Journal of Hazardous Materials 270 (2014) 176–186
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Silica nanoparticles induce autophagy and autophagic cell death in HepG2 cells triggered by reactive oxygen species Yongbo Yu a,b,1 , Junchao Duan a,b,1 , Yang Yu a,b , Yang Li a,b , Xiaomei Liu c , Xianqing Zhou a,b , Kin-fai Ho d , Linwei Tian d,∗∗ , Zhiwei Sun a,b,∗ a
School of Public Health, Capital Medical University, Beijing, 100069, P.R. China Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing, 100069, P.R. China c School of Public Health, Jilin University, Changchun, Jilin, 130021, P.R. China d School of Public Health and Primary Care, Chinese University of Hong Kong, Hong Kong, China b
h i g h l i g h t s • Silica nanoparticles (SNPs) induce autophagy and autophagic cell death. • The typically morphological changes of whole autophagy process are observed. • ROS scavenger and autophagy inhibitor effectively suppress the SNPs-induced autophagy and autophagy cell death.
a r t i c l e
i n f o
Article history: Received 30 September 2013 Received in revised form 22 December 2013 Accepted 7 January 2014 Available online 24 January 2014 Keywords: Nanotoxicity Silica nanoparticles Autophagy Autophagic cell death Reactive oxygen species
a b s t r a c t Silica nanoparticles (SNPs) are becoming favorable carriers for drug delivery or gene therapy, and in turn, the toxic effect of SNPs on biological systems is gaining attention. Currently, autophagy is recognized as an emerging toxicity mechanism triggered by nanomaterials, yet there have been scarcely research about the mechanisms of autophagy and autophagic cell death associated with SNPs. In this study, we verified the activation of SNPs-induced autophagy via the MDC-staining and LC3-I/LC3-II conversion, resulted in a dose-dependent manner. The typically morphological characteristics (autophagosomes and autolysosomes) of the autophagy process were observed in TEM ultrastructural analysis. In addition, the autophagic cell death was evaluated by cellular co-staining assay. And the underlying mechanisms of autophagy and autophagic cell death were performed using the intracellular ROS detection, autophagy inhibitor and ROS scavenger. Results showed that the elevated ROS level was in line with the increasing of autophagy activation, while both the 3-MA and NAC inhibitors effectively suppressed the autophagy and cell death induced by SNPs. In summary, our findings demonstrated that the SNPs-induced autophagy and autophagic cell death were triggered by the ROS generation in HepG2 cells, suggesting that exposure to SNPs could be a potential hazardous factor for maintaining cellular homeostasis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Silica nanoparticles (SNPs) have been widely used as additives to cosmetics, paints, varnishes, and food [1]. The emerging commercialization of the SNPs products has increased the environment and human exposure. In addition, the unique physicochemical properties of SNPs make it attractive for a variety of biomedical and
∗ Corresponding author at: Zhiwei Sun, School of Public Health, Capital Medical University, Beijing, 100069, P.R. China. Tel.: +86 010 83911507; fax: +86 010 83911507. ∗∗ Corresponding author at: Linwei Tian, School of Public Health and Primary Care, Chinese University of Hong Kong, Hong Kong, China. Tel.: +852 22528879; fax: +852 26063500. E-mail addresses: [email protected] (L. Tian), [email protected] (Z. Sun). 1 The first two authors contribute equally to this work. 0304-3894/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2014.01.028
biotechnological applications such as cancer therapy, drugs and gene delivery and biosensors [2,3]. SNPs as carriers of drug delivery systems are generally injected into the body intravenously and translated to organs via the systemic circulation [4]. It was reported that the liver is the target organ of SNPs in which the SNPs could be accumulated and retained for over 30 days [5,6]. Therefore, it is essential to understand the biological effects and potential hepatotoxicity of SNPs either in vivo or in vitro. Currently, several studies have reported that the SNPs can penetrate the cytomembrane, deposit in mitochondria or even the nucleus, and eventually lead to cell death [7,8]. According to the recommendations of Nomenclature Committee on Cell Death (NCCD), cell death can be classified into necrosis, apoptosis, autophagic cell death, mitotic catastrophe and other atypical modalities [9]. Unfortunately, most previous studies focus on the apoptosis and necrosis induced by SNPs in vitro [10–12]; little is known about the autophagy and its
Y. Yu et al. / Journal of Hazardous Materials 270 (2014) 176–186
toxicological consequence, which is called autophagic cell death or type II programmed cell death [13,14]. Autophagy is a highly regulated intracellular process for the lysosomal degradation and recycling of proteins and organelles [15]. In the process of autophagy, autophagosomes (twomembraned and containing degenerating cytoplasmic organelles or parts of the cytoplasm) are finally delivered to lysosomes for bulk degradation [16,17]. A basal level of autophagy is a homeostatic cytoprotective mechanism; however, the autophagy dysfunction can lead to severe pathological states, such as neurodegenerative diseases, cardiomyopathy, and particularly cancer [18,19]. Recently, autophagy was proposed as an emerging toxicity mechanisms occurred in several nanomaterials, such as nano-sized fullerene, rare earth oxides and titanium dioxide and copper oxide [20–23]. However, there exists scarcely literature focused on the biological response of the SNPs associated with autophagy, which provides limited evidences to confirm their relationship. Moreover, these studies failed to consider the underlying mechanisms between autophagy and autophagic cell death [24,25]. Based on the issues above, an insight evaluation of SNPs-induced autophagy and autophagic cell death was then conducted in vitro with human hepatocellular carcinoma HepG2 cells. The HepG2 cells line was generally used to assess the hepatotoxicity in vitro studies [26]. Prior to undertaking in vitro toxicity experiments, the characterization of SNPs, which is essential for nanotoxicity studies, was performed by transmission electron microscope (TEM) and dynamic light scattering (DLS) measurements. To investigate the mechanisms of autophagy and autophagic cell death induced by SNPs, we conducted a sequence of assessments including cellular uptake and ultrastructural analysis, cell viability, intracellular ROS generation, MDC-staining, LC3-I/LC3-II conversion and cellular co-staining after HepG2 cells exposure to SNPs for 24 h. We also used the autophagy inhibitor 3-Methyladenine (3-MA) and the ROS scavenger N-acetyl cystiene (NAC) to analyze the underlying mechanisms of the SNPs-induced autophagy and autophagic cell death. 2. Materials and methods 2.1. Synthesis of the amorphous SNPs The SNPs were prepared using the Stöber method [27]. Briefly, 2.5 mL of tetraethylorthosilicate (TEOS) (Sigma, USA) was added to premixed ethanol solution (50 mL) containing ammonia (2 mL) and water (1 mL). The reaction mixture was kept at 40 ◦ C for 12 h with continuous stirring (150 r/min). The resulting particles were isolated by centrifugation (12,000 r/min, 15 min) and washed three times with deionized water and then dispersed in 50 mL of deionized water. 2.2. Characterization of the SNPs The particle size and distribution of the SNPs were measured by transmission electron microscope (TEM) (JEOL, Japan) and ImageJ software. The hydrodynamic sizes and zeta potential of SNPs in ultrapure water and serum-free DMEM were examined by Zetasizer (Malvern Nano-ZS90, Britain). Suspensions of SNPs were dispersed by sonicator (160 W, 20 kHz, 5 min) (Bioruptor UDC-200, Belgium) before addition to dispersion media in order to minimize their aggregation.
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Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin at 37 ◦ C in a humidified incubator with 5% CO2 . For experiments, the cells were seeded in culture plates and treated with SNPs suspended in DMEM of certain concentrations for another 24 h. Suspension of SNPs was sterilized by an autoclave and dispersed by sonicator (160 W, 20 KHz, 5 min) before added into culture medium to minimize their aggregation. Cells maintained in DMEM without SNPs were used as control group. 2.4. Cellular internalization of the SNPs HepG2 cells were seeded in 35 mm-diameter glass bottom cell culture dishes and were cultured in DMEM as above. After 24 h of cell attachment, the cells were treated with Ruthenium (II) hydrate (Ru(phen)3 2+ ) interior-labeled SNPs (50 g/mL) for 24 h at 37 ◦ C in serum-free medium. These red fluorescent SNPs were prepared by a modified Stöber method and characterized as described before [28]. Cells were then washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. The cells were washed with 0.1% Triton X-100 three times and incubated with Phalloidin-FITC Actin-Tracker Green (Jiancheng, China) at room temperature for 30 min. The Actin-Tracker was dissolved in the mixture of 0.1% Triton X-100 and 3% bovine serum albumin (BSA) (Sigma, USA) for staining the actin filamentous skeleton. After that, the nucleus was stained with 2 g/mL 4,6-diamidino-2phenylindole (DAPI) (Sigma, USA) in PBS for 5 min. Cellular uptake were observed by a laser scanning confocal microscopy (LSCM) (Leica TCS SP5, Germany). 2.5. Cell viability assay The cytotoxicity of the SNPs was determined using MTT assay. Cell morphology was observed by optical microscope (Olympus IX81, Japan). Briefly, 1 × 104 HepG2 cells were seeded into a 96-well plate in a volume of 100 L DMEM and incubated for 24 h at 37 ◦ C. Cells were pretreated with or without 5 mM autophagy inhibitor 3Methyladenine (3-MA) or ROS scavenger N-acetyl cystiene (NAC) for 1 h and then incubated with varying concentrations of SNPs (25, 50, 75 and 100 g/mL) for 24 h at 37 ◦ C. After 24 h incubation, 10 L MTT was added to each well at 5 mg/mL and further incubated for 4 h. After which 150 L of dimethylsulfoxide (DMSO) was added in and mixed thoroughly for 5 min. Optical density was then measured with a microplate reader (Themo Multiscan MK3, USA) at 492 nm. 2.6. Autophagy measurement To detect whether autophagy was induced in HepG2 cells exposed to the SNPs, Monodansylcadaverine (MDC), a specific marker of autophagic vacuoles, was employed to stain autophagosomes. Briefly, bout 3 × 105 cells were seeded into a 6-well plate for 24 h. After pretreated with or without 5 mM 3-MA for 1 h, the cells were treated with series concentrations of SNPs (25–100 g/mL) for 24 h. The cells were then stained with 0.05 mM MDC (Sigma, USA) in fresh DMEM and incubated for 30 min at 37 ◦ C in dark. After three times washing with PBS, the cells were harvested and rinsed with PBS. Intracellular MDC intensity was measured using a flow cytometry (Becton-Dickison, USA) at 360 nm excitation, 530 nm emission and observed with fluorescence microscope (Olympus IX81, Japan). The visualization of MDC-stained autophagosomes and autolysosomes were observed by a laser scanning confocal microscopy (LSCM) (Leica TCS SP5, Germany).
2.3. Cell culture and the SNPs exposure 2.7. Immunoblot assay Human hepatocellular carcinoma HepG2 cells were purchased from Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in
Briefly, after exposure to different concentrations of SNPs (25–100 g/mL) for 24 h, HepG2 cells were washed once with
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ice-cold PBS, and lysed in ice-cold RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (DingGuo, China) and phosphatase inhibitor for 30 min. After centrifuging the lysates at 12,000 rpm, 4 ◦ C for 10 min, the total cellular protein extracts were determined by performing the bicinchoninic acid (BCA) protein assay (Pierce, USA). The equal amounts of proteins (40 g) were loaded onto 12–15% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) for 1 h, the membrane was incubated with LC3 and GAPDH antibodies [1:1000, rabbit antibodies, Cell Signaling Technology (CST), USA] overnight at 4 ◦ C. The membrane was then washed with TBST, and incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (CST, USA) for 1 h at room temperature. After washed three times with TBST, the antibody-bound proteins were detected using the ECL chemiluminescence reagent (Pierce, USA). 2.8. The TEM observation To detect whether autophagy was induced in SNPs treated HepG2 cells, transmission electron microscopy (TEM), the gold standard method to determine double-membrane vacuole structures, was applied. After HepG2 cells were incubated for 24 h with SNPs (50 g/mL), the cells were washed with PBS and then centrifuged at 1500 rpm for 10 min. The supernatants were removed. The cell pellets were fixed in a 0.1 M PBS solution containing 2.5% glutaraldehyde for 3 h. They were then washed with 0.1 M PBS, embedded in 2% agarosegel, postfixed in 4% osmium tetroxide solution for 1 h, washed with distilled water, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol (30%, 60%, 70%, 90%, and 100%), and embedded in epoxy resin. The resin was polymerized at 60 ◦ C for 48 h. Ultrathin sections obtained with a ultramicrotome were stained with 5% aqueous uranyl acetate and 2% aqueous lead citrate, air dried, and imaged under a transmission electron microscope (TEM) (JEOL JEM2100, Japan). 2.9. Autophagic cell death detection After HepG2 cells were exposed to SNPs (50 g/mL) for 24 h, the cells were stained with 0.05 mM MDC (Sigma, USA) in fresh DMEM and incubated for 30 min at 37 ◦ C in dark. Subsequently, Propidium Iodide (PI) was used to visualize dead cells for another 30 min in dark. Ultimately, the cells were washed three times with PBS, double-stained cells were visualized as cell death by autophagy by a laser scanning confocal microscopy (LSCM) (Leica TCS SP5, Germany). 2.10. Intracellular reactive oxygen species (ROS) measurement Intracellular reactive oxygen species (ROS) were measured by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) (Beyotime, China) as a probe. About 3 × 105 cells were seeded into a 6-well plate for 24 h. After HepG2 cells were exposed to series concentrations of SNPs (25–100 g/mL) for 24 h, the cells were washed twice with PBS and co-incubated with serum-free DMEM containing10 M DCFH-DA for 30 min at 37 ◦ C in dark. Subsequently, the cells were harvested and rinsed with PBS. Fluorescent intensities were measured at 488 nm excitation, 525 nm emission using a flow cytometer (Becton Dickison, USA). 2.11. Statistical analysis Data were expressed as means ± S.D. and significance was determined by using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test to compare the differences
between groups. Student’s t-test was performed to draw statistical comparisons between two treatment groups and p < 0.05 was considered to be statistically significant. 3. Results 3.1. Characterization of the SNPs As shown in Fig. 1A, the TEM image of SNPs appeared nearspherical and well dispersed. The size distribution of particles was calculated by ImageJ software and showed approximately normal distribution with an average diameter of 62.26 nm (Fig. 1B). The hydrodynamic sizes of the SNPs were measured in ultrapure water as stock media and in DMEM as culture media by Dynamic Light Scattering (DLS) at different time points to reflect their dispersibility (Table 1). The SNPs exhibited very good monodispersity both in ultrapure water and DMEM. Owing to the Van der Waals force and hydrophobic interaction with surrounding media, the hydrodynamic size is generally larger than original. Zeta potentials provide quantitative information on the stability of the particles. It is well documented that the particles are more likely to remain dispersed if the absolute value of zeta potential is higher than 30 mV [29,30]. The SNPs tested in our study had the absolute value of zeta potential that was higher than 30 mV, which ensured the stability of the particles. Our results showed that the SNPs in culture medium possessed uniform shape and showed relatively favorable dispersibility. 3.2. The SNPs internalization Since the intracellular localization may play an important role in the SNPs-induced biological effects, we examined the uptake of Ru(phen)3 2+ -labeled SNPs (62 nm) by HepG2 cells. The LSCM images showed that after 24 h of SNPs treatment, the fluochromelabeled SNPs were highly aggregated in cell cytoplasm (Fig. 2). In line with the LSCM results, the internalization of SNPs was also found in the TEM images (Fig. 3). A number of SNPs were dispersed as electron dense material either free or as membrane-bound aggregates in cytoplasm (Fig. 3C). The SNPs were found to deposit in mitochondria (Fig. 3D), inducing destruction of mitochondrial structures, such as mitochondrial swelling, cristae rupturing and disappearance (Fig. 3E). The particle clusters were also accumulated in vesicles of endosomes and lysosomes in cytoplasm (Fig. 3F). 3.3. Cytotoxicity induced by the SNPs To evaluate the possible toxicity of SNPs on HepG2 cells, cell viability was determined after the cultured cells exposed to various concentrations of SNPs (25, 50, 75, and 100 g/mL) for 24 h. As shown in Fig. 4, the cell viability was gradually decreased in a dose-dependent manner. We also pretreated the HepG2 cells with autophagy inhibitor 3-MA and the ROS scavenger NAC to assess the possible role of autophagy and ROS in SNPs-induced cytotoxicity. The cell viability was significantly elevated when compared to the corresponding SNPs treatment alone, indicating that both autophagy inhibitor and ROS scavenger increased cell viability (Fig. 4). 3.4. Monodansylcadaverine staining The fluorescent dye Monodansylcadaverine (MDC) was used to measure autophagic vacuoles. As shown in Fig. 5A and B, both the numbers and the fluorescence intensity of MDC-labeled vesicles were increased in a dose-dependent manner. In addition, the fluorescence intensity of MDC staining in HepG2 cells were relatively sparse and weak after pretreated with 3-MA or NAC compared
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Fig. 1. Characterization of the SNPs. (A) The SNPs that appeared in the TEM image showed spherical and well dispersed. (B) The size distribution of the SNPs that was calculated by ImageJ software showed approximately normal distribution. Data are expressed as means ±S.D. n = 500 (Scale bar: 0.1 m).
to those treated with SNPs only (Fig. 6). Our data suggested that either the 3-MA or NAC could effectively inhibit the SNPs-induced autophagy. 3.5. The LC3-I/LC3-II conversion LC3 protein, as the standard marker of autophagy, is reliably associated with autophagosomes and widely used in assessing autophagy. The SNPs-induced autophagy was further verified by assessing the expression levels of LC3-I and LC3-II. LC3 protein usually exhibits a molecular form conversion from cytosolic LC3-I into its enzymatic counterpart LC3-II, which is bound to the membrane of autophagosomes during autophagy. Our results showed that the level of LC3-II protein gradually increased, suggesting that the autophagy occurred in SNPs-treated HepG2 cells with a dosedependent way (Fig. 7A). Furthermore, the immunoblot results also demonstrated that the conversion of LC3-I to LC3-II was obviously suppressed by both 3-MA and NAC (Fig. 7B).
contained membrane-bound cytoplasmic material or organelles (mitochondria) while the late autophagic vacuoles (AVd) contained electron-dense cytoplasmic materials which are partially degraded (Fig. 8F). These representative TEM images proved the clear evidences that the SNPs induced autophagy in HepG2 cells. 3.7. Autophagic cell death induced by the SNPs Autophagy dysfunction has been considered to associate with autophagic cell death. Change of cellular morphology was directly reflected to the effects of SNPs. As shown in the bright field of Fig. 9, the morphological changes of HepG2 cells treated with the SNPs became obviously such as rounding cell shape and cellular shrinkage. The cellular co-staining assay was performed with MDC and Propidium Iodide (PI) to determine the SNPs-induced autophagic cell death. The double-stained cells were visualized as cell death associated with autophagy (Fig. 9). These results confirmed that the SNPs could lead to autophagic cell death in HepG2 cells.
3.6. The observation of autophagic ultrastructural features 3.8. Intracellular ROS generation induced by the SNPs In order to confirm the activation of SNPs-induced autophagy, the TEM ultrastructural analysis as golden standard was then performed. These typical autophagic ultrastructural features were shown in Fig. 8: a double-membranous phagophore engulfing portions of the cytoplasm, indicating the activation of autophagy (Fig. 8B); an autophagosome formation with cytoplasmic contents after engulfing by a phagophore (Fig. 8C); the fusion of autophagosomes with lysosomes or endosomes (Fig. 8D); late autophagic vacuoles (autolysosomes) with cytoplasmic contents inside (Fig. 8E); autophagic vacuoles at different stages of the complete process where the early or initial autophagic vacuoles (AVi)
To get a closer insight into the possible mechanism of SNPsinduced autophagy, the intracellular ROS levels were determined by using the DCFH-DA probe. As shown in Fig. 10A, the ROS levels were elevated gradually in HepG2 cells with increasing concentrations of SNPs. The generation of ROS in all SNPs-treated groups was significant differences compared to control group. Moreover, there was a significant positive correlation between the intracellular ROS levels and autophagy (R2 = 0.9855) (Fig. 10B). An overview of the putative mechanisms involved the SNPs-induced autophagy and autophagic cell death was presented in Fig. 11.
Table 1 The hydrodynamic size and zeta potential of the SNPs in ultrapure water and DMEM at different time points. Ultrapure water
1h 3h 6h 12 h 24 h
DMEM
Hydrodynamic sizes (nm)
Zeta potential (mV)
Hydrodynamic sizes (nm)
Zeta potential (mV)
107.5 ± 1.20 105.9 ± 1.51 106.8 ± 0.62 108.3 ± 2.99 106.0 ± 1.69
43.7 ± 2.40 41.3 ± 3.00 42.9 ± 0.87 46.1 ± 2.77 44.2 ± 1.40
114.4 ± 1.11 116.2 ± 0.95 113.6 ± 0.44 113.6 ± 2.09 116.3 ± 1.10
35.5 ± 1.65 36.2 ± 2.11 35.0 ± 1.90 35.5 ± 1.55 38.3 ± 0.49
Data are expressed as means ±S.D. n = 3.
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Fig. 2. Confocal fluorescence images of the cellular internalization of the SNPs. (A) The control cells and magnification of the selected area. (B) The cells treated with Ru(phen)3 2+ -labeled SNPs (50 g/mL, red) of size 62 nm and magnification of the selected area. The cell skeleton was stained with Phalloidin-FITC (green), and the cell nucleus with 4,6-diamidino-2-phenylindole (DAPI; blue) (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).
Fig. 3. The TEM images of cellular uptake of HepG2 cells exposed to the SNPs (50 g/mL) for 24 h. (A) Control group. (B) The magnification of selected area of control showed evidently intact mitochondrias (white arrows) and Golgi apparatus (black arrows). (C) Electron-dense SNPs dispersed in cytoplasm either free or as membrane-bound aggregates (arrows). (D) The SNPs deposited in mitochondria (arrows). (E) The SNPs localization in lysosomes (black arrows) and induction of mitochondrial swelling, cristae rupturing and disappearance (white arrows). (F) The SNPs accumulated in endosomes (white arrows) or lysosomes (black arrows) in cytoplasm.
Y. Yu et al. / Journal of Hazardous Materials 270 (2014) 176–186
Fig. 4. Cell viability of HepG2 cells treated with the SNPs or pretreated with inhibitors was measured by MTT assay after 24 h exposure. Cell viability was significantly decreased in a dose-dependent manner after the SNPs treatment. SNPs + 3-MA or SNPs + NAC co-treatment significantly suppressed HepG2 cell death, when compared to corresponding SNPs treatment alone, *p
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Silica nanoparticles induce autophagy and autophagic cell death in HepG2 cells triggered by reactive oxygen species Yongbo Yu a,b,1 , Junchao Duan a,b,1 , Yang Yu a,b , Yang Li a,b , Xiaomei Liu c , Xianqing Zhou a,b , Kin-fai Ho d , Linwei Tian d,∗∗ , Zhiwei Sun a,b,∗ a
School of Public Health, Capital Medical University, Beijing, 100069, P.R. China Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing, 100069, P.R. China c School of Public Health, Jilin University, Changchun, Jilin, 130021, P.R. China d School of Public Health and Primary Care, Chinese University of Hong Kong, Hong Kong, China b
h i g h l i g h t s • Silica nanoparticles (SNPs) induce autophagy and autophagic cell death. • The typically morphological changes of whole autophagy process are observed. • ROS scavenger and autophagy inhibitor effectively suppress the SNPs-induced autophagy and autophagy cell death.
a r t i c l e
i n f o
Article history: Received 30 September 2013 Received in revised form 22 December 2013 Accepted 7 January 2014 Available online 24 January 2014 Keywords: Nanotoxicity Silica nanoparticles Autophagy Autophagic cell death Reactive oxygen species
a b s t r a c t Silica nanoparticles (SNPs) are becoming favorable carriers for drug delivery or gene therapy, and in turn, the toxic effect of SNPs on biological systems is gaining attention. Currently, autophagy is recognized as an emerging toxicity mechanism triggered by nanomaterials, yet there have been scarcely research about the mechanisms of autophagy and autophagic cell death associated with SNPs. In this study, we verified the activation of SNPs-induced autophagy via the MDC-staining and LC3-I/LC3-II conversion, resulted in a dose-dependent manner. The typically morphological characteristics (autophagosomes and autolysosomes) of the autophagy process were observed in TEM ultrastructural analysis. In addition, the autophagic cell death was evaluated by cellular co-staining assay. And the underlying mechanisms of autophagy and autophagic cell death were performed using the intracellular ROS detection, autophagy inhibitor and ROS scavenger. Results showed that the elevated ROS level was in line with the increasing of autophagy activation, while both the 3-MA and NAC inhibitors effectively suppressed the autophagy and cell death induced by SNPs. In summary, our findings demonstrated that the SNPs-induced autophagy and autophagic cell death were triggered by the ROS generation in HepG2 cells, suggesting that exposure to SNPs could be a potential hazardous factor for maintaining cellular homeostasis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Silica nanoparticles (SNPs) have been widely used as additives to cosmetics, paints, varnishes, and food [1]. The emerging commercialization of the SNPs products has increased the environment and human exposure. In addition, the unique physicochemical properties of SNPs make it attractive for a variety of biomedical and
∗ Corresponding author at: Zhiwei Sun, School of Public Health, Capital Medical University, Beijing, 100069, P.R. China. Tel.: +86 010 83911507; fax: +86 010 83911507. ∗∗ Corresponding author at: Linwei Tian, School of Public Health and Primary Care, Chinese University of Hong Kong, Hong Kong, China. Tel.: +852 22528879; fax: +852 26063500. E-mail addresses: [email protected] (L. Tian), [email protected] (Z. Sun). 1 The first two authors contribute equally to this work. 0304-3894/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2014.01.028
biotechnological applications such as cancer therapy, drugs and gene delivery and biosensors [2,3]. SNPs as carriers of drug delivery systems are generally injected into the body intravenously and translated to organs via the systemic circulation [4]. It was reported that the liver is the target organ of SNPs in which the SNPs could be accumulated and retained for over 30 days [5,6]. Therefore, it is essential to understand the biological effects and potential hepatotoxicity of SNPs either in vivo or in vitro. Currently, several studies have reported that the SNPs can penetrate the cytomembrane, deposit in mitochondria or even the nucleus, and eventually lead to cell death [7,8]. According to the recommendations of Nomenclature Committee on Cell Death (NCCD), cell death can be classified into necrosis, apoptosis, autophagic cell death, mitotic catastrophe and other atypical modalities [9]. Unfortunately, most previous studies focus on the apoptosis and necrosis induced by SNPs in vitro [10–12]; little is known about the autophagy and its
Y. Yu et al. / Journal of Hazardous Materials 270 (2014) 176–186
toxicological consequence, which is called autophagic cell death or type II programmed cell death [13,14]. Autophagy is a highly regulated intracellular process for the lysosomal degradation and recycling of proteins and organelles [15]. In the process of autophagy, autophagosomes (twomembraned and containing degenerating cytoplasmic organelles or parts of the cytoplasm) are finally delivered to lysosomes for bulk degradation [16,17]. A basal level of autophagy is a homeostatic cytoprotective mechanism; however, the autophagy dysfunction can lead to severe pathological states, such as neurodegenerative diseases, cardiomyopathy, and particularly cancer [18,19]. Recently, autophagy was proposed as an emerging toxicity mechanisms occurred in several nanomaterials, such as nano-sized fullerene, rare earth oxides and titanium dioxide and copper oxide [20–23]. However, there exists scarcely literature focused on the biological response of the SNPs associated with autophagy, which provides limited evidences to confirm their relationship. Moreover, these studies failed to consider the underlying mechanisms between autophagy and autophagic cell death [24,25]. Based on the issues above, an insight evaluation of SNPs-induced autophagy and autophagic cell death was then conducted in vitro with human hepatocellular carcinoma HepG2 cells. The HepG2 cells line was generally used to assess the hepatotoxicity in vitro studies [26]. Prior to undertaking in vitro toxicity experiments, the characterization of SNPs, which is essential for nanotoxicity studies, was performed by transmission electron microscope (TEM) and dynamic light scattering (DLS) measurements. To investigate the mechanisms of autophagy and autophagic cell death induced by SNPs, we conducted a sequence of assessments including cellular uptake and ultrastructural analysis, cell viability, intracellular ROS generation, MDC-staining, LC3-I/LC3-II conversion and cellular co-staining after HepG2 cells exposure to SNPs for 24 h. We also used the autophagy inhibitor 3-Methyladenine (3-MA) and the ROS scavenger N-acetyl cystiene (NAC) to analyze the underlying mechanisms of the SNPs-induced autophagy and autophagic cell death. 2. Materials and methods 2.1. Synthesis of the amorphous SNPs The SNPs were prepared using the Stöber method [27]. Briefly, 2.5 mL of tetraethylorthosilicate (TEOS) (Sigma, USA) was added to premixed ethanol solution (50 mL) containing ammonia (2 mL) and water (1 mL). The reaction mixture was kept at 40 ◦ C for 12 h with continuous stirring (150 r/min). The resulting particles were isolated by centrifugation (12,000 r/min, 15 min) and washed three times with deionized water and then dispersed in 50 mL of deionized water. 2.2. Characterization of the SNPs The particle size and distribution of the SNPs were measured by transmission electron microscope (TEM) (JEOL, Japan) and ImageJ software. The hydrodynamic sizes and zeta potential of SNPs in ultrapure water and serum-free DMEM were examined by Zetasizer (Malvern Nano-ZS90, Britain). Suspensions of SNPs were dispersed by sonicator (160 W, 20 kHz, 5 min) (Bioruptor UDC-200, Belgium) before addition to dispersion media in order to minimize their aggregation.
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Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin at 37 ◦ C in a humidified incubator with 5% CO2 . For experiments, the cells were seeded in culture plates and treated with SNPs suspended in DMEM of certain concentrations for another 24 h. Suspension of SNPs was sterilized by an autoclave and dispersed by sonicator (160 W, 20 KHz, 5 min) before added into culture medium to minimize their aggregation. Cells maintained in DMEM without SNPs were used as control group. 2.4. Cellular internalization of the SNPs HepG2 cells were seeded in 35 mm-diameter glass bottom cell culture dishes and were cultured in DMEM as above. After 24 h of cell attachment, the cells were treated with Ruthenium (II) hydrate (Ru(phen)3 2+ ) interior-labeled SNPs (50 g/mL) for 24 h at 37 ◦ C in serum-free medium. These red fluorescent SNPs were prepared by a modified Stöber method and characterized as described before [28]. Cells were then washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. The cells were washed with 0.1% Triton X-100 three times and incubated with Phalloidin-FITC Actin-Tracker Green (Jiancheng, China) at room temperature for 30 min. The Actin-Tracker was dissolved in the mixture of 0.1% Triton X-100 and 3% bovine serum albumin (BSA) (Sigma, USA) for staining the actin filamentous skeleton. After that, the nucleus was stained with 2 g/mL 4,6-diamidino-2phenylindole (DAPI) (Sigma, USA) in PBS for 5 min. Cellular uptake were observed by a laser scanning confocal microscopy (LSCM) (Leica TCS SP5, Germany). 2.5. Cell viability assay The cytotoxicity of the SNPs was determined using MTT assay. Cell morphology was observed by optical microscope (Olympus IX81, Japan). Briefly, 1 × 104 HepG2 cells were seeded into a 96-well plate in a volume of 100 L DMEM and incubated for 24 h at 37 ◦ C. Cells were pretreated with or without 5 mM autophagy inhibitor 3Methyladenine (3-MA) or ROS scavenger N-acetyl cystiene (NAC) for 1 h and then incubated with varying concentrations of SNPs (25, 50, 75 and 100 g/mL) for 24 h at 37 ◦ C. After 24 h incubation, 10 L MTT was added to each well at 5 mg/mL and further incubated for 4 h. After which 150 L of dimethylsulfoxide (DMSO) was added in and mixed thoroughly for 5 min. Optical density was then measured with a microplate reader (Themo Multiscan MK3, USA) at 492 nm. 2.6. Autophagy measurement To detect whether autophagy was induced in HepG2 cells exposed to the SNPs, Monodansylcadaverine (MDC), a specific marker of autophagic vacuoles, was employed to stain autophagosomes. Briefly, bout 3 × 105 cells were seeded into a 6-well plate for 24 h. After pretreated with or without 5 mM 3-MA for 1 h, the cells were treated with series concentrations of SNPs (25–100 g/mL) for 24 h. The cells were then stained with 0.05 mM MDC (Sigma, USA) in fresh DMEM and incubated for 30 min at 37 ◦ C in dark. After three times washing with PBS, the cells were harvested and rinsed with PBS. Intracellular MDC intensity was measured using a flow cytometry (Becton-Dickison, USA) at 360 nm excitation, 530 nm emission and observed with fluorescence microscope (Olympus IX81, Japan). The visualization of MDC-stained autophagosomes and autolysosomes were observed by a laser scanning confocal microscopy (LSCM) (Leica TCS SP5, Germany).
2.3. Cell culture and the SNPs exposure 2.7. Immunoblot assay Human hepatocellular carcinoma HepG2 cells were purchased from Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in
Briefly, after exposure to different concentrations of SNPs (25–100 g/mL) for 24 h, HepG2 cells were washed once with
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ice-cold PBS, and lysed in ice-cold RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (DingGuo, China) and phosphatase inhibitor for 30 min. After centrifuging the lysates at 12,000 rpm, 4 ◦ C for 10 min, the total cellular protein extracts were determined by performing the bicinchoninic acid (BCA) protein assay (Pierce, USA). The equal amounts of proteins (40 g) were loaded onto 12–15% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) for 1 h, the membrane was incubated with LC3 and GAPDH antibodies [1:1000, rabbit antibodies, Cell Signaling Technology (CST), USA] overnight at 4 ◦ C. The membrane was then washed with TBST, and incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (CST, USA) for 1 h at room temperature. After washed three times with TBST, the antibody-bound proteins were detected using the ECL chemiluminescence reagent (Pierce, USA). 2.8. The TEM observation To detect whether autophagy was induced in SNPs treated HepG2 cells, transmission electron microscopy (TEM), the gold standard method to determine double-membrane vacuole structures, was applied. After HepG2 cells were incubated for 24 h with SNPs (50 g/mL), the cells were washed with PBS and then centrifuged at 1500 rpm for 10 min. The supernatants were removed. The cell pellets were fixed in a 0.1 M PBS solution containing 2.5% glutaraldehyde for 3 h. They were then washed with 0.1 M PBS, embedded in 2% agarosegel, postfixed in 4% osmium tetroxide solution for 1 h, washed with distilled water, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol (30%, 60%, 70%, 90%, and 100%), and embedded in epoxy resin. The resin was polymerized at 60 ◦ C for 48 h. Ultrathin sections obtained with a ultramicrotome were stained with 5% aqueous uranyl acetate and 2% aqueous lead citrate, air dried, and imaged under a transmission electron microscope (TEM) (JEOL JEM2100, Japan). 2.9. Autophagic cell death detection After HepG2 cells were exposed to SNPs (50 g/mL) for 24 h, the cells were stained with 0.05 mM MDC (Sigma, USA) in fresh DMEM and incubated for 30 min at 37 ◦ C in dark. Subsequently, Propidium Iodide (PI) was used to visualize dead cells for another 30 min in dark. Ultimately, the cells were washed three times with PBS, double-stained cells were visualized as cell death by autophagy by a laser scanning confocal microscopy (LSCM) (Leica TCS SP5, Germany). 2.10. Intracellular reactive oxygen species (ROS) measurement Intracellular reactive oxygen species (ROS) were measured by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) (Beyotime, China) as a probe. About 3 × 105 cells were seeded into a 6-well plate for 24 h. After HepG2 cells were exposed to series concentrations of SNPs (25–100 g/mL) for 24 h, the cells were washed twice with PBS and co-incubated with serum-free DMEM containing10 M DCFH-DA for 30 min at 37 ◦ C in dark. Subsequently, the cells were harvested and rinsed with PBS. Fluorescent intensities were measured at 488 nm excitation, 525 nm emission using a flow cytometer (Becton Dickison, USA). 2.11. Statistical analysis Data were expressed as means ± S.D. and significance was determined by using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test to compare the differences
between groups. Student’s t-test was performed to draw statistical comparisons between two treatment groups and p < 0.05 was considered to be statistically significant. 3. Results 3.1. Characterization of the SNPs As shown in Fig. 1A, the TEM image of SNPs appeared nearspherical and well dispersed. The size distribution of particles was calculated by ImageJ software and showed approximately normal distribution with an average diameter of 62.26 nm (Fig. 1B). The hydrodynamic sizes of the SNPs were measured in ultrapure water as stock media and in DMEM as culture media by Dynamic Light Scattering (DLS) at different time points to reflect their dispersibility (Table 1). The SNPs exhibited very good monodispersity both in ultrapure water and DMEM. Owing to the Van der Waals force and hydrophobic interaction with surrounding media, the hydrodynamic size is generally larger than original. Zeta potentials provide quantitative information on the stability of the particles. It is well documented that the particles are more likely to remain dispersed if the absolute value of zeta potential is higher than 30 mV [29,30]. The SNPs tested in our study had the absolute value of zeta potential that was higher than 30 mV, which ensured the stability of the particles. Our results showed that the SNPs in culture medium possessed uniform shape and showed relatively favorable dispersibility. 3.2. The SNPs internalization Since the intracellular localization may play an important role in the SNPs-induced biological effects, we examined the uptake of Ru(phen)3 2+ -labeled SNPs (62 nm) by HepG2 cells. The LSCM images showed that after 24 h of SNPs treatment, the fluochromelabeled SNPs were highly aggregated in cell cytoplasm (Fig. 2). In line with the LSCM results, the internalization of SNPs was also found in the TEM images (Fig. 3). A number of SNPs were dispersed as electron dense material either free or as membrane-bound aggregates in cytoplasm (Fig. 3C). The SNPs were found to deposit in mitochondria (Fig. 3D), inducing destruction of mitochondrial structures, such as mitochondrial swelling, cristae rupturing and disappearance (Fig. 3E). The particle clusters were also accumulated in vesicles of endosomes and lysosomes in cytoplasm (Fig. 3F). 3.3. Cytotoxicity induced by the SNPs To evaluate the possible toxicity of SNPs on HepG2 cells, cell viability was determined after the cultured cells exposed to various concentrations of SNPs (25, 50, 75, and 100 g/mL) for 24 h. As shown in Fig. 4, the cell viability was gradually decreased in a dose-dependent manner. We also pretreated the HepG2 cells with autophagy inhibitor 3-MA and the ROS scavenger NAC to assess the possible role of autophagy and ROS in SNPs-induced cytotoxicity. The cell viability was significantly elevated when compared to the corresponding SNPs treatment alone, indicating that both autophagy inhibitor and ROS scavenger increased cell viability (Fig. 4). 3.4. Monodansylcadaverine staining The fluorescent dye Monodansylcadaverine (MDC) was used to measure autophagic vacuoles. As shown in Fig. 5A and B, both the numbers and the fluorescence intensity of MDC-labeled vesicles were increased in a dose-dependent manner. In addition, the fluorescence intensity of MDC staining in HepG2 cells were relatively sparse and weak after pretreated with 3-MA or NAC compared
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Fig. 1. Characterization of the SNPs. (A) The SNPs that appeared in the TEM image showed spherical and well dispersed. (B) The size distribution of the SNPs that was calculated by ImageJ software showed approximately normal distribution. Data are expressed as means ±S.D. n = 500 (Scale bar: 0.1 m).
to those treated with SNPs only (Fig. 6). Our data suggested that either the 3-MA or NAC could effectively inhibit the SNPs-induced autophagy. 3.5. The LC3-I/LC3-II conversion LC3 protein, as the standard marker of autophagy, is reliably associated with autophagosomes and widely used in assessing autophagy. The SNPs-induced autophagy was further verified by assessing the expression levels of LC3-I and LC3-II. LC3 protein usually exhibits a molecular form conversion from cytosolic LC3-I into its enzymatic counterpart LC3-II, which is bound to the membrane of autophagosomes during autophagy. Our results showed that the level of LC3-II protein gradually increased, suggesting that the autophagy occurred in SNPs-treated HepG2 cells with a dosedependent way (Fig. 7A). Furthermore, the immunoblot results also demonstrated that the conversion of LC3-I to LC3-II was obviously suppressed by both 3-MA and NAC (Fig. 7B).
contained membrane-bound cytoplasmic material or organelles (mitochondria) while the late autophagic vacuoles (AVd) contained electron-dense cytoplasmic materials which are partially degraded (Fig. 8F). These representative TEM images proved the clear evidences that the SNPs induced autophagy in HepG2 cells. 3.7. Autophagic cell death induced by the SNPs Autophagy dysfunction has been considered to associate with autophagic cell death. Change of cellular morphology was directly reflected to the effects of SNPs. As shown in the bright field of Fig. 9, the morphological changes of HepG2 cells treated with the SNPs became obviously such as rounding cell shape and cellular shrinkage. The cellular co-staining assay was performed with MDC and Propidium Iodide (PI) to determine the SNPs-induced autophagic cell death. The double-stained cells were visualized as cell death associated with autophagy (Fig. 9). These results confirmed that the SNPs could lead to autophagic cell death in HepG2 cells.
3.6. The observation of autophagic ultrastructural features 3.8. Intracellular ROS generation induced by the SNPs In order to confirm the activation of SNPs-induced autophagy, the TEM ultrastructural analysis as golden standard was then performed. These typical autophagic ultrastructural features were shown in Fig. 8: a double-membranous phagophore engulfing portions of the cytoplasm, indicating the activation of autophagy (Fig. 8B); an autophagosome formation with cytoplasmic contents after engulfing by a phagophore (Fig. 8C); the fusion of autophagosomes with lysosomes or endosomes (Fig. 8D); late autophagic vacuoles (autolysosomes) with cytoplasmic contents inside (Fig. 8E); autophagic vacuoles at different stages of the complete process where the early or initial autophagic vacuoles (AVi)
To get a closer insight into the possible mechanism of SNPsinduced autophagy, the intracellular ROS levels were determined by using the DCFH-DA probe. As shown in Fig. 10A, the ROS levels were elevated gradually in HepG2 cells with increasing concentrations of SNPs. The generation of ROS in all SNPs-treated groups was significant differences compared to control group. Moreover, there was a significant positive correlation between the intracellular ROS levels and autophagy (R2 = 0.9855) (Fig. 10B). An overview of the putative mechanisms involved the SNPs-induced autophagy and autophagic cell death was presented in Fig. 11.
Table 1 The hydrodynamic size and zeta potential of the SNPs in ultrapure water and DMEM at different time points. Ultrapure water
1h 3h 6h 12 h 24 h
DMEM
Hydrodynamic sizes (nm)
Zeta potential (mV)
Hydrodynamic sizes (nm)
Zeta potential (mV)
107.5 ± 1.20 105.9 ± 1.51 106.8 ± 0.62 108.3 ± 2.99 106.0 ± 1.69
43.7 ± 2.40 41.3 ± 3.00 42.9 ± 0.87 46.1 ± 2.77 44.2 ± 1.40
114.4 ± 1.11 116.2 ± 0.95 113.6 ± 0.44 113.6 ± 2.09 116.3 ± 1.10
35.5 ± 1.65 36.2 ± 2.11 35.0 ± 1.90 35.5 ± 1.55 38.3 ± 0.49
Data are expressed as means ±S.D. n = 3.
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Fig. 2. Confocal fluorescence images of the cellular internalization of the SNPs. (A) The control cells and magnification of the selected area. (B) The cells treated with Ru(phen)3 2+ -labeled SNPs (50 g/mL, red) of size 62 nm and magnification of the selected area. The cell skeleton was stained with Phalloidin-FITC (green), and the cell nucleus with 4,6-diamidino-2-phenylindole (DAPI; blue) (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).
Fig. 3. The TEM images of cellular uptake of HepG2 cells exposed to the SNPs (50 g/mL) for 24 h. (A) Control group. (B) The magnification of selected area of control showed evidently intact mitochondrias (white arrows) and Golgi apparatus (black arrows). (C) Electron-dense SNPs dispersed in cytoplasm either free or as membrane-bound aggregates (arrows). (D) The SNPs deposited in mitochondria (arrows). (E) The SNPs localization in lysosomes (black arrows) and induction of mitochondrial swelling, cristae rupturing and disappearance (white arrows). (F) The SNPs accumulated in endosomes (white arrows) or lysosomes (black arrows) in cytoplasm.
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Fig. 4. Cell viability of HepG2 cells treated with the SNPs or pretreated with inhibitors was measured by MTT assay after 24 h exposure. Cell viability was significantly decreased in a dose-dependent manner after the SNPs treatment. SNPs + 3-MA or SNPs + NAC co-treatment significantly suppressed HepG2 cell death, when compared to corresponding SNPs treatment alone, *p
