The role of reactive oxygen species in the genotoxicity of surface-modified magnetite nanoparticles

Toxicology Letters 226 (2014) 303–313

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

The role of reactive oxygen species in the genotoxicity of surface-modified magnetite nanoparticles Monika Mesároˇsová a , Katarína Kozics a , Andrea Bábelová a , Eva Regendová a , Michal Pastorek a , Dominika Vnuková b , Barbora Buliaková a , Filip Rázga b , Alena Gábelová a,∗ a b

Cancer Research Institute, SAS, Vlárska 7, 833 91 Bratislava, Slovakia Polymer Institute, SAS, Dúbravská cesta 9, 845 41 Bratislava, Slovakia

h i g h l i g h t s • • • •

Magnetite nanoparticles (MNPs) induced variable levels of iROS in human lung cell lines. No oxidative damage to DNA was detected in MNP-treated human lung cell lines. No substantial changes in the TAC, iGSH or GPx activity were found in either of the cell lines. Oxidative stress generation plays, at most, only a marginal role in MNP genotoxicity.

a r t i c l e

i n f o

Article history: Received 23 July 2013 Received in revised form 26 February 2014 Accepted 27 February 2014 Available online 12 March 2014 Keywords: Magnetite nanoparticles Human lung cells Reactive oxygen species Oxidative DNA damage Antioxidant enzymes

a b s t r a c t The generation of reactive oxygen species (ROS) has been proposed as the underlying mechanism involved in the genotoxicity of iron oxide nanoparticles. The data published to date are, however, inconsistent, and the mechanism underlying ROS formation has not been completely elucidated. Here, we investigated the capacity of several surface-modified magnetite nanoparticles (MNPs) to generate ROS in A549 human lung adenocarcinoma epithelial cells and HEL 12469 human embryonic lung fibroblasts. All MNPs, regardless of the coating, induced significant levels of DNA breakage in A549 cells but not in HEL 12469 cells. Under the same treatment conditions, variable low levels of intracellular ROS were detected in both A549 and HEL 12469 cells, but compared with control treatment, none of the coated MNPs produced any significant increase in oxidative damage to DNA in either of these cell lines. Indeed, no significant changes in the total antioxidant capacity and intracellular glutathione levels were observed in MNPs-treated human lung cell lines regardless of surface coating. In line with these results, none of the surface-modified MNPs increased significantly the GPx activity in A549 cells and the SOD activity in HEL 12469 cells. The GPx activity was significantly increased only in SO-Fe3 O4 -treated HEL 12469 cells. The SOD activity was significantly increased in SO-PEG-PLGA-Fe3 O4 -treated A549 cells but significantly decreased in SO-Fe3 O4 -treated A549 cells. Our data indicate that oxidative stress plays, at most, only a marginal role in the genotoxicity of surface-modified MNPs considered in this study in human lung cells. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Magnetic iron oxide nanoparticles, including ␥-Fe2 O3 (maghemite), ␣-Fe2 O3 (hematite) and Fe3 O4 (magnetite), are increasingly being investigated for use in a variety of biomed-

∗ Corresponding author at: Department of Genetics, Laboratory of Mutagenesis and Carcinogenesis, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovakia. Tel.: +421 2 59327512; fax: +421 2 59327250. E-mail address: [email protected] (A. Gábelová). http://dx.doi.org/10.1016/j.toxlet.2014.02.025 0378-4274/© 2014 Elsevier Ireland Ltd. All rights reserved.

ical applications, both diagnostic and therapeutic (Corchero and Villaverde, 2009; Wahajuddin and Arora, 2012). Because iron metabolism is well controlled and excess iron is efficiently removed from the body (Jomova and Valko, 2011), iron oxide nanoparticles are considered to be biocompatible and nontoxic. Magnetite is one of the most frequently used forms of iron oxide in nanoparticles. Magnetite nanoparticles (MNPs) have great potential as magnetic resonance imaging (MRI) contrast agents (Singh et al., 2009; Triantafyllou et al., 2013), heating mediators in hyperthermia-based cancer therapy (Jordan et al., 2001) and

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nanocarriers in targeted drug/gene delivery (Duguet et al., 2006). Moreover, the superparamagnetic properties of MNPs allow the delivery and trapping of drug-loaded MNPs in the target site via an external magnetic field (Marszall, 2011). The coating of MNPs with synthetic and natural chemical moieties minimizes hydrophobic interactions, thus enhancing their desirable properties, such as colloid stability and internalization efficiency, and enabling their functionalization with ligands, drugs, genes or antibodies that enhance target-specific interactions with tumor cells, thus increasing their therapeutic benefit (Babic et al., 2008; Gupta and Gupta, 2005). MNPs are also being utilized for a plethora of biotechnological applications, including enzyme immobilization, targeted cell/macromolecule separation and purification or magnetofection (Corchero and Villaverde, 2009). Although the benefit of MNPs is obvious, the impact of MNPs on basic cellular processes such as the cell cycle, cell signaling, apoptosis, oxidative stress and inflammation has not been sufficiently explored (Singh et al., 2009, 2010). MNPs have already been approved as contrast agents for MRI analysis (Duncan and Gaspar, 2011). In addition, they are frequently being utilized in cellular therapy such for cell labeling and sorting (Andreas et al., 2012; Elias and Tsourkas, 2009; Marek et al., 2008). Therefore, a thorough investigation of the impact of MNPs on diploid cells and gaining an understanding of nanoparticle–cell interactions are necessary. The number of studies of MNP bio-safety in non-tumor (diploid cells) and tissues is, however, limited. Several studies have shown that MNPs can cause serious damage to healthy cells. Strong cytotoxicity, the disruption of cytoskeletal structures, apoptosis, and oxidative stress have been detected in both human and mammalian diploid cells treated with MNPs (Buyukhatipoglu and Clyne, 2011; Guichard et al., 2012; Hanini et al., 2011; Mesarosova et al., 2012; Wu et al., 2010). The generation of reactive oxygen species (ROS) has been proposed as the underlying mechanism involved in the genotoxicity of metal oxide nanoparticles, particularly iron oxide nanoparticles. Several mechanisms have been proposed for ROS generation. Iron ions released into the cytosol due to lysosomal enzymatic degradation can participate in the Fenton reaction, producing the hydroxyl radicals (Valko et al., 2006), or the particle surface per se may act as a catalyst (Klein et al., 2012). Alternatively, ROS formation may occur due to MNP-mediated damage to the mitochondrial membrane (Sioutas et al., 2005), or MNPs may interact with NADPH oxidase in the plasma membrane during entry into the cell (Bedard and Krause, 2007). Although free iron ions are thought to be stored in the iron-storage proteins ferritin and hemosiderin and progressively re-used (Elias and Tsourkas, 2009), the iron-binding capacity of cellular ferritin is limited (Ganz, 2003). Efforts to achieve the maximum therapeutic effect can lead to iron overload at the target site and the disruption of iron homeostasis, promoting ROS generation. However, cells possess an effective inherent antioxidant defense system composed of non-enzymatic and enzymatic antioxidants; therefore, the contribution of MNP-mediated oxidative stress to the toxicity of MNPs is poorly understood (Soenen et al., 2011). The particle size and surface chemistry have been shown to have great importance in the ROS-mediated activity of MNPs (Hong et al., 2011; Hoskins et al., 2012). Bare iron oxide nanoparticles might be significantly more toxic than coated nanoparticles because the surface iron ions are more efficient inducers of ROS production (Voinov et al., 2011) while the coating may function as a barrier and attenuate the potential toxic effects (Auffan et al., 2006). Data publishing until now are, however, controversial. The bare MNPs were shown to induce ROS generation in human diploid and tumor cells (Choi et al., 2009; Hoskins et al., 2012; Karlsson et al., 2009; Zhu et al., 2008), rat lung epithelial cells (Ramesh et al., 2012), and Chinese hamster ovary cells (Kawanishi et al., 2013) but not in Syrian hamster embryo cells (Guichard et al.,

2012), Cos-1 cells (Magdolenova et al., 2012) and human lung adenocarcinoma epithelial A549 cells (Kain et al., 2012; Konczol et al., 2011). On the other hand, variable levels of ROS formation was detected in human and mammalian cell lines treated with surface-modified MNPs. The MNPs-mediated ROS generation was dependent on surface coating (Guichard et al., 2012; Hong et al., 2011; Konczol et al., 2011; Liu et al., 2011; Magdolenova et al., 2012; Sharma et al., 2014) and cell type (Guadagnini et al., 2013; Liu et al., 2011). The citrate-coated MNPs did not induce any ROS formation in L-929 cells in contrast to tetraethyl orthosilicate- and (3aminopropyl)trimethoxysilane-modified MNPs (Hong et al., 2011). A dose-dependent increase in ROS formation was determined in SH-SY5Y cells treated with polyethyleneimine coated MNPs (MNPPEI), however, coating the MNP-PEI particles with polyethylene glycol (MNP-PEI PEG) resulted in ROS production consistent with the control cells (Hoskins et al., 2012). The sodium oleate coated MNPs induced ROS generation in Cos-1 cells (Magdolenova et al., 2012) and in 16HBE cells but not in A549 cells (Guadagnini et al., 2013). Accordingly, increased ROS production was determined in several human and mammalian cell lines but not in HeLa cells after treatment with DMSA-coated MNPs (Liu et al., 2011). The goals of this study were as follows: (i) to investigate the role of ROS generation in the genotoxicity of coated MNPs, (ii) to compare the sensitivities of human lung cancer and diploid cells to MNP treatment, and (iii) to assess the contribution of the surface chemistry of MNPs to oxidative stress and ROS generation. Magnetic nanoparticles with a 7.6 nm magnetite core and different hydrophilic shells were characterized in depth using different physicochemical assays (Mesarosova et al., 2012). The MNPs used in this study were coated with the following: (i) sodium oleate (SO prevents aggregation and makes MNPs stable; SO-Fe3 O4 ,), (ii) SO + polyethylene glycol (PEG reduces interactions with plasma proteins and thus minimizes MNP internalization and clearance by macrophages; SO-PEG-Fe3 O4 ), and (iii) SO + PEG + poly[lactide-coglycolic acid] (PLGA prevents degradation and aids in the regulation of drug release from nanoparticles; SO-PEG-PLGA-Fe3 O4 ). DNA breakage, oxidative damage to the DNA, the intracellular ROS levels and the activities of antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)) were assessed in both A549 and HEL 12469 cells under control and MNP-exposed conditions. 2. Materials and methods 2.1. Chemicals Poly(lactide-co-glycolic acid) [PLGA, d,l-lactide to glycolide ratio 85:15, Mw = 50–75 kDa], Pluronic F68, poly(ethylene glycol) [PEG, 1 kDa, Mw = 1000], 2 ,7 -dichlorodihydrofluorescein diacetate (H2 DCFH-DA, CAS 4091-99-0), ethidium bromide (EtBr, CAS 1239-45-8), low-melting-point (LMP) agarose, Triton X-100, HEPES, propidium iodide (PI, CAS 25535-16-4), and hydrogen peroxide (H2 O2 ) were purchased from Sigma–Aldrich (Lambda Life, Slovakia). Sodium oleate was purchased from Riedel-de Haën (Hannover, Germany), formamidopyrimidine-DNA glycosylase/AP nuclease (FPG) was purchased from Biolabs (BioTech, Slovakia), and methanol and glycine were purchased from SERVA (BioTech, Slovakia). (R)-1-[(10-Chloro-4-oxo-3-phenyl-4H-benzo(a)quinolizin-1yl)carbonyl]-2-pyrrolidine-methanol (Ro 19-8022; RO) was provided by Hoffmann – LaRoche AG (Basel, Switzerland), and the RANSOD kit was purchased from Randox Laboratories (Crumlin, UK). Culture media, fetal bovine serum (FBS), antibiotics and other chemicals used for cell cultivation were purchased from GIBCO (KRD Ltd., Slovakia). All other chemicals and solvents were of analytical grade. 2.2. Magnetite nanoparticles The spherical magnetic iron oxide (Fe3 O4 ) nanoparticles with a 7.6 nm magnetite core and different hydrophilic shells were kindly provided by Dr. M. Timko, PhD, Institute of Experimental Physics, SAS, Koˇsice, Slovakia. Three types of magnetite nanoparticles (MNPs) were used in these experiments: (i) MNPs coated with sodium oleate (SO-Fe3 O4 ), (ii) MNPs coated with SO + polyethylene glycol (SOPEG-Fe3 O4 ), and (iii) MNPs coated with SO + PEG + poly[lactide-co-glycolic acid], PLGA (SO-PEG-PLGA-Fe3 O4 ). The physico-chemical characteristics of the surface-

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305

Table 1 Particle characteristics in terms of particle shape and surface, particle size, z-potential, magnetism, surface area.

Magnetite inner core Particle shape Surface of particle Particle size (DH ) Is at 295 K Zeta potential () Fe3 O4 concentration The surface area per particle The number of particles per ml

SO-Fe3 O4

SO-PEG-Fe3 O4

SO-PEG-PLGA-Fe3 O4

7.6 nm Nearly spherical Smooth 44 nm 7.7 Am2 kg−1 −41.8 mV 134 mg ml−1 6.079 × 10−11 cm2 1.13 × 1017

7.6 nm Nearly spherical Smooth 76 nm 6.0 Am2 kg−1 −42.3 mV 100 mg ml−1 18.137 × 10−11 cm2 8.4 × 1016

7.6 nm Nearly spherical Smooth 155 nm 0.88 Am2 kg−1 −50 mV 13.7 mg ml−1 75.439 × 10−11 cm2 1.15 × 1016

modified MNPs considered in this study are shown in Table 1. The synthesis and surface modifications of these surface-modified magnetite nanoparticles have already been published (Mesarosova et al., 2012). 2.2.1. Particle size distribution and zeta potential in culture media Particle size distribution and zeta potential of surface modified MNPs in particular culture media at different concentrations were determined by dynamic laser light scattering (DLS) using Zetasizer Nano-ZS (Malvern Instruments, UK) equipped with a 4-mW helium/neon laser ( = 633 nm) and a thermoelectric temperature controller at 37 ◦ C. The characteristics of nanoparticles and individual culture media (Table S1) were obtained using the following methods: (i) refractive index using Differential refractometer BP-2000-V (Phoenix Precision, USA), (ii) absorption using UV-VIS spectrophotometer UV-1650 (Shimadzu, JPN) and (iii) dielectric constant using Solartron 1255A frequency analyzer (Farnborough, UK). The measurements were performed at ∼3 min intervals during 24 h (details are given in Figs. S1–S6); every data point was recorded as the average of at least 11 repetitions (note that

the number of repetitions was automatically defined by the Zetasizer Nano-ZS software). Zeta potential () was approximated from the electrophoretic mobility of MNPs according to the theory of Helmholtz-von Smoluchowski. Particle size value corresponds to hydrodynamic diameter and is referred as value at the maximum of obtained distribution (Figs. S1–S6; dmax ). Particle size distribution and -potential of surface-modified MNPs determined in Dulbecco’s modified Eagle medium (DMEM) and Minimum essential medium with Eagle’s salts (MEM) supplemented with 2% fetal bovine serum (FBS) at different concentrations are shown in Table 2 and Figs. S1–S6. In general, the hydrodynamic particle size of each type of MNPs increased substantially in both DMEM and MEM medium due to absorption of serum protein(s) on particle surface. The sodium oleate-coated MNPs were stable in both DMEM and MEM medium at all concentrations considered in this study. In contrast, the polymer-coated MNPs were less stable in MEM than in DMEM medium and were prone to precipitation after 24 h treatment. Multimodal distribution of polymer-coated MNPs indicated colloidal instability. Multimodal distribution of SO-PEG-Fe3 O4 particles was determined in

Table 2 Particle size distribution and zeta potential of particular surface-modified MNPs determined in 2% DMEM and 2% MEM at different concentrations. MNPs

Conc. (mM)

2% DMEM

2% MEM

Particle size distribution (%)

Zeta potential (mV) 2% DMEM

2% MEM

SO-Fe3 O4

0.3 0.35 0.4 0.5

244 ± 6 nm (100%) – 245 ± 6 nm (100%) 251 ± 6 nm (100%)

261 ± 5 nm (100%) 265 ± 8 nm (100%) 262 ± 7 nm (100%) –

−14.6 ± 0.9 – −14.8 ± 1.0 −15.1 ± 0.8

−14.1 ± 1.1 −14.6 ± 1.0 −15.6 ± 1.4 –

SO-PEG-Fe3 O4

0.1 0.15 0.2

243 ± 7 nm (100%) – 281 ± 4 nm (100%)

−15.8 ± 1.0 – −13.8 ± 0.9

−14.7 ± 1.4 −14.7 ± 1.1 −14.3 ± 1.0

0.3

345 ± 9 nm (100%)

288 ± 7 nm (100%) 312 ± 8 nm (100%) 62 ± 9 nm (0.8 ± 0.8%) 369 ± 24 nm (99.2 ± 0.8%) 4732 ± 297 nm (0.1 ± 0.1%) 67 ± 10 nm (1.1 ± 1.0%) 430 ± 33 nm (98.8 ± 1.2%) 4731 ± 495 nm (0.1 ± 0.1%)

−14.1 ± 0.9

−15.4 ± 0.9

0.05

216 ± 3 nm (100%)

−14.0 ± 1.0

−13.0 ± 0.8

0.1

196 ± 81 nm (9.1 ± 8.4%) 932 ± 189 nm (90.0 ± 9.8%) 5077 ± 254 nm (0.8 ± 0.5%) 115 ± 49 nm (4.2 ± 4.2%) 555 ± 161 nm (95.6 ± 4.4%) 5032 ± 270 nm (0.6 ± 0.6%) 168 ± 53 nm (4.9 ± 3.8%) 884 ± 311 nm (94.9 ± 5.1%) 5051 ± 1433 nm (0.1 ± 0.1%)

24 ± 21 nm (5.4% ± 5.0%) 215 ± 49 nm (92.5 ± 7.5%) 2702 ± 2432 nm (1.4 ± 1.1%) 28 ± 24 nm (1.3 ± 0.9%) 218 ± 72 nm (92.1 ± 7.9%) 4654 ± 1473 nm (5.8 ± 4.3%)

−12.8 ± 0.9

−12.3 ± 0.8

37 ± 27 nm (3.1 ± 3.1%) 287 ± 145 nm (95.8 ± 4.2%) 4048 ± 2251 nm (0.3 ± 0.2%)

−14.7 ± 1.2

−12.2 ± 1.0

–

−12.8 ± 0.9

–

SO-PEG-PLGA-Fe3 O4

0.15

0.2

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Table 3 The IC50 values determined in A549 and HEL 12469 cells treated with surfacemodified MNPs for 4 h and 24 h. The IC50 values were calculated from dose–response curves using CalcuSyn software. Nanoparticles

IC50 values (mM) A549 cells

SO-Fe3 O4 SO-PEG-Fe3 O4 SO-PEG-PLGA-Fe3 O4

HEL 12469 cells

4h

24 h

4h

24 h

5.726 1.183 1.611

0.759 0.344 0.184

0.820 0.593 0.282

0.414 0.311 0.129

MEM medium at concentrations higher than 0.2 mM but no agglomeration was found in DMEM medium. On the other hand, multimodal distribution of SO-PEGPLGA-Fe3 O4 particles was observed at all concentrations considered in MEM and DMEM medium except the lowest concentration (0.05 mM). The -potential of all surface-modified MNPs increased in both DMEM and MEM but no substantial differences in z-potential values were detected between DMEM and MEM; the values of -potential ranged from −12.8 to −15.1 mV. 2.3. Cell cultures The human lung adenocarcinoma epithelial cell line A549 was maintained in DMEM supplemented with 10% FBS and antibiotics (penicillin, 100 U/ml; streptomycin and kanamycin, 100 ␮g/ml). HEL 12469 human embryo lung cells were cultivated in MEM supplemented with 10% FBS, 1% non-essential amino acids and antibiotics (penicillin, 50 U/ml; streptomycin, 50 ␮g/ml). The cell lines were cultured in a humidified atmosphere of 5% CO2 at 37 ◦ C. In all experiments, cells were exposed to nanoparticles in medium supplemented with 2% FBS.

time intervals (30 min, 2 h, 4 h and 24 h). After treatment, the cells were incubated with the fluorescence probe H2 DCFH-DA (10 ␮M) in serum-free culture medium for 30 min at 37 ◦ C in the dark and then washed twice with PBS. The change in fluorescence resulting from the oxidation of DCFH-DA was measured at 485/520 nm (excitation and emission, respectively) using a PolarStar Optima fluorescence microplate reader (BMG Labtech, Germany), and the values were expressed as fluorescence units. To avoid false positive/negative results, the viability of the exposed cells was determined by propidium iodide staining. Cells were fixed with 2% paraformaldehyde for 15 min, incubated with 0.1 M glycine for 5 min, permeabilized with 0.1% Triton X-100 for 1 min, stained with propidium iodide (PI, 62.5 ␮g/ml) for 10 min and then washed with PBS. The fluorescence of PI was measured at 544/615 nm (excitation and emission, respectively). The iROS levels produced by particular MNPs per living cell were then calculated. Hydrogen peroxide (500 ␮M in HEL 12469, 1000 ␮M in A549, 10 min) was used as a positive control in these experiments.

2.7. Antioxidant enzyme activities The total superoxide dismutase (SOD, EC 1.15.1.1) activity was determined using the RANSOD kit following the manufacturer’s instructions. The SOD activity is measured by the degree of inhibition of the formation of a red formazan dye due to the reaction of superoxide radicals with 2-(4-iodophenyl)-3-(4-nitrophenol)-5phenyltetrazolium chloride (I.N.T.). The decrease in the absorbance at 505 nm was measured using a PolarStar Optima microplate reader (BMG Labtech, Germany). One unit of SOD was defined as the amount that caused a 50% inhibition of the rate of reduction of INT under the conditions of the assay. The glutathione peroxidase (GPx, EC 1.11.1.9) activity was assessed according to the method of Paglia and Valentine (Paglia and Valentine 1967) using cumene hydroperoxide as a substrate. The changes in absorbance at 505 nm were measured using a PolarStar Optima microplate reader (BMG Labtech, Germany). One unit of GPx activity was defined as the amount of enzyme that catalyzes the conversion of one micromole of NADPH per minute under standard conditions.

2.4. Treatment of cells Exponentially growing cells were exposed to different concentrations of surfacemodified MNPs nanoparticles for 30 min, 2 h, 4 h and 24 h depending on the endpoint measured. Concentrations of particular surface-modified MNPs used in experiments on HEL 12469 and A549 cells were chosen based on the cell viability determined by MTT (Mesarosova et al., 2012). The IC50 values were calculated from the dose–response curves using CalcuSyn software (Table 3). The unit mM (mmol/l) of iron oxide was used to express the MNP concentrations; under these conditions, equal numbers of particles per surface culture dish and equal amount of magnetite per surface culture dish were applied to human lung cells regardless of the surface coating (Table S2). The treatment of the cells was finished by removing the medium and washing the cells twice with phosphate-buffered saline (PBS).

2.8. The glutathione levels The intracellular glutathione (iGSH) was measured by flow cytometry (CANTO II; Becton Dickinson, Franklin Lakes, NJ, USA) using monochlorobimane (MCB; Sigma–Aldrich) staining for iGSH. 1–2 × 105 control and MNPs-treated A549 and HEL12469 cells were stained with 40 ␮M MCB at room temperature for 20 min. Cells were chilled by addition of ice-cold PBS to stop the enzyme-dependent staining reaction, washed twice with PBS and kept at 4 ◦ C. Five ␮l of propidium iodide (PI; 1 mg ml−1 ) were added to all samples to exclude the dead cells from the analysis. Fluorescence of MCB–GSH conjugate, representing iGSH content, was detected using 405 nm excitation laser and 450/50 emission bandpass filter (Violet 1-A). Viable cells were analyzed by FCS Express 4.0 (de Novo) software. The iGSH levels are expressed as the ratio of iGSH in MNPs-treated to untreated cells.

2.5. Single-cell gel electrophoresis (SCGE, the comet assay) The procedure of Collins et al. (Collins et al., 1995) as modified by Gabelova et al. (1997) was followed. Briefly, cells were embedded in 0.75% LMP agarose in PBS buffer (Ca2+ and Mg2+ free) and placed in a lysis solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris–HCl, pH 10 and 1% Triton X-100) at 4 ◦ C for 1 h. The slides were then transferred to an electrophoresis box and immersed in an alkaline solution (300 mM NaOH, 1 mM Na2 EDTA, pH > 13). After 20 min unwinding time, a voltage of 25 V (300 mA) was applied for 30 min at 4 ◦ C. The slides were neutralized with three 5 min washes with Tris–HCl (0.4 M, pH 7.4) and stained with 20 ␮l of ethidium bromide (EtBr, 5 ␮g/ml). EtBr-stained nucleoides were examined with an Axio Imager fluorescence microscope and Metafer 3.6 software (MetaSystems GmbH, Altlussheim, Germany). The percentage of DNA in the tail (% tail DNA) was used as a measure of DNA damage (DNA strand breaks). Three hundred nucleoides were scored per each sample in each electrophoretic run. 2.5.1. Oxidative damage to DNA identified by SCGE (FPG-sensitive sites) After lysis, slides were washed three times for 5 min in endonuclease buffer (40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, pH 8.0) and then incubated with formamidopyrimidine-DNA glycosylase/AP nuclease (FPG, 0.2 U/slide) for 30 min in a humidified atmosphere at 37 ◦ C. The slides were then transferred to an electrophoresis box and immersed in an alkaline solution. SCGE was then performed as described above. The photosensitiser RO 19-8022 (0.5 ␮M in HEL 12469, 1 ␮M in A549) was used as a positive control in these experiments. The cells were treated with RO 19-8022 in PBS-G buffer (140 mM NaCl, 3 mM KCl, 8 mM Na2 HPO4 , 1 mM KH2 PO4 , 1 mM CaCl2 , 0.5 mM MgCl2 , 0.1% glucose, pH 7.4) for 2 min, and then the cells were irradiated on ice using a 1000 W halogen lamp (Philips PF811) at a distance of 33 cm for 2 min (Collins et al., 2003). 2.6. Intracellular reactive oxygen species formation The kinetics of intracellular ROS (iROS) generation was determined in each human lung cell line treated with surface-modified MNPs at several sampling

2.9. Total antioxidant capacity Total antioxidant capacity was determined using OxiSelect TM Total Antioxidant Capacity (TAC) Assay Kit (Cell Biolabs) according to the manufacturer recommendations. Briefly, A549 and HEL12469 cells were treated with given concentrations of surface modified MNPs for 24 h and cell homogenates were prepared in PBS. After sonication and centrifugation (10,000 × g, 4 ◦ C, 10 min), cell supernatants were stored at −80 ◦ C. Results from the TAC assay based on the reduction of copper (II) to copper (I) were normalized to the protein concentration measured by Bradford assay. Standards of known uric acid concentrations were used to create a calibration curve. Results are expressed as ␮M Copper Reducing Equivalents (CRE) per mg proteins.

2.10. Eletrophoretic determination of apoptosis (DNA ladder) The procedure of Repicky et al. (2009) was followed with some modifications. In brief, treated cells were harvested in PBS and lysed in 100 ␮l of lysis solution (10 mM Tris, 10 mM EDTA, and 0.5% Triton X-100) supplemented with proteinase K (1 mg ml−1 ). Samples were then incubated at 37 ◦ C for 1 h and heated at 70 ◦ C for 10 min. Following lysis, RNAse (200 ␮g/ml) was added followed by incubation at 37 ◦ C for 1 h. The samples were subjected to electrophoresis at 40 V for ∼3 h in 2% (w/v) agarose gel. After electrophoresis, the gel was stained with ethidium bromide (EtBr, 0.5 ␮g/ml) for 2 h. 2.11. Statistical analysis The data are given as the mean values ± SD. The differences between control cells and treated cells were evaluated by Student’s t-test and one-way analysis of variance (ANOVA). Dose–response data were fitted by a linear or exponential regression. The threshold of statistical significance was set at p < 0.05.

M. Mesároˇsová et al. / Toxicology Letters 226 (2014) 303–313

A

A549 30 25

tail DNA [%]

307

***

*

**

**

20

**

**

**

***

0.2

0.3

0.4

*** **

15 10 5 0 0

0.3

0.4

0.5

0.1

SO-Fe3O4 [mM]

B

SO-PEG-Fe3O4 [mM]

0.1

0.15

0.2

SO-PEG-PLGAFe3O4 [mM]

HEL 12469 30

tail DNA [%]

25

*

20 15 10 5 0 0

0.3

0.35

0.4

SO-Fe3O4 [mM]

0.1

0.15

0.2

SO-PEG-Fe3O4 [mM]

0.05

0.1

0.15

SO-PEG-PLGA-Fe3O4 [mM]

Fig. 1. The levels of DNA strand breaks induced by coated MNPs in A549 cells (A) and HEL 12469 cells (B). Cells were exposed to MNPs with different surface coatings for 24 h. The columns represent the means ± S.D. from at least two independent experiments run in triplicate. Significant difference with respect to the control group at *p < 0.05; **p < 0.01; ***p < 0.001.

3. Results 3.1. DNA strand break formation induced by surface-modified MNPs Because DNA strand breaks are readily formed upon exposure to genotoxic compounds, they have been proposed as a standard biomarker of DNA damage (Andreoli et al., 2003). The capacity of individual MNPs to produce DNA breakage was evaluated after 24 h treatment with MNPs at concentrations of 0.05–0.5 mM. In A549 human lung tumor cells, all MNPs, regardless of the surface coating, induced a significant increase in the level of DNA strand breaks compared with the level in control cells (Fig. 1A). Neither a dosedependent increase in DNA migration nor significant differences in the levels of DNA breakage among individual MNPs were detected in these cells. Despite the stronger cytotoxicity of individual MNPs to HEL 12469 human diploid lung cells (Mesarosova et al., 2012), the levels of DNA strand breaks detected in MNP-treated cells were nearly equal to those observed in control cells except for the level for the 0.35 mM SO-Fe3 O4 treatment (Fig. 1B). 3.2. The oxidative damage to DNA produced by surface-modified MNPs Although the suitable methods for precise measurement of oxidative lesions introduced into DNA are still being discussed, it has been proposed that modified alkaline single-cell gel electrophoresis might be an appropriate approach to detect such damage, particularly if low levels of oxidative damage to DNA are being assessed (Collins et al., 2003). The bacterial base excision repair enzyme formamidopyrimidine DNA glycosylase (FPG), which converts oxidized purines into DNA breaks, did not reveal any base modifications in A549 or HEL 12469 human lung cells treated with different surface-modified MNPs for 24 h (Fig. 2).

Fig. 2. The levels of oxidative damage to DNA detected in A549 (A) and HEL 12469 (B) cells treated with different MNPs for 24 h. The data are expressed as the net FPG sensitive sites, i.e., the number of DNA strand breaks detected in the presence of FPG endonuclease (FPG-sensitive sites) minus the number of DNA strand breaks detected immediately after treatment in the absence of FPG endonuclease. RO – the positive control; cells were exposed to the photosensitiser RO 19-8022 (0.5 ␮M for HEL 12469 cells, 1 ␮M for A549 cells) for 2 min and irradiated on ice with a 1000 W halogen lamp (Philips PF811) at a distance of 33 cm for 2 min. The columns represent the means ± S.D. from at least two independent experiments run in triplicate. Significant difference with respect to the control group at *p < 0.05; **p < 0.01; ***p < 0.001.

Significant levels of 8-oxo-deoxyguanosine (8-oxo dG) were detected only in cells exposed to the positive control, RO 19-8022 treatment plus irradiation with visible light. 3.3. The levels of ROS formation in cells exposed to surface-modified MNPs The DCFH-DA assay has been shown to be a useful tool for the quantitative measurement of nanoparticle-induced oxidative stress (Aranda et al., 2013). This approach was used in this study to assess the capacity of surface-modified MNPs to generate reactive oxygen species (ROS) in human lung cell lines at various treatment times (30 min, 1 h, 2 h, 4 h and 24 h). Based on the cytotoxicity of individual surface-modified MNPs (Mesarosova et al., 2012), concentrations of 0.1–2 mM were selected for the shortterm treatment intervals (30 min, 1 h, 2 h, 4 h), and concentrations of 0.05–0.3 mM were selected for the long-term treatment interval (24 h). Surprisingly, the steady-state ROS level per viable cell was at least 3-fold higher in HEL 12469 human diploid lung cells (Fig. 3B) than in A549 human lung tumor cells (Fig. 3A). A significant dose-dependent increase in iROS compared with the control was found in A549 cells exposed to SO-Fe3 O4 particles for 4 h (Fig. 3A). A significant increase in ROS generation was also found in SO-PEG-Fe3 O4 -treated A549 cells, but only at the 0.3 mM concentration. In contrast, SO-PEG-PLGA-Fe3 O4 particles did not induce any ROS formation in A549 cells during the short-term treatment

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Fig. 3. The intracellular reactive oxygen species (ROS) level produced by particular MNPs per living cell in A549 (A) and HEL 12469 (B) cells treated with different MNPs for 30 min, 2 h and 4 h. Hydrogen peroxide (500 ␮M for HEL 12469 cells, 1000 ␮M for A549 cells, 10 min) was used as a positive control in these experiments. The columns represent the means ± S.D. from at least two independent experiments run in triplicate. Significant difference with respect to the control group at *p < 0.05; **p < 0.01; ***p < 0.001.

intervals. In HEL 12469 human diploid lung cells, none of the individual MNPs, regardless of the surface modification, produced any significant increase in ROS formation during the short-term treatment intervals. For the 24 h treatment interval, both SO-Fe3 O4 and SO-PEGFe3 O4 particles but not SO-PEG-PLGA-Fe3 O4 particles induced significant levels of ROS in A549 cells (Fig. 4A). Likewise, significant levels of ROS were detected in HEL 12469 cells exposed to SO-PEG-Fe3 O4 and SO-PEG-PLGA-Fe3 O4 particles but not in cells exposed to SO-Fe3 O4 particles (Fig. 4B). 3.4. The activities of superoxide dismutase and glutathione peroxide in cells exposed to surface-modified MNPs The harmful effects of reactive oxygen species are balanced by the antioxidant functions of low-molecular-weight scavengers and antioxidant enzymes. The most efficient enzymatic antioxidants are superoxide dismutase, catalase and glutathione peroxidase (Valko et al., 2006). The activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) were assessed in human lung cells treated with surface-modified MNPs for 24 h (Figs. 5 and 6, respectively). The basal level of GPx activity was at least 2-fold higher in HEL 12469 cells than in A 549 cells (Fig. 5A). Despite this difference, no significant changes in GPx activity between control and MNPs-treated cells except one concentration (0.2 mM SO-PEGPLGA-Fe3 O4 particles) were found in A549 cells. In HEL 12469

cells, a significant increase in GPx activity was determined only in SO-Fe3 O4 -treated cells (Fig. 5B). Neither SO-PEG-Fe3 O4 nor SOPEG-PLGA-Fe3 O4 particles influenced the GPx activity substantially compared to control cells. In contrast, a significant decrease in SOD activity was determined in SO-Fe3 O4 -treated A549 cells while a significant increase in SOD activity was detected in SO-PEG-PLGA-Fe3 O4 -treated A549 cells (Fig. 6A). No changes in the SOD activity was found in A549 cells treated with SO-PEG-PLGA-Fe3 O4 particles. In HEL 12469 cells, no significant differences in SOD activity were detected between MNPs-treated and control HEL 12469 cells except two concentrations; a decrease in SOD activity was determined at 0.3 mM SO-Fe3 O4 and an increase in SOD activity was detected at 0.05 mM SO-PEG-PLGA-Fe3 O4 (Fig. 6B). 3.5. Total antioxidant capacity and intracellular glutathione level Besides the activity of antioxidant enzymes, the assessment of the non-enzymatic antioxidant capacity (TAC) and the intracellular glutathione levels (iGSH) was performed in control and MNP-treated cells. These measurements provide an indication of the overall ability to eliminate ROS and resist oxidative damage. The basal TAC determined in A549 cells were at least 3-fold higher than in HEL 12469 cells (Fig. 7). Despite this fact, no significant differences in TAC were detected between MNP-treated and control cells in either of the human lung cell lines after 24 h treatment

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Fig. 4. The intracellular reactive oxygen species (ROS) level produced by particular MNPs per living cell in A549 (A) and HEL 12469 (B) cells treated with different MNPs for 24 h. Hydrogen peroxide (500 ␮M for HEL 12469 cells, 1000 ␮M for A549 cells, 10 min) was used as a positive control in these experiments. The columns represent the means ± S.D. from at least two independent experiments (in triplicate per sample). Significant difference with respect to the control group at *p < 0.05; **p < 0.01; ***p < 0.001.

(Fig. 7A and B) except one concentration. A significant decrease in TAC was observed in HEL 12469 cells treated with 0.15 mM SOPEG-PLGA-Fe3 O4 particles (Fig. 7B). Consistent with TAC, no significant changes in iGSH were determined between MNP-treated and control cells in either of the human lung cell lines (Fig. 8A and B). The iGSH levels in cells treated with surface-modified MNPs were expressed as the ratio of iGSH in MNP-treated to untreated cells. 3.6. Apoptotic DNA fragmentation The electrophoretic approach was used to investigate the capacity of surface-modified MNPs to induce apoptosis in A549 and HEL 12469 cells. No intranucleosomal fragmentation of cellular DNA was detected in MNP-treated A549 cells and HEL 12469 cells (Fig. 9). 4. Discussion MNPs are one of the most promising types of nanoparticles for diagnostic and therapeutic purposes; they are easily manufactured and their cytotoxicity is relatively low (Choi et al., 2009; Hong et al., 2011; Liu et al., 2011). Taking into account the potential benefits of MNP use, human exposure to MNPs will increase, primarily in the context of nanomedicine-based diagnostics and therapy, thus the bio-safety of MNPs is a great concern.

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Fig. 5. The activity of glutathione peroxidase (GPx) in A549 cells (A) and HEL 12469 cells (B) treated with different surface-modified MNPs for 24 h. The columns represent the means ± S.D. from at least three independent experiments (in triplicate per sample). Significant difference with respect to the control group at *p < 0.05; **p < 0.01; ***p < 0.001. Results are expressed as U GPx per mg proteins.

The cell-based in vitro assays play increasing important role in risk assessment of existing and emerging nanomaterials prior to toxicity testing by more expensive, time-consuming and ethically challenging in vivo tests. Detailed physicochemical characteristics of nanoparticles’ behavior in the cell culture media is hence a prerequisite for the evaluation of nano:bio biological effects. Differences in the composition of the culture medium, serum and even cytosolic proteins arising from cells were shown to cause that identical nanoparticles form different sizes of agglomerates (Jiang et al., 2014; Lesniak et al., 2010; Maiorano et al., 2010; Sharma et al., 2014). Apparent differences in stability of the surfacemodified MNPs considered in this study were observed in particular culture media. The SO-Fe3 O4 particles were the most stable particles in both DMEM and MEM culture medium; no agglomeration was detected at each of the concentrations considered during 24 h. In contrast, the SO-PEG-PLGA-Fe3 O4 particles were the least stable ones and agglomerated relatively rapidly in both culture media, nevertheless these MNPs were the most cytotoxic particles in both A549 cells and HEL 12469 cells (Mesarosova et al., 2012). Accordingly, the agglomerated silica nanoparticles produced more potent inflammatory responses in BEAS-2B cells than the non-agglomerated particles (Gualtieri et al., 2012). The colloidal stability of nanoparticles in culture media can influence particle uptake into cells. More rapid accumulation of the larger agglomerates compared to the smaller agglomerates was observed in C10 cells (Sharma et al., 2014) and mature human macrophages (Muller et al., 2014). Moreover, the agglomeration was shown to govern the mode of particle uptake (Muller et al., 2014). In general, a sharp increase in the hydrodynamic sizes from 60 nm to nearly 350 nm was determined for all surface-modified

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Fig. 6. The activity of superoxide dismutase (SOD) in A549 cells (A) and HEL 12469 cells (B) treated with different surface-modified MNPs for 24 h. The columns represent the means ± S.D. from at least three independent experiments. Significant increase with respect to the control group at *p < 0.05; **p < 0.01; ***p < 0.001. Significant decrease with respect to the control group at # p < 0.05; ## p < 0.01; ### p < 0.001. Results are expressed as U SOD per ␮g proteins.

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Fig. 7. Total antioxidant capacity (TAC) of A549 cells (A) and HEL 12469 cells (B) treated with different surface-modified MNPs for 24 h. The columns represent the means ± S.D. from at least two independent experiments with triplicate per sample. Significant difference with respect to the control group at *p < 0.05. Results are expressed as ␮M Copper Reducing Equivalents (CRE) per mg proteins.

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MNPs in the culture media due to adsorption of amino acids and serum proteins onto the particle surface. The adsorbed proteins (protein corona) affect not only the dispersion of nanoparticles, but facilitate their intracellular uptake and biological activity, as well as stabilize the agglomerates (Aggarwal et al., 2009; Jiang et al., 2014; Ruh et al., 2012; Singh et al., 2012). In serum free culture medium, all surface-modified MNPs considered in this study deposited immediately at the bottom of the culture dishes and none particles entered the cells. Supplementation of culture medium with FBS stopped the rapid agglomeration and allowed the surfacemodified MNPs to internalize into the cells (Mesarosova et al., 2012). Indeed, protein corona brought a shift in the zeta potentials of all MNPs toward a slight negative charge. These results corresponded with other studies that found changes in particles’ net zeta potentials in culture medium due to nanoparticle–protein interactions (Chen et al., 2008; Jiang et al., 2014; Sharma et al., 2014). ROS generation represents one of the primary mechanisms involved in the genotoxicity of iron oxide nanoparticles. It is believed that oxidative stress could be induced either by iron ions released from the particle surface or by the particles per se. Iron oxides were shown to be of very low solubility at neutral pH (Schwertmann, 1991), characteristic for cell culture condition. Voinov et al. (2011) have revealed that iROS are produced mostly by the catalytic reactions at the nanoparticles’ surface and the surfacemediated ROS generation is not suppressed by surface coating with serum albumin or oleate. Consistent with these results, the oleatecoated MNPs induced ROS generation in Cos-1 cells (Magdolenova et al., 2012) and 16HBE cells but not in A549 cells (Guadagnini et al.,

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Fig. 8. Intracellular glutathione (iGSH) determined in A549 cells (A) and HEL 12469 cells (B) treated with different surface-modified MNPs for 24 h. The columns represent the means ± S.D. from at least three independent experiments. The iGSH levels are expressed as the ratio of iGSH in MNPs-treated to untreated cells.

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Fig. 9. Apoptotic DNA fragmentation in A549 cells (A) and HEL 12469 cells (B) after exposure to surface-modified MNPs. Cells were treated with surface-modified MNPs for 24 h and postcultivated in fresh medium for 72 h and 96 h (A549 cells) and 72 h and 120 h (HEL 12469 cells) before electrophoretic detection of intranucleosomal fragmentation of cellular DNA. M – size marker, NC – negative control; 1 – 0.8 mM SO-Fe3 O4 particles (72 h); 2 – 0.8 mM SO-Fe3 O4 particles (96 h); 3 – 0.35 mM SO-PEG-Fe3 O4 particles (72 h); 4 – 0.35 mM SO-PEG-Fe3 O4 particles (96 h); 5 – 0.3 mM SO-PEG-PLGA-Fe3 O4 particles (72 h); 6 – 0.3 mM SO-PEG-PLGA-Fe3 O4 particles (96 h); PC – positive control (cells treated with 0.5 ␮M B[a]P for 1 h and irradiated with 2.4 J/m2 and postcultivated for 48 h); 1 – 0,4 mM SO-Fe3 O4 particles (72 h); 2 – 0,4 mM SO-Fe3 O4 particles (120 h); 3 – 0,2 mM SO-PEG-Fe3 O4 particles (72 h); 4 – 0,2 mM SO-PEG-Fe3 O4 particles (120 h); 5 – 0,15 mM SO-PEG-PLGA-Fe3 O4 particles (72 h); 6 – 0,15 mM SO-PEG-PLGA-Fe3 O4 particles (120 h).

2013). In our experiments, the SO-Fe3 O4 particles induced significant levels of ROS in A549 cells already after 4 h treatment whereas no significant increase in iROS compared to control cells were detected in HEL 12469 cells. Although a PEGylation diminished ROS generation by MNP-PEI particles in SH-SY5Y cells (Hoskins et al., 2012), the SO-PEG-Fe3 O4 particles produced iROS generation in both human lung cell lines regardless of their stability in particular culture medium. The agglomeration of nanoparticles per se cannot affect iROS formation in treated cells. Sharma et al. (2014) have shown recently that the agglomerates of carboxylated MNPs increased expression of several genes involved in redox-regulated pathways while the agglomerates of amine-modified MNPs of the same sizes failed to up-regulate the expression of these genes. Pretreatment of cells with ROS scavengers was shown, for example, to increase the viability of MNP-treated cells (Sharma et al., 2014) and reverse the suppression of cytokine production and glutathione depletion (Shen et al., 2011). These studies, indeed, confirmed the contribution of oxidative stress in adverse cellular response to MNPs treatment, but it is difficult to specify whether the protective effects of antioxidants are mediated through intracellular or extracellular mechanisms. Shen et al. (2011) did not detect any increased levels of iROS in MNPs-treated cells. Despite of differences in iROS generation between individual cell lines, no oxidative damage to DNA compared with control cells was detected in either of the human lung cell lines. The lack of oxidative DNA damage could not be caused by the interference of surface-modified MNPs with Fpg because Fe(II) ions were shown not to compete for the zinc-binding site in the catalytic center of Fpg (O’Connor et al., 1993). We believe that none of the MNPs particles per se could influence the Fpg activity because no interference between Fpg and Fe3 O4 nanoparticles ((Kain et al., 2012)) or silica nanoparticles (Magdolenova et al., 2012) has been detected. Cell response to oxidative stress is a complex process in which both the non-enzymatic and enzymatic antioxidant defense systems are involved. These antioxidant defense systems play a key role in regulation of cellular redox homeostasis and cell protection against excess of free radicals (Valko et al., 2006). The basal level of total antioxidant capacity (TAC) was at least 4-fold higher in A549 cells than in HEL 12469 cells and vice versa nearly 4-fold lower SOD and GPx activities were determined in A549 cells compared to HEL 12469 cells. Cell exposure to surface-modified MNPs did not induce substantial changes in TAC and iGSH in either of the cell lines. Accordingly, none of the surface-modified MNPs except

SO-Fe3 O4 particles in HEL 12469 cells produced considerable changes in GPx activity in human lung cell lines. Consistent with these results, no significant increase in SOD activity was determined in HEL 12469 cells. On the other hand, a significant increase in SOD activity was detected only in A549 cells treated with SOPEG-PLGA-Fe3 O4 particles while the SO-Fe3 O4 particles caused a decrease in SOD activity in these cells. A slight fluctuation in SOD activity that, however, failed to reach the significance, was detected in six mammalian cell lines treated with DMSA-MNPs (Liu et al., 2011). On the other hand, a depletion of antioxidants, SOD activity and GSH, and induction of apoptosis was found in rat lung epithelial cells treated with polyacrylate-coated MNPs (Ramesh et al., 2012). Based on our results, we hypothesized that oxidative stress induced by surface-modified MNPs used in this study could be balanced by the intrinsic antioxidant defense systems of particular cell lines prior to induction of the oxidative damage to DNA. Therefore we are convinced that oxidative stress plays, at most, only a marginal role in the genotoxicity of surface-modified MNPs investigated in this study in human lung cells. In general, the biological effects of any engineered nanoparticles depend not only on their chemical nature and internalized amount into the cell, but also on the composition of protein corona, intracellular localization and their biostability inside the cells (Muller et al., 2014). Although the internalized amount of surface-modified MNPs detected in A549 cells was much lower than that determined in HEL 12469 cells (Mesarosova et al., 2012), DNA breakage was detected only in MNPs-treated A549 cells. It is tempting to speculate that not only internalized amount of MNPs, but also interaction of MNPs with the cytoskeletal proteins can lead to disruption of cell homeostasis and physiological cellular processes. Depolymerization of amyloid fibrils by albumin-modified MNPs has been recently revealed (Siposova et al., 2012). Indeed, we found visible changes in cell morphology already during cell exposure to surfacemodified MNPs (data not shown). This together with the higher amount of internalized MNPs might, at least in part, explain higher cytotoxicity of MNPs toward HEL 12469 cells when compared to A549 cells. We can assume that the surface-modified MNPs might have induced severe disruption of cytoskeleton structure and consequently DNA lesions incompatible with cell survival in HEL 12469 cells. The highly damaged cells might be later lost via necrosis as none of the surface-modified MNPs induced apoptosis in either of the cell lines. Moreover, highly damaged DNA is much too small to be detected by the comet assay; short DNA pieces can diffuse

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away from the gel already during lysis (Collins et al., 2008). Interestingly, great cytoskeleton changes, disorganization of actin fiber and tubulin networks were observed also in human umbilical vein endothelial cells treated with coated MNPs (Wu et al., 2010). In conclusion, our study demonstrated that MNP-mediated ROS formation plays, at most, only a marginal role in genotoxicity of surface-modified MNPs used in this study. Although variable increases in the iROS levels were observed in both HEL12469 and A549 cells, no oxidative damage to DNA was found in either of these cell lines. None of the surface-modified MNPs affected substantially the TAC, iGSH or GPx activity in either of the cell lines. The surface chemistries of MNPs did not significantly affect the cell response to exposure. Further studies are required to thoroughly investigate the MNPs’ interactions with cytoskeleton structure in both human tumor and diploid cells. Conflict of interest statement The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments The authors express their appreciation to Dr. Milan Timko, PhD, Institute of Experimental Physics, SAS, Koˇsice, Slovakia, for the preparation and detailed characterization of the surface-modified MNPs, and Mrs. Angela Perrin, F. Hoffmann-La Roche Ltd., Pharma Research (pRED), Switzerland, for providing us with the RO 19-8022. The authors wish to thank Mrs. Anna Morávková for excellent technical assistance. This study was supported by the VEGA grants 2/0051/09, 2/0143/13 and 2/0163/12, and APVV-0658-11 grant. This publication is the result of the project implementation TRANSMED, ITMS 26240120008, and TRANSMED 2, ITMS 26240120030, supported by the Research and Development Operational Programme funded by the ERDF. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet. 2014.02.025. References Aggarwal, P., Hall, J.B., McLeland, C.B., Dobrovolskaia, M.A., McNeil, S.E., 2009. Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv. Drug Deliv. Rev. 61, 428–437. Andreas, K., Georgieva, R., Ladwig, M., Mueller, S., Notter, M., Sittinger, M., Ringe, J., 2012. Highly efficient magnetic stem cell labeling with citrate-coated superparamagnetic iron oxide nanoparticles for MRI tracking. Biomaterials 33, 4515–4525. Andreoli, C., Gigante, D., Nunziata, A., 2003. A review of in vitro methods to assess the biological activity of tobacco smoke with the aim of reducing the toxicity of smoke. Toxicol. In Vitro 17, 587–594. Aranda, A., Sequedo, L., Tolosa, L., Quintas, G., Burello, E., Castell, J.V., Gombau, L., 2013. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay: a quantitative method for oxidative stress assessment of nanoparticle-treated cells. Toxicol. In Vitro 27, 954–963. Auffan, M., Decome, L., Rose, J., Orsiere, T., De, M.M., Briois, V., Chaneac, C., Olivi, L., Berge-Lefranc, J.L., Botta, A., Wiesner, M.R., Bottero, J.Y., 2006. In vitro interactions between DMSA-coated maghemite nanoparticles and human fibroblasts: a physicochemical and cyto-genotoxical study. Environ. Sci. Technol. 40, 4367–4373. Babic, M., Horak, D., Trchova, M., Jendelova, P., Glogarova, K., Lesny, P., Herynek, V., Hajek, M., Sykova, E., 2008. Poly(l-lysine)-modified iron oxide nanoparticles for stem cell labeling. Bioconjug. Chem. 19, 740–750.

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