Mutat Res Gen Tox En xxx (xxxx) xxxx Contents lists available at ScienceDirect Mutat Res Gen Tox En journal homepage: www.elsevier.com/locate/gentox Kidney nanotoxicity studied in human renal proximal tubule epithelial cell line TH1 Monika Sramkovaa, , Katarina Kozicsa, Vlasta Masanovab, Iveta Uhnakovab, Filip Razgac,i, ⁎ Veronika Nemethovac,i, Petra Mazancovac,i, Lucyna Kapka-Skrzypczakd,e, Marcin Kruszewskid,e,f, Marta Novotovag, Victor F. Puntesh, Alena Gabelovaa a Cancer Research Institute, Biomedical Research Center SAS, Dubravska cesta 9, 845 05, Bratislava, Slovakia b Slovak Medical University, Limbova 12, 833 03, Bratislava, Slovakia c Polymer Institute SAS, Dubravska cesta 9, 845 41, Bratislava, Slovakia d Department of Molecular Biology and Translational Research, Institute of Rural Health, Jaczewskiego 2, 20-090, Lublin, Poland e Department of Medical Biology and Translational Research, Faculty of Medicine, University of Information Technology and Management, Sucharskiego 2, 35-225, Rzeszów, Poland f Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195, Warsaw, Poland g Institute of Experimental Endocrinology, Biomedical Research Center SAS, Dubravska cesta 9, 845 05, Bratislava, Slovakia h Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, 08193, Barcelona, Spain i Selecta Biotech SE, Heydukova 2138/1, 811 08, Bratislava, Slovakia ARTICLE INFO ABSTRACT Keywords: Progressive expansion of nanomaterials in our everyday life raises concerns about their safety for human health. Inorganic nanoparticles Although kidneys are the primary organs of xenobiotic elimination, little attention has been paid to the kidneys Human renal proximal tubule cells in terms of nanotoxicological studies up to now. Here we investigate the cytotoxic and genotoxic potential of Comet assay four solid-core uncoated inorganic nanoparticles (TiO2NPs, SiO2NPs, Fe3O4NPs and AuNPs) using the human TEM analysis renal proximal tubule epithelial TH1 cells. To mimic the in vivo conditions more realistic, TH1 cells were exposed AAS analysis ICP-MS in vitro to inorganic NPs under static as well as dynamic conditions for 3 h and 24 h. The medium throughput alkaline comet assay (12 minigels per slide) was employed to evaluate the impact of these NPs on genome integrity and their capacity to produce oxidative lesions to DNA. The accumulation and localization of studied inorganic NPs inside the cells was monitored by transmission electron microscopy (TEM) and the efficacy of internalization of particular NPs was determined by atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). From all the tested NPs, only Fe3O4NPs induced a slight cyto- toxicity in TH1 cells exposed to high concentrations (> 700 μg/ml) for 24 h. On the other hand, the inorganic NPs did not increase significantly the level of DNA strand breaks or oxidative DNA damage regardless of the treatment mode (static vs. dynamic conditions). Interestingly, substantial differences were observed in the in- ternalized amount of inorganic NPs in TH1 cells exposed to equivalent (2.2 μg/ml) concentration. Fe3O4NPs were most efficiently taken up while the lowest quantity of particles was determined in TiO2NPs-treated cells. As the particle size and shape of individual inorganic NPs in culture medium was nearly identical, it is reasonable to suppose that the chemical composition may contribute to the differences in the efficacy of NPs uptake. 1. Introduction supply and ability to concentrate toxins, makes the kidneys particularly susceptible to xenobiotics, including nanoparticles (NPs), which reach The kidneys are vital organs that perform several important phy- the bloodstream after intentional or unintentional exposure. Generally, siological functions such as regulation of blood pressure, control of liver and kidneys are the most common accumulation secondary organs plasma concentrations of nutrients and electrolytes, reabsorption of regardless of exposure routes, animal models and physicochemical water and small molecules, production of hormones, as well as detox- properties of NPs [3]. In comparison to lungs, the most important target ification and excretion of xenobiotics from the body [1,2]. High blood organ in the case of occupational exposure [4], the impact of NPs in ⁎ Corresponding author. E-mail address: monika.sramkova@savba.sk (M. Sramkova). https://doi.org/10.1016/j.mrgentox.2019.01.012 Received 26 July 2018; Received in revised form 27 November 2018; Accepted 17 January 2019 1383-5718/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). Please cite this article as: Monika Sramkova, et al., Mutat Res Gen Tox En, https://doi.org/10.1016/j.mrgentox.2019.01.012 M. Sramkova, et al. Mutat Res Gen Tox En xxx (xxxx) xxxx kidneys has though received only little attention. So far, limited number 2.2. Inorganic nanoparticles (NPs) of in vitro and in vivo studies has proved the capacity of NPs to exert various adverse effects on kidneys, thus affecting their physiological All solid-core inorganic NPs (TiO2NPs, SiO2NPs, Fe3O4NPs and functions [5]. Among them, mainly metal NPs and silica, have been AuNPs) were synthesized and characterized in-depth by group of Prof. already shown to induce nephrotoxicity and different levels of adverse Victor F. Puntes (Institute of Nanoscience and Nanotechnology, renal effects [6–13]. However, up to the date there are no validated or Barcelona, Spain). AuNPs were stabilized by sodium citrate (2.2 mM) regulatory approved in vitro models available for predicting ne- while dispersant used for TiO2NPs and Fe3O4NPs was TMAOH (tetra- phrotoxicity, which means there is still a great need for the develop- methylammonium hydroxide, 10 mM). SiO2NPs were kept in Mili-Q ment of new in vitro renal cell models. water. All inorganic NPs were stored in the dark and dry place. Renal clearance of waste products and xenobiotics is a multifaceted process involving glomerular filtration, tubular reabsorption and se- 2.3. Dynamic light scattering (DLS) cretion, and elimination of the compound through urinary excretion, thus employing all the kidney compartments. Differences in the impact Particle size distribution and colloidal stability of inorganic NPs in of NPs on particular nephron structure, the basic structural and func- culture medium at 37 °C was determined by DLS using Zetasizer Nano- tional unit of the kidney, have been detected (reviewed in Du et al., ZS (Malvern Instruments, UK) equipped with a 4 mW helium/neon laser 2018). For example, signs of glomerulonephritis have been observed in (λ = 633 nm) and a thermoelectric temperature controller. The mea- mice after exposure to 25 nm and 80 nm TiO2NPs [12] as well as surements were performed at ˜3 min intervals during 1 h at three time 23.5 nm CuNPs [6], both applied by oral gavage. On the other hand, no intervals – 0 h (0–1 h), 3 h (3–4 h) and 24 h (23–24 h); every data point significant changes in the glomeruli were found after exposure of mice was recorded as the average of at least 11 repetitions. Instrument set-up to various sizes of AuNPs (10, 20, 50 nm, i.p. exposure) [13]. In contrast was done with respect to the physico-chemical characteristics (ab- to glomeruli alterations upon NPs exposure, structural changes (renal sorption, refractive index, viscosity, temperature) of the studied col- fibrosis) and pathological degeneration within the renal proximal loidal solutions [16]. convoluted tubules have been determined in mice exposed to TiO2NPs and CuNPs [6,12]. These results indicated that the tubular region of the 2.4. Cell culture kidneys might be a preferential site of NPs toxicity. This suggestion supports the fact that a greater sensitivity of tubular LLC-PK1 cells The human renal proximal tubule epithelial TH1 cell line cell line compared to mesangial IP15 cells was detected in vitro after treatment was obtained from Kerafast Inc. (Boston, USA). The cells were cultured with carbon black nanoparticles and TiO2NPs [14]. Moreover, experi- in DMEM medium with high glucose supplemented with 10% fetal calf ments on rats revealed that even the proximal renal convoluted tubules serum (FCS) and antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ are more susceptible to AuNPs toxicity than the distal ones [13]. ml) on plastic Petri dishes (Ø 100 mm) at 37 °C in humidified atmo- Therefore, the aim of this study was evaluate the nephrotoxicity of sphere of 5% CO2. four inorganic solid-core uncoated NPs using immortalized human renal proximal tubule epithelial TH1 cell line which displays several 2.5. Treatment of cells characteristics of the proximal tubule [15]. Tested NPs (titanium di- oxide/TiO2NPs, silica dioxide/SiO2NPs, magnetite/Fe3O4NPs and gold Exponentially growing cells were exposed to different concentra- nanoparticles/AuNPs) are routinely used in biomedical applications or tions of inorganic NPs for 3 h and 24 h. All concentrations were pre- consumer products. The proximal tubule epithelial cells are naturally pared freshly before exposure using procedure provided by Prof. Victor exposed to fluid shear stress generated by continuously filtrate move- F. Puntes (Institute of Nanoscience and Nanotechnology, Barcelona, ment from glomeruli to urinary bladder. In order to more realistically Spain) [17]. Briefly, 1 ml of the inorganic NPs was initially mixed with recapitulate the in vivo conditions, TH1 cells were in vitro exposed to 1 ml of FCS and then culture medium with FCS (9 ml) was added. From model inorganic NPs under static as well as dynamic conditions. The this basic stock dispersion of NPs in medium, further dilutions were cytotoxicity of selected inorganic NPs was evaluated by alamarBlue® prepared. The solvents/dispersants (sodium citrate, TMAOH) were also assay and the alkaline comet assay was used to investigate their impact tested for potential cyto- and genotoxic effects. The treatment of the on genome integrity and capacity to induce oxidative DNA lesions. cells was ended by removing the medium with inorganic NPs and Intracellular localization of NPs and ultrastructural changes in mor- washing the cells twice with phosphate-buffered saline (PBS). phology of TH1 cells upon NPs treatment were studied by transmission electron microscopy (TEM) and the internalized amount of particular 2.6. alamarBlue® assay inorganic NPs inside the cells was determined by flame atomic ab- sorption spectroscopy (FAAS) and inductively coupled plasma mass TH1 cells were seeded into the series of 96 well plates in a density of spectrometry (ICP-MS). 2 × 104/well and cultured in complete culture medium. Exponentially growing cells were then pre-incubated either in the presence of broad 2. Materials and methods range of inorganic NPs, solvents/dispersants or untreated (control) cells for 3 h and 24 h. After treatment, cells were incubated with 100 μl of 2.1. Chemicals working solution of alamarBlue® (Invitrogen, USA) for 4 h according to the manufacturer’s protocol. The fluorescence (excitation 530 nm, Ethidium bromide (EtBr), low-melting-point (LMP) agarose, emission 590 nm) in each well was measured on a microplate reader - normal-melting-point (NMP) agarose, Triton X-100, and HEPES were POLARStar OPTIMA- BMG LABTECH. purchased from Sigma-Aldrich (Lambda Life, Slovakia), and for- mamidopyrimidine-DNA glycosylase/AP nuclease (Fpg) was purchased 2.7. Single-cell gel electrophoresis (SCGE, the comet assay) from New England Biolabs (BioTech, Slovakia). (R)-1-[(10-Chloro-4- oxo-3-phenyl-4H-benzo(a)quinolizin-1-yl)-carbonyl]-2-pyrrolidine-me- The capacity of inorganic NPs to induce DNA strand breaks was thanol (Ro 19-8022; RO) was kindly provided by F. Hoffmann – analyzed using a medium-throughput format of comet assay (12 mini- LaRoche AG (Basel, Switzerland). Culture media, fetal bovine serum gels per slide) according to Shaposhnikov et al. [18] with minor mod- (FBS), antibiotics and other chemicals used for cell cultivation were ifications. Briefly, TH1 cells (1.2 × 104) were gently resuspended and purchased from GIBCO (Lambda Life, Slovakia). All other chemicals mixed in 1% LMP agarose in PBS (Ca2+ and Mg2+ free); the final and solvents were of analytical grade from commercial suppliers. concentration of LMP was 0.75%. Twelve drops of 10 μl (one thousand 2 M. Sramkova, et al. Mutat Res Gen Tox En xxx (xxxx) xxxx cells per drop) were placed on 1% NMP agarose pre-coated microscopic cell was finally calculated from the internalized amount of NPs nor- slides. After solidification of the gels, the slides were placed in lysis malized to the total number of treated cells according to the following solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, pH = 10 and formula: 1% Triton X-100, at 4 °C) for 1 h to remove cellular proteins. After lysis, Internalized amount of NPs per cell = internalized amount of NPs/ slides were transferred to an electrophoresis box and immersed in an number of cells alkaline solution (300 mM NaOH, 1 mM Na2EDTA, pH > 13). After 30 min of unwinding time, a voltage of 25 V (0.7 V/cm) was applied for 2.9. Transmission electron microscopy (TEM) 20 min at 4 °C. The slides were neutralized with three 5 min washes with Tris–HCl (0.4 M, pH = 7.4) and dried with ethanol. Before scoring, The uptake and accumulation of inorganic NPs in TH1 cells was slides were stained with ethidium bromide (EtBr, 5 μg/ml). EtBr-stained evaluated by TEM. Cells were plated on Petri dishes (Ø 60 mm, nucleoids were examined with Zeiss Axio Imager.Z2 fluorescence mi- 3–5 × 105 cells/dish) and exposed to inorganic NPs at exponential croscope (Zeiss, Germany) using the computerized image analysis phase of growth. After treatment, cells were washed twice with PBS and (Metafer 3.6, MetaSystems GmbH, Altlussheim, Germany). The per- trypsinized. TH1 cellular suspension (treated with TiO2NPs, Fe3O4NPs, centage of DNA in the tail (% of tail DNA) was used as a parameter for AuNPs or untreated) was centrifuged at 1000 rpm for 5 min in PBS. The DNA damage measurement. One hundred comets were scored per each samples were then fixed with 4% formaldehyde in PBS for 45 min. After sample in each gel. post-fixation with 1% osmium tetraoxide (OsO4,) in cacodylate buffer for 30 min, samples were contrasted overnight with a saturated aqueous 2.7.1. Oxidative damage to DNA identified by SCGE (Fpg-sensitive sites) solution of uranyl acetate at room temperature. The samples were then After lysis, slides were washed three times for 5 min in endonuclease dehydrated in graded ethanol series and propylene oxide, and em- buffer (40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, pH 8.0) and then bedded into Durcupan (Fluka AG, Switzerland). Ultrathin (60 nm) incubated with formamidopyrimidine-DNA glycosylase/AP nuclease sections were cut by Power-Tome MT-XL (RMC/Sorvall, Tucson, USA) (Fpg, 0.2 U/slide) for 30 min in a humidified atmosphere at 37 °C. The ultramicrotome, placed on copper grids covered with formvar mem- slides were then transferred to an electrophoresis box and immersed in branes and contrasted by lead citrate. The sections were examined with an alkaline solution. SCGE was then performed as described above. The JEM 1200 electron microscope (Jeol, Tokyo, Japan) at 7500–50000× photosensitizer RO 19-8022 was utilized as a positive control in these magnification. Images were recorded using a Gatan Dual Vission 300 W experiments. The working concentration (1 μM in PBS) was prepared CCD camera (Gatan Inc., Pleasanton, USA). freshly before use. The cells were treated with RO 19-8022 in PBS-G buffer (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4, 1 mM 2.10. Statistical analysis 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 Data are given as mean values with ± SD. The differences between PF811) at a distance of 33 cm for 2 min [19]. control cells and treated cells were evaluated by Student's t-test and one-way analysis of variance (ANOVA). The threshold of statistical 2.8. Quantification of the internalized amount of inorganic NPs significance was set to p < 0.05. Flame atomic absorption spectrometry (FAAS) and inductively 3. Results coupled plasma mass spectrometry (ICP-MS) were adapted to quantify the cellular uptake of inorganic NPs. Au and Fe contents in cell samples 3.1. Characteristics of inorganic NPs, particle size distribution and colloidal were determined by FAAS (AA240FS, Varian, Australia) after sonica- stability in culture medium tion with concentrated nitric acid (Suprapure, Merck, Slovakia). Si and Ti contents in cells were determined by ICP-MS (XSeries 2, Thermo As physico-chemical properties of NPs are critical to understand Fisher Scientific, Germany) after sonication and high-pressure micro- their biological activity, prior to nanotoxicity testing, the character- wave digestion with concentrated nitric acid and hydrogen peroxide ization of concentration, particle size and ζ-potential of tested NPs has using standard and collision cell modes, respectively. Sc, Y and In were been performed. The basic physico-chemical characteristics of tested chosen as internal standards. The detection limits (LODs) of the in- inorganic NPs after preparation are summarized in Table 1. Except struments for Au, Fe, Si, and Ti were 0.010 mg/l; 0.008 mg/l; 0.40 μg/l; SiO2NPs, the core size of all tested NPs was nearly identical. and 0.046 μg/l, respectively. The limits of quantification (LOQs) of the The particle size distribution and colloidal stability of particular NPs instruments for Au, Fe, Si, and Ti were 0.033 mg/l; 0.027 mg/l; 1.3 μg/ in culture medium was measured at three concentrations (Table 2). The l; and 0.15 μg/l, respectively. hydrodynamic particle size of each type of NPs increased substantially The cellular uptake of NPs expressed as pg of particular element per in DMEM medium due to agglomeration of particles and absorption of Table 1 Basic physico-chemical properties of tested inorganic NPs. Nanoparticles AuNPs SiO2NPs TiO2NPs Fe3O4NPs inner core diameter [nm] 13.8 ± 1.71 27.6 ± 3.8 5 – 10 7-15 Particle size (DH) diameter 13.8 ± 0.58 (sodium 30.19 ± 10.65 (Mili-Q 23.67 ± 7.93 (tetramethylammonium 19.12 ± 3.91 (tetramethylammonium in solvent [nm] citrate, 2.2 mM) water) hydroxide, 10 mM) hydroxide, 10 mM) PDI 0.219 ± 0.29 0.217 ± 0.01 0.174 ± 0.005 0.437 ± 0.038 Zeta (ξ) potential [mV] −40.9 ± 2.76 −52.1 ± 10.4 −42.0 ± 15.4 −35.9 ± 3.87 Concentration in stock [mg/ 0.066 10.15 5 11.6 ml] Number of particles/ml ˜3.02 × 1012 ˜3 × 1014 ˜5 × 1015 ˜5 × 1015 TEM image 3 M. Sramkova, et al. Mutat Res Gen Tox En xxx (xxxx) xxxx Table 2 Size distribution of TiO2NPs, SiO2NPs, Fe3O4NPs and AuNPs in culture medium. TiO2NPs 165 μg/ml 22 μg/ml 2.2 μg/ml 0h 3h 24 h 0h 3h 24 h 0h 3h 24 h PdI 0.32 ± 0.03 0.32 ± 0.03 0.29 ± 0.01 0.53 ± 0.06 0.46 ± 0.04 0.39 ± 0.06 0.71 ± 0.18 0.42 ± 0.08 0.26 ± 0.01 Z-Ave 251 ± 5 251 ± 5 251 ± 5 204 ± 11 223 ± 4 234 ± 11 161 ± 45 238 ± 10 231 ± 5 Size [nm] (Number) 83 ± 42 72 ± 47 83 ± 45 88 ± 61 91 ± 57 81 ± 53 89 ± 47 80 ± 61 120 ± 38 Size [nm] (Intensity) 367 ± 17 368 ± 26 350 ± 21 357 ± 26 365 ± 29 356 ± 14 357 ± 37 376 ± 22 310 ± 14 Intensity peaks 367 ± 17 368 ± 26 350 ± 21 6 ± 1; 356 ± 24 6 ± 1; 365 ± 29 356 ± 14 6 ± 1; 357 ± 37 376 ± 22 310 ± 14 SiO2NPs 165 μg/ml 22 μg/ml 2.2 μg/ml 0h 3h 24 h 0h 3h 24 h 0h 3h 24 h PdI 0.56 ± 0.10 0.26 ± 0.01 0.19 ± 0.01 0.53 ± 0.20 0.27 ± 0.02 0.22 ± 0.01 0.55 ± 0.14 0.30 ± 0.06 0.17 ± 0.01 Z-Ave 68 ± 20 130 ± 3 148 ± 2 83 ± 22 132 ± 1 140 ± 2 58 ± 29 124 ± 6 146 ± 2 Size [nm] (Number) 6 ± 1* 87 ± 9 111 ± 6 6 ± 1* 94 ± 8 102 ± 8 6 ± 1* 93 ± 9 110 ± 5 Size [nm] (Intensity) 143 ± 17 174 ± 4 179 ± 4 153 ± 22 178 ± 7 176 ± 4 141 ± 51 174 ± 5 174 ± 4 Intensity peaks 143 ± 17 174 ± 4 179 ± 4 24 ± 4; 153 ± 22 178 ± 7 176 ± 4 141 ± 51 174 ± 5 174 ± 4 Fe3O4NPs 165 μg/ml 22 μg/ml 2.2 μg/ml 0h 3h 24 h 0h 3h 24 h 0h 3h 24 h PdI 0.40 ± 0.10 0.43 ± 0.08 0.28 ± 0.01 0.61 ± 0.15 0.54 ± 0.08 0.38 ± 0.0.06 0.53 ± 0.15 0.46 ± 0.07 0.28 ± 0.01 Z-Ave 720 ± 27 617 ± 16 355 ± 4 485 ± 70 415 ± 28 180 ± 6 166 ± 63 174 ± 21 145 ± 1 Size [nm] (Number) 495 ± 302 317 ± 188 222 ± 87 20 ± 33 127 ± 89 66 ± 54 7 ± 0.6* 62 ± 30 110 ± 10 Size [nm] (Intensity) 973 ± 281 921 ± 196 488 ± 18 628 ± 61 671 ± 184 275 ± 16 390 ± 112 250 ± 15 197 ± 4 AuNPs 2.2 μg/ml 0.66 μg/ml 0.22 μg/ml 0h 3h 24 h 0h 3h 24 h 0h 3h 24 h PdI 0.36 ± 0.03 0.67 ± 0.07 0.29 ± 0.01 0.45 ± 0.13 0.62 ± 0.09 0.33 ± 0.01 0.59 ± 0.28 0.40 ± 0.09 0.27 ± 0.01 Z-Ave 17 ± 1 52 ± 15 139 ± 2 18 ± 10 87 ± 12 126 ± 2 56 ± 36 119 ± 8 136 ± 2 Size [nm] 6 ± 0.3* 6 ± 1* 124 ± 7 6 ± 1* 6 ± 0.4* 117 ± 8 6 ± 1* 7 ± 1* 111 ± 7 (Number) Size [nm] 26 ± 11 173 ± 26 192 ± 3 23 ± 13 183 ± 10 185 ± 4 127 ± 64 190 ± 3 182 ± 3 (Intensity) Intensity peaks 12 ± 2; 6 ± 1; 190 ± 4 7 ± 1; 198 ± 4 8 ± 1; 58 ± 11 8 ± 1; 198 ± 3 185 ± 6 7 ± 1; 58 ± 12 7 ± 1;196 ± 6 183 ± 5 59 ± 14 * Particles from serum proteins, probably BSA. serum protein(s) on particle surface (protein corona formation). TH1 cells for 3 h and 24 h. Under these treatment conditions, none of Fe3O4NPs that manifested polydisperse distribution were colloidally the tested inorganic NPs, except Fe3O4NPs at higher concentrations unstable at 165 and 22 μg/ml concentrations and their aggregation and (> 700 μg/ml), manifested any cytotoxic effects either after 3 h or 24 h sedimentation were visible already after visual inspection. At the lowest (Fig. 1A–D). No cytotoxic effect was determined after treatment of TH1 2.2 μg/ml concentration, the colloid dispersion was relatively stable cells with the highest concentration of particular solvents/dispersants within the tested time frame. All other types of NPs: AuNPs, TiO2NPs after 24 h (data not shown). and SiO2NPs were colloidally stable in culture medium at all three concentrations used. At the low concentrations (≤ 2.2 μg/ml), DLS measurements were strongly affected by the presence of small particles 3.3. Genotoxicity representing the serum proteins (probably BSA) as they are in high excess in culture medium in comparison to inorganic NPs. In line with Based on the recommendation of EU-funded NaNoReg and Ji et al. [20], DLS analysis of NPs-free medium (DMEM + 10% FBS + NanoTest projects [21], the concentrations 2.2, 22, and 165 μg/ml ATB) confirmed the presence of 6 ± 1 nm particles. (corresponding to 1, 10 and 75 μg/cm2) were selected for SiO2NPs, TiO2NPs and Fe3O4NPs. None of these concentrations had any cytotoxic effect as shown earlier (Fig. 1A–D). In the case of AuNPs, the con- 3.2. Cytotoxicity centrations 0.22, 0.66 and 2.2 μg/ml (corresponding to 0.1, 0.3 and 1 μg/cm2) were used as the concentration of stock solution was sub- A broad range of concentrations of particular inorganic NPs was stantially lower. The impact of inorganic NPs on genome stability was utilized to evaluate their effect on viability of TH1 cells. In order to determined by the alkaline comet assay. In order to simulate more cover as wide concentration range as possible for the cytotoxicity realistic in vivo kidney conditions, TH1 cells were seeded on Transwell testing, the highest concentration of particular NP has been calculated polycarbonate membrane cell culture inserts (3 μm pore density) and based upon the stock concentration (see Material and Methods), hence exposed to inorganic NPs under static and dynamic conditions for 3 h the differences between tested NPs. Concentrations ranging from 0.1 to and 24 h. Medium flow rate - 20 μl/min, corresponded in our experi- 3 μg/ml (AuNPs); from 6 to 924 μg/ml (SiO2NPs), from 58 to 1056 μg/ mental setup to ˜ 0.2 dyn/cm−2 which reflects the in vivo situation in ml (Fe3O4NPs); and from 83 to 454 μg/ml (TiO2NPs) were applied to kidneys according to Ingber et al. [22]. In addition to DNA breakage, 4 M. Sramkova, et al. Mutat Res Gen Tox En xxx (xxxx) xxxx Fig. 1. Cell viability of TH1 cells treated with inorganic NPs. Fig. 3. The impact of inorganic NPs on genome stability of TH1 cells under dynamic conditions after 3 h (A) and 24 h (B) of treatment. Fig. 2. The impact of inorganic NPs on genome stability of TH1 cells under static conditions after 3 h (A) and 24 h (B) of treatment. in control TH1 cells were observed upon dynamic exposure [6.98% ± 2.36% (3 h) or 10.13% ± 2.67% (24 h)] when compared to the capacity of inorganic NPs to induce oxidative DNA damage was static conditions 11.63% ± 3.15% (3 h) or 10.79% ± 2.97% (24 h). assessed as well. None of the inorganic NPs increased significantly the level of DNA strand breaks and oxidative DNA damage in TH1 cells 3.4. Quantification of internalized NPs compared to control cells when treated under static conditions (Fig. 2A and B). Similar results were obtained when TH1 cell were treated with For the nanosafety assessment, the quantification of internalized inorganic NPs under dynamic conditions. No significant increase in the amount of NPs is critical step and supports the association between the level of DNA strand breaks and oxidative damage to DNA were de- amount of internalized nanoparticles and their biological effect. In termined (Fig. 3A and B). Also no changes in basal level of DNA damage order to explain the absence of any impact on cell viability and the level 5 M. Sramkova, et al. Mutat Res Gen Tox En xxx (xxxx) xxxx Table 3 The amount of elements (Ti, Si, Fe and Au) detected in TH1 cells exposed to TiO2NPs, SiO2NPs, Fe3O4NPs (2.2 μg/ml, 24 h) and the uptake efficiency in TH1 cells. The amount of particular element [pg/cell] is expressed as the net value. Ti [pg/cell] Si [pg/cell] Fe [pg/cell] Au [pg/cell] Cell number [x106] Total applied dose [μg] Internalized amount Total [μg] [%] from total applied dose Control cells 0.0018 0.0991 0.0228 0.0 13.20 – – – TiO2NPs 0.0107 – – – 10.25 33.0 0.128 0.389 SiO2NPs – 0.0430 – – 8.90 33.0 1.265 3.833 Fe3O4NPs – – 1.4672 – 8.83 33.0 13.190 39.970 AuNPs – – – 0.8652 7.42 33.0 6.420 19.455 of DNA damage (strand breaks and oxidative DNA damage), the FAAS phagolysosomes. The size of the phagolysosomes and their occurrence and ICP-MS were employed to rule out the possibility of the failure of in the cytosol of the cells varied. The occasionally observed fusion of studied NPs to enter the cells. Additionally, to compare the efficacy of phagolysosomes (Fig. 5C), may explain the presence of large phagoly- NPs uptake based on their chemical composition, exponentially sosomes filled with accumulated NPs (Fig. 5D). Phagolysosomes con- growing TH1 cells were exposed to equivalent concentration (2.2 μg/ tained nanoparticles in dense condensed form together with the grain ml) of the tested inorganic NPs for 24 h. The basal level of particular matter (Fig. 5A–C). Lipofuscin granules and autophagosomes (Fig. 5D) elements (Fe, Si, Ti and Au) was determined also in control (untreated) were rarely present. These observations indicate that TH1 cells cannot cells to avoid any false positive results. The Table 3 summarizes the process excess of NPs, for instance by exocytosis or encapsulation, since internalized amount of different elements (expressed as pg of Ti, Si, Fe, these processes were not observed. An excessive accumulation of NPs or Au per cell). destroys the structure of cells to the extent that prevents their normal As small amounts of Ti, Si and Fe were detected also in control function and, in effect, may lead to cell inactivity, death and formation (untreated) TH1, these quantities were subtracted from the total of tissue scars. amounts of particular elements detected in exposed cells, and the data presented in the Table 3 represent the net quantity. Taking into the account the initial amount of NPs that has been used to treat TH1 cell, 4. Discussion the efficacy of the uptake has been calculated and showed that TH1 cells internalized more efficiently Fe3O4NPs and AuNPs compared to Renal clearance of nanoparticles, which have reached the systemic SiO2NPs and TiO2NPs. As the hydrodynamic size of all inorganic NPs in circulation, is an expected and possible elimination route in living or- culture medium was nearly the same (110–120 nm agglomerates) the ganisms. For this reason, kidneys might be one of the most important effect of chemical composition on NPs uptake cannot be excluded. secondary target organs; and the NPs-induced renal toxicity should be Moreover, the core size of inorganic NPs was nearly identical except carefully evaluated. SiO2NPs (Table 1). Several in vitro and in vivo studies have reported that different types of NPs are able to exert various toxic effects in renal cells/tissue [5], however the number of such studies is limited. Additionally, because of 3.5. The accumulation of NPs in TH1 cells the diversity of NPs, especially their physico-chemical properties that influence their characteristics and behavior, many nano-bio interac- Following the 24 h treatment with equivalent concentration (2.2 μg/ tions still remain to be understood. Therefore, to provide a compre- ml TiO2NPs, Fe3O4NPs and AuNPs), TH1 cells were processed for hensive and critical evaluation of the toxicity of NPs on the renal system electron microscopy to study the impact of inorganic NPs on their ul- further studies are inevitable. The employment of cell culture techni- trastructure, preferential accumulation inside the cells and possible ques in (nano)toxicology has gained more importance over the past mechanism of internalization. The electron microscopy analysis re- decades in order to minimize the experiments on animals. Cowie et al. vealed that all the tested NPs (regardless on the chemical composition) [23] studied the suitability of mammalian cells of different origin for are present inside the TH1 cells as of vesicles containing NPs were the assessment of genotoxicity of various NPs and the kidney cells used observed in cytoplasm (Fig. 4A–C). Based on the observation performed in their study (HEK293 and COS1) seemed to be one of the most reliable with cells exposed to 165 μg/ml Fe3O4NPs, it is reasonable to predict to detect a dose-response upon NPs treatment. The currently available that the inorganic NPs are internalized by a process similar to phago- and used in vitro human renal cell models such as HK-2 cells and cytosis that manifested at the active surface of the cell (Fig. 5A–B). The HEK293 cells (reviewed in [24], this Special Issue), however, lack small aggregates of NPs at the cell surface were surrounded by the sufficient in vivo physiological relevance as the cells are in the majority protrusions of plasma membrane and engulfed in vesicles to the cell of studies cultivated under static conditions as two dimensional (2D) (Fig. 5B–D). Phagosomes with NPs interact with lysosomes and form cultures. Fig. 4. Accumulation of NPs in the TH1 cells treated with 2.2 μg/ml TiO2NPs, AuNPs, and Fe3O4NPs for 24h. 6 M. Sramkova, et al. Mutat Res Gen Tox En xxx (xxxx) xxxx Fig. 5. Electron-microscopic images of ultrastructure TH1 cells exposed to 165 μg/ml Fe3O4NPs for 24 h. In order to evaluate the impact of selected inorganic NPs under cells [33] after exposure to inorganic NPs (AuNPs, TiO2NPs, ZnONPs, more realistic in vivo conditions, the human renal TH1 cells were grown SiO2NPs, CdNPs, AgNPs). Oxidative stress and generation of reactive in this study on Transwell membrane inserts and treated with inorganic oxygen species (ROS) has been proposed as one of the main mechan- NPs under continuous medium flow rate. It has been suggested that isms underlying NP-induced toxicity [34]. ROS formation may occur combination of in vitro methods simulating as closely as possible in vivo due to NP-mediated damage to the mitochondrial membrane [35] or conditions should be used to access the possible toxic effects of NPs NPs may affect the NADPH oxidase in the plasma membrane during [25]. Moreover, culturing TH1 cells on permeable supports also pro- entry into the cell [36]. In the case of iron oxide NPs, it is supposed that motes apical-basal polarization that is an important characteristic of iron ions released from the particle surface due to lysosomal enzymatic mature epithelial layers, thereby simulating in vivo conditions. degradation can participate in the Fenton reaction, producing the hy- Four inorganic solid-core NPs (TiO2NPs, SiO2NPs, Fe3O4NPs and droxyl radicals [37]. Treatment of HEK293 cells with Fe2O3NP-condi- AuNPs) that can be found in everyday life commercial products were tioned medium revealed that the cytotoxic effect induced by Fe2O3NPs evaluated in this study. All inorganic NPs except Fe3O4NPs at higher per se was much higher so the authors concluded that the contribution concentrations were colloidally stable in culture medium during 24 h, of metal ion release to Fe2O3NPs cytotoxicity was fairly low [38]. though the particle size of individual NPs increased substantially The glomerular filtration apparatus possesses an effective size cutoff probably due to particle agglomeration and protein corona formation of 10 nm, in case of metal-based NPs the threshold of 5.5 nm was de- [26,27]. None of the inorganic NPs induced any DNA strand breaks and fined by investigation of the renal clearance of quantum dots of dif- oxidative DNA lesions regardless of the exposure (static and dynamic ferent sizes [39]. In contrast to glomerular filtration membrane, which conditions). Even with the use of unrealistically high concentrations acts as a physical barrier (size and charge barrier), the proximal tubules (with respect to the common human exposure), no cytotoxicity was behave as a chemical barrier for NPs because of their active endocytic observed, with the exception of Fe3O4NPs (> 700 μg/ml). Despite the machinery. After glomerular filtration, nanoparticles are concentrated fact that the surface coating of NPs is a widely used procedure to in- in the proximal tubules, which can lead to internalization by tubular crease the biocompatibility and reduce the potential toxicity, there are cells through endocytosis [40]. However, despite the size cutoff, it has also studies [28] showing no (or low) cytotoxic effect of uncoated in- been shown by Zuckerman et al. [41] that polycationic cyclodextrin organic NPs (etc. Fe3O4NPs) which is in agreement with our findings. NPs containing siRNA (˜ 70 nm) accumulated in glomerular mesangium Moreover, our results were in line with L'Azou et al. [14], Halamoda and Williams et al. [42] reported that the “mesoscale” nanoparticles Kenzaoui et al. [11] and Kermanizadeh et al. [29] who did not detect (PLGA-PEG, 400 nm, i.v. administration) localized selectively in renal any significant increase in ROS formation in human or mammalian proximal tubules. Also study by Oroojalian et al. [43] showed that renal cells after exposure to TiO2NPs, Fe3O4NPs and SiO2NPs. On the modified-polymyxin-PEI/DNA nanoparticles can effectively target other hand, intracellular ROS generation was detected in human renal megalin-expressing kidney cells (megalin being one of the markers for HK-2, HEK293 cells and human mesangial IP15 cells [7,30,31], porcine proximal tubule cells). 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Henriksson, S. Meier, I. Gaivão, N.H. Huskisson, A. Smart, G. Brunborg, M. Nilsson, A.R. Collins, Twelve-gel slide format optimised The authors declare that there are no conflicts of interests. for comet assay and fluorescent in situ hybridisation, Toxicol. Lett. 195 (2010) 31–34, https://doi.org/10.1016/j.toxlet.2010.02.017. [19] A. Collins, C. Gedik, N. Vaughan, S. Wood, A. White, J. Dubois, J.F. Rees, S. Loft, Acknowledgements P. Møller, H. Poulsen, J. Cadet, T. Douki, J.L. Ravanat, S. Sauvaigo, H. Faure, I. Morel, B. Morin, B. Epe, N. Phoa, A. Hartwig, T. Schwerdtle, P. Dolara, L. Giovannelli, M. Lodovici, R. Olinski, K. Bialkowski, M. Foksinski, D. Gackowski, The authors would like to thank to Prof. Victor F. Puntes (Institute Z. Duračková, L. Hlinčiková, P. Korytar, M. Sivonová, M. Dušinská, C. Mislanová, of Nanoscience and Nanotechnology, Barcelona, Spain) who kindly J. Viña, L. Möller, T. Hofer, J. Nygren, E. Gremaud, K. Herbert, J. Lunec, C. 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