agglomerated cerium oxide nanoparticles in an in vitro 3D airway model: The influence of mucociliary clearance

Toxicology in Vitro 29 (2015) 389–397

Contents lists available at ScienceDirect

Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit

Toxicity assessment of aggregated/agglomerated cerium oxide nanoparticles in an in vitro 3D airway model: The influence of mucociliary clearance C. Frieke Kuper a,⇑, Mariska Gröllers-Mulderij a, Thérèse Maarschalkerweerd b, Nicole M.M. Meulendijks c, Astrid Reus d, Frédérique van Acker d, Esther K. Zondervan-van den Beuken a, Mariëlle E.L. Wouters c, Sabina Bijlsma a, Ingeborg M. Kooter b a

TNO, Utrechtseweg 48, 3704 HE Zeist, The Netherlands TNO, Princetonlaan 6, 3584 CB Utrecht, The Netherlands c TNO, De Rondom 1, 5612 AP Eindhoven, The Netherlands d TNO Triskelion bv, Utrechtseweg 48, 3704 HE Zeist, The Netherlands b

a r t i c l e

i n f o

Article history: Received 8 June 2014 Accepted 21 October 2014 Available online 4 November 2014 Keywords: Cerium oxide In vitro 3D human bronchial epithelial model Mucociliary apparatus Mucociliary clearance

a b s t r a c t We investigated the toxicity of aggregated nanoparticles of cerium oxide (CeO2) using an in vitro 3D human bronchial epithelial model that included a mucociliary apparatus (MucilAir™). CeO2 was dispersed in saline and applied to the apical surface of the model. CeO2 did not induce distinct effects in the model, whereas it did in BEAS-2B and A549 cell cultures. The absence of effects of CeO2 was not because of the model’s insensitivity. Nanoparticles of zinc oxide (ZnO) elicited positive responses in the toxicological assays. Respiratory mucus (0.1% and 1%) added to dispersions increased aggregation/ agglomeration to such an extent that most CeO2 sedimented within a few minutes. Also, the mucociliary apparatus of the model removed CeO2 from the central part of the apical surface to the borders. This ‘clearance’ may have prevented the majority of CeO2 from reaching the epithelial cells. Chemical analysis of cerium in the basal tissue culture medium showed only minimal translocation of cerium across the 3D barrier. In conclusion, mucociliary defence appeared to prevent CeO2 reaching the respiratory epithelial cells in this 3D in vitro model. This model and approach can be used to study compounds of specific toxicological concern in airway defence mechanisms in vitro. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Nanomaterials are included in a wide variety of industrial and commercial products as they can confer novel properties and functionalities. Nevertheless, concerns have been raised about their Abbreviations: CeO2, cerium oxide; EDX, energy-dispersive X-ray spectrometry; LDH, Lactate dehydrogenase; TNFa, Tumour necrosis factor alpha; IL-1a, Interleukine 1alpha; IL-8, Interleukine 8; MCP-1, Monocyte chemotactic protein 1 (CCL2); sICAM-1, Soluble intercellular adhesion molecule 1 (CD54); SEM, scanning electron microscopy; TEER, Transepithelial electrical resistance; ZnO, zinc oxide; LPS, lipopolysaccharide; MMS, methyl methanesulfonate. ⇑ Corresponding author. Tel.: +31 (0)88 866 17 36. E-mail addresses: [email protected] (C. Frieke Kuper), [email protected] (M. Gröllers-Mulderij), [email protected] (T. Maarschalkerweerd), [email protected] (N.M.M. Meulendijks), [email protected] (A. Reus), [email protected] (F. van Acker), [email protected] (E.K. Zondervan-van den Beuken), [email protected] (M.E.L. Wouters), [email protected] (S. Bijlsma), [email protected] (I.M. Kooter). http://dx.doi.org/10.1016/j.tiv.2014.10.017 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.

safety profiles. One particular area of concern is that of airborne nanomaterials and the potential harms that may result in the respiratory tract (Oberdörster, 2001). Examples of exposure to such airborne nanomaterials include additives in diesel post-combustion and occupational exposure during fabrication of nanomaterials. In vivo inhalation studies in animals are representative ways of safety evaluation, but the costs, time required and political and societal pressures to replace, refine and reduce (3Rs) the amount of animals used in research mean that other approaches are needed. In vitro reconstructed respiratory tract human tissues cultured at air–liquid interfaces are increasingly being used for safety assessments of inhaled substances (Randell et al., 2011; Nichols et al., 2013; Huang et al., 2008, 2011, 2013). They consist of fully differentiated (human) cells that are morphologically and functionally similar to those found in vivo. These include characteristics such as ciliary beating, mucus production and representative metabolic activities. As a result they are considered to be more representative than monolayer cell cultures such as BEAS-2B, an

390

C. Frieke Kuper et al. / Toxicology in Vitro 29 (2015) 389–397

immortalized human bronchial epithelial cell line, and A549, an adenocarcinomatous human alveolar epithelial cell line. The A549 cell line is the most widely applied in vitro system to test (nano) particle inhalation toxicity. This makes sense because the alveolar epithelial lining covers a far greater surface area than the respiratory lining. Moreover, respiratory tract toxicity evaluation in general has long been focused on lungs, because this was the site of most concern in humans. In addition, particles are reported to exert effects especially in the alveolar region (reviewed by Gradon and Sosnowski (2014)). However, particles may deposit predominantly on the respiratory epithelium (Lippmann et al., 1980; Oberdörster et al., 2005) that covers nasal passages, larynx, trachea and extra-pulmonary bronchi. These tissues are not routinely examined in inhalation studies, which may explain why knowledge surrounding particle deposition in these anatomical regions is relatively poor. The new OECD412 guideline for inhalation toxicity highlights the significance of these organs (OECD guideline on subacute inhalation toxicity, 2009). Cerium oxide (CeO2) nanomaterials have widespread use as (automotive) catalysts, in solar cells, gas sensors, and in metallurgic and glass and ceramic applications (Murray et al., 1999; Zheng et al., 2005). Many of these applications are dispersive in nature and are associated with increased risk of exposure with unknown health and safety implications. CeO2 has been studied in vivo in various sub-acute inhalation studies. This includes an examination of the entire respiratory tract and the potential toxicity of CeO2 nanoparticles (Cassee et al., 2011; Gosens et al., 2014; Landsiedel et al., 2014). These studies confirmed that the main target for cerium oxide is the alveolar region and not the respiratory epithelium despite high deposition rates. Moreover, numerous data are available on in vitro toxicity of CeO2, with these data typically having been obtained via immersion exposure to cell lines (reviewed by US EPA, 2009). Despite this wealth of data, such cell lines lack key features (such as mucociliary apparatus) meaning that there is a risk that in vitro cellular assessments of toxicity may overestimate potential effects of compounds. We investigated the behaviour of CeO2 nanoparticles in respiratory mucus and assessed toxicity in an in vitro 3D human airway model (MucilAir™; Epithelix Sàrl) that included a mucociliary apparatus. Nano particles of zinc oxide (with its toxicity being related to its solubility (Cho et al., 2013)) were used as a positive control particle. ZnO exerts in vivo toxicity in the upper respiratory tract as well as in the alveolar region (Vandebriel and De Jong, 2012; Landsiedel et al., 2014). We also assessed CeO2 toxicity in A549 and BEAS-2B cell lines to benchmark our data. 2. Materials and methods Commercially available CeO2 nanoparticles (product number 544841) and ZnO nanoparticles (purity 99.99%, CAS #1314-13-2) were obtained from Sigma–Aldrich Chemie BV (Zwijndrecht, the Netherlands). Solutions of methyl methanesulfonate (MMS, 3 lg/ cm2; Sigma Aldrich), lipopolysaccharide (LPS (30 lg/cm2; Sigma Aldrich) and Triton X100 (0.3 lg/cm2; Sigma Aldrich) were also used as positive controls in the toxicity assays (see Table 1). Respiratory mucus was obtained from Epithelix Sàrl (Geneva, Switzerland). 2.1. Dispersions and dextran tablet exposure methods Particle dispersions were prepared in saline (containing 10 mM HEPES and 1.25 mM CaCl2), or Dulbecco’s modified eagle medium (DMEM) and DMEM/fetal calf serum (FCS) at concentrations of 1 or 2 mg/ml, and sonicated on ice twice for 150 s with an amplitude of 210 lm on ice (Bransonic Sonifier S-250 D/S-250 A, 1/800 tapered

tip). The dispersions were vortexed for 5 s and put in a 37 °C water bath. Instead of via dispersions, particles can also be embedded into an inert and neutral substance as a carrier to avoid the problem of unstable dispersions. Dextran has been used as carrier to deliver particles onto the apical surfaces of MucilAir™. It is a bacterial by-product with the dextran macromolecule consisting of glycan groups linked end to end. It was tested by the supplier and found to be inert to MucilAir™ (Huang et al., 2013). The preparation of dextran tablets with CeO2 and ZnO was done by Epithelix Sàrl (Geneva, Switzerland). In this experiment ZnO nanoparticles were used as a control. 2.2. Characterisation of CeO2

2.2.1. Surface free energy, dynamic light scattering and zeta potential CeO2 was characterised under the exposure conditions, namely the dispersion of particles in the BEAS-2B and A549 media (DMEM and DMEM/FCS) and saline for the 3D model, at a temperature of 37 °C and at a representative concentration range. A Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) was used to determine zeta potential and hydrodynamic size distribution (via dynamic light scattering (DLS)). Average size and polydispersity index (PDI) were determined according to ISO22412. The PDI scale was 0–1 with 0 representing a monodisperse state and 1 representing a polydisperse state. Due to high conductivity of the dispersions (because of the high salt concentration in the media; measured with Cyberscan PCD 6500, EutechÒ), and the influence of proteins in the media (which have their own zeta potential), measurement settings of the Zetasizer were adjusted to use an applied voltage of 50 V and a relaxation time between measurements of 45 s. 2.2.2. Scanning electron microscopy and element analysis CeO2 particle shape and size were determined with a Field Emission SEM (FEG-SEM/EDX; Tescan MIRA-LMH FEG-SEM). Dry powders of the particles were prepared by placing them onto conducting carbon tape for examination by SEM. CeO2 particle dispersions (3D saline, with or without respiratory mucus) were filtered and dried on SEM stubs, and were analysed in Quantomix capsules, with the backscattered electron technique (BEI). Quantomix capsules are made from stainless steel coated with a thin polymer film, and can contain about 15 ll of dispersions. Element analysis was performed with energy-dispersive X-ray spectrometry (EDX) (Bruker AXS spectrometer with a Quantax 400 workstation and an XFlash 5030 detector with an active area of 30 mm2 and super light element window (SLEW)), which allows X-ray detection of elements higher than boron (Z > 5). 2.3. 3D bronchial cell culture model (MucilAir™) and BEAS-2B and A549 cell cultures 2.3.1. 3D model Fully differentiated bronchial epithelial MucilAir™ models (Epithelix Sárl, Geneva, Switzerland), reconstituted from primary human cells, were used for the 3D bronchial cell culture experiments. The models were maintained on 24-well TranswellÒ culture supports at an air–liquid interface using MucilAir™ culture medium (Epithelix Sàrl, Geneva, Switzerland; supplemented with 1% amphotericin, 1% penicillin/streptomycin and 0.5% gentamicin). The models were maintained in a humidified incubator at 37 °C and 5% CO2. Upon receipt, the MucilAir™ models were maintained in culture for at least one week prior to performing the experiments. Culture medium was refreshed every 2–3 days. The basolateral culture medium was refreshed approximately one hour before exposure to the test compounds. The MucilAir™ models were also rinsed with saline containing 10 mM HEPES and 1.25 mM CaCl2 to

C. Frieke Kuper et al. / Toxicology in Vitro 29 (2015) 389–397

391

Table 1 Experimental protocols for testing of toxicity of CeO2 and respective controls. Test compound BEAS-2B and A549 cell culture CeO2 MMS (positive control for Comet assay) 3D tissue culture (MucilAir™) CeO2

ZnO (positive particle control for toxicity)

MMS (positive control Comet)

LPS (positive control HO-1 expression and cytokine production) Triton-X100 (positive control for cytotoxicity)

Experimental conditions

Parameters

Immersion 33, 100 and 333 lg/cm2 Dissolved in DMEM + FCS 3 lg/cm2

Cytotoxicity (LDH; ghost cells) Comet and HO-1 expression Comet and HO-1 expression

Droplet exposure (3, 24, 48 h) Dextran tablet exposure (24 h) Mucus removed 24 h + just before, or 24 h before exposure 30 (for cytokines) or 33, 100, 333 lg/cm2 Dextran tablet exposure (24 h) Mucus removed 24 h before exposure 9 lg/cm2 Mucus removed 24 h + just before, or 24 h before exposure Droplet (3 h) Dissolved in saline 3 lg/cm2 Droplet (3, 24, 48 h) Dissolved in saline 30 lg/cm2 Dissolved in saline (24 h) 0.3 lg/cm2

Cytotoxicity (TEER, LDH, ghost cells) Cytokines (IL-8, MCP-1, sICAM-1, IL-1a, TNFa) Comet and HO-1 expression. No HO-1 in dextran tablet exposure Chemical analysis of cerium in basal medium Cytotoxicity (TEER, LDH, ghost cells) Comet Cytotoxicity (TEER, LDH, ghost cells) Cytokines (IL-8, MCP-1, sICAM-1, IL-1alpha) Comet Cytotoxicity (LDH) Cytokines (IL-8, MCP-1, sICAM-1, IL-1alpha) HO-1 Cytotoxicity (LDH, TEER)

Effect of mucus on dispersions and behaviour of particles on surface of 3D tissue culture (MucilAir™) CeO2 Dispersions in water, saline, saline with 0.1% or Effect of mucus on dispersion by visual inspection (photographs) of 1% mucus, saline with FCS sedimentation over 24 h and SEM/EDX 333 lg/cm2; 50 ll droplet (mucus in dispersion) Behaviour on surface of 3D model by visual inspection over 24 h 2 and 0.02 lg/ml (particle distribution)

ensure that each individual model contained a mucus layer of comparable thickness. 2.3.2. Cell lines: BEAS-2B and A549 The BEAS-2B cell line (CRL-9609) and the A549 cell line (CCL-185) were obtained from ATCC (Wesel, Germany). The cell lines were cultured in DMEM with glutamax (Lonza BioWhittaker, Breda, the Netherlands), 10% FCS and 1% penicillin and streptomycin, and kept in a humidified incubator at 37 °C with 5% CO2. The cells were subcultured every 2–3 days in 96-wells plates. Per well 1.104 cells were used for exposure. 2.4. Toxicity assays The different test protocols are presented in Table 1. MucilAir™ cells (inserts) in 24-well plates were cultured for 24 h at an air– liquid interface. Once prepared, 50 ll of dispersions of CeO2 were added to each insert. Five experiments were performed with droplet exposure to CeO2 with three models/dose/time of exposure (the doses were 30 (cytokine assays) or 33, 100 and 333 lg/cm2; the duration of exposure was 3, 24 and 48 h; number of independent replicates for the different exposure durations was respectively 3, 4 and 1). All experiments were at least performed in duplicate for each test condition while most were performed in triplicate. BEAS-2B and A549 cells were exposed to particle-cell culture medium dispersions in the presence of 1 wt%. FCS for 24 h. Cells were treated in 96-well plates with 100 ll culture medium for the cytotoxicity assay. 2.4.1. Cytotoxicity (TEER and LDH) Trans Epithelial Electric Resistance (TEER) was measured using a Millicell ERS-2 device (Millipore). After exposure, MucilAir™ inserts were transferred to 24-well plates with 600 ll saline containing 16.2 mM CaCl2. Cells were washed with 200 ll saline containing 16.2 mM CaCl2 and TEER was measured. LDH release

was determined via a commercially available kit (LDH cytotoxicity detection kit, Roche Diagnostics, Mannheim, Germany) and was performed according to manufacturer instructions. The assay was performed in 96 well plates on 50 ll cell free supernatants from each treatment concentration. After 2 h of incubation at 37 °C, absorbance was measured at 490 nm using a microplate reader (Benchmark Plus, Bio-Rad, Hercules, USA). 2.4.2. Cytokines A panel of cytokines were analysed in the exposure medium using a Bender MedSystems Flow Cytomix™ and according to the manufacturer’s instructions. The following cytokines were analysed with flow cytometry: IL-1a, IL-8, TNFa, MCP-1 and sICAM-1 (soluble cell adhesion molecule). 2.4.3. Comet assay and HO-1 analyses After exposure, cells were washed with saline and harvested. Two thirds of the cells were frozen at 70 °C for subsequent use in the HO-1 expression analysis. The balance was used for the Comet Assay. For HO-1 expression analysis, RNA was isolated from frozen cells using a Nucleospin RNA II spin kit (Machery-Nagel GmbH & Co. KG, Düren, Germany). RNA concentration was determined using a Nanodrop (ND-1000, Isogen Life Science, Meern, The Netherlands). qPCR was performed according to manufacturer instructions (Invitrogen, SYBR GreenER™ qPCR supermix for iCycler; Cat. No. 11761-100) with 5 ll of cDNA product and 1 ll DNA primers (forward mus HMOX-1: TGC CCC AGGATT TGT CTGA; reverse mus HMOX-1-R: CTG AAA GTT CTT CAT GAA CTC AGC ATT CTC). A reference response curve of mouse genomic DNA was used to measure the amplification of HO-1. Two slides were prepared for the Comet assay from each culture. In short, cell suspension was mixed with 0.5% low-melting agarose (37 °C) and spread onto each of the slides. The slides were then immersed in chilled lysis buffer and incubated at 2–10 °C

392

C. Frieke Kuper et al. / Toxicology in Vitro 29 (2015) 389–397

overnight. Electrophoresis was performed at 25 V and 300 mA for 30 min in electrophoresis buffer, and then rinsed in chilled neutralisation buffer for 3–5 min. The slides were immersed in ethanol for at least 5 min and air-dried. For scoring, each slide was stained with ethidium bromide. 50 randomly chosen cells were analysed per slide (Comet Assay IV, Perceptive Instruments Ltd., Suffolk, UK). In total 300 cells per treatment concentration were scored. Average median for each treatment concentration was calculated from the median score calculated from each slide. The presence of ghost cells was scored as well, as an indication of cytotoxicity (necrosis or apoptosis). 2.5. Effect of respiratory mucus on particle dispersions and visualisation of particle distribution on apical surface of MucilAir™ 2.5.1. Mucus CeO2 agglomeration/aggregation Stock nanoparticle solutions (2.2 mg/ml) were prepared in saline containing 16.2 mM CaCl2 by sonicating twice for 30 s on ice (ultrasonication using a microtip with an amplitude of 24). Dispersions in a dose range of 0, 27, 54, 108 and 1000 lg/ml were divided over glass tubes (total volume of each tube was 2 ml). Four conditions were compared: the saline dispersion only, the dispersion with the addition of 0.1% or 1% mucus, and the saline dispersion with the addition of 1% FCS (last condition tested at 1000 lg/ml CeO2 only). After preparation the dispersions were mixed on a vortex and photographed. Photographs were taken immediately after preparation and at 3 and 24 h. This experiment was performed at room temperature. 2.5.2. Visual observation of particle distribution on apical surface of the 3D airway model An Olympus Colorview IIU camera on an Olympus CKX41 inverted microscope was used to record the distribution of droplets with 33, 100 and 333 lg/cm2 CeO2 on the mucus layer that covered the apical surface of the MucilAir™ cells. Photographs were taken at 0, 1, 3, 6 and 24 h after application of the droplets. The experiment was performed in duplicate. 2.6. Translocation of cerium across 3D model: Chemical analysis of cerium in basal medium of exposed 3D model Total cerium content in the basolateral 3D tissue culture medium was measured by inductively coupled plasma mass spectrometry (ICP-MS, Element 2, Thermo Scientific, Breda, the Netherlands). Cerium concentrations were measured using an external calibration on an inductively coupled plasma sector field mass spectrometer as described before (Limbach et al., 2005; Gosens et al., 2014). 2.7. Statistical analysis For each parameter a model validation was performed using vehicle, MMS or LPS (if MMS was not available). MMS and LPS were excluded for the test model. In all models a one-way ANOVA model was performed on the data with group as fixed factor and experiments as random factor. The residual plots were always inspected. If these plots revealed a low variation for low values of the parameter of interest and a high variation for high values of the parameter of interest, the data were LOG transformed and the ANOVA assumptions were checked again. The transformation was performed on the original data set. Statistical outliers were removed and missing values were excluded from analysis. For all analyses the null hypothesis (no effect of group) was rejected at the 0.5 level of significance. P-values between 0.05 and 0.1 were considered to represent a trend. A Tukey Kramer correction was used to correct for multiple comparisons in the analysis on differences between

groups. The statistical tests were performed in SAS V9.3 Copyright Ó by SAS Institute Inc., Cary, NC, USA. 3. Results 3.1. Characterisation of CeO2 particles

3.1.1. 3D saline The characteristics of CeO2, as provided by the supplier, indicated that the primary particle size in dry powder was 13.8 nm and the specific surface area was 61 m2/g. Surface shape was given as spherical and surface structure as smooth. Their data also indicated that CeO2 particles are hydrophobic in aqueous solutions. Our SEM analysis of the dry powder indicated that particle size was approximately 15 nm. The characteristics of CeO2 in tissue culture medium are summarised in Table 2. Due to sedimentation of the samples in the matrix over time, and the high conductivity of the matrix (saline used for MucilAir™), the obtained particle sizes by DLS are not fully reliable. DLS measurements underestimate the smaller-sized particles in the presence of (a few) large-sized particles. As a result, only the size values are given, and not the calculated average diameter (Z-avg) of the particles with the PDI. SEM and EDX analysis confirmed the results of the DLS measurements that almost all CeO2 was present as agglomerates/aggregates at the high concentration, but in addition, nano-sized particles were observed with a size of about 15 nm. A clear trend in the size of the CeO2 dispersions as a function of concentration was observed. 3.1.2. BEAS-2B and A549 medium (DMEM + FCS) The agglomerate/aggregated particle sizes of CeO2 in the medium were comparable with those found for CeO2 in the 3D saline at doses of 0.66 and 0.20 mg/ml. In the medium there was no effect of dose of CeO2 on the particle size (Table 2). 3.2. Toxicity in 3D model The toxicity data are summarised in Table 3 and the actual data are given in the Supplementary files. 3.2.1. Droplet exposure (3, 24 and 48 h exposure) CeO2 (33, 100 or 333 lg/cm2) did not induce cytotoxicity (TEER, LDH release) in MucilAir™, after 3, 24 or 48 h of exposure (except for a tendency towards increased number of ghost cells in the Comet assay in the 3D models at 3 h exposed to 333 lg/cm2, which suggests that cell death was induced by the high concentration of CeO2). Removal of most of the mucus from the apical surface of the 3D model immediately or 24 h before exposure did not influence the results. Triton X100 consistently induced total cytotoxicity in the first experiments and was therefore not tested in later experiments. CeO2 (30, 100 or 333 lg/cm2) induced no IL-1a, sICAM-1 or TNFa production after 3 or 24 h of exposure. MCP-1 tended to be increased after 3 h exposure to CeO2 at the mid-dose of 100 lg/ cm2, and was significantly increased at the low dose of 30 lg/ cm2. The low dose also induced increased IL-8 at 3 h. The control substance LPS did not induce any effects. CeO2 did not influence HO-1 expression after 3 or 24 h of exposure. Overall, no effect on percentage tail DNA (Comet formation) was observed upon exposure to CeO2 in MucilAir™. However, CeO2 exposure induced a minimal increase in comet formation at the low- and high-dose at 3 h. The positive control MMS induced a distinct comet formation, without affecting TEER and LDH parameters, i.e. in the absence of cytotoxicity.

393

C. Frieke Kuper et al. / Toxicology in Vitro 29 (2015) 389–397 Table 2 Characteristics of cerium oxide (CeO2) in cell culture media. Particle conditions

Concentration (mg/ml)

Peak size (DLS) (nm)A

Zeta potential (mV)B

Conductivity (mS/cm)

3D saline at 37 °C

2.200 0.660 0.200

3012 1968 1174

21.5 26.1 nm

19.7 20.1 nm

DMEM + 1% FCS at 37 °C

0.175 0.100 0.050 0.025

1106 814 1269 1254

12.8 nm nm nm

nm nm nm nm

nm = not measured. A SEM analysis of CeO2 in 3D saline on a filter showed a peak size of around 1000 nm with a cut-off at 2000 nm, and a lower boundary at about 100 nm. PDI of CeO2 in DMEM + FCS ranged from 0.60 to 0.90 for the different doses. B Zeta potential for DMEM + 1% FCS without test substance was 9.5 mV. Zeta potential for CeO2 in DMEM alone was 4.8 mV. Zeta potential for CeO2 in MilliQ water was 31 mV.

3.2.2. Dextran tablet exposure (24 h exposure) CeO2 did not induce cytotoxicity (TEER, LDH), whereas the positive control ZnO induced a distinct decrease in TEER. ZnO exposure led also to an increased presence of ghost cells in the Comet assay, which suggests cell death. The cytokines MCP-1 and IL-8 were slightly increased in the 100 lg/cm2 CeO2 group. CeO2 did not induce comet formation. ZnO was positive in the Comet assay, most probably because of its cytotoxicity. The control MMS was positive in the Comet assay, but did not affect the other parameters. The control LPS was negative for all parameters. 3.3. Toxicity in BEAS-2B and A549 CeO2 has been tested in numerous in vitro systems. We tested CeO2 as used in the 3D airway model in a number of cultures of BEAS-2B and A549 cells to check the conformity of our results with those reported in literature. Exposure of BEAS-2B cells to 33, 100 or 333 lg/cm2 CeO2 did not induce cytotoxicity as measured by LDH release at 24 h exposure, but did induce a dose-related and distinct response in % tail DNA (comet formation). CeO2 induced a two- to threefold induction in HO-1 expression. Exposure of A549 cells to 33, 100 or 333 lg/cm2 CeO2 induced a minimal increase in LDH, albeit only at the mid-dose, and a distinct increase in % tail DNA at all three doses, although a dose–response relationship was absent. No effect on HO-1 expression was observed. CeO2 did not induce an increase in ghost cells in BEAS-2B and A549 cells. 3.4. Effect of respiratory mucus and FCS on CeO2 particle dispersions Dispersions of CeO2 in saline at concentrations of 54 lg/ml and above were distinctly turbid. Mucus (1% and 0.1%) added to the saline dispersions induced instantaneous sedimentation of the dispersed material (Fig. 1). The effect of mucus was dose-dependent (0.1% less so than 1% mucus). The effect was still noticeable after 24 h, because the dispersion in saline without mucus was still slightly turbid above the sediment, whereas the saline/mucus dispersions were clear above the sediment. No effect of mucus was observed with the lower concentrations of CeO2 because sedimentation was hardly seen at all (dispersions were clear). FCS (1%) added to the saline did not have an effect on the sedimentation rate. SEM analysis of the particles in saline or saline with mucus, at 1000 lg/ml showed that the agglomerates/aggregates in saline with mucus were generally larger (Fig. 2). This indicates that respiratory mucus increased agglomeration/aggregation of the particles, thereby accelerating the sedimentation. 3.5. Behaviour of CeO2 particles on the surface of MucilAir™ During the 24 h of exposure of the 3D model to 2 mg/ml CeO2 particles in 50 ll droplets, the grossly visible (at 40 magnification)

part of the CeO2 material was removed from the centre to the borders of the 3D airway models (Fig. 3). At 3 h the material was still relatively well distributed over the entire apical surface; at 6 h most of the material was removed and at 24 h exposure all grossly visible CeO2 aggregates were transferred to the border of the 3D models. MucilAir™ 3D models were washed 24 h before exposure or at 24 h and just before exposure, but this did not influence strongly the clearance of the visible part of the aggregates/agglomerates from the centres of the inserts/models. The 0.02 mg/ml dispersion of CeO2 was not grossly visible on the surface of the 3D model and thus the effect of ciliary beating on the distribution of the particles could not be examined. 3.6. Translocation across 3D model: Analysis of cerium (Ce) in basal medium of MucilAir Minimal amounts of Ce were detected in the basal tissue culture medium of the 3D models (Fig. 4A). Approximately 1 lg/L Ce above background was found in the medium of 3D models exposed for 24 h to 333 lg/cm2 CeO2. When the levels of cerium in the medium were expressed as a percentage of the amount of Ce in the applied dose of CeO2, the dose–response relationship was inverse, namely the highest ratio was observed with the lowest exposure dose (Fig. 4B). 4. Discussion We studied the toxicity of largely aggregated/agglomerated, nano-sized CeO2 particles in a three-dimensional (3D) tissue model of respiratory epithelium, cultured at an air–liquid interface (MucilAir™). CeO2 was applied in saline droplets (with added CaCl2) to the 3D model. The effects of CeO2 on the 3D model were limited to a minimal induction of ghost cells (which suggests dead cells) at the highest dose level of 333 lg/cm2 and increased cytokine MCP-1 production especially at the lower-doses of 30 and 100 lg/cm2 and % tail DNA formation (Comet assay, genotoxicity) at the low- and high-dose. The response upon exposure of CeO2 via dextran tablets did not lead to different effects. Again MCP-1 was increased at the mid-dose, and so was IL-8, but cytotoxicity and genotoxicity was not observed. The positive control LPS for cytokine induction was negative; Grellet et al. (2013) reported that LPS needs stimulants such as TNFa and simian foamy virus (SFV) to induce a cytokine response in this 3D airway model. The limited toxicity in the 3D model was apparently not because of insensitivity of the 3D model, as was shown by the cytotoxicity and genotoxicity of a very low concentration of 9 lg/cm2 of the control particle ZnO in dextran. In vitro toxicity has been reported for CeO2 in cell lines, although CeO2 was not highly toxic (reviewed by Cassee et al., 2011). We found genotoxicity was induced in BEAS-2B and A549 cells

394

C. Frieke Kuper et al. / Toxicology in Vitro 29 (2015) 389–397

Table 3 Summary of results from the toxicity tests. Parameter

lg/cm2

CeO2 3D airway model

MMS 3D airway model

ZnO 3D airway model

3D tabletC

3 lg/cm2

9 lg/cm2

Immersion

Immersion

48 hC

24 h

3h

24 h

24 h

24 h

–

–

– – –

– 4% increase –

–

6-fold

– – –

– – –

12-fold

14-fold

CeO2 3D droplet 3h LDH release

A

24 h

B

33 100

– –

– –

– –

– –

CeO2 BEAS2B

CeO2 A549

333

–

–

–

–

Increase in ghost cells

33 100 333

– – 1.8-fold

– – –

– – –

– – –

Comet: increase in % tail DNAE

33

2-fold@

–

–

–

100 333

– 1.5fold@

– –

– –

– –

5-fold

2-fold

20-fold 22-fold

10-fold 12-fold

TEER

33 100 333

– – –

– – –

– – –

– – –

–

55 % decrease

naD

na

HO-1 expression

33 100 333

– – –

– – –

nmD nm nm

nm nm nm

–

nm

– 2-fold 3-fold

– – –

30

–

nm

–

nm

nm

100

MCP-1 2-fold# IL-8 1.5fold# MCP-1

–

nm

nm

nm

333

2-fold& –

–

nm

MCP-1; IL8 1.5–2-fold –

nm

nm

Increase in cytokines

–

nm

A

3 Independent replicates; 3 inserts or wells/dose/replicate. B 4 Independent replicates; 3 inserts or wells/dose/replicate. C 1 Replicate; 3 inserts or wells/dose. D nm = not measured; na = not applicable. E The positive control MMS (3 h exposure) had 5-fold increase in % tail DNA in the 3D airway model (mean of the medians of three experiments; statistically different from vehicle control); 45-fold increase in BEAS-2B cells; 55-fold increase in A549 cells. MMS did not induce an increase in ghost cells. # p < 0.05. & p < 0.07 (trend), both analyses on log transformed data. @ p < 0.05 analyses on untransformed data.

Fig. 1. Photographs of CeO2 dispersions in 3D saline without (top row) and with (bottom row) 1% respiratory mucus directly after preparation. Concentrations of CeO2 from left to right: 0, 27, 54, 108 and 1000 lg/ml. Almost instantaneous sedimentation of 108 and 1000 lg/ml CeO2 occurred in the presence of saline + mucus.

C. Frieke Kuper et al. / Toxicology in Vitro 29 (2015) 389–397

395

Fig. 2. Scanning electron microscopy (SEM; top line) and element analysis (EDX; bottom line) of CeO2 in dispersions of 3D saline and saline with 0.1% and 1% respiratory mucus (left to right). Larger agglomerates/aggregates occur in the presence of saline and mucus.

(measured as % tail DNA formation) with all three doses of CeO2. ZnO on the other hand induced cytotoxicity, genotoxicity and cytokine production in several cell lines at quite low doses (current study; Sauer et al., 2013; Gilbert et al., 2012; Vandebriel and De Jong, 2012; Hackenberg et al., 2011). The high toxicity of ZnO particles has been ascribed to the dissolution of the particles and their oxidative potential (Xia et al., 2008). The difference in response induced by CeO2 in the monolayer cell lines and the 3D MucilAir™ model may be due to (1) tumour cell lines such as A549 having less intact repair mechanisms; (2) the interaction between respiratory

mucus (3D MucilAir™) or DMEM + FCS (A549 and BEAS 2B) with CeO2 particles most probably differs as shown by the effects on sedimentation rate in our experiments; and (3) the 3D model has a mucociliary system that can prevent or diminish the amount of CeO2 from reaching the underlying epithelium. The effects of CeO2 and ZnO particles on the 3D airway model reflect the local toxicity observed in in vivo studies. CeO2 particles induced toxicity only at the alveolar region, leading to accumulation and aggregation of alveolar macrophages (Cassee et al., 2011; Gosens et al., 2014; Landsiedel et al., 2014), whereas ZnO

Fig. 3. The distribution of grossly visible CeO2 agglomerates/aggregates on the apical surface of the 3D airway models (50 ll of 333 lg CeO2/cm2). Top from left to right: centre of model before, shortly after start of exposure, and at 1 h. Bottom from left to right: centre at 6 h and 24 h, and border of model at 6 h. All grossly visible material was eventually located at the borders of the inserts.

396

C. Frieke Kuper et al. / Toxicology in Vitro 29 (2015) 389–397

A

B

Fig. 4. Translocation of cerium across the 3D model: Analysis of cerium in basal medium of 3D model MucilAir™ by ICP-MS. (A) Actual amount of cerium. (B) Amount of cerium measured in medium as percentage of the amount applied on the apical surface.

particles affected both the alveolar region, leading to increased macrophages, granulocytes and necrosis of olfactory epithelium in the nasal passages and broncho-alveolar hyperplasia (the latter restricted to microscale ZnO) (Vandebriel and De Jong, 2012; Landsiedel et al., 2014). Unfortunately, several in vivo studies do not include a proper examination of a large part of the respiratory epithelium, meaning that effects in these regions may be underestimated. The 3D model possesses a mucociliary apparatus. Mucociliary clearance of inhaled substances by the respiratory epithelium is an important physiological process that protects respiratory surfaces from damage and it reduces transport across the biobarrier. As such, effects may not be observed despite expected deposition (Nahar et al., 2013). Respiratory mucus may increase aggregation and agglomeration of nanoparticles as found with CeO2 at the highest dose. Particle size (agglomerates and aggregates) influences the ability of particles to reach the epithelial cells underneath the mucus and to transport across the respiratory mucosa (Brooking et al., 2001). We found that the particle (agglomerate) size of CeO2 was dose-dependent, with the largest particles observed at the high dose. This may explain why the relative amount of cerium that had passed the 3D tissue layer was low at the high dose compared to the amount of cerium in CeO2 applied on the 3D apical surface. This might also explain why the minimal effects in the 3D model did not show a linear dose–effect relationship, the high dose being almost ineffective. However, characteristics such as surface chemistry, charge and hydrophilicity are also a determinant for transport across the respiratory barrier (Henning et al., 2010; Mura et al., 2011; Kirch et al., 2012). More information with respect to different types of particles is needed to examine the applicability domain of the 3D airway model. In addition, results in the 3D airway model can direct the selection of observations in in vivo studies. In summary, the 3D airway model may more realistically predict local toxicity and systemic availability of inhaled particles than monolayer cell lines, which may overestimate toxicity of particles. The interaction of particles with biomatrices such as mucus appears to play an important factor in toxicity.

Conflict of Interest The authors state that they have no conflicts of interest in relation to this work. The work used an in vitro cell model of the human bronchial epithelium (MucilAir™) that has been developed, produced and sold by Epithelix. The guidance and support of

Epithelix had no influence on the results and the objectivity of the interpretation of the results. Transparency Document The Transparency document associated with this article can be found in the online version.

Acknowledgements This work is supported by NanoNextNL, a micro and nanotechnology programme of the Dutch Government and 130 other partners. The authors gratefully acknowledge Max Bingham ([email protected]) for his editorial assistance with the manuscript prior to submission, and Peter Tromp ([email protected]) and Fariza Bouallala ([email protected]) for their technical assistance. Appendix A. Supplementary material Toxicity data, summarised in Table 3, are presented as supplementary data. Details on characterisation of CeO2 are given as supplementary data as well. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.tiv.2014.10.017. References Brooking, J., Davis, S.S., Illum, L., 2001. Transport of nanoparticles across the rat nasal mucosa. J. Drug Target 9, 267–279. http://dx.doi.org/10.3109/ 10611860108997935. Cassee, F.R., van Balen, E.C., Singh, C., Green, D., Muijser, H., Weinstein, J., Dreher, K., 2011. Exposure, health and ecological effects review of engineered nanoscale cerium and cerium oxide associated with its use as a fuel additive. Crit. Rev. Toxicol. 41, 213–229. http://dx.doi.org/10.3109/10408444.2010.529105. Cho, W.-S., Duffin, R., Bradley, M., Megson, I.L., MacNee, W., Lee, J.K., Jeong, J., Donaldson, K., 2013. Predictive value of in vitro assays depends on the mechanism of toxicity of metal oxide nanoparticles. Part. Fibre Toxicol. 10, 55. http://dx.doi.org/10.1186/1743-8977-10-55. Gilbert, B., Fakra, S.C., Xia, T., Pokhrel, S., Madler, L., Nel, A.E., 2012. The fate of ZnO nanoparticles administered to human bronchial epithelial cells. ACS Nano 6, 4921–4930. http://dx.doi.org/10.1021/nn300425a. Gosens, I., Mathijssen, L.E., Bokkers, B.G., Muijser, H., Cassee, F.R., 2014. Comparative hazard identification of nano- and micro-sized cerium oxide particles based on 28-day inhalation studies in rats. Nanotoxicology 8, 643–653. http://dx.doi.org/ 10.3109/17435390.2013.815814. Gradon, L., Sosnowski, T.R., 2014. Formation of particles for dry powder inhalers. Adv. Powder Technol. 25, 43–55. http://dx.doi.org/10.1016/j.apt.2013.09.012.

C. Frieke Kuper et al. / Toxicology in Vitro 29 (2015) 389–397 Grellet, S., Momas, I., Seta, N., Bardet, G., Achard, S., 2013. Reconstituted human airway epithelium 3D-model to assess the impact of indoor air pollutants on the inflammatory response. Toxicol. Lett. 221, S112. http://dx.doi.org/10.1016/ j.toxlet.2013.05.184. Hackenberg, S., Scherzed, A., Technau, A., Kessler, M., Froehlich, K., Ginzkey, C., Koelher, C., Burghartz, M., Hagen, R., Kleinsasser, N., 2011. Cytotoxic, genotoxic and pro-inflammatory effects of zinc oxide nanoparticles in human nasal mucosa cell in vitro. Toxicol. In Vitro 25, 657–663. http://dx.doi.org/10.1016/ j.tiv.2011.01.003. Henning, A., Schneider, M., Nafee, N., Muijs, L., Rytting, E., Wang, X., Kissel, T., Grafahrend, D., Klee, D., Lehr, C.-M., 2010. Influence of particle size and material properties on mucociliary clearance from the airways. J. Aerosol Med. Pulm. Drug Deliv. 23, 233–241. http://dx.doi.org/10.1089/jamp.2009.0806. Huang, S., Constant, S., Wisniewski, L., Derouette, J.P., 2008. MucilAir: a novel human 3D airway epithelium model for acute or long term toxicity testing of chemicals. ALTEX 25 (Suppl. 1), 30–31 . Huang, S., Wisniewski, L., Constant, S., 2011. The use of in vitro 3D cell models in drug development for respiratory diseases. In: Kapetanovic, I.M. (Ed.), Drug Discovery and Development – Present and Future. InTech – Open Access Company, pp. 169–190, http://dx.doi.org/10.5772/28132. Huang, S., Wisniewskia, L., Constant, S., Roggen, E., 2013. Potential of in vitro reconstituted 3D human airway epithelia (MucilAir™) to assess respiratory sensitizers. Toxicol. In Vitro 27, 1151–1156. http://dx.doi.org/10.1016/ j.tiv.2012.10.010. Kirch, J., Guenther, M., Doshi, N., Schaefer, U.F., Schneider, M., Mitragotri, S., Lehr, C.M., 2012. Mucociliary clearance of micro- and nanoparticles is independent of size, shape and charge – an ex vivo and in silico approach. J. Control Release 159, 128–134. http://dx.doi.org/10.1016/j.jconrel.2011.12.015. Landsiedel, R., Ma-Hock, L., Hofmann, T., Wiemann, M., Strauss, V., Treumann, S., Wohlleben, W., Gröters, S., Wiench, K., van Ravenzwaay, B., 2014. Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials. Part. Fibre Toxicol. 11, 1–26. Limbach, L.K., Li, Y., Grass, R.N., Brunner, T.J., Hintermann, M.A., Muller, M., Gunther, D., Stark, W.J., 2005. Oxide nanoparticle uptake in human lung fibroblasts: effects of particle size, agglomeration, and diffusion at low concentrations. Environ. Sci. Technol. 39, 9370–9376. http://dx.doi.org/10.1021/es051043o. Lippmann, M., Yeates, D.B., Albert, R.E., 1980. Deposition, retention, and clearance of inhaled particles. Br. J. Ind. Med. 37, 337–362. Mura, S., Hillaireau, H., Nicolas, J., Kerdine-Römer, S., Le Droumaguet, B., Deloménie, C., Nicolas, V., Pallardy, M., Tsapis, N., Fattal, E., 2011. Biodegradable nanoparticles meet the bronchial airway barrier: how surface properties

397

affect their interaction with mucus and epithelial cells. Biomacromolecules 12, 4136–4143. http://dx.doi.org/10.1021/bm201226x. Murray, E.P., Tsai, T., Barnett, S.A., 1999. A direct methane fuel cell with ceria based a node. Nature 400, 649–651. Nahar, K., Gupta, N., Gauvin, R., Absar, S., Patel, B., Gupta, V., Khademhosseini, A., Ahsan, F., 2013. In vitro, in vivo and ex vivo models for studying particle deposition and drug absorption of inhaled pharmaceuticals. Eur. J. Pharm. Sci. 49, 805–818. http://dx.doi.org/10.1016/j.ejps.2013.06.004. Nichols, J.E., Niles, J.A., Vega, S., Cortiella, J., 2013. Novel in vitro respiratory models to study lung development, physiology, pathology and toxicology. Stem Cell Res. Ther. 4 (Suppl. 1), S7. http://dx.doi.org/10.1186/scrt368. Oberdörster, G., 2001. Pulmonary effects of inhaled ultrafine particles. Int. Arch. Occup Envrion. Health 74, 1–8. http://dx.doi.org/10.1007/s004200000185. Oberdörster, G., Oberdörster, E., Oberdörster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 113, 823–839. http://dx.doi.org/10.1289/ehp.7339. OECD Guidelines for the Testing of Chemicals, Section 4. Test No. 412: Subacute Inhalation Toxicity: 28-Day Study, http://dx.doi.org/10.1787/9789264070783en. Randell, S.H., Fulcher, M.L., O’Neal, W., Olsen, J.C., 2011. Primary epithelial cell models for cystic fibrosis research. Methods Mol. Biol. 742, 285–310. http:// dx.doi.org/10.1007/978-1-61779-120-8_18. Sauer, U., Vogel, S., Hess, A., Kolle, S.N., Ma-Hock, L., van Ravenzwaay, B., Landsiedel, R., 2013. In vivo–in vitro comparison of acute respiratory tract toxicity using human 3D airway epithelial models and human A549 and murine 3T3 monolayer cell systems. Toxicol. In Vitro 27, 174–190. http://dx.doi.org/ 10.1016/j.tiv.2012.10.007. U.S. Environmental Protection Agency, Washington DC, 2009. Toxicological review of Cerium oxide and Cerium compounds. In support of summary information on the integrated risk information system (IRIS). . Vandebriel, R.J., de Jong, W.H., 2012. A review of mammalian toxicity of ZnO nanoparticles. Nanotechnol. Sci. Appl. 5, 61–71. http://dx.doi.org/10.2147/ NSA.S23932. Xia, T., Kovochich, M., Liong, M., Madlers, L., Gilbert, B., Shi, H., Yeh, J.I., Zink, J.I., Nel, A.E., 2008. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2, 2121–2134. http://dx.doi.org/10.1012/nn800511k. Zheng, X., Zhang, X., Wang, X., Wang, S., Wu, S., 2005. Preparation and characterization of Cu/CeO2 catalysts and their applications in lowtemperature CO oxidation. Appl. Catal. A: Gen. 295, 142–149.