stress [16,17]. NanoAg-mediated generation of ROS was reported in in vitro study [14]. In our data, expressions of both mt-2A and ho-1 genes were up-regulated (Fig. 4), and these genes were induced by oxidative stress [18–20]. These results indicate that apoptosis induction by nanoAg may be created by ROS generation. Cd induced expressions of ho-1, mt-2A, and hsp70 genes However, both nanoAg and AgNO3 had little or no effect of hsp70 gene expression (Fig. 4). This result indicates, as is obvious, that the mechanism of nanoAg-induced biological effects is different from Cd. In comparison with AgNO3, nanoAg seems to use the similar mechanism as which AgNO3 to use. It has been problem how nanoAg might affect in living cells [21], our results show the possibility that silver ion releases from nanoAg and this ion induces cytotoxicity, apoptosis and induction of stress-response genes, because these biological effects were similar in both nanoAg and AgNO3. Although the cytotoxic effect of nanoAg was weaker than that of AgNO3 in our results, contrary result using silver carbonate as a silver ion source is exit [22]. There are various differences in experimental conditions such as silver compounds, nanoAg, and cell line. Especially, silver carbonate is generally considered to be non-hazardous; the EC50 value was 408 lg/ml and no significant effect on cell viability was observed up to 100 lg/ml [22]. To estimate and comparison the toxicity of nanoparticles including nanoAg, the investigators including us should be harmonize these conditions. In conclusion, nanoAg possesses the potential for cytotoxiciy in the case of high concentration exposure. Apoptosis is associated with this cell death. Further, both nanoAg and AgNO3 up-regulate the expression levels of stress genes, ho-1 and mt-2A. Since nanoAg is often used as a paste form, it is necessary to consider the skin exposure in addition to inhalation exposure. If percutaneous absorption occurred, apoptosis may be induced in inner skin (dermal layer), not in outer skin (epidermal layer). Therefore, we are recognizing the necessity for the animal study. We should consider the nanoAg toxicity by several exposure routes and protection from its toxicity.
Acknowledgment This study was supported by a project research of JNIOSH (Project No. P19-01).
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References [1] M.S. Cohen, J.M. Stern, A.J. Vanni, R.S. Kelley, E. Baumgart, D. Field, J.A. Libertino, I.C. Summerhayes, In vitro analysis of a nanocrystalline silver-coated surgical mesh, Surg. Infect. (Larchmt) 8 (2007) 397–403. [2] J. Fu, J. Ji, D. Fan, J. Shen, Construction of antibacterial multilayer films containing nanosilver via layer-by-layer assembly of heparin and chitosansilver ions complex, J. Biomed. Mater. Res. A 79 (2006) 665–674. [3] X. Xu, Q. Yang, J. Bai, T. Lu, Y. Li, X. Jing, Fabrication of biodegradable electrospun poly(L-lactide-co-glycolide) fibers with antimicrobial nanosilver particles, J. Nanosci. Nanotechnol. 8 (2008) 5066–5070. [4] T.M. Benn, P. Westerhoff, Nanoparticle silver released into water from commercially available sock fabrics, Environ. Sci. Technol. 42 (2008) 4133–4139. [5] N. Vigneshwaran, A.A. Kathe, P.V. Varadarajan, R.P. Nachane, R.H. Balasubramanya, Functional finishing of cotton fabrics using silver nanoparticles, J. Nanosci. Nanotechnol. 7 (2007) 1893–1897. [6] Y. Matsumura, K. Yoshikata, S. Kunisaki, T. Tsuchido, Mode of bactericidal action of silver zeolite and its comparison with that of silver nitrate, Appl. Environ. Microbiol. 69 (2003) 4278–4281. [7] E. Navarro, F. Piccapietra, B. Wagner, F. Marconi, R. Kaegi, N. Odzak, L. Sigg, R. Behra, Toxicity of silver nanoparticles to Chlamydomonas reinhardtii, Environ. Sci. Technol. 42 (2008) 8959–8964. [8] V. Sambhy, M.M. MacBride, B.R. Peterson, A. Sen, Silver bromide nanoparticle/ polymer composites: dual action tunable antimicrobial materials, J. Am. Chem. Soc. 128 (2006) 9798–9808. [9] R.M. Slawson, M.I. Van Dyke, H. Lee, J.T. Trevors, Germanium and silver resistance, accumulation, and toxicity in microorganisms, Plasmid 27 (1992) 72–79. [10] C.N. Lok, C.M. Ho, R. Chen, Q.Y. He, W.Y. Yu, H. Sun, P.K. Tam, J.F. Chiu, C.M. Che, Proteomic analysis of the mode of antibacterial action of silver nanoparticles, J. Proteome Res. 5 (2006) 916–924. [11] A. Nel, T. Xia, L. Madler, N. Li, Toxic potential of materials at the nanolevel, Science 311 (2006) 622–627. [12] I. Sondi, B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria, J. Colloid Interface Sci. 275 (2004) 177–182. [13] N. Miura, Y. Kanayama, W. Nagai, T. Hasegawa, Y. Seko, T. Kaji, A. Naganuma, Characterization of an immortalized hepatic stellate cell line established from metallothionein-null mice, J. Toxicol. Sci. 31 (2006) 391–398. [14] Y.H. Hsin, C.F. Chen, S. Huang, T.S. Shih, P.S. Lai, P.J. Chueh, The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T 3 cells, Toxicol. Lett. 179 (2008) 130–139. [15] K. Cha, H.W. Hong, Y.G. Choi, M.J. Lee, J.H. Park, H.K. Chae, G. Ryu, H. Myung, Comparison of acute responses of mice livers to short-term exposure to nanosized or micro-sized silver particles, Biotechnol. Lett. 30 (2008) 1893–1899. [16] M. Ott, V. Gogvadze, S. Orrenius, B. Zhivotovsky, Mitochondria, oxidative stress and cell death, Apoptosis 12 (2007) 913–922. [17] S.V. Rana, Metals and apoptosis: recent developments, J. Trace Elem. Med. Biol. 22 (2008) 262–284. [18] M. Kondoh, Y. Inoue, S. Atagi, N. Futakawa, M. Higashimoto, M. Sato, Specific induction of metallothionein synthesis by mitochondrial oxidative stress, Life Sci. 69 (2001) 2137–2146. [19] M. Sato, I. Bremner, Oxygen free radicals and metallothionein, Free Radic. Biol. Med. 14 (1993) 325–337. [20] T. Takahashi, K. Morita, R. Akagi, S. Sassa, Heme oxygenase-1: a novel therapeutic target in oxidative tissue injuries, Curr. Med. Chem. 11 (2004) 1545–1561. [21] N. Lubick, Nanosilver toxicity: ions, nanoparticles – or both? Environ. Sci. Technol. 42 (2008) 8617. [22] L. Braydich-Stolle, S. Hussain, J.J. Schlager, M.C. Hofmann, In vitro cytotoxicity of nanoparticles in mammalian germline stem cells, Toxicol. Sci. 88 (2005) 412–419.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Cytotoxic effect and apoptosis induction by silver nanoparticles in HeLa cells Nobuhiko Miura a,*, Yasushi Shinohara b a b
Division of Health Effects Research, National Institute of Occupational Safety and Health (JNIOSH), 6-21-1 Nagao, Tama-ku, Kawasaki 214-8585, Japan Division of Work Environment Research, National Institute of Occupational Safety and Health (JNIOSH), 6-21-1 Nagao, Tama-ku, Kawasaki 214-8585, Japan
a r t i c l e
i n f o
Article history: Received 5 October 2009 Available online 21 October 2009 Keywords: Silver nanoparticles Silver nitrate Apoptosis Metallothionein Heme oxygenase
a b s t r a c t Nanosilver has well-known antibacterial properties, and is widely used in daily life as various medical and general products. In comparison with silver ion, there is serious lacking of information concerning the biological effects of nanoAg. In this study, we observed the cytotoxic effect of nanoAg in HeLa cells. The nanoAg-induced cytotoxicity was lower than that of AgNO3, used as a silver ion source. Apoptosis evaluated by flowcytometric analysis was associated with this cell death. Further, the expressions of ho-1 and mt-2A, well-known oxidative stress-related genes, were up-regulated by nanoAg treatment. Our results showed that nanoAg possesses the potential for cytotoxicity, therefore, in the case of exposure at high concentrations, we should consider to protect from nanoAg-induced toxicity. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Nanosilver (nanoAg) is one of the most commonly used nanomaterials because of its strong antibacterial properties. For example, nanoAg is used for medical purpose by coating or embedding for wound dressings, surgical instruments, implants and bone prostheses [1–3]. NanoAg coatings are also used on various textiles for manufacture of clothing and socks [4,5]. Further, nanoAg is marketed as deodorants, room sprays, water cleaners, laundry detergents and wall paints. Therefore daily exposure to nanoAg is occurred to these consumers, in addition to workers who labor at manufacturing factory of these products. The inhibitory effect of ionic silver, well known highly toxic to bacteria, is due to several biological events such as its adsorption to the negatively charged bacterial cell wall, generating reactive oxygen species (ROS) and de-activating cellular enzymes [6–9]. In comparison, possible mechanisms by which nanoAg inhibit bacterial growth include nanoAg attachment to cell membranes, changes of membrane permeability and intracellular ROS accumulation [10–12]. Despite the widespread use of nanoAg products, relatively few studies have been undertaken to determine the biological effects of nanoAg exposure to mammalian cells. In this paper, we evaluated the toxicity of nanoAg and examined influences on the
Abbreviations: ho-1, heme oxygenase-1; mt-2A, metallothionein-2A; HSP70, heat shock protein 70 kDa; NanoAg, silver nanoparticles; EDS, energy dispersive spectrometer. * Corresponding author. Fax: +81 44 865 6124. E-mail address: [email protected] (N. Miura). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.039
expression levels of several stress-response genes by nanoAg in cultured human cells. Materials and methods Materials. NanoAg solution was purchased from Mitsuboshi Belting Ltd. (Kobe, Japan); this solution was supplied as 10% (w/ v) in pure water. Silver nitrate (AgNO3) was purchased from nacalai tesque (Tokyo, Japan). Annexin V fluorescein conjugate (ApoScreen Annexin V) was purchased from SouthernBiotech (Birmingham, USA). Other reagents were purchased from Wako Pure Chemical (Tokyo, Japan) unless otherwise stated. Transmission electron microscopy (TEM). The sizes and aggregational states of nanoAg were examined using a TEM (JEM-2100 equipped with JED-2300T energy dispersive spectrometer (EDS) system, JEOL, Tokyo). NanoAg stock solution (10%, 200 ll) was sonicated in water-bath using Bioruptor (Diagenode, Belgium) with a maximum power of cycling of ON (5 s)–OFF (10 s) for 10 cycles, and diluted to 120 lg Ag/ml with ultra pure water (UPW). The samples were deposited on carbon-coated nickel grids and were air dried overnight before TEM analysis. Cell culture and cell survival rate. HeLa S3 cells were maintained in Eagle’s minimum essential medium (Nissui pharmaceutical) supplemented with 10% calf serum and non-essential amino acids. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in air. For estimation of the cell viability against nanoAg and AgNO3, cells were seeded at 1 104 cells/90 ll in 96-well plates and precultured for 3 days. Before treatment, nanoAg solution was sonicated as mentioned above. NanoAg or AgNO3 were diluted to appropriate concentration with UPW, and then treated to
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HeLa cells (10 ll, n = 3). The cell survival rates were estimated after 24 h incubation with Ag reagents using AlamarBlue reagent (Invtrogen, Carlsbad, USA) as previously described [13]. All experiments were performed at least on three separate occasions. Apoptosis analysis. HeLa S3 cells were seeded at 1 105 cells/ 650 ll in 12-well plates and cultured for 24 h. Cells were then treated with various concentrations of nanoAg, AgNO3, or H2O2 (6.5 ll, n = 3) and incubated for 3 h. Cells were washed with 1 ml of PBS twice and scraped. The cell pellet was resuspended in binding buffer (10 mM Hepes (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA) and stained with ApoScreen Annexin V (FITC-conjugated) for 15 min followed by staining with propidium iodide (PI; final 1 lg/ml). Apoptosis was analyzed by flow cytometry using Epics XL-MCL instrument and Epics XL EXPO32 analysis software (Beckman Coulter, Japan). Expression levels of stress genes. HeLa S3 cells were seeded at 1 105 cells/650 ll in 12-well plates and cultured for 3 days. Cells were then treated with various concentrations of nanoAg, AgNO3, or CdSO4 (6.5 ll, n = 3) and incubated for 4 h. Total RNA from HeLa cells was isolated by MagNA Pure LC instrument (Roche Diagnostics, Germany) using MagNA Pure LC RNA isolation kit III (Roche) according to the manufacture’s instructions. Concentrations of total RNA were estimated by measuring absorbance at 260 nm using NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, USA). The oligo (dT)-based first strand cDNA was synthesized from 1 lg of total RNA using Transcriptor first strand cDNA synthesis kit (Roche) according to the manufacture’s instructions. The expression levels of stress genes were estimated by real-time PCR using LightCycler 480 system (Roche). The sequences of primer sets used were: heme oxygenase-1 (ho-1) sense, 50 -CAGGCAGAGAATGCTGAGTTC-30 and antisense, 50 -GCTTCACATAGCGCTGCA-30 ; metallothionein-2A (mt-2A) sense, 50 -AGTCCCAGCGAACCC-30 and antisense, 50 -ATTATCATTCACATTATTTCATAG-30 ; heat shock protein 70 (hsp70) sense 50 -AGCTGGAGCAGGTGTGTAAC-30 and antisense, 50 -CTCCTGACCT
CAAGTGATCC-30 . PCR reactions were performed using LightCycler 480 SYBR green I master kit (Roche) according to manufacture’s instructions. Statistical analysis. All experiments were done in triplicate, and all of data were presented as mean ± standard deviation (SD). Statistical significance was determined by Student’s t-test. Results NanoAg induced cytotoxicity The data sheet from Mitsuboshi Belting Ltd. indicates that the properties of nanoAg are: mean diameter sizes of 5–10 nm; included protectant (patented material) to suppress ionizing of Ag; supplied as 10% (w/v) in pure water; free of Pb, Cd, halogen, and sulfide. In our observation, nanoAg was diluted to 120 lg Ag/ml (a maximum final concentration for cell viability assay) in UPW, then sonicated and deposited on carbon-coated nickel grids (see Materials and methods). TEM observation showed that the mean diameter of nanoAg particles was in the comparatively uniform nanosize range (2–5 nm) (Fig. 1A). NanoAg particles were well dispersed in both UPW (Fig. 1A and C) and the cell-free culture medium whereas some particles seemed to be caught in some polymeric masses existed in cell-free culture medium (Fig. 1B). These nanoAg particles were identified from their EDS spectra (Fig. 1D). Using this nanoAg dispersion, we assessed the effect of nanoAg on cell viability. HeLa cells were incubated with nanoAg or AgNO3 for 24 h and viable cells were measured by fluorometric method (see Materials and methods). NanoAg showed cytotoxicity at concentrations of 80 lg Ag/ml or higher (Fig. 2). AgNO3 showed stronger cytotoxicity compared to that of nanoAg: cell viability reduction was observed at concentrations of 12 lg Ag/ml. The IC50 values of nanoAg and AgNO3 were about 92 and 17 lg Ag/
Fig. 1. Well-dispersion of nanoAg in UPW and cell-free culture medium. NanoAg stock solution (10%) was sonicated and diluted to 120 lg Ag/ml with UPW. The samples were deposited on carbon-coated nickel grids, air dried overnight before TEM analysis. Images of nanoAg in UPW (A,C) and cell-free culture medium (B) were indicated. Arrow in (B) indicates some polymeric masses existed in cell-free culture medium. (D) EDS spectra of nanoAg particle in UPW (upper) and background area of carbon-coated support film (lower). Si is a contaminant from carbon evapolator at the time of grid preparation.
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120
nanoAg
100
Cell viability (%)
Cell viability (%)
120
*
80 60 40
*
20
*
AgNO3
100 80
*
60 40 20
0
* * * * *
0 0
20
40
60
80 100 120
0
Concentration (µg Ag/ml)
8
16
24
32
40
Concentration (µg Ag/ml)
Overlay Cell viability (%)
120
nanoAg
100 80 60
AgNO3
40 20 0
0
20
40
60
80
100 120
Concentration (µg Ag/ml) Fig. 2. Comparison of cytotoxicic effect by nanoAg and AgNO3 treatment. HeLa cells were precultured for 3 days, and then incubated with various concentrations (lg Ag/ml) of nanoAg or AgNO3 for 24 h. Cell viability was estimated by AlamarBlue reagents. Each value indicates mean ± SD. Lower ‘‘Overlay” panel: overlaid upper panels to compare the cell viabilities of both reagents. *Significantly different from control (p < 0.01).
Cells / total cells (%)
(A) Total apoptosis
(B) Early apoptosis
*
100
*
*
100 80
80
60
60
*
40 20
*
*
40
*
20
*
*
*
#
*
* 0
0 C None
30 60 90 120 8 nanoAg (µ µg Ag/ml)
16 27
C
32
30 60 90 120 8
None
AgNO3 (µg Ag/ml)
nanoAg (µg Ag/ml)
16 27
32
AgNO3 (µg Ag/ml)
Cells / total cells (%)
(C) Late apoptosis 100
*
*
80
*
60 40
*
20
#
#
*
0 C None
30 60 90 120 8 nanoAg (µg Ag/ml)
16 27
32
AgNO3 (µg Ag/ml)
Fig. 3. Induction of apoptosis by nanoAg treatment. HeLa cells were precultured overnight, and then incubated with various concentrations (lg Ag/ml) of nanoAg or AgNO3 for 3 h. Cells were washed with PBS, resuspended in binding buffer containing 2.5 mM CaCl2 followed by staining with FITC-conjugated Annexin V and PI. The distribution of apoptotic cells was analyzed by flow cytometry. *,#Significantly different from control (p < 0.01 and p < 0.05, respectively).
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ml, respectively (Fig. 2). These results demonstrate that nanoAg possesses cytotoxicity though it is not stronger than AgNO3.
binding, oxidative stress and protein homeostasis. As typical genes of these biological responses, we selected mt-2A, ho-1 and hsp70 genes for this analysis. NanoAg remarkably induced mt-2A and ho-1 gene expressions. However, nanoAg gave little or no effect on hsp70 gene expression (Fig. 4A). Similar induction pattern was recognized by AgNO3 treatment (Fig. 4B), though induction ratios were higher in AgNO3. In the case of CdSO4, a strong inducer of these genes, hsp70 gene expression was markedly induced in addition to mt-2A and ho-1 gene (Fig. 4C). Therefore, nanoAg seems to possess similar biological effects or mechanisms to AgNO3.
NanoAg triggered apoptosis To further analyze the feature of cell death induced by nanoAg, we performed apoptosis analysis using flow cytometry. Cells were double-stained with Annexin V-FITC and propidium iodide at 3 h after the silver treatment. As shown in Fig. 3A, nanoAg induced apoptosis in a dose dependent manner. Although the ratio of early apoptosis to total apoptosis was frequent at lower doses (<60 lg Ag/ml: Fig. 3B), the late-apoptosis ratio became remarkable at higher dose (120 lg Ag/ml: Fig. 3C); each ratio of early- and lateapoptosis to total apoptosis was summarized under Fig. 3B and C, respectively. The apoptosis induction by AgNO3 was more strongly than by nanoAg as well as the result of cytotoxicity (Fig. 3). These results indicated that apoptosis took part in the mechanism of cell death by nanoAg.
Discussion Our results showed that nanoAg possessed cytotoxic effect to human cells. The IC50 value of nanoAg was higher than that of AgNO3 (about 5.4-fold different; Fig. 2). Therefore, although nanoAg does not have strong cytotoxicity like AgNO3, nanoAg seem to show clear cytotoxicity when exposing it to cells in higher concentrations. Both nanoAg and AgNO3 induced apoptosis (total-, early- and late-apoptosis) in a dose dependent manner, and the strength of apoptosis induction by AgNO3 was strong compared to nanoAg as well as cytotoxicity. The apoptotic effect of nanoAg was reported using mouse NIH3T 3 cells [14]. Further, apoptosis-related genes
NanoAg induced stress-response genes The biological effect of nanoAg was estimated by gene expressions as an index. It is well known that heavy metals such as cadmium (Cd) induced several stress-response genes related for metal
nanoAg Induction (fold)
(A)
mt-2A gene
ho-1 gene
hsp70 gene
50
600
60
40
500
50
400
40
300
30
200
20
30
*
20 10
*
** *
* 0 **
0
(B)
50
AgNO3 Induction (fold)
0
40
20
40
60
*
80
100
*
30
* *
(C)
50
CdSO4 Induction (fold)
40
8
12
100
50
500
*
*
*
*
0
0
4
* *
*
8
12
16
0
50
100
150
200
*
0
4
60
80
100
8
12
16
*
50
*
40
*
30
200
20
100
10
*
* *
*
0
0
0
*
60
20 10
*
300
10
40
30
*
400
20
20
40
*
200
600
*
0
500
16
**
30
80
60
0 4
60
600
100
0 0
40
300
*
10 0
20
400
20 10
0
*
*
100
0
50
100
150
200
0
50
100
150
200
Fig. 4. Up-regulation of mt-2A and ho-1 genes by nanoAg treatment. HeLa cells were precultured for 3 days, and then incubated with various concentrations of nanoAg or AgNO3 (lg Ag/ml) or CdSO4 (lM) for 4 h. Total RNA (1 lg) was reverse transcribed to cDNA in the presence of oligo (dT) primers. The expression levels of stress genes (mt-2A, ho-1 and hsp70) were estimated by real-time PCR using LightCycler 480 system. Values were indicated as fold-induction to control. *Significantly different from control (p < 0.01).
N. Miura, Y. Shinohara / Biochemical and Biophysical Research Communications 390 (2009) 733–737
were altered in the liver of nanoAg-fed mice [15]. Therefore, apoptosis takes part in the mechanisms of the cell death by nanoAg. It has long been known that apoptosis occurs in response to various biological stresses including oxidative stress [16,17]. NanoAg-mediated generation of ROS was reported in in vitro study [14]. In our data, expressions of both mt-2A and ho-1 genes were up-regulated (Fig. 4), and these genes were induced by oxidative stress [18–20]. These results indicate that apoptosis induction by nanoAg may be created by ROS generation. Cd induced expressions of ho-1, mt-2A, and hsp70 genes However, both nanoAg and AgNO3 had little or no effect of hsp70 gene expression (Fig. 4). This result indicates, as is obvious, that the mechanism of nanoAg-induced biological effects is different from Cd. In comparison with AgNO3, nanoAg seems to use the similar mechanism as which AgNO3 to use. It has been problem how nanoAg might affect in living cells [21], our results show the possibility that silver ion releases from nanoAg and this ion induces cytotoxicity, apoptosis and induction of stress-response genes, because these biological effects were similar in both nanoAg and AgNO3. Although the cytotoxic effect of nanoAg was weaker than that of AgNO3 in our results, contrary result using silver carbonate as a silver ion source is exit [22]. There are various differences in experimental conditions such as silver compounds, nanoAg, and cell line. Especially, silver carbonate is generally considered to be non-hazardous; the EC50 value was 408 lg/ml and no significant effect on cell viability was observed up to 100 lg/ml [22]. To estimate and comparison the toxicity of nanoparticles including nanoAg, the investigators including us should be harmonize these conditions. In conclusion, nanoAg possesses the potential for cytotoxiciy in the case of high concentration exposure. Apoptosis is associated with this cell death. Further, both nanoAg and AgNO3 up-regulate the expression levels of stress genes, ho-1 and mt-2A. Since nanoAg is often used as a paste form, it is necessary to consider the skin exposure in addition to inhalation exposure. If percutaneous absorption occurred, apoptosis may be induced in inner skin (dermal layer), not in outer skin (epidermal layer). Therefore, we are recognizing the necessity for the animal study. We should consider the nanoAg toxicity by several exposure routes and protection from its toxicity.
Acknowledgment This study was supported by a project research of JNIOSH (Project No. P19-01).
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