- Email: [email protected]
Toxicity of nanoparticles embedded in paints compared to pristine nanoparticles, in vitro study
Toxicology Letters 232 (2015) 333–339
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Toxicity of nanoparticles embedded in paints compared to pristine nanoparticles, in vitro study Stijn Smulders a , Katrien Luyts a , Gert Brabants b , Luana Golanski c, Johan Martens b , Jeroen Vanoirbeek a , Peter H.M. Hoet a, * a b c
Occupational and Environmental Toxicology, KU Leuven, Leuven, Belgium Centre for Surface Chemistry and Catalysis, KU Leuven, Leuven, Belgium CEA-Grenoble, Liten, 17 Rue Des Martyrs, France
H I G H L I G H T S
Toxic effects of pristine ENPs were compared to those embedded in a paint matrix. The paint matrix was aged, whether or not containing ENPs (TiO2, Ag and SiO2). Toxicity was assessed in a tri-culture model. Pristine ENPs show some toxic effects in our in vitro model. No additional significant toxic effects were observed in paints containing ENPs.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 April 2014 Received in revised form 8 November 2014 Accepted 27 November 2014 Available online 28 November 2014
The unique physicochemical properties of nanomaterials has led to an increased use in the paint and coating industry. In this study, the in vitro toxicity of three pristine ENPs (TiO2, Ag and SiO2), three aged paints containing ENPs (TiO2, Ag and SiO2) and control paints without ENPs were compared. In a first experiment, cytotoxicity was assessed using a biculture consisting of human bronchial epithelial (16HBE14o-) cells and human monocytic cells (THP-1) to determine subtoxic concentrations. In a second experiment, a new coculture model of the lung–blood barrier consisting of 16HBE14o- cells, THP-1 and human lung microvascular endothelial cells (HLMVEC) was used to study pulmonary and extrapulmonary toxicity. The results show that the pristine TiO2 and Ag ENPs have some cytotoxic effects at relative high dose, while pristine SiO2 ENPs and all aged paints with ENPs and control paints do not. In the complex triculture model of the lung–blood barrier, no considerable changes were observed after exposure to subtoxic concentration of the different pristine ENPs and paint particles. In conclusion, we demonstrated that although pristine ENPs show some toxic effects, no significant toxicological effects were observed when they were embedded in a complex paint matrix. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Nanotoxicology Nanoparticles Paint In vitro Cytotoxicity
1. Introduction
* Corresponding author at: Department of Public Health, Occupational, Environmental and Insurance Medicine, Occupational and Environmental Toxicology, Herestraat 49 mailbox 706 B-3000 Leuven, Belgium. Tel.: +32 16 33 01 97; fax: +32 16 33 08 06. E-mail addresses: [email protected] (S. Smulders), [email protected] (K. Luyts), [email protected] (G. Brabants), [email protected] (L. Golanski), [email protected] (J. Martens), [email protected] (J. Vanoirbeek), [email protected] (P.H.M. Hoet). http://dx.doi.org/10.1016/j.toxlet.2014.11.030 0378-4274/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Nanomaterials are increasingly being used in a broad range of applications in the construction industry (Hanus and Harris, 2013). Addition of metal oxide engineered nanoparticles (ENPs) and carbon nanotubes to concrete can improve structural efficiency, durability and strength, while incorporation of nanomaterials in solar cells reduces costs and increases the energy conversion efficiency (Guo, 2011). Lately, extensive research has been done on the development of new coating and paint systems for wood, metals, ceramics, natural stone, concrete, composites and plastics
334
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
(Hanus and Harris, 2013). Incorporation of ENPs give rise to paints and coatings with for example anti-UV (TiO2, ZnO), self-cleaning (TiO2), antimicrobial (Ag, TiO2), fire-resistant (SiO2, CNT) and scratch-resistant (SiO2, CNT) effects (Hanus and Harris, 2013; Lee et al., 2010; Pacheco-Torgal and Jalali, 2011). In order to reduce the prevalence of illness and discomfort, antimicrobial coatings already have applications in hospital environments, childcare centers and nursing homes (Hanus and Harris, 2013). Currently, coatings, paints and pigments are the most important applications of ENPs in terms of overall use (Keller et al., 2013). A study of Mueller and Nowack in 2008 showed that 35% of the nano-Ag production and 25% of the nano-TiO2 production in Europe are used by the paint and coating industry, making this sector the first end-user of nano-Ag and the second end-user of nano-TiO2 (Mueller and Nowack, 2008). Exposure to ENPs used in paints and coatings can occur during production, handling and application of the paint on a surface or after aging. Different inhalation and instillation studies already have shown the infiltration of inflammatory cells, release of pro-inflammatory cytokines and oxidative stress in the lung after exposure to ENPs (Napierska et al., 2010; Shi et al., 2013). Moreover, ENPs have pro-trombotic effects and are able to induce endothelial dysfunction (Vesterdal et al., 2010). Both monoculture and complex coculture in vitro models of the lung are being used or are in development to study respiratory toxicity. An overview on the state-of-the-art of relevant lung cel6l-based in vitro assays, which are currently under development for the evalutation of particles and chemicals can be found in the review paper of Klein et al. (2011). Recently, in our lab, a new in vitro coculture model of the lung–blood barrier was developed and validated by Luyts et al. (2014). The triculture model consists of human bronchial epithelial cells (16HBE14o-) and human monocytic THP-1 cells representing the pulmonary epithelium and macrophages respectively. Human lung microvascular endothelial cells (HLMVEC) were used as a model for the cells of the pulmonary circulation. Epithelial and endothelial cells were grown on opposite sides of a Transwell insert membrane until confluence while the monocytes were added before the experiment. A schematic overview of the model setup can be found in Fig. 1. The combination of the different cell types in this model form a good representation of the lung–blood barrier as it is in vivo. The model can be used to assess the toxicity of ENPs; different parameters can be measured to assess pulmonary and extrapulmonary toxicity including barrier functionality, inflammation, oxidative stress and endothelium activation. Although a lot of research has been performed regarding the toxic effects of pristine ENPs, little is known about their toxicity when they are embedded in a complex paint or coating matrix. In this study, we compared the in vitro toxicity of three pristine ENPs
(TiO2, Ag and SiO2), three aged paints containing ENPs (TiO2, Ag and SiO2) and corresponding control paints without ENPs. In a first experiment, the cytotoxicity of the different particles was assessed using a biculture consisting of 16HBE14o- cells and THP-1 cells. In a second experiment, a coculture model of the lung–blood barrier consisting of 16HBE14o- cells, THP-1 and HLMVEC, developed and validated by Luyts et al. (2014), was used to study pulmonary and extrapulmonary toxicity. 2. Materials and methods 2.1. Materials Pristine ENPs (TiO2, Ag and SiO2), paints containing these ENPs and control paints without ENPs were provided by industrial project partners. Dulbecco’s Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12), Roswell Park Memorial Institute 1640 (RPMI) medium, Hank’s balanced salt solution (HBSS), phosphate buffered saline (PBS), penicillin–streptomycin, fungizone, L-glutamine, fetal calf serum (FCS), and Transwell1 membrane inserts were purchased from Sigma–Aldrich (Bornem, Belgium). Human low density lipoprotein was purchased from Stemcell Technologies (Grenoble, France). 2.2. Manufacturing aged powder paint particles Paints were applied on a plastic panel using a film applicator creating a uniform film of 200 mm thickness. After drying for 24 h at room temperature (20 C), paints were removed manually using a metallic spatula to obtain a powder paint. Then, powders were milled using a planetary ball mill PM 100 (Retsch, Haan, Germany) and finally exposed to UV-A (Philips TL20W/09N) as an aging process. 2.3. Experimental strategy The in vitro cytotoxicity was assessed using a biculture consisting of human bronchial epithelial (16HBE14o-) cells and human monocytic cells (THP-1). The cells were exposed to particles (3-fold serial dilution: 0, 1, 3, 9, 27, 81 and 243 mg/ml) during 24 h. Cytotoxicity was assessed with two different assays: lactate dehydrogenase (LDH) release (Napierska et al., 2009) and WST-1 assay. Subsequently, based on the previous experiment, 2 subtoxic concentrations were determined for each particle that were used in a triculture model of the lung–blood barrier consisting of human bronchial epithelial (16HBE14o-) cells, human monocytic cells (THP-1) and endothelial cells (HLMVEC) (Fig. 1). Different parameters including barrier integrity, inflammation and oxidative stress were investigated. 2.4. Particle preparation All particles were dispersed in Baxter water (stock concentration: 4.86 mg/ml) and sonicated using a MicrosonTM ultrasonic cell disruptor (Misonix, Newtown, USA) during 16 min at 400 W. Subsequently, the particles were diluted 10-fold in the culture medium to obtain final concentrations. 2.5. Cell culture
Fig. 1. In vitro triculture model setup. Triculture model of the lung–blood barrier consisting of human bronchial epithelial (16HBE14o-) cells, human monocytic cells (THP-1) and endothelial cells (HLMVEC).
16HBE14o- cells (16HBE), kindly provided by Dr. Gruenert (University of California, San Fransisco, USA) were cultured in DMEM/F12 medium supplemented with 5% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 2.5 mg/ml fungizone. Human lung microvascular endothelial cells (HLMVEC)
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
were purchased from the European collection of cell cultures (Salisbury, UK) and cultured in microvascular endothelial cell basal medium supplemented with the supplement from the kit, 100 U/ ml penicillin, 100 mg/ml streptomycin and 0.5 mg/ml fungizone. THP-1 cells were cultured in RPMI medium supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 2.5 mg/ml fungizone. The cells were incubated at 37 C, in a 100% humidified atmosphere containing 5% CO2. 2.6. Cytotoxicity 16HBE cells were seeded at a density of 200,000 cells/cm2 in a 48-well plate. After an incubation period of 24 h, THP-1 cells were added in a 1:10 ratio. After 4 h of incubation, the cells were exposed to particles during 24 h. For the LDH assay, after particle incubation, the supernatant was removed, the cells were washed twice with PBS and then lysed using 0.2% Triton-X. The LDH activity of the supernatant (LDHsupernatant) and the cell lysate (LDHcells) were determined by monitoring the reduction of pyruvate spectrophotometrically. Cell viability was calculated according to the formula: %viability = (LDHcells/(LDHcells + LDHsupernatant)) 100. For the WST-1 assay, after particle incubation, the supernatant was removed, the cells were washed and incubated with WST-1 reagent (1:20 diluted) for 2 h. The absorbance was measured spectrophotometrically at 450 nm. 2.7. Triculture model setup A detailed description and validation of the triculture model can be found in the paper of Luyts et al. (2014). HLMVEC were seeded on the lower side (basolateral compartment) of a non-coated Transwell1 insert membrane (0.4 mm pore density and 0.33 cm2 surface area) at a density of 45,500 cells/cm2. After a 2 h incubation period the inserts were returned to their original orientation and afterwards 16HBE cells were seeded on the upper side (apical compartment) of the membrane at a density of 100,000 cells/cm2. When the biculture reaches submaximal transepithelial electrical resistance (TEER) (at day four), THP-1 cells were added to the apical compartment in a 1:10 ratio. The medium of the apical compartment consisted of a mix of 16HBE (9/10) and THP-1 (1/10) medium with 0.2% FBS.
335
concentrations were measured in the supernatant, according to the manufacturer’s instructions. 2.10. ELISA In the culture medium of the apical compartment concentrations of IL-8 and TNF-a were measured using an ELISA (R&D systems, Abingdon, UK) according to the manufacturer’s instructions. In the culture medium of the basolateral compartment concentrations of IL-6 and sICAM (R&D systems) were measured. 2.11. Statistical analysis All results are expressed as mean S.D. Normality distribution was assessed using the D’Agostino and Pearson omnibus normality test. When normally distributed, a one-way ANOVA followed by Dunnett’s multiple-comparison test was performed. When the data were not normally distribusted, the Kruskal–Wallis test followed by a Dunn’s multiple comparison test was performed (Graphpad Prism 4.01, Graphpad Software Inc., San Diego, USA). A level of p < 0.05 was considered significant. 3. Results 3.1. Particle characterization A detailed characterization of all pristine ENPs has been published earlier (Smulders et al., 2012). Transmission electron microscopy (TEM) analysis showed average particle sizes of 15 nm (TiO2), 25–85 nm (Ag), and 19 nm (SiO2). Dynamic light scattering (DLS) analysis showed single populations of 396 nm (TiO2), 90 nm (Ag) and 192 nm (SiO2). Zeta potentials are respectively 25 mV (TiO2), 42 mV (Ag) and 40 (SiO2). An overview of the composition and characterization of the different aged paints and corresponding control paints can be found in the paper of Smulders et al. (2014). Scanning electron microscopy (SEM) images show a very heterogeneous composition, showing both very large (>10 mm) and smaller particles (<1 mm). DLS analysis showed single populations of 313 nm (TiO2 paint), 652 nm (Ag paint) and 530 nm (SiO2 paint). Zeta potentials are respectively 4 mV (TiO2 paint), 4 mV (Ag paint) and 2 mV (SiO2 paint). 3.2. Cytotoxicity
2.8. Triculture exposure Triculture systems were exposed on day 5 in culture. Before the exposures, TEER measurements were made to serve as baseline values. After exposure, TEER values were measured at 6 different time points between 30 min and 24 h after exposure. After 24 h incubation, the experiments were ended and the culture media of the apical and basolateral compartment were stored at 80 C for cytokine measurements. The cells of the apical compartment were used for the measurement of total glutathione. 2.9. Total glutathione measurements Concentrations of total glutathione were measured in the cells of the apical compartment using a (total) glutathione detection kit (Enzo Life Sciences, Antwerpen, Belgium). The cells of the apical compartment were enzymatically removed from the Transwell1 insert membrane, centrifuged (10 min, 300 g, 4 C) and afterwards rinsed in ice cold PBS. The cells were counted, centrifuged and resuspended in 500 ml cold 5% (w/v) metaphosphoric acid per 2 million cells. Afterwards, the cells were sonicated (10 min) and centrifuged (5 min, 12,000 g, 4 C). Finally, total glutathione
The in vitro toxicity of both pristine and aged paint particles was assessed using a biculture consisting of human bronchial epithelial (16HBE14o-) cells and human monocytic cells (THP-1). The results of the in vitro cytotoxic effects of TiO2 ENPs/paints, Ag ENPs/paints and SiO2 ENPs/paints are shown in Fig. 2. Subtle cytotoxicity of pristine TiO2 ENPs was observed at the 2 highest applied concentrations (81 and 243 mg/ml) in the LDH and WST-1 assay. No cytotoxicity was observed neither in the aged paint containing TiO2 ENPs nor in the control paint without TiO2 ENPs. Concerning the pristine Ag ENPs, the LDH assay showed cytotoxicity starting at a concentration of 27 mg/ml. Since Ag ENPs interfered with the spectrophotometric measurements at the highest concentration, no results were obtained for this concentration. Similar results were observed in the WST-1 assay for pristine Ag ENPs, where cytotoxicity started at a concentration of 81 mg/ml. An increased mitochondrial activity was observed at lower concentrations (3, 9 and 27 mg/ml). The aged paint containing Ag ENPs and control paint did not induce cytotoxic effects. Both pristine SiO2 ENPs, paint containing SiO2 ENPs and control paint did not induce cytotoxicity in both assays.
336
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
Fig. 2. Viability of cells 24 h after exposure to pristine ENPs, aged paints containing ENPs or control paints. Bicultures of HBE and THP-1 cells were exposed (24 h) to increasing concentrations (0, 1, 3, 9, 27, 81 and 243 mg/ml) of pristine ENPs or paint particles and viability was assessed with LDH (A) and WST-1 (B) assays. Pristine TiO2 ENPs, aged paint with TiO2 ENPs (P(TiO2)) and control paint without ENPs (P(contr)) (left graphs). Pristine Ag ENPs, aged paint with Ag ENPs (P(Ag)) and control paint without ENPs (P(contr)) (middle graphs). Pristine SiO2 ENPs, aged paint with SiO2 ENPs (P(SiO2)) and control paint without ENPs (P(contr)) (right graphs). Mean + SD. **p<0.01, ***p < 0.001.
3.3. TEER measurements
4. Discussion
Based on the cytotoxicity experiments, 2 subtoxic concentrations (viability >90%) were selected for each particle that were subsequently used in the triculture model. After particle exposure, TEER was measured at different time points up to 24 h after the exposures as a measure of the barrier integrity. Results of TEER of TiO2 ENPs/paints, Ag ENPs/paints and SiO2 ENPs/paints are shown in Fig. 3. None of the tested particles caused significant TEER changes at any of the time points. A non-significant decrease in TEER was observed at the latest time point (24 h) for all particles at the highest tested dose, except for the SiO2 ENPs.
In this study, we showed that the pristine TiO2 and Ag ENPs show some cytotoxic effects at relative high dose, while pristine SiO2 ENPs and all aged paints with ENPs and control paints do not. In the complex triculture model of the lung–blood barrier, no considerable changes were observed after apical exposure to the different pristine ENPs and paint particles. ENPs are increasingly being incorporated in paints and coatings since they can improve vital characteristics such as fire-resistance and self-cleaning properties (Hanus and Harris, 2013; Lee et al., 2010). Besides their beneficial properties, ENPs can be released in the environment as single particles or attached to larger paint particles and can potentially exert harmful effects on human health. Exposure can occur during production, handling and application of the paint on a surface or after aging. The aged paint particles used in our studies were generated after processing the liquid paints. Liquid paints were consecutively applied on a panel, dried, scratched off with a metallic spatula, milled and finally exposed to UV-A as an aging process. In this way, we simulate real-life situations as for example pets or children scratching walls, drilling, exposure during demolition and release after long-term exposure to UV and heat. Very little attention has focused on the release of ENPs from nano-containing paints and nanocomposites (Froggett et al., 2014). Kaegi et al. demonstrated that TiO2 ENPs are detached from new and aged facade paints by natural weather conditions (rain) (Kaegi et al., 2008). Microscopic analysis revealed that most of the TiO2 ENPs are released as aggregates consisting of a few particles embedded in the organic binder of the paint. Similar results were found using a Ag ENP containing paint (Kaegi et al., 2010). Analyses of the water samples obtained after each rain event revealed that more than 30% of the Ag ENPs were released to the environment after one year. Göhler et al. compared the release of particles from
3.4. Cytokine and total glutathione measurements Results of cytokines and total glutathione, measured in the apical and basolateral compartment after exposure to TiO2 ENPs/paints, Ag ENPs/paints or SiO2 ENPs/paints are shown in Tables 1–3 respectively. Total glutathione was measured in the cells of the apical compartment as a measure of oxidative stress 24 h after exposure. Significant decreases of total glutathione were observed after exposure to paint containing SiO2 ENPs at the lowest (81 mg/ml) and highest (243 mg/ml) concentrations applied. The inflammatory cytokines TNF-a and IL-8 were measured in the apical compartment, while IL-6 and sICAM-1 were measured in the basolateral compartment. A decrease of IL-8 was observed after TiO2 ENPs exposure, probably caused by interference of the ENPs on the ELISA. Increases of IL-8 were observed after exposure to the control paint without Ag ENPs (243 mg/ml), paint with SiO2 ENPs (243 mg/ml) and control paint without SiO2 ENPs (81 mg/ml and 243 mg/ml). An increase of sICAM was observed after exposure to control paint without Ag ENPs (243 mg/ml).
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
337
Fig. 3. TEER measurements of tricultures exposed to pristine ENPs, aged paints containing ENPs or control paints. Tricultures of HBE, THP-1 and HLMVEC cells were exposed to pristine ENPs or aged paint particles and transepithelial resistance (TEER) was measured at different time points up to 24 h. Pristine TiO2 ENPs, aged paint with TiO2 ENPs (P(TiO2)) and control paint without ENPs (P(contr)) (left graphs). Pristine Ag ENPs, aged paint with Ag ENPs (P(Ag)) and control paint without ENPs (P(contr)) (middle graphs). Pristine SiO2 ENPs, aged paint with SiO2 ENPs (P(SiO2)) and control paint without ENPs (P(contr)) (right graphs). The TEER values are expressed as percentages compared to the baseline value. n = 5–6.
paints with ZnO ENPs and control paints without ENPs after sanding (Gohler et al., 2010). They showed a considerable generation of ENPs during the sanding process, however, no differences were found between ENP-containing paints and control paints. Furthermore, they demonstrated that the generated particles are predominantly made up from matrix material, which contain the embedded ZnO ENPs. Similar results were obtained by Koponen et al., they observed no significant differences in the size distributions of dust released by sanding between paints with and without ENPs (Koponen et al., 2011). Nowadays, many studies have been published concerning the toxic effects of ENPs, while data about toxicity when incorporated
in a complex paint matrix is lacking. Only a few studies have been performed investigating the in vitro toxicity of ENPs-containing paints. In this study, we used a biculture consisting of human bronchial epithelial cells and monocytes to evaluate cytotoxicity of pristine ENPs and aged paints containing ENPs. We showed that the pristine TiO2 and Ag ENPs have some cytotoxic effects at relative high dose, while pristine SiO2 ENPs and all aged paints with ENPs and control paints do not. Similar results were obtained by Kaiser et al., they studied the cytotoxic effects of the same pristine ENPs and aged paint particles as used in our study on gastrointestinal cells (CaCo-2) and immune system cells (Jurkat) (Kaiser et al., 2013). They showed that only pristine Ag ENPs had
Table 1 Total glutathione and cytokine concentrations measured after exposure of the tricultures to pristine TiO2 ENPs, aged paint with TiO2 ENPs (P(TiO2)) or control paint without ENPs (P(contr)). Total glutathione concentrations were measured in the cells of the apical compartment. TNF-a and IL-8 concentrations were measured in the supernatant of the apical compartment whereas IL-6 and sICAM-1 concentrations were measured in the supernatant of the basolateral compartment. n = 5–6. Apical
Control 27 mg/ml TiO2 ENPs 81 mg/ml TiO2 ENPs 81 mg/ml P(TiO2) 243 mg/ml P(TiO2) 81 mg/ml P(contr) 243 mg/ml P(contr) *
p < 0.05 compared to control.
Basolateral
Total glutathione (pmol)
TNF-a (pg/ml)
335 72 310 122 306 40 294 45 315 46 374 89 268 74
133 211 131 66 63 103 33
227 404 113 75 74 72 16
IL-8 (pg/ml) 1177 951 498 1330 1383 1291 1408
IL-6 (pg/ml) 278 293 99* 357 246 156 111
620 605 641 648 665 597 627
65 95 143 125 134 113 90
sICAM-1 (ng/ml) 1.47 1.49 1.29 1.84 1.54 1.41 1.54
0.24 0.29 0.24 0.24 0.40 0.57 0.14
338
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
Table 2 Total glutathione and cytokine concentrations measured after exposure of the tricultures to pristine Ag ENPs, aged paint with Ag ENPs (P(Ag)) or control paint without ENPs (P (contr)). Total glutathione concentrations were measured in the cells of the apical compartment. TNF-a and IL-8 concentrations were measured in the supernatant of the apical compartment whereas IL-6 and sICAM-1 concentrations were measured in the supernatant of the basolateral compartment. n = 5–6. Basolateral
Apical
Control 9 mg/ml Ag ENPs 27 mg/ml Ag ENPs 81 mg/ml P(Ag) 243 mg/ml P(Ag) 81 mg/ml P(contr) 243 mg/ml P(contr) *
Total glutathione (pmol)
TNF-a (pg/ml)
335 72 336 53 311 77 298 61 275 85 354 121 279 46
133 30 42 92 91 48 69
IL-8 (pg/ml)
227 40 61 58 69 24 59
1177 1224 1189 1404 1447 1623 1663
IL-6 (pg/ml) 278 196 178 354 308 346 260*
620 540 557 590 602 631 685
sICAM-1 (ng/ml)
65 127 144 71 49 100 111
1.47 1.34 1.49 1.39 1.58 1.28 1.70
0.24 0.43 0.17 0.29 0.19 0.22 0.25*
p < 0.05 compared to control.
significant cytotoxic effects on both cell types, while no cytotoxicity was observed after exposure to the other pristine ENPs (TiO2 and SiO2) and all aged paint particles with ENPs. Furthermore, Saber et al. studied the toxicity of paint dusts with different ENPs (TiO2, CB or SiO2) using a monoculture of FE1-Muta Mouse Lung (MML) epithelial cells (Saber et al., 2011). For all dusts, the cell death was below 25% at a concentration of 200 mg/ml. Mikkelsen et al. showed that sanding dust from ENP-containing paint did not generate more oxidative stress or expression of cell adhesion molecules in human umbilical vein endothelial cells than sanding dust from paint without ENPs (Mikkelsen et al., 2013). Although ENP exposure can occur via different routes (inhalation, ingestion, dermal and injection), inhalation is considered to be the major route of exposure for ENPs (Oberdorster et al., 2005). Epidemiological studies show a high deposition efficiency of ultrafine particles in the total respiratory tract of healthy subjects, from the upper airways and bronchi to the alveoli, due to diffusion. After inhalation, the particles will come in contact with the highly differentiated airway/alveolar epithelium and macrophages. The lung epithelium is separated from the subepithelial connective tissue containing the blood and lymphatic vessels by a basement membrane (Geiser and Kreyling, 2010). The inner surface of the lung functions as a physical, biochemical and immunological barrier, separating the outside from inside. We used a complex in vitro model of the lung–blood barrier consisting of human epithelial cells, monocytes and endothelial cells. This model is a good representation as it is in vivo and allows us to study the toxicity of (nano) particles (and chemicals). Different endpoints were measured to study pulmonary and extrapulmonary toxicity. Similar models are already used in nanotoxicology research; Snyder-Talkington et al. studied endothelial effects after epithelial exposure to MWCNT using a
biculture of human small airway epithelial cells (SAEC) and human microvascular endothelial cells (HMVEC) (Snyder-Talkington et al., 2013). Farcal et al. used a coculture of epithelial human cell line NCI-H441 with endothelial human cell line ISO-HAS1 to evaluate the toxic effects of SiO2 ENPs, in the presence or absence of THP-1 cells (monocytes) (Farcal et al., 2013). In our study, both paint particles and pristine ENPs had no effect on the TEER values, indicating that none of the tested particles influences the barrier integrity/functionality. Oxidative stress was assessed measuring total glutathione in the epithelial cells and monocytes in the basolateral compartment. Only the aged paint containing SiO2 ENPs showed a decrease in total glutathione, while all other tested particles had no effects on total glutathione. Since the paint containing SiO2 caused no general changes in TEER and inflammatory cytokines, we assume this decrease in total glutathione is a chance finding. The pro-inflammatory cytokines TNF-a and IL-8 were measured in the apical compartment, while IL-6 was measured in the basolateral compartment. IL-8, predominantly produced by macrophages and moncoytes, is a chemokine responsible for the recruitment of neutrophils, and thus a main regulator of the acute inflammatory response (Remick, 2005). IL-6 is produced by a wide variety of cells including endothelial cells and is an important marker of inflammation (Rincon, 2012). No relevant changes in any of the measured cytokines were observed in our study. Similarly, no considerable effects were seen on the levels of sICAM, a biomarker of endothelial activation and inflammation. In a previous study, we compared the in vivo toxicity of ENPs embedded in paints to pristine ENPs (Smulders et al., 2014), using the same particles as in the in vitro study. Mice were exposed by oropharyngeal aspiration and local (lung), systemic inflammation and body distribution was evaluated. We showed that the pristine
Table 3 Total glutathione and cytokine concentrations measured after exposure of the tricultures to pristine SiO2 ENPs, aged paint with SiO2 ENPs (P(SiO2)) or control paint without ENPs (P(contr)). Total glutathione concentrations were measured in the cells of the apical compartment. TNF-a and IL-8 concentrations were measured in the supernatant of the apical compartment whereas IL-6 and sICAM-1 concentrations were measured in the supernatant of the basolateral compartment. n = 5–6. Basolateral
Apical
Control 81 mg/ml SiO2 ENPs 243 mg/ml SiO2 ENPs 81 mg/ml P(SiO2) 243 mg/ml P(SiO2) 81 mg/ml P(contr) 243 mg/ml P(contr) * **
p < 0.05 compared to control. p < 0.01 compared to control. p < 0.001 compared to control.
***
Total glutathione (pmol)
TNF-a (pg/ml)
335 72 355 73 304 125 250 35* 246 69* 347 85 319 49
133 127 76 220 67 41 141
227 256 72 463 69 35 109
IL-8 (pg/ml) 1177 1236 1309 1491 2257 1701 2241
IL-6 (pg/ml) 278 112 303 290 553** 269* 517***
620 665 598 673 699 658 639
65 101 115 74 49 91 84
sICAM-1 (ng/ml) 1.47 1.44 1.51 1.36 1.55 1.56 1.67
0.24 0.22 0.26 0.29 0.20 0.14 0.25
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
ENPs had a subtle toxic effect in the lungs and no effects on the blood parameters (systemic effects). The most pronounced effects were observed with the pristine Ag ENPs in the lung; increase in total BAL cells and neutrophils, 2-fold increase in pro-inflammatory cytokines KC and IL-1b. The paints containing ENPs did not show significant toxicity. The results of the in vivo study correlate very well with the in vitro results. The pristine Ag ENPs showed in both studies the most pronounced effects, while the aged paints containing ENPs had no toxic effects at all. In another in vivo study conducted by Saber et al., mice were exposed to paint dusts with different ENPs and several toxic parameters were analyzed (Saber et al., 2011, 2012). They did not observe any changes in BAL cells and inflammatory cytokines and concluded that the level of pulmonary inflammation in mice exposed to sanding dusts was not affected by the addition of ENPs to the paint. In conclusion, we demonstrated that although pristine ENPs show some toxic effects, no significant toxicological effects were observed when they were embedded in a complex paint matrix. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgement The work is financially supported by the Seventh Framework Program of the European Commission NanoHouse-Grant (Agreement No. 207816). 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.2014.11.030 References Hanus, M.J., Harris, A.T., 2013. Nanotechnology innovations for the construction industry. Prog. Mater. Sci. 58, 1056–1102. Guo, K.W., 2011. Green nanotechnology of trends in future energy. Recent Patents Nanotechnol. 5, 76–88. Lee, J., Mahendra, S., Alvarez, P.J.J., 2010. Nanomaterials in the construction industry: a review of their applications and environmental health and safety considerations. ACS Nano 4, 3580–3590. Pacheco-Torgal, F., Jalali, S., 2011. Nanotechnology advantages and drawbacks in the field of construction and building materials. Constr. Build. Mater. 25, 582–590. Keller, A.A., McFerran, S., Lazareva, A., Suh, S., 2013. Global life cycle releases of engineered nanomaterials. J. Nanopart. Res. 15. Mueller, N.C., Nowack, B., 2008. Exposure modeling of engineered nanoparticles in the environment. Environ. Sci. Technol. 42, 4447–4453. Napierska, D., Thomassen, L.C., Lison, D., Martens, J.A., Hoet, P.H., 2010. The nanosilica hazard: another variable entity. Part. Fibre Toxicol. 7, 39. Shi, H., Magaye, R., Castranova, V., Zhao, J., 2013. Titanium dioxide nanoparticles: a review of current toxicological data. Part. Fibre Toxicol. 10, 15.
339
Vesterdal, L.K., Folkmann, J.K., Jacobsen, N.R., Sheykhzade, M., Wallin, H., Loft, S., Moller, P., 2010. Pulmonary exposure to carbon black nanoparticles and vascular effects. Part. Fibre Toxicol. 7, 33. Klein, S.G., Hennen, J., Serchi, T., Blomeke, B., Gutleb, A.C., 2011. Potential of coculture in vitro models to study inflammatory and sensitizing effects of particles on the lung. Toxicol. In Vitro 25, 1516–1534. Luyts, K., Napierska, D., Dinsdale, D., Klein, S.G., Serchi, T., Hoet, P.H.M., 2014. A coculture model of the lung–blood barrier: the role of activated phagocytic cells. Toxicol. In Vitro Available online 4 November. Napierska, D., Thomassen, L.C., Rabolli, V., Lison, D., Gonzalez, L., Kirsch-Volders, M., Martens, J.A., Hoet, P.H., 2009. Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells. Small 5, 846–853. Smulders, S., Kaiser, J.P., Zuin, S., Van Landuyt, K.L., Golanski, L., Vanoirbeek, J., Wick, P., Hoet, P.H., 2012. Contamination of nanoparticles by endotoxin: evaluation of different test methods. Part. Fibre Toxicol. 9, 41. Smulders, S., Luyts, K., Brabants, G., Landuyt, K.V., Kirschhock, C., Smolders, E., Golanski, L., Vanoirbeek, J., Hoet, P.H., 2014. Toxicity of nanoparticles embedded in paints compared with pristine nanoparticles in mice. Toxicol. Sci. 141, 132–140. Froggett, S.J., Clancy, S.F., Boverhof, D.R., Canady, R.A., 2014. A review and perspective of existing research on the release of nanomaterials from solid nanocomposites. Part. Fibre Toxicol. 11, 17. Kaegi, R., Ulrich, A., Sinnet, B., Vonbank, R., Wichser, A., Zuleeg, S., Simmler, H., Brunner, S., Vonmont, H., Burkhardt, M., Boller, M., 2008. Synthetic TiO2 nanoparticle emission from exterior facades into the aquatic environment. Environ. Pollut. 156, 233–239. Kaegi, R., Sinnet, B., Zuleeg, S., Hagendorfer, H., Mueller, E., Vonbank, R., Boller, M., Burkhardt, M., 2010. Release of silver nanoparticles from outdoor facades. Environ. Pollut. 158, 2900–2905. Gohler, D., Stintz, M., Hillemann, L., Vorbau, M., 2010. Characterization of nanoparticle release from surface coatings by the simulation of a sanding process. Ann. Occup. Hyg. 54, 615–624. Koponen, I.K., Jensen, K.A., Schneider, T., 2011. Comparison of dust released from sanding conventional and nanoparticle-doped wall and wood coatings. J. Exposure Sci. Environ. Epidemiol. 21, 408–418. Kaiser, J.P., Roesslein, M., Diener, L., Wick, P., 2013. Human health risk of ingested nanoparticles that are added as multifunctional agents to paints: an in vitro study. PLoS One 8, e83215. Saber, A.T., Koponen, I.K., Jensen, K.A., Jacobsen, N.R., Mikkelsen, L., Moller, P., Loft, S., Vogel, U., Wallin, H., 2011. Inflammatory and genotoxic effects of sanding dust generated from nanoparticle-containing paints and lacquers. Nanotoxicology . Mikkelsen, L., Jensen, K.A., Koponen, I.K., Saber, A.T., Wallin, H., Loft, S., Vogel, U., Moller, P., 2013. Cytotoxicity, oxidative stress and expression of adhesion molecules in human umbilical vein endothelial cells exposed to dust from paints with or without nanoparticles. Nanotoxicology 7, 117–134. Oberdorster, G., Oberdorster, E., Oberdorster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 113, 823–839. Geiser, M., Kreyling, W.G., 2010. Deposition and biokinetics of inhaled nanoparticles. Part. Fibre Toxicol. 7, 2. Snyder-Talkington, B.N., Schwegler-Berry, D., Castranova, V., Qian, Y., Guo, N.L., 2013. Multi-walled carbon nanotubes induce human microvascular endothelial cellular effects in an alveolar-capillary co-culture with small airway epithelial cells. Part. Fibre Toxicol. 10, 35. Farcal, L.R., Uboldi, C., Mehn, D., Giudetti, G., Nativo, P., Ponti, J., Gilliland, D., Rossi, F., Bal-Price, A., 2013. Mechanisms of toxicity induced by SiO2 nanoparticles of in vitro human alveolar barrier: effects on cytokine production, oxidative stress induction, surfactant proteins A mRNA expression and nanoparticles uptake. Nanotoxicology 7, 1095–1110. Remick, D.G., 2005. Interleukin-8. Crit. Care Med. 33, S466–S467. Rincon, M., 2012. Interleukin-6: from an inflammatory marker to a target for inflammatory diseases. Trends Immunol. 33, 571–577. Saber, A.T., Jacobsen, N.R., Mortensen, A., Szarek, J., Jackson, P., Madsen, A.M., Jensen, K.A., Koponen, I.K., Brunborg, G., Gutzkow, K.B., Vogel, U., Wallin, H., 2012. Nanotitanium dioxide toxicity in mouse lung is reduced in sanding dust from paint. Part. Fibre Toxicol. 9, 4.
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Toxicity of nanoparticles embedded in paints compared to pristine nanoparticles, in vitro study Stijn Smulders a , Katrien Luyts a , Gert Brabants b , Luana Golanski c, Johan Martens b , Jeroen Vanoirbeek a , Peter H.M. Hoet a, * a b c
Occupational and Environmental Toxicology, KU Leuven, Leuven, Belgium Centre for Surface Chemistry and Catalysis, KU Leuven, Leuven, Belgium CEA-Grenoble, Liten, 17 Rue Des Martyrs, France
H I G H L I G H T S
Toxic effects of pristine ENPs were compared to those embedded in a paint matrix. The paint matrix was aged, whether or not containing ENPs (TiO2, Ag and SiO2). Toxicity was assessed in a tri-culture model. Pristine ENPs show some toxic effects in our in vitro model. No additional significant toxic effects were observed in paints containing ENPs.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 April 2014 Received in revised form 8 November 2014 Accepted 27 November 2014 Available online 28 November 2014
The unique physicochemical properties of nanomaterials has led to an increased use in the paint and coating industry. In this study, the in vitro toxicity of three pristine ENPs (TiO2, Ag and SiO2), three aged paints containing ENPs (TiO2, Ag and SiO2) and control paints without ENPs were compared. In a first experiment, cytotoxicity was assessed using a biculture consisting of human bronchial epithelial (16HBE14o-) cells and human monocytic cells (THP-1) to determine subtoxic concentrations. In a second experiment, a new coculture model of the lung–blood barrier consisting of 16HBE14o- cells, THP-1 and human lung microvascular endothelial cells (HLMVEC) was used to study pulmonary and extrapulmonary toxicity. The results show that the pristine TiO2 and Ag ENPs have some cytotoxic effects at relative high dose, while pristine SiO2 ENPs and all aged paints with ENPs and control paints do not. In the complex triculture model of the lung–blood barrier, no considerable changes were observed after exposure to subtoxic concentration of the different pristine ENPs and paint particles. In conclusion, we demonstrated that although pristine ENPs show some toxic effects, no significant toxicological effects were observed when they were embedded in a complex paint matrix. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Nanotoxicology Nanoparticles Paint In vitro Cytotoxicity
1. Introduction
* Corresponding author at: Department of Public Health, Occupational, Environmental and Insurance Medicine, Occupational and Environmental Toxicology, Herestraat 49 mailbox 706 B-3000 Leuven, Belgium. Tel.: +32 16 33 01 97; fax: +32 16 33 08 06. E-mail addresses: [email protected] (S. Smulders), [email protected] (K. Luyts), [email protected] (G. Brabants), [email protected] (L. Golanski), [email protected] (J. Martens), [email protected] (J. Vanoirbeek), [email protected] (P.H.M. Hoet). http://dx.doi.org/10.1016/j.toxlet.2014.11.030 0378-4274/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Nanomaterials are increasingly being used in a broad range of applications in the construction industry (Hanus and Harris, 2013). Addition of metal oxide engineered nanoparticles (ENPs) and carbon nanotubes to concrete can improve structural efficiency, durability and strength, while incorporation of nanomaterials in solar cells reduces costs and increases the energy conversion efficiency (Guo, 2011). Lately, extensive research has been done on the development of new coating and paint systems for wood, metals, ceramics, natural stone, concrete, composites and plastics
334
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
(Hanus and Harris, 2013). Incorporation of ENPs give rise to paints and coatings with for example anti-UV (TiO2, ZnO), self-cleaning (TiO2), antimicrobial (Ag, TiO2), fire-resistant (SiO2, CNT) and scratch-resistant (SiO2, CNT) effects (Hanus and Harris, 2013; Lee et al., 2010; Pacheco-Torgal and Jalali, 2011). In order to reduce the prevalence of illness and discomfort, antimicrobial coatings already have applications in hospital environments, childcare centers and nursing homes (Hanus and Harris, 2013). Currently, coatings, paints and pigments are the most important applications of ENPs in terms of overall use (Keller et al., 2013). A study of Mueller and Nowack in 2008 showed that 35% of the nano-Ag production and 25% of the nano-TiO2 production in Europe are used by the paint and coating industry, making this sector the first end-user of nano-Ag and the second end-user of nano-TiO2 (Mueller and Nowack, 2008). Exposure to ENPs used in paints and coatings can occur during production, handling and application of the paint on a surface or after aging. Different inhalation and instillation studies already have shown the infiltration of inflammatory cells, release of pro-inflammatory cytokines and oxidative stress in the lung after exposure to ENPs (Napierska et al., 2010; Shi et al., 2013). Moreover, ENPs have pro-trombotic effects and are able to induce endothelial dysfunction (Vesterdal et al., 2010). Both monoculture and complex coculture in vitro models of the lung are being used or are in development to study respiratory toxicity. An overview on the state-of-the-art of relevant lung cel6l-based in vitro assays, which are currently under development for the evalutation of particles and chemicals can be found in the review paper of Klein et al. (2011). Recently, in our lab, a new in vitro coculture model of the lung–blood barrier was developed and validated by Luyts et al. (2014). The triculture model consists of human bronchial epithelial cells (16HBE14o-) and human monocytic THP-1 cells representing the pulmonary epithelium and macrophages respectively. Human lung microvascular endothelial cells (HLMVEC) were used as a model for the cells of the pulmonary circulation. Epithelial and endothelial cells were grown on opposite sides of a Transwell insert membrane until confluence while the monocytes were added before the experiment. A schematic overview of the model setup can be found in Fig. 1. The combination of the different cell types in this model form a good representation of the lung–blood barrier as it is in vivo. The model can be used to assess the toxicity of ENPs; different parameters can be measured to assess pulmonary and extrapulmonary toxicity including barrier functionality, inflammation, oxidative stress and endothelium activation. Although a lot of research has been performed regarding the toxic effects of pristine ENPs, little is known about their toxicity when they are embedded in a complex paint or coating matrix. In this study, we compared the in vitro toxicity of three pristine ENPs
(TiO2, Ag and SiO2), three aged paints containing ENPs (TiO2, Ag and SiO2) and corresponding control paints without ENPs. In a first experiment, the cytotoxicity of the different particles was assessed using a biculture consisting of 16HBE14o- cells and THP-1 cells. In a second experiment, a coculture model of the lung–blood barrier consisting of 16HBE14o- cells, THP-1 and HLMVEC, developed and validated by Luyts et al. (2014), was used to study pulmonary and extrapulmonary toxicity. 2. Materials and methods 2.1. Materials Pristine ENPs (TiO2, Ag and SiO2), paints containing these ENPs and control paints without ENPs were provided by industrial project partners. Dulbecco’s Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12), Roswell Park Memorial Institute 1640 (RPMI) medium, Hank’s balanced salt solution (HBSS), phosphate buffered saline (PBS), penicillin–streptomycin, fungizone, L-glutamine, fetal calf serum (FCS), and Transwell1 membrane inserts were purchased from Sigma–Aldrich (Bornem, Belgium). Human low density lipoprotein was purchased from Stemcell Technologies (Grenoble, France). 2.2. Manufacturing aged powder paint particles Paints were applied on a plastic panel using a film applicator creating a uniform film of 200 mm thickness. After drying for 24 h at room temperature (20 C), paints were removed manually using a metallic spatula to obtain a powder paint. Then, powders were milled using a planetary ball mill PM 100 (Retsch, Haan, Germany) and finally exposed to UV-A (Philips TL20W/09N) as an aging process. 2.3. Experimental strategy The in vitro cytotoxicity was assessed using a biculture consisting of human bronchial epithelial (16HBE14o-) cells and human monocytic cells (THP-1). The cells were exposed to particles (3-fold serial dilution: 0, 1, 3, 9, 27, 81 and 243 mg/ml) during 24 h. Cytotoxicity was assessed with two different assays: lactate dehydrogenase (LDH) release (Napierska et al., 2009) and WST-1 assay. Subsequently, based on the previous experiment, 2 subtoxic concentrations were determined for each particle that were used in a triculture model of the lung–blood barrier consisting of human bronchial epithelial (16HBE14o-) cells, human monocytic cells (THP-1) and endothelial cells (HLMVEC) (Fig. 1). Different parameters including barrier integrity, inflammation and oxidative stress were investigated. 2.4. Particle preparation All particles were dispersed in Baxter water (stock concentration: 4.86 mg/ml) and sonicated using a MicrosonTM ultrasonic cell disruptor (Misonix, Newtown, USA) during 16 min at 400 W. Subsequently, the particles were diluted 10-fold in the culture medium to obtain final concentrations. 2.5. Cell culture
Fig. 1. In vitro triculture model setup. Triculture model of the lung–blood barrier consisting of human bronchial epithelial (16HBE14o-) cells, human monocytic cells (THP-1) and endothelial cells (HLMVEC).
16HBE14o- cells (16HBE), kindly provided by Dr. Gruenert (University of California, San Fransisco, USA) were cultured in DMEM/F12 medium supplemented with 5% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 2.5 mg/ml fungizone. Human lung microvascular endothelial cells (HLMVEC)
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
were purchased from the European collection of cell cultures (Salisbury, UK) and cultured in microvascular endothelial cell basal medium supplemented with the supplement from the kit, 100 U/ ml penicillin, 100 mg/ml streptomycin and 0.5 mg/ml fungizone. THP-1 cells were cultured in RPMI medium supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 2.5 mg/ml fungizone. The cells were incubated at 37 C, in a 100% humidified atmosphere containing 5% CO2. 2.6. Cytotoxicity 16HBE cells were seeded at a density of 200,000 cells/cm2 in a 48-well plate. After an incubation period of 24 h, THP-1 cells were added in a 1:10 ratio. After 4 h of incubation, the cells were exposed to particles during 24 h. For the LDH assay, after particle incubation, the supernatant was removed, the cells were washed twice with PBS and then lysed using 0.2% Triton-X. The LDH activity of the supernatant (LDHsupernatant) and the cell lysate (LDHcells) were determined by monitoring the reduction of pyruvate spectrophotometrically. Cell viability was calculated according to the formula: %viability = (LDHcells/(LDHcells + LDHsupernatant)) 100. For the WST-1 assay, after particle incubation, the supernatant was removed, the cells were washed and incubated with WST-1 reagent (1:20 diluted) for 2 h. The absorbance was measured spectrophotometrically at 450 nm. 2.7. Triculture model setup A detailed description and validation of the triculture model can be found in the paper of Luyts et al. (2014). HLMVEC were seeded on the lower side (basolateral compartment) of a non-coated Transwell1 insert membrane (0.4 mm pore density and 0.33 cm2 surface area) at a density of 45,500 cells/cm2. After a 2 h incubation period the inserts were returned to their original orientation and afterwards 16HBE cells were seeded on the upper side (apical compartment) of the membrane at a density of 100,000 cells/cm2. When the biculture reaches submaximal transepithelial electrical resistance (TEER) (at day four), THP-1 cells were added to the apical compartment in a 1:10 ratio. The medium of the apical compartment consisted of a mix of 16HBE (9/10) and THP-1 (1/10) medium with 0.2% FBS.
335
concentrations were measured in the supernatant, according to the manufacturer’s instructions. 2.10. ELISA In the culture medium of the apical compartment concentrations of IL-8 and TNF-a were measured using an ELISA (R&D systems, Abingdon, UK) according to the manufacturer’s instructions. In the culture medium of the basolateral compartment concentrations of IL-6 and sICAM (R&D systems) were measured. 2.11. Statistical analysis All results are expressed as mean S.D. Normality distribution was assessed using the D’Agostino and Pearson omnibus normality test. When normally distributed, a one-way ANOVA followed by Dunnett’s multiple-comparison test was performed. When the data were not normally distribusted, the Kruskal–Wallis test followed by a Dunn’s multiple comparison test was performed (Graphpad Prism 4.01, Graphpad Software Inc., San Diego, USA). A level of p < 0.05 was considered significant. 3. Results 3.1. Particle characterization A detailed characterization of all pristine ENPs has been published earlier (Smulders et al., 2012). Transmission electron microscopy (TEM) analysis showed average particle sizes of 15 nm (TiO2), 25–85 nm (Ag), and 19 nm (SiO2). Dynamic light scattering (DLS) analysis showed single populations of 396 nm (TiO2), 90 nm (Ag) and 192 nm (SiO2). Zeta potentials are respectively 25 mV (TiO2), 42 mV (Ag) and 40 (SiO2). An overview of the composition and characterization of the different aged paints and corresponding control paints can be found in the paper of Smulders et al. (2014). Scanning electron microscopy (SEM) images show a very heterogeneous composition, showing both very large (>10 mm) and smaller particles (<1 mm). DLS analysis showed single populations of 313 nm (TiO2 paint), 652 nm (Ag paint) and 530 nm (SiO2 paint). Zeta potentials are respectively 4 mV (TiO2 paint), 4 mV (Ag paint) and 2 mV (SiO2 paint). 3.2. Cytotoxicity
2.8. Triculture exposure Triculture systems were exposed on day 5 in culture. Before the exposures, TEER measurements were made to serve as baseline values. After exposure, TEER values were measured at 6 different time points between 30 min and 24 h after exposure. After 24 h incubation, the experiments were ended and the culture media of the apical and basolateral compartment were stored at 80 C for cytokine measurements. The cells of the apical compartment were used for the measurement of total glutathione. 2.9. Total glutathione measurements Concentrations of total glutathione were measured in the cells of the apical compartment using a (total) glutathione detection kit (Enzo Life Sciences, Antwerpen, Belgium). The cells of the apical compartment were enzymatically removed from the Transwell1 insert membrane, centrifuged (10 min, 300 g, 4 C) and afterwards rinsed in ice cold PBS. The cells were counted, centrifuged and resuspended in 500 ml cold 5% (w/v) metaphosphoric acid per 2 million cells. Afterwards, the cells were sonicated (10 min) and centrifuged (5 min, 12,000 g, 4 C). Finally, total glutathione
The in vitro toxicity of both pristine and aged paint particles was assessed using a biculture consisting of human bronchial epithelial (16HBE14o-) cells and human monocytic cells (THP-1). The results of the in vitro cytotoxic effects of TiO2 ENPs/paints, Ag ENPs/paints and SiO2 ENPs/paints are shown in Fig. 2. Subtle cytotoxicity of pristine TiO2 ENPs was observed at the 2 highest applied concentrations (81 and 243 mg/ml) in the LDH and WST-1 assay. No cytotoxicity was observed neither in the aged paint containing TiO2 ENPs nor in the control paint without TiO2 ENPs. Concerning the pristine Ag ENPs, the LDH assay showed cytotoxicity starting at a concentration of 27 mg/ml. Since Ag ENPs interfered with the spectrophotometric measurements at the highest concentration, no results were obtained for this concentration. Similar results were observed in the WST-1 assay for pristine Ag ENPs, where cytotoxicity started at a concentration of 81 mg/ml. An increased mitochondrial activity was observed at lower concentrations (3, 9 and 27 mg/ml). The aged paint containing Ag ENPs and control paint did not induce cytotoxic effects. Both pristine SiO2 ENPs, paint containing SiO2 ENPs and control paint did not induce cytotoxicity in both assays.
336
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
Fig. 2. Viability of cells 24 h after exposure to pristine ENPs, aged paints containing ENPs or control paints. Bicultures of HBE and THP-1 cells were exposed (24 h) to increasing concentrations (0, 1, 3, 9, 27, 81 and 243 mg/ml) of pristine ENPs or paint particles and viability was assessed with LDH (A) and WST-1 (B) assays. Pristine TiO2 ENPs, aged paint with TiO2 ENPs (P(TiO2)) and control paint without ENPs (P(contr)) (left graphs). Pristine Ag ENPs, aged paint with Ag ENPs (P(Ag)) and control paint without ENPs (P(contr)) (middle graphs). Pristine SiO2 ENPs, aged paint with SiO2 ENPs (P(SiO2)) and control paint without ENPs (P(contr)) (right graphs). Mean + SD. **p<0.01, ***p < 0.001.
3.3. TEER measurements
4. Discussion
Based on the cytotoxicity experiments, 2 subtoxic concentrations (viability >90%) were selected for each particle that were subsequently used in the triculture model. After particle exposure, TEER was measured at different time points up to 24 h after the exposures as a measure of the barrier integrity. Results of TEER of TiO2 ENPs/paints, Ag ENPs/paints and SiO2 ENPs/paints are shown in Fig. 3. None of the tested particles caused significant TEER changes at any of the time points. A non-significant decrease in TEER was observed at the latest time point (24 h) for all particles at the highest tested dose, except for the SiO2 ENPs.
In this study, we showed that the pristine TiO2 and Ag ENPs show some cytotoxic effects at relative high dose, while pristine SiO2 ENPs and all aged paints with ENPs and control paints do not. In the complex triculture model of the lung–blood barrier, no considerable changes were observed after apical exposure to the different pristine ENPs and paint particles. ENPs are increasingly being incorporated in paints and coatings since they can improve vital characteristics such as fire-resistance and self-cleaning properties (Hanus and Harris, 2013; Lee et al., 2010). Besides their beneficial properties, ENPs can be released in the environment as single particles or attached to larger paint particles and can potentially exert harmful effects on human health. Exposure can occur during production, handling and application of the paint on a surface or after aging. The aged paint particles used in our studies were generated after processing the liquid paints. Liquid paints were consecutively applied on a panel, dried, scratched off with a metallic spatula, milled and finally exposed to UV-A as an aging process. In this way, we simulate real-life situations as for example pets or children scratching walls, drilling, exposure during demolition and release after long-term exposure to UV and heat. Very little attention has focused on the release of ENPs from nano-containing paints and nanocomposites (Froggett et al., 2014). Kaegi et al. demonstrated that TiO2 ENPs are detached from new and aged facade paints by natural weather conditions (rain) (Kaegi et al., 2008). Microscopic analysis revealed that most of the TiO2 ENPs are released as aggregates consisting of a few particles embedded in the organic binder of the paint. Similar results were found using a Ag ENP containing paint (Kaegi et al., 2010). Analyses of the water samples obtained after each rain event revealed that more than 30% of the Ag ENPs were released to the environment after one year. Göhler et al. compared the release of particles from
3.4. Cytokine and total glutathione measurements Results of cytokines and total glutathione, measured in the apical and basolateral compartment after exposure to TiO2 ENPs/paints, Ag ENPs/paints or SiO2 ENPs/paints are shown in Tables 1–3 respectively. Total glutathione was measured in the cells of the apical compartment as a measure of oxidative stress 24 h after exposure. Significant decreases of total glutathione were observed after exposure to paint containing SiO2 ENPs at the lowest (81 mg/ml) and highest (243 mg/ml) concentrations applied. The inflammatory cytokines TNF-a and IL-8 were measured in the apical compartment, while IL-6 and sICAM-1 were measured in the basolateral compartment. A decrease of IL-8 was observed after TiO2 ENPs exposure, probably caused by interference of the ENPs on the ELISA. Increases of IL-8 were observed after exposure to the control paint without Ag ENPs (243 mg/ml), paint with SiO2 ENPs (243 mg/ml) and control paint without SiO2 ENPs (81 mg/ml and 243 mg/ml). An increase of sICAM was observed after exposure to control paint without Ag ENPs (243 mg/ml).
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
337
Fig. 3. TEER measurements of tricultures exposed to pristine ENPs, aged paints containing ENPs or control paints. Tricultures of HBE, THP-1 and HLMVEC cells were exposed to pristine ENPs or aged paint particles and transepithelial resistance (TEER) was measured at different time points up to 24 h. Pristine TiO2 ENPs, aged paint with TiO2 ENPs (P(TiO2)) and control paint without ENPs (P(contr)) (left graphs). Pristine Ag ENPs, aged paint with Ag ENPs (P(Ag)) and control paint without ENPs (P(contr)) (middle graphs). Pristine SiO2 ENPs, aged paint with SiO2 ENPs (P(SiO2)) and control paint without ENPs (P(contr)) (right graphs). The TEER values are expressed as percentages compared to the baseline value. n = 5–6.
paints with ZnO ENPs and control paints without ENPs after sanding (Gohler et al., 2010). They showed a considerable generation of ENPs during the sanding process, however, no differences were found between ENP-containing paints and control paints. Furthermore, they demonstrated that the generated particles are predominantly made up from matrix material, which contain the embedded ZnO ENPs. Similar results were obtained by Koponen et al., they observed no significant differences in the size distributions of dust released by sanding between paints with and without ENPs (Koponen et al., 2011). Nowadays, many studies have been published concerning the toxic effects of ENPs, while data about toxicity when incorporated
in a complex paint matrix is lacking. Only a few studies have been performed investigating the in vitro toxicity of ENPs-containing paints. In this study, we used a biculture consisting of human bronchial epithelial cells and monocytes to evaluate cytotoxicity of pristine ENPs and aged paints containing ENPs. We showed that the pristine TiO2 and Ag ENPs have some cytotoxic effects at relative high dose, while pristine SiO2 ENPs and all aged paints with ENPs and control paints do not. Similar results were obtained by Kaiser et al., they studied the cytotoxic effects of the same pristine ENPs and aged paint particles as used in our study on gastrointestinal cells (CaCo-2) and immune system cells (Jurkat) (Kaiser et al., 2013). They showed that only pristine Ag ENPs had
Table 1 Total glutathione and cytokine concentrations measured after exposure of the tricultures to pristine TiO2 ENPs, aged paint with TiO2 ENPs (P(TiO2)) or control paint without ENPs (P(contr)). Total glutathione concentrations were measured in the cells of the apical compartment. TNF-a and IL-8 concentrations were measured in the supernatant of the apical compartment whereas IL-6 and sICAM-1 concentrations were measured in the supernatant of the basolateral compartment. n = 5–6. Apical
Control 27 mg/ml TiO2 ENPs 81 mg/ml TiO2 ENPs 81 mg/ml P(TiO2) 243 mg/ml P(TiO2) 81 mg/ml P(contr) 243 mg/ml P(contr) *
p < 0.05 compared to control.
Basolateral
Total glutathione (pmol)
TNF-a (pg/ml)
335 72 310 122 306 40 294 45 315 46 374 89 268 74
133 211 131 66 63 103 33
227 404 113 75 74 72 16
IL-8 (pg/ml) 1177 951 498 1330 1383 1291 1408
IL-6 (pg/ml) 278 293 99* 357 246 156 111
620 605 641 648 665 597 627
65 95 143 125 134 113 90
sICAM-1 (ng/ml) 1.47 1.49 1.29 1.84 1.54 1.41 1.54
0.24 0.29 0.24 0.24 0.40 0.57 0.14
338
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
Table 2 Total glutathione and cytokine concentrations measured after exposure of the tricultures to pristine Ag ENPs, aged paint with Ag ENPs (P(Ag)) or control paint without ENPs (P (contr)). Total glutathione concentrations were measured in the cells of the apical compartment. TNF-a and IL-8 concentrations were measured in the supernatant of the apical compartment whereas IL-6 and sICAM-1 concentrations were measured in the supernatant of the basolateral compartment. n = 5–6. Basolateral
Apical
Control 9 mg/ml Ag ENPs 27 mg/ml Ag ENPs 81 mg/ml P(Ag) 243 mg/ml P(Ag) 81 mg/ml P(contr) 243 mg/ml P(contr) *
Total glutathione (pmol)
TNF-a (pg/ml)
335 72 336 53 311 77 298 61 275 85 354 121 279 46
133 30 42 92 91 48 69
IL-8 (pg/ml)
227 40 61 58 69 24 59
1177 1224 1189 1404 1447 1623 1663
IL-6 (pg/ml) 278 196 178 354 308 346 260*
620 540 557 590 602 631 685
sICAM-1 (ng/ml)
65 127 144 71 49 100 111
1.47 1.34 1.49 1.39 1.58 1.28 1.70
0.24 0.43 0.17 0.29 0.19 0.22 0.25*
p < 0.05 compared to control.
significant cytotoxic effects on both cell types, while no cytotoxicity was observed after exposure to the other pristine ENPs (TiO2 and SiO2) and all aged paint particles with ENPs. Furthermore, Saber et al. studied the toxicity of paint dusts with different ENPs (TiO2, CB or SiO2) using a monoculture of FE1-Muta Mouse Lung (MML) epithelial cells (Saber et al., 2011). For all dusts, the cell death was below 25% at a concentration of 200 mg/ml. Mikkelsen et al. showed that sanding dust from ENP-containing paint did not generate more oxidative stress or expression of cell adhesion molecules in human umbilical vein endothelial cells than sanding dust from paint without ENPs (Mikkelsen et al., 2013). Although ENP exposure can occur via different routes (inhalation, ingestion, dermal and injection), inhalation is considered to be the major route of exposure for ENPs (Oberdorster et al., 2005). Epidemiological studies show a high deposition efficiency of ultrafine particles in the total respiratory tract of healthy subjects, from the upper airways and bronchi to the alveoli, due to diffusion. After inhalation, the particles will come in contact with the highly differentiated airway/alveolar epithelium and macrophages. The lung epithelium is separated from the subepithelial connective tissue containing the blood and lymphatic vessels by a basement membrane (Geiser and Kreyling, 2010). The inner surface of the lung functions as a physical, biochemical and immunological barrier, separating the outside from inside. We used a complex in vitro model of the lung–blood barrier consisting of human epithelial cells, monocytes and endothelial cells. This model is a good representation as it is in vivo and allows us to study the toxicity of (nano) particles (and chemicals). Different endpoints were measured to study pulmonary and extrapulmonary toxicity. Similar models are already used in nanotoxicology research; Snyder-Talkington et al. studied endothelial effects after epithelial exposure to MWCNT using a
biculture of human small airway epithelial cells (SAEC) and human microvascular endothelial cells (HMVEC) (Snyder-Talkington et al., 2013). Farcal et al. used a coculture of epithelial human cell line NCI-H441 with endothelial human cell line ISO-HAS1 to evaluate the toxic effects of SiO2 ENPs, in the presence or absence of THP-1 cells (monocytes) (Farcal et al., 2013). In our study, both paint particles and pristine ENPs had no effect on the TEER values, indicating that none of the tested particles influences the barrier integrity/functionality. Oxidative stress was assessed measuring total glutathione in the epithelial cells and monocytes in the basolateral compartment. Only the aged paint containing SiO2 ENPs showed a decrease in total glutathione, while all other tested particles had no effects on total glutathione. Since the paint containing SiO2 caused no general changes in TEER and inflammatory cytokines, we assume this decrease in total glutathione is a chance finding. The pro-inflammatory cytokines TNF-a and IL-8 were measured in the apical compartment, while IL-6 was measured in the basolateral compartment. IL-8, predominantly produced by macrophages and moncoytes, is a chemokine responsible for the recruitment of neutrophils, and thus a main regulator of the acute inflammatory response (Remick, 2005). IL-6 is produced by a wide variety of cells including endothelial cells and is an important marker of inflammation (Rincon, 2012). No relevant changes in any of the measured cytokines were observed in our study. Similarly, no considerable effects were seen on the levels of sICAM, a biomarker of endothelial activation and inflammation. In a previous study, we compared the in vivo toxicity of ENPs embedded in paints to pristine ENPs (Smulders et al., 2014), using the same particles as in the in vitro study. Mice were exposed by oropharyngeal aspiration and local (lung), systemic inflammation and body distribution was evaluated. We showed that the pristine
Table 3 Total glutathione and cytokine concentrations measured after exposure of the tricultures to pristine SiO2 ENPs, aged paint with SiO2 ENPs (P(SiO2)) or control paint without ENPs (P(contr)). Total glutathione concentrations were measured in the cells of the apical compartment. TNF-a and IL-8 concentrations were measured in the supernatant of the apical compartment whereas IL-6 and sICAM-1 concentrations were measured in the supernatant of the basolateral compartment. n = 5–6. Basolateral
Apical
Control 81 mg/ml SiO2 ENPs 243 mg/ml SiO2 ENPs 81 mg/ml P(SiO2) 243 mg/ml P(SiO2) 81 mg/ml P(contr) 243 mg/ml P(contr) * **
p < 0.05 compared to control. p < 0.01 compared to control. p < 0.001 compared to control.
***
Total glutathione (pmol)
TNF-a (pg/ml)
335 72 355 73 304 125 250 35* 246 69* 347 85 319 49
133 127 76 220 67 41 141
227 256 72 463 69 35 109
IL-8 (pg/ml) 1177 1236 1309 1491 2257 1701 2241
IL-6 (pg/ml) 278 112 303 290 553** 269* 517***
620 665 598 673 699 658 639
65 101 115 74 49 91 84
sICAM-1 (ng/ml) 1.47 1.44 1.51 1.36 1.55 1.56 1.67
0.24 0.22 0.26 0.29 0.20 0.14 0.25
S. Smulders et al. / Toxicology Letters 232 (2015) 333–339
ENPs had a subtle toxic effect in the lungs and no effects on the blood parameters (systemic effects). The most pronounced effects were observed with the pristine Ag ENPs in the lung; increase in total BAL cells and neutrophils, 2-fold increase in pro-inflammatory cytokines KC and IL-1b. The paints containing ENPs did not show significant toxicity. The results of the in vivo study correlate very well with the in vitro results. The pristine Ag ENPs showed in both studies the most pronounced effects, while the aged paints containing ENPs had no toxic effects at all. In another in vivo study conducted by Saber et al., mice were exposed to paint dusts with different ENPs and several toxic parameters were analyzed (Saber et al., 2011, 2012). They did not observe any changes in BAL cells and inflammatory cytokines and concluded that the level of pulmonary inflammation in mice exposed to sanding dusts was not affected by the addition of ENPs to the paint. In conclusion, we demonstrated that although pristine ENPs show some toxic effects, no significant toxicological effects were observed when they were embedded in a complex paint matrix. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgement The work is financially supported by the Seventh Framework Program of the European Commission NanoHouse-Grant (Agreement No. 207816). 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.2014.11.030 References Hanus, M.J., Harris, A.T., 2013. Nanotechnology innovations for the construction industry. Prog. Mater. Sci. 58, 1056–1102. Guo, K.W., 2011. Green nanotechnology of trends in future energy. Recent Patents Nanotechnol. 5, 76–88. Lee, J., Mahendra, S., Alvarez, P.J.J., 2010. Nanomaterials in the construction industry: a review of their applications and environmental health and safety considerations. ACS Nano 4, 3580–3590. Pacheco-Torgal, F., Jalali, S., 2011. Nanotechnology advantages and drawbacks in the field of construction and building materials. Constr. Build. Mater. 25, 582–590. Keller, A.A., McFerran, S., Lazareva, A., Suh, S., 2013. Global life cycle releases of engineered nanomaterials. J. Nanopart. Res. 15. Mueller, N.C., Nowack, B., 2008. Exposure modeling of engineered nanoparticles in the environment. Environ. Sci. Technol. 42, 4447–4453. Napierska, D., Thomassen, L.C., Lison, D., Martens, J.A., Hoet, P.H., 2010. The nanosilica hazard: another variable entity. Part. Fibre Toxicol. 7, 39. Shi, H., Magaye, R., Castranova, V., Zhao, J., 2013. Titanium dioxide nanoparticles: a review of current toxicological data. Part. Fibre Toxicol. 10, 15.
339
Vesterdal, L.K., Folkmann, J.K., Jacobsen, N.R., Sheykhzade, M., Wallin, H., Loft, S., Moller, P., 2010. Pulmonary exposure to carbon black nanoparticles and vascular effects. Part. Fibre Toxicol. 7, 33. Klein, S.G., Hennen, J., Serchi, T., Blomeke, B., Gutleb, A.C., 2011. Potential of coculture in vitro models to study inflammatory and sensitizing effects of particles on the lung. Toxicol. In Vitro 25, 1516–1534. Luyts, K., Napierska, D., Dinsdale, D., Klein, S.G., Serchi, T., Hoet, P.H.M., 2014. A coculture model of the lung–blood barrier: the role of activated phagocytic cells. Toxicol. In Vitro Available online 4 November. Napierska, D., Thomassen, L.C., Rabolli, V., Lison, D., Gonzalez, L., Kirsch-Volders, M., Martens, J.A., Hoet, P.H., 2009. Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells. Small 5, 846–853. Smulders, S., Kaiser, J.P., Zuin, S., Van Landuyt, K.L., Golanski, L., Vanoirbeek, J., Wick, P., Hoet, P.H., 2012. Contamination of nanoparticles by endotoxin: evaluation of different test methods. Part. Fibre Toxicol. 9, 41. Smulders, S., Luyts, K., Brabants, G., Landuyt, K.V., Kirschhock, C., Smolders, E., Golanski, L., Vanoirbeek, J., Hoet, P.H., 2014. Toxicity of nanoparticles embedded in paints compared with pristine nanoparticles in mice. Toxicol. Sci. 141, 132–140. Froggett, S.J., Clancy, S.F., Boverhof, D.R., Canady, R.A., 2014. A review and perspective of existing research on the release of nanomaterials from solid nanocomposites. Part. Fibre Toxicol. 11, 17. Kaegi, R., Ulrich, A., Sinnet, B., Vonbank, R., Wichser, A., Zuleeg, S., Simmler, H., Brunner, S., Vonmont, H., Burkhardt, M., Boller, M., 2008. Synthetic TiO2 nanoparticle emission from exterior facades into the aquatic environment. Environ. Pollut. 156, 233–239. Kaegi, R., Sinnet, B., Zuleeg, S., Hagendorfer, H., Mueller, E., Vonbank, R., Boller, M., Burkhardt, M., 2010. Release of silver nanoparticles from outdoor facades. Environ. Pollut. 158, 2900–2905. Gohler, D., Stintz, M., Hillemann, L., Vorbau, M., 2010. Characterization of nanoparticle release from surface coatings by the simulation of a sanding process. Ann. Occup. Hyg. 54, 615–624. Koponen, I.K., Jensen, K.A., Schneider, T., 2011. Comparison of dust released from sanding conventional and nanoparticle-doped wall and wood coatings. J. Exposure Sci. Environ. Epidemiol. 21, 408–418. Kaiser, J.P., Roesslein, M., Diener, L., Wick, P., 2013. Human health risk of ingested nanoparticles that are added as multifunctional agents to paints: an in vitro study. PLoS One 8, e83215. Saber, A.T., Koponen, I.K., Jensen, K.A., Jacobsen, N.R., Mikkelsen, L., Moller, P., Loft, S., Vogel, U., Wallin, H., 2011. Inflammatory and genotoxic effects of sanding dust generated from nanoparticle-containing paints and lacquers. Nanotoxicology . Mikkelsen, L., Jensen, K.A., Koponen, I.K., Saber, A.T., Wallin, H., Loft, S., Vogel, U., Moller, P., 2013. Cytotoxicity, oxidative stress and expression of adhesion molecules in human umbilical vein endothelial cells exposed to dust from paints with or without nanoparticles. Nanotoxicology 7, 117–134. Oberdorster, G., Oberdorster, E., Oberdorster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 113, 823–839. Geiser, M., Kreyling, W.G., 2010. Deposition and biokinetics of inhaled nanoparticles. Part. Fibre Toxicol. 7, 2. Snyder-Talkington, B.N., Schwegler-Berry, D., Castranova, V., Qian, Y., Guo, N.L., 2013. Multi-walled carbon nanotubes induce human microvascular endothelial cellular effects in an alveolar-capillary co-culture with small airway epithelial cells. Part. Fibre Toxicol. 10, 35. Farcal, L.R., Uboldi, C., Mehn, D., Giudetti, G., Nativo, P., Ponti, J., Gilliland, D., Rossi, F., Bal-Price, A., 2013. Mechanisms of toxicity induced by SiO2 nanoparticles of in vitro human alveolar barrier: effects on cytokine production, oxidative stress induction, surfactant proteins A mRNA expression and nanoparticles uptake. Nanotoxicology 7, 1095–1110. Remick, D.G., 2005. Interleukin-8. Crit. Care Med. 33, S466–S467. Rincon, M., 2012. Interleukin-6: from an inflammatory marker to a target for inflammatory diseases. Trends Immunol. 33, 571–577. Saber, A.T., Jacobsen, N.R., Mortensen, A., Szarek, J., Jackson, P., Madsen, A.M., Jensen, K.A., Koponen, I.K., Brunborg, G., Gutzkow, K.B., Vogel, U., Wallin, H., 2012. Nanotitanium dioxide toxicity in mouse lung is reduced in sanding dust from paint. Part. Fibre Toxicol. 9, 4.