Apoptosis induction by silica nanoparticles mediated through reactive oxygen species in human liver cell line HepG2

https://doi.org/10.1016/j.taap.2011.12.020Get rights and content

Abstract

Silica nanoparticles are increasingly utilized in various applications including agriculture and medicine. In vivo studies have shown that liver is one of the primary target organ of silica nanoparticles. However, possible mechanisms of hepatotoxicity caused by silica nanoparticles still remain unclear. In this study, we explored the reactive oxygen species (ROS) mediated apoptosis induced by well-characterized 14 nm silica nanoparticles in human liver cell line HepG2. Silica nanoparticles (25–200 μg/ml) induced a dose-dependent cytotoxicity in HepG2 cells. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of ROS and lipid peroxidation and depletion of glutathione (GSH). Quantitative real-time PCR and immunoblotting results showed that both the mRNA and protein expressions of cell cycle checkpoint gene p53 and apoptotic genes (bax and caspase-3) were up-regulated while the anti-apoptotic gene bcl-2 was down-regulated in silica nanoparticles treated cells. Moreover, co-treatment of ROS scavenger vitamin C significantly attenuated the modulation of apoptotic markers along with the preservation of cell viability caused by silica nanoparticles. Our data demonstrated that silica nanoparticles induced apoptosis in human liver cells, which is ROS mediated and regulated through p53, bax/bcl-2 and caspase pathways. This study suggests that toxicity mechanisms of silica nanoparticles should be further investigated at in vivo level.

Highlights

► We explored the mechanisms of toxicity caused by silica NPs in human liver HepG2 cells. ► Silica NPs induced a dose-dependent cytotoxicity in HepG2 cells. ► Silica NPs induced ROS generation and oxidative stress in a dose-dependent manner. ► Silica NPs were also modulated apoptosis markers both at mRNA and protein levels. ► ROS mediated apoptosis induced by silica NPs was preserved by vitamin C.

Introduction

Silica nanoparticles are increasingly used in various applications including chemical industry, cosmetic and medicine (Bottini et al., 2007, Slowing et al., 2008, Zhu et al., 2010). The wide-spread applications of silica nanoparticles, however, increase the environmental and human exposure concern and thus the potential risk related to their short- and long-term toxicity. There are studies reported the toxicity of silica nanoparticles to bacteria (Bacillus subtilis and Escherichia coli), green algae (Pseudokirchneriella subcapitata) and zebrafish (Danio rerio) (Adams et al., 2006, Fent et al., 2010, Hoecke et al., 2008).

Silica nanoparticles can enter human body via different routes such as inhalation, ingestion, dermal contact and injection (Lam et al., 2004, Oberdoerster et al., 2005, Warheit et al., 2004, Zhou and Yokel, 2005). Silica nanoparticles injected to mice could distribute nearly in all organs and mainly accumulate, retain and induce toxic effects in lung, liver and spleen (Kaewamatawong et al., 2006, Liu et al., 2011, Park and Park, 2009). Investigators also observed that liver was one of the most target organs for silica nanoparticles (Lu et al., 2011, Nishimori et al., 2009, Xie et al., 2010). There are few important studies evaluating the toxicity of silica nanoparticles in cultured human liver cells (Li et al., 2011, Ye et al., 2010). However, possible mechanisms of hepatotoxicity due to silica nanoparticles exposure still remain unclear.

Apoptosis is induced by extracellular or intracellular signals, which trigger onset of signaling cascade with characteristic biochemical and cytological signatures including nuclear condensation and DNA fragmentation (Gopinath et al., 2010). There are several genes known to involve in apoptotic pathways. The p53 gene is able to activate cell cycle checkpoints, DNA repair and apoptosis to maintain genomic stability (Sherr, 2004). The ratio of bax/bcl-2 proteins represent a cell death switch, which determines the life or death of cells in response to an apoptotic stimulus; an increased bax/bcl-2 ratio decreases the cellular resistance to apoptotic stimuli, leading to apoptosis (Chougule et al., 2011, Gao and Wang, 2009). Also destabilization of the mitochondrial integrity by apoptotic stimuli precedes activation of caspases leading to apoptosis (Timmer and Salvesen, 2007, Youle and Strasser, 2008).

The potential mechanisms of nanoparticles toxicity are not fully explored. One mechanism often discussed is the induction of oxidative damage of cellular constituents due to the generation of reactive oxygen species (ROS) (Nel et al., 2006). Studies demonstrated that nanoparticles have potential to induce ROS mediated DNA damage and apoptosis (Ahamed et al., 2011a, Asharani et al., 2009, Park and Park, 2009). In the present study, we investigated the cytotoxicity, ROS generation and oxidative stress induced by well-characterized 14 nm amorphous silica nanoparticles in human liver cell line HepG2. Apoptosis induction by silica nanoparticles was explored via p53, bax/bcl-2, and caspase-3 pathways. We further examined the role of ROS in silica nanoparticles induced apoptosis using vitamin C, a potent ROS scavenger. This study provides molecular evidence for the ROS mediated apoptosis in human liver cells due to silica nanoparticles exposure.

Section snippets

Silica nanoparticles and chemicals

Silica (SiOx) nanopowder (Product No. 4850MR, Average particle size: 15 nm, specific surface area: 640 m2/g, Purity: 99.5% trace metals basis) purchased from Nanostructured & Amorphous Materials, Inc. (Houston, TX).

Fetal bovine serum (FBS), penicillin-streptomycin and DMEM/F-12 medium were obtained from Invitrogen Co. (Carlsbad, CA). MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide], 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH), anti-p53 antibody,

Silica nanoparticles characterization

X-ray diffraction (XRD) analysis exhibits the amorphous nature of silica nanoparticles (Fig. 1A). Fig. 1B and C depicts the typical SEM and TEM images of silica nanoparticles respectively. TEM average diameter was calculated from measuring over 100 particles in random fields of TEM view. The average TEM diameter of silica nanoparticles was 14.23 ± 2.16 nm. The EDS spectrum of silica nanoparticles is given in Fig. 1D. The presence of Cu and C signals was from the carbon-coated Cu grid. The EDS

Discussion

It has been suggested that the physicochemical properties of nanoparticles should be appropriately characterized before their nanotoxicity research (Balbus et al., 2007, Li et al., 2011, Murdock et al., 2008, Yu et al., 2009). We utilized XRD, SEM, TEM, EDS and DLS techniques to characterize the present silica nanoparticles. Amorphous nature of silica nanoparticles was confirmed by XRD. SEM and TEM showed that nanoparticles were almost spherical in shape, smooth surface and with an average

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by King Abdulaziz City for Science and Technology (KACST) under the National Plan for Science and Technology (NPST) (Grant No.: 10-NAN1201-02).

References (62)

  • S. Barillet et al.

    In vitro evaluation of SiC nanoparticles impact on A549 pulmonary cells: cyto-, genotoxicity and oxidative stress

    Toxicol. Lett.

    (2010)
  • C. Berasain et al.

    Novel role for amphiregulin in protection from liver injury

    J. Biol. Chem.

    (2005)
  • M.M. Bradford

    A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

    Anal. Biochem.

    (1976)
  • G.I. Ellman

    Tissue sulfhydryl groups

    Arch. Biochem. Biophys.

    (1959)
  • K. Fent et al.

    Assessment of uptake and toxicity of fluorescent silica nanoparticles in zebrafish (Danio rerio) early life stages

    Aquat. Toxicol.

    (2010)
  • P. Gopinath et al.

    Signaling gene cascade in silver nanoparticle induced apoptosis

    Colloids Surf. B

    (2010)
  • R.U. Janicke et al.

    Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis

    J. Biol. Chem.

    (1998)
  • K. Jomova et al.

    Advances in metal-induced oxidative stress and human disease

    Toxicology

    (2011)
  • D.C. Julien et al.

    In vitro proliferating cell models to study cytotoxicity of silica nanowires

    Nanomedicine

    (2010)
  • Y. Li et al.

    Size-dependent cytotoxicity of amorphous silica nanoparticles in human hepatoma HepG2 cells

    Toxicol. In Vitro

    (2011)
  • T. Liu et al.

    Single and repeated dose toxicity of mesoporous hollow silica nanoparticles in intravenously exposed mice

    Biomaterials

    (2011)
  • M. Mahmoudi et al.

    Cell toxicity of superparamagnetic iron oxide nanoparticles

    J. Colloid Interface Sci.

    (2009)
  • H. Nishimori et al.

    Silica nanoparticles as hepatotoxicants

    Eur. J. Pharm. Biopharm.

    (2009)
  • H. Ohkawa et al.

    Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

    Anal. Biochem.

    (1979)
  • E.J. Park et al.

    Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro

    Toxicol. Lett.

    (2009)
  • C.J. Sherr

    Principles of tumor suppression

    Cell

    (2004)
  • I.I. Slowing et al.

    Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers

    Adv. Drug Deliv. Rev.

    (2008)
  • F. Wang et al.

    Oxidative stress contributes to silica nanoparticle-induced cytotoxicity in human embryonic kidney cells

    Toxicol. In Vitro

    (2009)
  • J.P. Wise et al.

    Silver nanospheres are cytotoxic and genotoxic to fish cells

    Aquat. Toxicol.

    (2010)
  • Y. Ye et al.

    Nano-SiO2 induces apoptosis via activation of p53 and Bax mediated by oxidative stress in human hepatic cell line

    Toxicol. in Vitro

    (2010)
  • Y. Yuan et al.

    Size-mediated cytotoxicity and apoptosis of hydroxyapatite nanoparticles in human hepatoma HepG2 cells

    Biomaterials

    (2010)
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