Cobalt iron oxide nanoparticles induce cytotoxicity and regulate the apoptotic genes through ROS in human liver cells (HepG2)

Colloids and Surfaces B: Biointerfaces 148 (2016) 665–673

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Cobalt iron oxide nanoparticles induce cytotoxicity and regulate the apoptotic genes through ROS in human liver cells (HepG2) Maqusood Ahamed a,∗ , Mohd Javed Akhtar a , M.A. Majeed Khan a , Hisham A. Alhadlaq a,b , Aws Alshamsan a,c,d a

King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, Saudi Arabia Department of Physics and Astronomy, College of Science, King Saud University, Riyadh, Saudi Arabia c Nanomedicine Research Unit, Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia d Life Science and Environment Research Institute, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia b

a r t i c l e

i n f o

Article history: Received 25 June 2016 Received in revised form 8 September 2016 Accepted 29 September 2016 Available online 29 September 2016 Keywords: Cobalt iron oxide (CoFe2 O4 ) Cytotoxicity Apoptosis ROS Biomedical applications

a b s t r a c t Cobalt iron oxide (CoFe2 O4 ) nanoparticles (CIO NPs) have been one of the most widely explored magnetic NPs because of their excellent chemical stability, mechanical hardness and heat generating potential. However, there is limited information concerning the interaction of CIO NPs with biological systems. In this study, we investigated the reactive oxygen species (ROS) mediated cytotoxicity and apoptotic response of CIO NPs in human liver cells (HepG2). Diameter of crystalline CIO NPs was found to be 23 nm with a band gap of 1.97 eV. CIO NPs induced cell viability reduction and membrane damage, and degree of induction was dose- and time-dependent. CIO NPs were also found to induce oxidative stress revealed by induction of ROS, depletion of glutathione and lower activity of superoxide dismutase enzyme. Realtime PCR data has shown that mRNA level of tumor suppressor gene p53 and apoptotic genes (bax, CASP3 and CASP9) were higher, while the expression level of anti-apoptotic gene bcl-2 was lower in cells following exposure to CIO NPs. Activity of caspase-3 and caspase-9 enzymes was also higher in CIO NPs exposed cells. Furthermore, co-exposure of N-acetyl-cysteine (ROS scavenger) efficiently abrogated the modulation of apoptotic genes along with the prevention of cytotoxicity caused by CIO NPs. Overall, we observed that CIO NPs induced cytotoxicity and apoptosis in HepG2 cells through ROS via p53 pathway. This study suggests that toxicity mechanisms of CIO NPs should be further investigated in animal models. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Due to unique properties magnetic nanoparticles (NPs) are being utilized in numerous applications including drug delivery, tissue imaging, cancer hyperthermia and magnetic resonance imaging [1,2]. Cobalt iron oxide (CIO, chemical formula CoFe2 O4 ) NPs have been one of the most widely explored magnetic nanomaterials because of their excellent chemical stability, mechanical hardness and heat generating potential besides being cost effective [3,4]. The demonstrated efficiency in cancer therapy has increased considerably the use of hyperthermia technique and CIO NPs have received great attention due to their slower magnetic moment relaxation as compared to magnetite (Fe3 O4 ) NPs [5]. Therefore,

∗ Corresponding author at: King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia. E-mail addresses: [email protected], [email protected] (M. Ahamed). http://dx.doi.org/10.1016/j.colsurfb.2016.09.047 0927-7765/© 2016 Elsevier B.V. All rights reserved.

it is crucial to explore the biological interaction CIO NPs at cellular and molecular level prior to their biomedical application. There are few studies reporting toxic potential of CIO NPs. Colognato et al. [6] observed that CIO NPs induce micronuclei formation in human peripheral lymphocytes. A different dose-response curve of cell viability for different types of cells was observed after CIO NPs exposure [7]. Kapilevich et al. [8] observed that CIO NPs cause respiratory problem in guinea pig. CIO NPs were also found to impair the lipid metabolism and embryogenesis [5,9]. However, Pasukoniene et al. [10] reported that CIO NPs were not toxic to human pancreatic and ovarian cancer cells and may serve as a powerful tool for tumor detection and magnetic field-assisted targeted chemotherapy or hyperthermia. In addition, CIO NPs were also found to exert toxicity to green algae and Zebrafish [11,12]. Possible mechanisms of NPs toxicity are not fully elucidated yet. Studies suggested that NPs might induce toxicity through disturbing the redox homeostasis. Imbalance of redox homeostasis is either due to excessive generation of reactive oxygen species (ROS) or by inactivation of antioxidant defense mechanism [13–15]. Stud-

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ies have shown that magnetic NPs induce toxicity to human cells through oxidative damage of cell biomolecules [1,16,17]. Our earlier reports demonstrated that nickel (Ni), copper (Cu) or zinc (Zn) iron oxide (XFe2 O4 where X = Ni, Cu or Zn) NPs induced cytotoxicity and apoptosis in human cells through ROS generation and oxidative stress [18–20]. However, studies that explore the underlying mechanisms of CIO NPs toxicity are largely lacking. Several genes are known to control the apoptotic pathway and serving as death switches. The p53 gene is known as the guard of the genome and is capable to activate cell cycle checkpoints, DNA repair or apoptosis to maintain the stability of cell genome [21,22]. Hence, p53 is used as a biomarker of genotoxicity [23]. The bcl-2 and bax are two distinct members of a gene family and play critical role in apoptosis. The bcl-2 gene has an anti-apoptotic effect, whereas the bax is known for pro-apoptotic effect [24]. A ratio of bax/bcl-2 expression plays an important role in mitochondrial outer-membrane permeabilization, release of cytochrome C in the cytosol and ultimately apoptotic cell death [25,26]. Signaling pathway of apoptosis is also involved the sequential activation of cysteine proteases termed as caspases [27,28]. This study was designed to explore the underlying mechanism of cytotoxicity and apoptosis induced by CIO NPs in human liver cancer cells (HepG2) cells through ROS via p53 pathway. To get this objective, we measured cell viability, membrane damage, ROS, glutathione (GSH), superoxide dismutase (SOD) enzyme, mitochondrial membrane potential (MMP) and regulation of apoptotic genes in HepG2 cells against CIO NPs exposure. We have chosen HepG2 cell line because it is a typical hepatic model system used to evaluate the hepatotoxicity of any particles or toxins [23,29,30]. 2. Materials and methods 2.1. Chemicals and reagents Dulbecco’s modified eagle’s medium (DMEM), penicillinstreptomycin and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Rhodamine-123 (Rh123), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), Nacetyl-cysteine (NAC), 2,7-dichlorofluorescin diacetate (DCFH-DA), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) and reduced glutathione (GSH) were bought from Sigma-Aldrich (St. Louis, MO). Caspase-3 & caspase-9 enzymes, and LDH assay kits were obtained from Bio-Vision Inc. (Milpitas, CA). Superoxide dismutase (SOD) enzyme assay kit was taken from Cayman Chemical Company (Ann Arbor, MI).

drop of NPs suspension was poured onto a carbon-coated Cu-grid, air-dried and FETEM measurement was performed. Elemental composition (purity) of CIO NPs was measured by energy dispersive X-ray spectroscopy (EDS). Hydrodynamic size and zeta potential of CIO NPs in water and culture medium were measured by dynamic light scattering (DLS) (Nano-ZetaSizer-HT, Malvern, UK) as suggested by Murdock et al. [31]. In brief, dry powder of CIO NPs was suspended in de-ionized water and complete cell culture media (DMEM+10% FBS). Suspension of NPs was further incubated at 37 ◦ C for 24 h. Then, suspension was sonicated at room temperature for 30 min at 40 W and performed the DLS experiment. 2.4. Cell culture Human liver carcinoma (HepG2) was bought from American Type Culture Collection (ATCC) (Manassas, VA). Cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin-streptomycin at 5% CO2 and 37 ◦ C. At 85% confluence, cells were harvested using 0.25% trypsin and were sub-cultured for toxicity studies. Cells were allowed to attach on the surface of culture flask for 24 h before exposure to NPs. 2.5. Exposure of nanoparticles to cells Dry powder of CIO NPs was suspended in DMEM medium at a concentration of 1 mg/ml and diluted to required concentrations (0–400 ␮g/ml). The different concentrations of NPs were then sonicated at room temperature for 15 min at 40 W to avoid agglomeration of NPs prior exposure to cells. In some experiments, cells were pre-exposed for 1 h with 10 mM of NAC before 24 h coexposure with or without CIO NPs. In some experiments, we have used ZnO NPs as a positive control. Cells not exposed to CIO NPs served as controls in each experiment. 2.6. MTT assay

Nanopowder of cobalt ferrite or cobalt iron oxide (CIO, chemical formula: CoFe2 O4 ) (Product No. 773352, 30 nm particle size, 98.5% trace metals basis) was obtained from Sigma-Aldrich (St. Louis, MO). We have done the physicochemical characterization of CIO NPs in our institute prior to biochemical studies.

Cell viability was measured following the procedures of Mossman [32] with some specific changes [18]. This assay measures the mitochondrial function by determining the ability of living cells to reduce MTT into blue formazon product. In brief, 1 × 104 cells/well were seeded in 96-well plates and exposed to different concentrations of CIO NPs (5–400 ␮g/ml) for 24, 48 & 72 h. After the treatment time completed, culture medium was discarded from each well to avoid interference of NPs and replaced with new medium containing MTT solution in an amount equal to 10% of culture volume and incubated for 3 h at 37 ◦ C until a purple-colored formazan product was formed. The resulting formazan product was dissolved in acidified isopropanol. Then, 96-well plate was centrifuged at 2300g for 5 min to settle down the remaining NPs. Further, 100 ␮l supernatant was transferred to new 96-well plate, and the absorbance was taken at 570 nm using a microplate reader (Synergy-HT, BioTek, USA).

2.3. Cobalt iron oxide nanoparticle characterization

2.7. LDH assay

The phase, purity and crystal structure of CIO NPs were determined by X-ray diffraction (XRD) technique. XRD spectra of NPs was acquired at room temperature with the help of a PANalytical X’Pert X-ray diffractometer equipped with an Ni filter using Cu K␣ (␭=1.54056 Å) radiations as an X-ray source. Morphology of CIO NPs were examined by field emission transmission electron microscopy (FETEM, JEM-2100F, JEOL Inc., Japan) at an accelerating voltage of 200 kV. In brief, a diluted suspension of CIO NPs was prepared in de-ionized water. Then suspension was sonicated at room temperature at 40W for 30 min to avoid agglomeration of NPs. Then, a

LDH assay was carried out using a BioVision LDH-cytotoxicity colorimetric assay kit. Briefly, 1 × 104 cells/well were seeded in 96well plates and exposed to different concentrations (5–400 ␮g/ml) of CIO NPs for 24, 48 & 72 h. After the treatment time completed, 96-well plate was centrifuged at 2300g for 10 min to settle the NPs. Then, 100 ␮l of the supernatant was transferred to a new 96-well plate that already contained 100 ␮l of the reaction mixture from the BioVision kit and incubated for 30 min at room temperature. At the end of incubation time, absorbance of the solution was measured at 340 nm using a microplate reader (Synergy-HT, BioTek, USA). LDH

2.2. Cobalt iron oxide nanoparticles

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Fig. 1. (A) Optical characterization and (B) X-ray diffraction (XRD) spectra of CIO NPs.

Fig. 2. Transmission electron microscopy (TEM) characterization of CIO NPs. (A & B) low resolution TEM images, (C) high resolution TEM image and (D) elemental composition of CIO NPs analyzed by energy dispersive spectroscopy (EDS).

level in culture medium versus those in the cells were quantified and compared with the controls according to the manufacturer’s instructions

2.8. Assay of reactive oxygen species ROS level in HepG2 cells after exposure to CIO NPs was measured using 2,7-dichlorofluorescin diacetate (DCFH-DA) probe as described by Wang and Joseph [33] with some specific modifications [34]. ROS level was determined by two methods; quantitative assay by a microplate reader and cell imaging by fluorescent microscopy. For quantitative assay, 1 × 104 cells/well were seeded in 96-well black-bottomed culture plate and allowed to attach on the surface for 24 h in a CO2 incubator at 37 ◦ C. Cells were further exposed to different concentrations of NPs for 24 h. After the completion of exposure time, cells were washed twice with HBSS before being incubated in 1 ml of working solution of DCFH-DA for 30 min

at 37 ◦ C. Then, cells were lysed in alkaline solution and centrifuged at 2300g for 15 min at room temperature. Further, 200 ␮l supernatant was transferred to a new 96-well plate, and fluorescence was measured at 485 nm excitation and 520 nm emission using a microplate reader (Synergy-HT, BioTek, USA). The values were presented as a percent of fluorescence intensity relative to controls. A parallel set of cells (5 × 104 cells/well in a 24-well transparent plate) were assayed for intracellular fluorescence using a fluorescence microscope (OLYMPUS CKX 41), with images captured at the magnification of 20×.

2.9. Assay of mitochondrial membrane potential MMP level of cells after exposure to CIO NPs was measured as described by Zhang et al. [35] with some specific modifications [34]. MMP level was determined by two methods; cell imaging by fluorescent microscopy and quantitative assay by microplate reader. In

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2.11. Assay of glutathione Intracellular GSH level was quantified by the method of Ellman [36]. Briefly, a mixture of 0.1 ml of crude cell extract and 0.9 ml of 5% trichloroacetic acid (TCA) was centrifuged at 2300g for 15 min at room temperature. After that 0.5 ml of the supernatant was added into 1.5 ml of 0.01% DTNB and the reaction was monitored at 412 nm. The content of GSH was presented in term of nanomole/mg protein.

2.12. Assay of superoxide dismutase enzyme SOD enzyme activity was evaluated using a commercially available kit (Cayman Chemical Company). This assay utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. Cell extract was used for SOD assay as per the manufacturer’s instruction.

2.13. Determination of apoptotic genes by real-time PCR

Fig. 3. Cytotoxic response of CIO NPs in HepG2 cells. Cells were treated with CIO NPs at the concentrations of 0, 5, 10, 25, 50, 100, 200 & 400 ␮g/ml for 24, 48 & 72 h. At the end of exposure time, cytotoxicity parameters were measured. (A) MTT assay and (B) LDH assay. Data represented are mean ± SD of three identical experiments made in three replicate. *Significant difference as compared to the control (p < 0.05).

brief, cells (5 × 104 cells/well) were exposed to NPs with or without NAC for 24 h. At the end of exposure time, cells were washed twice with PBS. Then, cells were treated with 10 ␮g/ml of Rh-123 fluorescent dye for 1 h at 37 ◦ C in dark. Cells were again washed with PBS. Then, fluorescence intensity of Rh-123 dye was measured using fluorescence microscope (OLYMPUS CKX 41) by capturing the images at 20× magnification. A parallel set of cells (1 × 104 cells/well) in 96-well plate were analyzed for quantification of Rh-123 using a microplate reader (Synergy-HT, BioTek, USA).

2.10. Cell extract preparation Crude cell extract was prepared as per the instructions of our previous publication [23]. Briefly, cells were cultured in 75-cm2 culture flask and treated with NPs with or without NAC for 24 h. At the end of exposure time, cells were harvested in ice-cold PBS by scraping and washed with PBS at 4 ◦ C. Then, cell pellets were lysed in cell lysis buffer [1 × 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1% Triton, 2.5 mM sodium pyrophosphate]. Buffer with lysed cells was centrifuged at 15000g for 10 min at 4 ◦ C to get supernatant without cell debris and NPs. Supernatant (cell extract) was stored in ice for further assays. This crude cell extract was used for glutathione (GSH), superoxide dismutase (SOD) enzyme, caspase-3 and caspase-9 enzyme assays.

Cells were cultured in 6-well plate and treated with CIO NPs at the concentration of 100 ␮g/ml with or without NAC (10 mM) for 24 h. At the end of exposure time, total RNA was isolated by Qiagen RNeasy mini Kit (Valencia, CA) as per the manufacturer’s instruction. The RNA content was estimated using Nanodrop 8000 spectrophotometer (Thermo-Scientific, Wilmington, DE), and the integrity of RNA was visualized on a 1% agarose gel using the gel documentation system (Universal Hood II, BioRad, Hercules, CA). The first strand of cDNA was synthesized from 1 ␮g of total RNA by the reverse transcriptase using M-MLV (Promega, Madison, WI) and oligo (dT) primers (Promega). Quantitative real-time PCR was performed by QuantiTect SYBR Green PCR kit (Qiagen) using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Two microliters of template cDNA was added to the final volume of 20 ␮l of reaction mixture. Real-time PCR cycle parameters included 10 min at 95 ◦ C followed by 40 cycles involving denaturation at 95 ◦ C for 15 s, annealing at 60 ◦ C for 20 s, and elongation at 72 ◦ C for 20 s. The sequences of the specific set of primers for p53, bax, bcl-2, CASP3, CASP9 and ␤-actin are reported in our earlier work [18]. Expression level of genes was normalized to the ␤-actin gene, which was used as control. 2.14. Assay of caspase-3 and −9 enzymes Activity of caspase-3 and −9 enzymes was evaluated by BioVision colorimetric assay kits. This assay was based on the principle that activated caspases in apoptotic cells cleave the synthetic substrates to release free chromophore p-nitroanilide (pNA), which is measured at 405 nm. The pNA was generated after specific action of caspase-3 and −9 on tertrapeptide substrates DEVD-pNA and LEHD-pNA, respectively [18,37]. In brief, reaction mixture consisted of 50 ␮l of cell extract protein, 50 ␮l of 2× reaction buffer (containing 10 mM dithiothreitol) and 5 ␮l of 4 mM DEVD-pNA (for caspase-3) or LEHD-pNA (for caspase-9) substrate in a total volume of 105 ␮l. The reaction mixture was incubated at 37 ◦ C for 1 h and absorbance of the product was measured using the microplate reader (Synergy-HT, BioTek, USA) at 405 nm as per the protocol of manufacturer.

2.15. Protein assay Protein level in cell extract was measured by the Bradford method [38] using bovine serum albumin as standard.

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Fig. 4. Oxidative stress response of CIO NPs in HepG2 cells. Cells were treated with CIO NPs at the concentrations of 0, 50, 100 & 200 ␮g/ml for 24 h. At the end of exposure time, ROS, SOD and GSH levels were measured. (A) Quantitative measurements of ROS. (B) Fluorescent microscopic images of ROS in treated and control cells. (C) SOD activity. (D) GSH level. Data represented are mean ± SD of three identical experiments made in three replicate. *Significant difference as compared to the control (p < 0.05).

Fig. 5. N-acetylcysteine (NAC, ROS scavenger) effectively prevented the oxidative stress and cytotoxicity of HepG2 cells caused by CIO NPs. Cells were treated with CIO NPs at the concentration of 100 ␮g/ml in the presence or absence of NAC (10 mM) for 24 h. After the exposure completed, oxidative stress and cytotoxicity parameters were determined. (A) ROS level, (B) SOD activity, (C) GSH level and (D) MTT cell viability. Data represented are mean ± SD of three identical experiments made in three replicate. *Significant difference as compared to the control (p < 0.05). # Significant inhibitory effect of NAC (p < 0.05).

2.16. Statistical analysis

3. Results

Statistical significance was measured by one-way analysis of variance followed by Dunnett’s multiple comparison tests. Significance was ascribed at p < 0.05.

3.1. Optical characterization In order to determine the optical band gap of CIO NPs, the absorbance spectra were recorded at room temperature (Fig. 1A).

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The optical band gap is related to absorption coefficient ␣, which can be obtained from the absorption spectrum according the following equation [39] ˛=

A d

(1)

Where ␣ is the absorption coefficient, A is the absorbance and d is the thickness of cuvette. The optical band gap of CIO NPs was determined from Tauc plots by analyzing the following relationship between absorption coefficient and photon energy for direct band transition [40] 2

(˛hv) = A(hv − Eg )

(2)

Where A is the constant, which does not depend on photon energy and Eg is the optical band gap energy. The (␣h)2 were plotted versus photon energy (Fig. 1A inset). The linear extrapolation to zero absorption leads for CIO NPs to the value of 1.97 eV. Optical band gap energy of CIO NPs obtained in this study was in agreement with other reports [41,42]. 3.2. XRD characterization Structural characterization and phase purity of CIO NPs were confirmed by powder XRD analysis. The XRD spectra of CIO NPs are given in Fig. 1B. The main diffraction peaks (2) appear at about 18.56◦ , 30.39◦ , 35.69◦ , 37.28◦ , 43.34◦ , 53.52◦ , 57.30◦ & 63.01◦ which are attributed to the (111), (220), (311), (222), (400), (422), (511) & (440) planes, respectively. The pattern matched those of cubic (Fd3m) CoFe2 O4 (JCPDS 22–1086). Peaks related to impurities were not detected for CIO NPs. The crystallite size (d) was measured using the Scherrer’s equation [39] d=

K ˇCos

(3)

where K = 0.9 is the shape factor, ␭ is the X-ray wavelength of Cu K␣ radiation (1.54 Å), ␪ is the Bragg diffraction angle and ␤ is the broadening of diffraction line measured at half of its maximum intensity (in radian). The crystallite size of CIO NPs was found to be 23 nm. 3.3. TEM characterization Shape, size and surface morphology of CIO NPs were determined by FETEM. Fig. 2A & B represent the low magnification images of TEM. These images suggested that most of the particles have almost spherical morphology with some amount of agglomeration. High resolution TEM image (Fig. 2C) shows clear lattice fringes and high crystallinity of CIO NPs, which are in agreement with XRD result. The average TEM size of CIO NPs was around 23.47 ± 3.15 nm that supporting the size calculated by Scherrer’s equation. Fig. 2D represents EDS spectrum of CIO NPs. The EDS spectrum indicated that Co, Fe & O were the main elemental composition in CIO NPs. Other elemental impurities were not detected. The presence of C and Cu signals was from the carbon-coated Cu-grid. 3.4. DLS characterization To get a realistic overview of CIO NPs behavior when they get interaction with the human cells, hydrodynamic size and zeta potential of NPs in de-ionized water and cell culture medium was measured by DLS (Table 1). DLS data revealed that CIO NPs aggregated to a hydrodynamic size of around 10–20 times of the primary size in aqueous suspension. Hydrodynamic size of CIO NPs in deionized water and complete cell culture medium (DMEM with 10% FBS) was 354 and 286 nm, respectively. Furthermore, zetapotential of CIO NPs in de-ionized water and culture medium was −11 mV and −16 mV, respectively.

3.5. Cobalt iron oxide nanoparticle-induced cytotoxicity Human liver (HepG2) cells were exposed to different concentrations (0–400 ␮g/ml) of CIO NPs for different time intervals (24–72 h) and cytotoxicity endpoints were examined by cell viability (MTT) and membrane damage (LDH) assays. Both MTT and LDH data indicated that CIO NPs induced dose- and time-dependent cytotoxicity in the dosage range of 50–400 ␮g/ml (p < 0.05). Below the concentration of 50 ␮g/ml, CIO NPs did not induce any toxicity to HepG2 cells. MTT data indicated that cell viability for 24 h exposure was reduced to 83, 69, 52 & 37%, whereas cell viability reduction for 72 h exposure was 73, 56, 41 & 24% for the dosages of 50, 100, 200 & 400 ␮g/ml, respectively (p < 0.05) (Fig. 3A). Fig. 3B represents that CIO NPs also induced dose- and time-dependent LDH leakage (an indicator of cell membrane damage) in HepG2 cells. LDH data were in agreement with MTT results. 3.6. Cobalt iron oxide nanoparticle-induced oxidative stress We explored the ability of CIO NPs to affect the redox status of cells by determining the ROS, SOD and GSH levels in HepG2 cells. Quantitative data indicated that CIO NPs induced intracellular ROS level in a dose-dependent manner (p < 0.05) (Fig. 4A). Microscopic images also revealed that CIO NPs treated cells expresses high intensity green fluoresce of DCF dye (an indicator of ROS production) as compared to the controls (Fig. 4B). SOD enzyme converted • superoxide (O2 − ) radicals into hydrogen peroxides (H2 O2 ). We observed that CIO NPs decrease the activity of SOD enzyme dosedependently (Fig. 4C). Over production of intracellular ROS is toxic to cells because of their ability to oxidize a range of biomolecules such as antioxidant GSH that plays crucial role in maintaining cellular redox homeostasis. Our results demonstrated that GSH level was decreases with increasing the concentrations of CIO NPs (Fig. 4D) 3.7. Cobalt iron oxide nanoparticle-induced cytotoxicity was mediated through ROS Oxidative stress has been proposed to be involved in NPmediated toxicity [13,40]. In this study, we further explored the role of ROS in cytotoxic response of CIO NPs. The HepG2 cells were treated with CIO NPs with or without N-acetyl-cysteine (NAC). We have also used ZnO NPs as a positive control. Results demonstrated that NAC efficiently averted the oxidant (ROS) generation and antioxidant (SOD & GSH) depletion caused by CIO or ZnO NPs (Fig. 5A–C) (p < 0.05). Moreover, we also found that co-exposure of NAC, effectively abolished the cytotoxicity induced CIO or ZnO NPs (Fig. 5D) (p < 0.05). Taken together, these observations indicated that ROS might play a crucial role in the cytotoxicity of CIO NPs in HepG2 cells. 3.8. Cobalt iron oxide nanoparticle-induced MMP loss was mediated through ROS Lowering of mitochondrial membrane potential (MMP) is an indicator of apoptosis [20,30]. We studied the effect of CIO NPs on MMP of HepG2 cells. Cells were exposed to CIO NPs (50–100 ␮g/ml) for 24 h and assayed for Rh123 dye uptake. Quantitative data showed that CIO NPs reduced the MMP level in a dose-dependent manner (Fig. 6A) (p < 0.05). Microscopic images also demonstrated that the brightness of the fluorescent intensity was reduced in cells treated with CIO NPs that suggest the loss of MMP (Fig. 6B). We also studied the role of ROS in CIO NP-induced MMP loss. Results showed that co-exposure of NAC effectively prevented the MMP loss caused by CIO NPs. MMP level was increased up to value of control for CIO NPs in the presence of NAC (Fig. 6) (p < 0.05).

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Table 1 Dynamic light scattering (DLS) characterization of cobalt iron oxide (CIO) NPs (mean ± SD, n = 3). Dispersant

Hydrodynamic size (nm)

PDI

Zeta potential (mV)

De-ionized water Complete cell culture medium (DMEM+10% FBS)

354 ± 39 286 ± 27

0.136 0.112

−11 ± 2.1 −16 ± 1.8

DMEM: Dulbecco’s modified eagle’s medium, FBS: fetal bovine serum.

Fig. 6. ROS mediated MMP loss in HepG2 cells after CIO NPs exposure. Cells were treated with CIO NPs at the concentration of 100 ␮g/ml in the presence or absence of NAC (10 mM) for 24 h. MMP level was measured using Rh123 fluorescent probe. (A) Quantitative measurements of MMP and (B) fluorescent microscopic images of cells with Rh123 probe. Data represented are mean ± SD of three identical experiments made in three replicate. *Significant difference as compared to the control (p < 0.05). # Significant inhibitory effect of NAC (p < 0.05).

3.9. ROS mediated apoptotic genes regulation due to cobalt iron oxide nanoparticles exposure The mRNA level of some apoptotic genes (p53, bax, bcl-2, CASP3 & CASP9) were analyzed in HepG2 cells treated with CIO NPs (100 ␮g/ml for 24 h) with or without NAC. We observed that CIO NPs significantly altered the regulation these apoptotic genes. The p53 gene and pro-apoptotic gene bax were up-regulated while the anti-apoptotic gene bcl-2 was down-regulated in NPs treated cells (Fig. 7A). The mRNA level of caspase genes (CASP3 & CASP9) was also higher in CIO NPs treated cells (Fig. 7A). We further noticed that CIO NPs induced alterations in apoptotic genes were efficiently abrogated by NAC, a potent ROS scavenger. 3.10. ROS mediated caspase-3 and −9 enzymes activity due to cobalt iron oxide nanoparticles exposure To validate mRNA data, we further investigated the activity of two apoptotic enzymes (Caspase-3 and −9) in HepG2 cells exposed to CIO NPs. Similar to mRNA data, activity of caspase-3 and −9 enzymes was induced by CIO NPs. Higher activity of these two apoptotic enzymes due to CIO NPs was also effectively prevented by NAC (Fig. 7B). These results suggest that CIO NPs altered the regulation of apoptotic genes via ROS generation. 4. Discussion Toxicity of magnetic NPs is very important considerations for several biomedical applications. Literature has shown the contradictory results regarding the toxicity of magnetic NPs. Some studies have shown that magnetic NPs induce toxicity [43–45] while others observed that these particles are biocompatible and exerted low or no toxicity to human cells [10,46,47]. The suitability of CIO NPs in biomedical application such as drug delivery and hyperthermia

must be supported by thorough investigations of their potential toxicity. This prompted us to investigate the toxic potential of CIO NPs in human liver cells (HepG2). This cell line holds the functions of fully differentiated primary hepatocytes and broadly utilized as a model system for hepatotoxicity [48,49]. Characterization of physicochemical properties of NPs before their biomedical or toxicological studies is necessary. Phase-purity, crystallinity, shape, size, surface morphology, hydrodynamic size and aggregation are primary parameters that may regulate the response of NPs at cellular or molecular level [13,50]. We have applied XRD, TEM, EDS and DLS techniques to characterize the CIO NPs. Low magnification TEM images revealed that CIO NPs were spherical with smooth surfaces. High magnification TEM image and XRD spectra indicated the crystalline nature of CIO NPs. TEM and XRD also measured that average primary particle size of CIO NPs was 23 nm. Hydrodynamic size of CIO NPs was 10–15 times higher than those of the primary particle size (size obtained from TEM and XRD). The higher size of NPs in aqueous suspension was due to the tendency of particles to form agglomerates. Our results are in agreement with other investigations [51,52] and briefly described in our previously published paper [18]. Moreover, we noticed that hydrodynamic size of CIO NPs was slightly lower in complete culture medium (DMEM+10% FBS) as compared to deionized water. Similar drop in the hydrodynamic size of metal and metal oxide NPs in culture medium than those of water is also reported elsewhere [53,54]. Size reduction might be due to the presence of serum in culture medium. Serum rapidly binds to the NPs and forms a protein corona on the surface of NPs [55]. This protein corona helps the NPs to disperse by providing steric hindrance and electrostatic repulsion between the NPs [56,57]. The absorption of protein on the surface of NP not only affects the size and physical properties of NP but also affect the interaction of NPs with cellular systems. These results suggested that in toxicity studies hydrodynamic size of NPs should be taken into account along with primary size.

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Fig. 7. ROS mediated apoptosis in HepG2 cells after CIO NPs exposure. (A) The mRNA level of apoptotic genes in HepG2 cells treated with CIO NPs at the concentration of 100 ␮g/ml in the presence or absence of NAC (10 mM) for 24 h. Quantitative real-time PCR (RT-PCRq) was performed by QuantiTect SYBR Green PCR kit using ABI PRISM 7900HT Sequence Detection System. The ␤-actin was used as the internal control to normalize the data. CIO NP-induced alterations in mRNA levels are expressed in relative quantity compared with those for the respective controls. (B) Activity of caspase-3 and −9 enzymes. Data represented are mean ± SD of three identical experiments made in three replicate. *Statistically significant difference as compared to the controls (p < 0.05). # Significant inhibitory effect of NAC (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

We employed a battery of tests to explore the possible mechanisms of toxicity caused by CIO NPs in HepG2 cells. MTT results demonstrated that CIO NPs significantly decreased the number of viable cells in a dose- and time-dependent manner. This assay served as a sensitive and integrated measure of cell integrity and inhibition of cell proliferation [19,58]. LDH leakage from cells into culture medium is another evidence of cytotoxicity [59,60]. Earlier studies have also shown that LDH level increases in medium after exposure to NPs [18,61]. When cell membrane integrity is disrupted, LDH leaks into the culture media and its extracellular level increases indicating the cytotoxicity of NPs. In the present study, we found that CIO NPs induced LDH leakage in a dose- and time-dependent manner. We further investigated the oxidative stress potential of CIO NPs in HepG2 cells. Oxidative stress has been proposed as a common mechanism of NPs toxicity [13,62]. Studies have shown that oxidative stress has been involved in the pathogenesis of a number of diseases such as stroke, inflammation and cancer [63,64]. We have shown in our earlier work that magnetic NPs have potential to induce oxidative damage to different types of human cells

[20,65,66]. In this study, we observed that intracellular ROS level was increases with increasing the concentrations of CIO NPs. Higher production of ROS is potentially harmful to cells because of their ability to oxidize cellular biomolecules including antioxidant GSH, which plays a vital role in preserving redox homeostasis. Exposure of CIO NPs to HepG2 cells induced a significant and dose-dependent depletion of GSH. We further observed that CIO NPs decreases the activity of antioxidant SOD enzyme dose-dependently. SOD enzyme is a first line of the antioxidant defense system that • converted highly toxic superoxide (O2 − ) radicals into hydrogen peroxides (H2 O2 ). Cell has a number of defense mechanisms against oxidative damage including direct interaction with antioxidants. Indeed, co-treatment with the antioxidant NAC (ROS scavenger) mitigated the ROS generation, GSH & SOD depletion as well as the cytotoxicity caused by CIO NPs in HepG2 cells, suggesting that oxidative stress might be accountable for the toxicity of such NPs. ROS may induce a number of physiological events such as inflammation, DNA damage and apoptosis [67,68]. A gene expression study indicated that CIO NPs were genotoxic to liver tissue of mice [69]. In this study, we found that mRNA expression of tumor suppressor gene p53 and pro-apoptotic gene bax were up-regulated, while the expression of anti-apoptotic gene bcl-2 was down-regulated in HepG2 cells exposed to CIO NPs. We also observed that CIO NPs induced MMP loss in HepG2 cells. Studies have shown that bax gene is up-regulated by p53 [70]. Since an increase in bax level was observed, the role of p53 in the upregulation of bax upon CIO NPs exposure can be postulated in this study. The insertion of bax into the mitochondrial membrane possibly leads to p53-mediated apoptosis [70]. We further observed that mRNA expression and activity of apoptotic enzymes caspase-3 and caspase-9 were higher in CIO NPs treated cells. Caspases are activated during apoptosis in many cells and are known to play a vital role in both initiation and execution of apoptosis. It was reported that caspase-3 and caspase-9 enzymes are is essential for apoptotic cell death [71]. At the end of this study we explored involvement of ROS in the regulation of apoptotic genes induced by CIO NPs using an antioxidant NAC. Interestingly, co-exposure of NAC significantly ameliorated the alterations of apoptotic genes caused by CIO NPs. We provide the evidence that CIO NPs induced apoptosis mediated through ROS via p53, bax/bcl-2 and caspase pathways in HepG2 cells. Results of this study are supported by our previously published work where different types of NPs altered the expression of several genes involved in apoptosis [20,65,66].

5. Conclusion We investigated the role of ROS in the toxic response of CIO NPs in HepG2 cells. Particle size of CIO NPs was around 23 nm with a band gap of 1.97 eV. Cytotoxicity studies have shown that CIO NPs induced cell viability (MTT) reduction and membrane damage (LDH leakage) in a dose- and time-dependent manner. CIO NPs was also found to induce oxidative stress indicated by ROS generation, GSH depletion and the lower activity of SOD enzyme. The mRNA level of tumor suppressor gene p53 and apoptotic gene bax were up-regulated whereas anti-apoptotic gene bcl-2 was down-regulated in HepG2 cells treated with CIO NPs. Activity of caspaspe-3 and caspas-9 enzymes was also higher in CIO NPs exposed cells. Furthermore, co-exposure of N-acetyl-cysteine (ROS scavenger) effectively attenuated the alteration of apoptotic genes and enzymes along with the prevention of cytotoxicity caused by CIO NPs. Taken together, our data suggested that CIO NPs induced apoptosis in HepG2 cells through oxidative stress via p53 pathway. This study warrants future research to explore if in vivo exposure consequences may exist for this nano-crystalline material.

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