Elsevier

Toxicology

Volume 283, Issues 2–3, 10 May 2011, Pages 101-108
Toxicology

Oxidative stress mediated apoptosis induced by nickel ferrite nanoparticles in cultured A549 cells

https://doi.org/10.1016/j.tox.2011.02.010Get rights and content

Abstract

Due to the interesting magnetic and electrical properties with good chemical and thermal stabilities, nickel ferrite nanoparticles are being utilized in many applications including magnetic resonance imaging, drug delivery and hyperthermia. Recent studies have shown that nickel ferrite nanoparticles produce cytotoxicity in mammalian cells. However, there is very limited information concerning the toxicity of nickel ferrite nanoparticles at the cellular and molecular level. The aim of this study was to investigate the cytotoxicity, oxidative stress and apoptosis induction by well-characterized nickel ferrite nanoparticles (size 26 nm) in human lung epithelial (A549) cells. Nickel ferrite nanoparticles induced dose-dependent cytotoxicity in A549 cells demonstrated by MTT, NRU and LDH assays. Nickel ferrite nanoparticles were also found to induce oxidative stress evidenced by generation of reactive oxygen species (ROS) and depletion of antioxidant glutathione (GSH). Further, co-treatment with the antioxidant l-ascorbic acid mitigated the ROS generation and GSH depletion due to nickel ferrite nanoparticles suggesting the potential mechanism of oxidative stress. Quantitative real-time PCR analysis demonstrated that following the exposure of A549 cells to nickel ferrite nanoparticles, the level of mRNA expressions of cell cycle checkpoint protein p53 and apoptotic proteins (bax, caspase-3 and caspase-9) were significantly up-regulated, whereas the expression of anti-apoptotic proteins (survivin and bcl-2) were down-regulated. Moreover, activities of caspase-3 and caspase-9 enzymes were also significantly higher in nickel ferrite nanoparticles exposed cells. To the best of our knowledge this is the first report showing that nickel ferrite nanoparticles induced apoptosis in A549 cells through ROS generation and oxidative stress via p53, survivin, bax/bcl-2 and caspase pathways.

Introduction

Spinel ferrite nanoparticles with the general formula MFe2O4 (where M is +2 cation of Ni, Mn, Zn or Co) are very important materials because of their interesting magnetic and electrical properties with good chemical and thermal stabilities (Willard et al., 2004). These nanocrystalline materials are used in many applications including magnetic extraction, magnetic resonance imaging, cell labeling, drug delivery and hyperthermia (Lee et al., 2007, Rana et al., 2007, Sun et al., 2008, Chertok et al., 2008). Hyperthermia is a method of cancer treatments which is designed to raise the temperature of cancer cells. Cancer cells more are susceptible to heat than normal cells. Hyperthermia treatment thus has the advantage of being less risky to the body, with fewer side effects (Tomitaka et al., 2010). Nickel ferrite (NiFe2O4) is one of the most important spinel ferrites. Despite the wide-spread application of nickel ferrite nanoparticles, there is a serious lack of information concerning the toxicity of these nanoparticles at the cellular and molecular level. Only a few significant studies reported potential cytotoxicity of spinet ferrite nanoparticles including nickel ferrite nanoparticles (Yin et al., 2005, Baldi et al., 2007, Beji et al., 2010). Tomitaka et al. (2009) reported that nickel ferrite nanoparticles showed only minimal changes on HeLa cell proliferation at concentrations of 10 μg/ml, but significantly low viability at concentrations of 100 μg/ml (Tomitaka et al., 2009).

The molecular mechanisms of toxicity of nanoparticles are still underway. One mechanism frequently discussed is the induction of oxidative damage of cellular constituents, either due to the generation of reactive oxygen species (ROS) or by inactivation of antioxidant defense system (Nel et al., 2006, Stone and Donaldson, 2006). Experimental evidence has shown that metal and metal oxide nanoparticles induced DNA damage and apoptosis through ROS generation and oxidative stress (Park et al., 2008, Asharani et al., 2009, Ahamed et al., 2010a, Ahamed et al., 2010b, Lunov et al., 2010). For example, our previous studies have shown that silver nanoparticles, copper oxide nanoparticles and silica nanoparticles induced cytotoxicity, DNA damage and apoptosis in cultured human cells through lipid peroxidation, ROS generation and oxidative stress (Ahamed et al., 2008, Ahamed et al., 2010c, Akhtar et al., 2010a).

Apoptosis is controlled by a large number of genes acting as death switches. The tumor suppressor gene p53 is regarded as the guardian of the cell genome is able to activate cell cycle checkpoints, DNA repair and apoptosis to maintain stability of genome (Sherr, 2004). In the presence of cellular stress, p53 triggers cell cycle arrest to provide time for the damage to be repaired or self-mediated apoptosis (Farnebo et al., 2010). Survivin, one member of inhibitor of apoptosis family, has recently been reported to play an important role in both cell proliferation and cell death. Down-regulation of survivin expression may cause a cell-cycle defect that leads to apoptotic cell death (Ryan et al., 2009). The bcl-2 and bax are two discrete members of a gene family also involved in the regulation of apoptosis. The bcl-2 blocks cell death following various stimuli, demonstrating a death-sparing effect; however, over expression of bax has a pro-apoptotic effect and bax also counters the anti-apoptotic activity of bcl-2 (Chougule et al., 2010). The ratio of bax to bcl-2 expression represents 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 increased apoptotic cell death (Bai and Meng, 2005, Gao and Wang, 2009). It has also been well documented that signaling pathway leading to apoptosis involved the sequential activation of cysteine proteases known as caspases (Takadera and Ohyashiki, 2007, Tang et al., 2010). The present study was designed to investigate the nickel ferrite nanoparticles induced apoptosis through ROS generation and oxidative stress via p53, survivin, bax/bcl-2 and caspase pathways in human lung epithelial A549 cells. This preliminary study provides insight into nickel ferrite nanoparticles induced apoptotic cell death in A549 cells and possible cellular and molecular mechanisms involved. Human lung epithelial A549 cells, derived from human lung carcinoma, have widely been used to study the cytotoxicity, ROS generation, oxidative stress and molecular mechanisms of apoptosis (Sánchez-Pérez et al., 2009, Ye et al., 2009, Ahamed et al., 2010c, Akhtar et al., 2010a, Akhtar et al., 2010b, Barillet et al., 2010, Zhang et al., 2010).

Section snippets

Nickel ferrite nanoparticles and reagents

Nickel ferrite (NiFe2O4) nanopowder (Product No. 637149, particle size: <50 nm and purity: ≥98% trace metals basis), MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide]), reduced glutathione (GSH), DTNB (5,5-dithio-bis-(2-nitrobenzoic acid)), DCFH-DA (2,7-dichlorofluorescin diacetate) and l-ascorbic acid (AA) were purchased from Sigma–Aldrich, USA. Fetal bovine serum (FBS), penicillin–streptomycin, DMEM/F-12 medium and HBSS were obtained from Invitrogen Co., USA. All other

Characterization of nickel ferrite nanoparticles

Fig. 1 shows the XRD pattern of nickel ferrite nanopowder that clearly exhibits crystalline nature of this material. The crystallite size has been estimated from the XRD pattern using the Scherrer's equation (Patterson, 1939).D=0.9λBcosθwhere D is the grain size, λ the wavelength of X-ray (1.54056 Å), B the full-width at half-maxima of the diffraction peak (in radian)

The average crystallite size corresponding to the highest peak observed in XRD was found to be 26 ± 17 nm. The presence of sharp

Discussion

Due to wide spread application of magnetic nanoparticles it is imperative to evaluate their potential risks to the environment and human health. Results published in the scientific literature concerning toxic potential of iron-based nanoparticles are conflicting. These nanoparticles have been shown to generate toxicity (Zhu et al., 2010, Apopa et al., 2009, Lunov et al., 2010, Park et al., 2010), while others report that they do not only show good biocompatibility but also exert very low

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgment

Financial assistance from King Abdullah Institute for Nanotechnology (KAIN) is thankfully acknowledged.

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