Genotoxicity of inhaled nanosized TiO2 in mice

Mutation Research 745 (2012) 58–64

Contents lists available at SciVerse ScienceDirect

Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Genotoxicity of inhaled nanosized TiO2 in mice Hanna K. Lindberg a,b , Ghita C.-M. Falck a,b , Julia Catalán a,b,c , Antti J. Koivisto a,b , Satu Suhonen a,b , Hilkka Järventaus a,b , Elina M. Rossi a,d , Heli Nykäsenoja a,d , Yrjö Peltonen a,b , Carlos Moreno c , Harri Alenius a,d , Timo Tuomi e , Kai M. Savolainen a , Hannu Norppa a,b,∗ a

Nanosafety Research Center, Finnish Institute of Occupational Health, FI-00250 Helsinki, Finland Safe New Technologies, Work Environment Development, Finnish Institute of Occupational Health, FI-00250 Helsinki, Finland Department of Anatomy, Embryology and Genetics, University of Zaragoza, 50013 Zaragoza, Spain d Immunotoxicology, Health and Work Ability, Finnish Institute of Occupational Health, FI-00250 Helsinki, Finland e Work Environment Development, Finnish Institute of Occupational Health, FI-00250 Helsinki, Finland b c

a r t i c l e

i n f o

Article history: Received 24 October 2011 Accepted 25 October 2011 Available online 7 November 2011 Keywords: Anatase Blood erythrocyte Comet assay Genotoxicity Lung Micronucleus

a b s t r a c t In vitro studies have suggested that nanosized titanium dioxide (TiO2 ) is genotoxic. The significance of these findings with respect to in vivo effects is unclear, as few in vivo studies on TiO2 genotoxicity exist. Recently, nanosized TiO2 administered in drinking water was reported to increase, e.g., micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs) and DNA damage in leukocytes. Induction of micronuclei in mouse PCEs was earlier also described for pigment-grade TiO2 administered intraperitoneally. The apparent systemic genotoxic effects have been suggested to reflect secondary genotoxicity of TiO2 due to inflammation. However, a recent study suggested that induction of DNA damage in mouse bronchoalveolar lavage (BAL) cells after intratracheal instillation of nanosized or fine TiO2 is independent of inflammation. We examined here, if inhalation of freshly generated nanosized TiO2 (74% anatase, 26% brookite; 5 days, 4 h/day) at 0.8, 7.2, and (the highest concentration allowing stable aerosol production) 28.5 mg/m3 could induce genotoxic effects in C57BL/6J mice locally in the lungs or systematically in peripheral PCEs. DNA damage was assessed by the comet assay in lung epithelial alveolar type II and Clara cells sampled immediately following the exposure. MN were analyzed by acridine orange staining in blood PCEs collected 48 h after the last exposure. A dosedependent deposition of Ti in lung tissue was seen. Although the highest exposure level produced a clear increase in neutrophils in BAL fluid, indicating an inflammatory effect, no significant effect on the level of DNA damage in lung epithelial cells or micronuclei in PCEs was observed, suggesting no genotoxic effects by the 5-day inhalation exposure to nanosized TiO2 anatase. Our inhalation exposure resulted in much lower systemic TiO2 doses than the previous oral and intraperitoneal treatments, and lung epithelial cells probably received considerably less TiO2 than BAL cells in the earlier intratracheal study. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Nanosized titanium dioxide (TiO2 ) is among the most widely used nanomaterials. Especially the rutile and anatase phases of nanosized TiO2 , available in variable sizes, shapes and coatings, are increasingly utilized in a number of industrial applications, cosmetics, plastics, catalysts, pharmaceuticals, and other products [1–5]. TiO2 nanoparticles are a protecting component of sunscreens, based on their function as microreflectors scattering UV light [6].

∗ Corresponding author at: Finnish Institute of Occupational Health, Nanosafety Research Center and Safe New Technologies, Topeliuksenkatu 41 aA, FI-00250 Helsinki, Finland. Tel.: +358 30 4742622; fax: +358 30 4742110. E-mail address: hannu.norppa@ttl.fi (H. Norppa). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.10.011

The toxicity of various forms of nanosized TiO2 has extensively been examined in vitro, and most studies have indicated that TiO2 is of relatively low toxicity, although anatase phase TiO2 often appears to be more cytotoxic than rutile phase TiO2 [2,3,7–10]. In mouse HEL-30 keratinocytes, anatase was reported to be cytotoxic through necrosis but rutile through apoptosis [8]. A number of studies have also suggested that nanoscale TiO2 is genotoxic in various cell systems in vitro. Different forms of nanosized TiO2 especially produced DNA damage in vitro [2,10–15], and nanosized TiO2 anatase (or P25, a 3:1 mixture of anatase and rutile) also induced micronuclei [2,11–14,16]. Studies of chromosomal aberrations were positive using a prolonged 48 h treatment with nanosized anatase in human lymphocytes [17] but negative in various cell lines treated with different forms of TiO2 for shorter periods [18–20]. The significance of the in vitro findings with respect to

H.K. Lindberg et al. / Mutation Research 745 (2012) 58–64

in vivo effects is, however, unclear, as very few in vivo studies on TiO2 genotoxicity are available. The question whether TiO2 is genotoxic in vivo is particularly interesting, because inhaled or intratracheally administered nanoand fine-sized TiO2 has been observed to induce lung cancer in rats [1,4,5]. Consequently, the International Agency for Research on Cancer has classified TiO2 as possibly carcinogenic to humans (class 2B), with sufficient evidence in experimental animals [1,4], and U.S. National Institute for Occupational Safety and Health (NIOSH) has determined ultrafine TiO2 as a potential occupational carcinogen [5]. It has been assumed that the rat lung carcinogenicity of TiO2 , and other poorly soluble, nonfibrous particles of low acute toxicity, is a high-dose over-loading effect due to particle deposition and retention on the respiratory epithelium, resulting in impairment of the clearance mechanism of the lung, inflammatory response, production of reactive oxygen and nitrogen species, epithelial cell injury and proliferation, and secondary genotoxic effects [1,4,5,21]. Yet, the mechanisms are not well understood, and it is not known, for instance, how important a role genotoxic events play in this process. It has been suggested that oxidants produced by inflammatory cells (neutrophils and macrophages), could induce genotoxic effects in lung epithelial cells [22]. Intratracheal (i.t.) instillation of 100 mg/kg body weight (b.wt) fine TiO2 anatase (median diameter 180 nm) to rats led to an increase in the frequency of hprt (hypoxanthine-guanine phosphoribosyl transferase) mutant alveolar epithelial cells 15 months after the treatment [22]. The TiO2 -treated rats also showed an increase in the proportion of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) fluid, indicative of a prolonged inflammation [22]. DNA damage was not, however, observed in lung cells of three rats that had inhaled AlOH- and polymer-coated TiO2 rutile nanoparticles (10 mg/m3 , 6 h/day for 5 days); however, the lung samples were collected 23 days after the last exposure which was probably too late, as most of the inflammation initially observed had subsided [23]. Similarly, oxidative DNA damage (8-oxoguanine) or inflammation was not detectable in rat lung samples 90 days after i.t. administration (0.15–1.2 mg) of nanosized uncoated (P25) or silanized (T805, phase not indicated) TiO2 [24]. TiO2 nanoparticles induced lung cancer in rats at lower exposure levels than larger particles [1,4,5], which agrees with the higher in vivo toxicity of nanosized than fine TiO2 , observed after i.t. [25] and oral administration [26]. Rat lung alveolar macrophages were reported to be inefficient in taking up 20 nm TiO2 , which suggested that the lungs or the rest of the body are less protected from inhaled nanosized than larger particles [27]. The higher tumor potency of nanoscale than fine TiO2 may be due to its greater translocation to the lung interstitium [5,28]. Studies on the carcinogenicity of TiO2 in mice are few. The only inhalation study available, concerning ultrafine P25 (about 10 mg/m3 for 13.5 months), was negative [29], but the high control incidence (30%) of lung tumors in the NMRI mice used may have compromised the sensitivity of the bioassay [5]. Intraperitoneal (i.p.) injection of TiO2 (particle size undefined) to mice did not induce cancer [30], and topical administration of noncoated or coated (Al(OH)3 and stearic acid) TiO2 nanoparticles of unknown phase did not produce skin cancer [31]. However, poorly tumorigenic and nonmetastatic fibrosarcoma QR-32 cells formed tumors and acquired a metastatic phenotype when subcutaneously transplanted into C57BL/6J mice at sites previously implanted with nanosized ZrO2 Al(OH)3 -doped hydrophilic TiO2 rutile; similar effects were not observed at sites implanted with hydrophobic nanosized rutile with additional stearic acid coating or if TiO2 was co-transplanted with QR-32 cells [32]. The authors explained the findings by the generation of ROS in the cells [32]. Similarly, TiO2 nanopowder (mixture of rutile and anatase) injected i.p. to C57BL/6J mice once a day for 28 days before subcutaneous

59

implantation of B16F10 melanoma cells, led to an enhancement of tumor growth, which was suggested to be due to an immunomodulatory effect of TiO2 [33]. Uncoated TiO2 nanoparticles (P25: 75% anatase, 25% rutile), administered in drinking water for 5 days, were observed to be genotoxic in mice, inducing micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs), DNA damage in blood leukocytes, and 8-hydroxy-2 -deoxyguanosine DNA adducts in the liver at a total dose of 500 mg/kg, foci positive for ␥-H2AX (phosporylated histone H2AX) in bone marrow cells at 50–500 mg/kg, and (after in utero exposure for days 8.5–18.5 post-coitum) deletions in the retina at 500 mg/kg [34]. The apparent systemic genotoxic effect was suggested to reflect secondary genotoxicity of TiO2 nanoparticles due to inflammation. It is of interest that also in an earlier study [35] an i.p. injection of pigment-grade TiO2 (size and phase undefined) was found to induce MN in mouse bone marrow and blood PCEs. A recent study showed an induction of DNA damage (measured by the comet assay) in BAL cells of female C57BL/6 mice 24 h after a single i.t. instillation of 54 ␮g (2.8 mg/kg) of fine rutile coated with Al and polyalcohol (average size 288 nm) or nanosized rutile coated with Al, Zr, and polyalcohol (20.6 nm) [36]; only the nanosized rutile produced pulmonary neutrophilia. However, similar treatment with uncoated anatase (9 nm; with 7.8%, w/w rutile, 12 nm) did not produce DNA damage, although a clear increase was seen in the number of neutrophils and total cells in BAL fluid [36]. Thus, these findings did not suggest correlation between the DNA damaging and inflammatory effects of the three types of TiO2 . In the present study, we examined, for the first time, if inhalation of nanosized TiO2 could induce genotoxic effects in mice locally in the lungs or systematically in peripheral PCEs. The studies were performed in C57BL/6J mice after a 5-day inhalation exposure to three concentrations of nanosized TiO2 (74% anatase, 26% brookite) from a gas-to-particle aerosol generator. DNA damage was assessed by the comet assay in lung alveolar type II and Clara cells, and MN were analyzed in peripheral blood PCEs. Although a dosedependent accumulation of Ti to lungs could be seen, and the highest exposure level showed an inflammatory effect, no significant increase in the level of DNA damage or MN was observed. 2. Materials and methods 2.1. Exposure, aerosol properties, particle characteristics, and lung deposition TiO2 particles were generated by the thermal decomposition of titanium tetraisopropoxide (Ti(C3 H7 O)4 , 97%, Sigma–Aldrich, St. Louis, MO) vapour in a laminar flow reactor [37] as explained in detail previously [38,39]. Briefly, the laminar flow reactor produced TiO2 particles by homogenous nucleation and condensation from supersaturated TiO2 vapour. Secondary particles were formed by primary particle agglomeration and by aggregation via TiO2 vapour condensation on the particles [39]. To facilitate direct exposure of mice to freshly produced TiO2 nanoparticles, the laminar flow reactor was attached to a whole-body inhalation exposure chamber. The experiments were carried out at three aerosol mass concentrations: 0.8 (SD, 0.2), 7.2 (SD, 0.7), and 28.5 (SD, 0.9) mg/m3 , 4 h/day for 5 days; the respective surface area concentrations were 0.049, 0.44 and 1.74 m2 /m3 . The largest concentration used (28.5 mg/m3 ) was about the highest exposure level that still allowed repeatable and stable generation of nanosized TiO2 , without excessive agglomeration and particle size distribution shifting to non-nanosized area. The intermediate concentration was near to the Finnish occupational exposure limit (OEL) of 10 mg/m3 for inorganic dust [40], applied to regulate TiO2 , and the lowest concentration was of the same order of magnitude as the recent suggestion of NIOSH (0.3 mg/m3 ) for the OEL of nanosized TiO2 [5]. The properties of the aerosol produced have been described before [39]. In short, mobility size distribution, measured with an aerosol mobility spectrometer, showed a geometric mean mobile diameter of 84.5 nm at 0.8 mg/m3 , 73.9 nm at 7.2 mg/m3 , and 89.2 nm at 28.5 mg/m3 TiO2 , with respective mean particle number concentrations of 0.4 × 106 /cm3 , 6.5 × 106 /cm3 , and 23.6 × 106 /cm3 . Condensation particle counter, used for the lower two TiO2 concentrations, gave similar particle number concentrations (0.40 × 106 /cm3 at 0.8 mg/m3 and 5.84 × 106 /cm3 at 7.2 mg/m3 TiO2 ) [39]. Aerodynamic size distribution, determined for the higher two concentrations using an electrical low pressure impactor, showed higher values than mobility size distribution, with mean diameters of 102 nm at 7.2 mg/m3 and 144 nm

60

H.K. Lindberg et al. / Mutation Research 745 (2012) 58–64

at 28.5 mg/m3 and respective mean particle concentrations of 5.7 × 106 /cm3 and 12.1 × 106 /cm3 . The difference between mobility and aerodynamic diameters was due to the effective density of the particles [39]. The characterization of the particles collected from the exposure chamber has been reported earlier [39,41]. In brief, X-ray diffraction analyses indicated that the particles comprised of two phases of TiO2 , anatase (74%, v/v) and brookite (26%, v/v), with respective crystallite sizes of 41 nm and 7 nm. Transmission electron microscopic analysis showed that the TiO2 particles consisted of agglomerates of 10–60 nm crystallites with an average primary particle size of 21 nm [39,41]. The specific surface area of the TiO2 powder, determined by the Brunauer–Emmet–Teller method using a gas adsorption analyzer, was 61 m2 /g [39]. The Z-potential of the freshly generated TiO2 nanoparticles was negative (approximately −40 mV) at pH 6, but positive at pH 4 (approximately 35 mV) and pH 2 [41]. The TiO2 powder generated was able to produce hydroxyl radicals in a dose-dependent manner (5–15 mg/mL) after 24 h or 90 h incubation (37 ◦ C) with benzoic acid probe in phosphate-buffered water, as detected chromatographically using UV detection [41]. Lung titanium content was determined by inductively coupled plasma mass spectrometry (ICP-MS, Thermo X Series II, Thermo Electro, Germany; Dr Mirja Kiilunen, Chemical Safety, Finnish Institute of Occupational Health, Helsinki; Finland) from lung tissue samples dried to constant weight and digested with HNO3 [39]. TiO2 deposition fraction (lung TiO2 mass content in comparison with aerosol TiO2 mass content inhaled by the mice during the experiment), estimated as described earlier [39], was 9%, 5%, and 8% at 0.8, 7.2 and 28.5 mg/m3 TiO2 , respectively. Ethylene oxide (>99.9%), used as a gaseous positive control exposure, was purchased from Oy Aga Ab (Espoo, Finland). Exposure to ethylene oxide was carried out by inhalation (600 mg/m3 ) in a whole-body exposure chamber for 4 h, in a similar manner as in a previous study [42]. The level of ethylene oxide remained constant throughout the exposure (data not shown) as measured by a Miran 1A-1395 infrared analyzer (Wilks Scientific). 2.2. Animals and exposure protocol Male C57BL/6J mice (9–11 weeks old) were purchased from Scanbur AB (Sollentuna, Sweden) and quarantined for 1 week. The mice were housed in groups of four in stainless steel cages bedded with aspen chip and were provided with standard mouse chow diet (Altromin no. 1314 FORTI, Altromin Spezialfutter GmbH & Co., Germany) and tap water ad libitum. The environment of the animal room was carefully controlled, with a 12 h dark/light cycle, temperature of 20–21 ◦ C, and relative humidity of 40–45%. The experiments were performed in agreement with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, March 18, 1986, adopted in Finland May 31, 1990). The study was approved by the Animal Experiment Board and the State Provincial Office of Southern Finland. The mice were exposed repeatedly, 4 h per day during 5 consecutive days, to three different concentrations of nanosized TiO2 (0.8, 7.2 and 28.5 mg/m3 ). Mice exposed to the positive control agent (600 mg/m3 of ethylene oxide) received a single inhalation exposure for 4 h. For each assay, 6 mice were simultaneously exposed to one of the doses, and another group of 6 unexposed mice served as negative controls. Although different animals were used for the micronucleus assay and for the comet assay, all the mice receiving the same treatment were exposed together. The ethylene oxide experiment was carried out separately from the TiO2 series and had a control group of its own. Macroscopic observations of the animals were conducted daily. Their weights were recorded before and after each exposure. 2.3. Inflammatory cells in bronchoalveolar lavage Immediately following the last exposure or 3 days later, the mice were anesthetized with i.p. pentobarbital (Mebunat, Orion Oyj, Espoo, Finland). The upper abdominal cavity and diaphragm were opened, the dorsal aorta was severed, and the heart and lungs were perfused with 0.15 M NaCl until the lungs became white. Tracheotomy was then performed, and an 18-gauge cannula was inserted in the trachea and tied. The lungs were lavaged with 800 ␮L of phosphate-buffered saline (PBS), and the BAL fluid was kept on ice during the collection. Cells in the lavage fluid were spun down, resuspended in 0.5 mL of PBS, and prepared to microscopic slides using a cytocentrifuge (Cyto-Tek, Sakura Finetek U.S.A., Inc., Torrance, USA). The slides were air-dried overnight and stained with May-Grünwald-Giemsa. The slides were coded, and the average number of cells in three different high-power fields was scored at 100× magnification. The cells were classified as macrophages, neutrophils, small lymphocytes, or monocytes, using an Axioplan 2E Universal Microscope (Zeiss, Jena, Germany).

collected (see above), the lungs were flushed 6 times with 0.15 M NaCl (2 mL at a time) to remove remaining macrophages, and once with 0.6 mL protease solution consisting of 0.25% trypsin (Biological Industries, Kibbutz Beit Haemek, Israel) in solution A containing 133 mM NaCl, 5.2 mM KCl, 1.89 mM CaCl2 , 1.29 mM MgSO4 (Merck, Darmstadt, Germany), 2.59 mM phosphate buffer, and 10.3 mM Hepes buffer (pH 7.4; PromoCell GmbH, Heidelberg, Germany), and 1 mg/mL glucose (Merck). The lungs and trachea were then disentangled and moved to a Petri dish containing 0.15 M NaCl at 37 ◦ C. The lungs were lavaged with ∼35 mL of the protease solution (0.25% trypsin in solution A) for 30 min at 37 ◦ C. Thereafter, the trachea and main bronchi were removed, and the lung was chopped into small pieces. The cell suspension was moved into a Falcon tube (Becton Dickinson, Discovery Labware, Bedford, MA, USA), and 5 mL of foetal bovine serum (Gibco, Paisley, Scotland, UK) was added. The solution was made up to 20 mL with solution B containing DNAase (250 ␮g/mL; Roche, Basel, Switzerland), and the tube was shaken by hand for 4 min in a water bath at 37 ◦ C. The suspension was then filtered through 150 ␮m and 30 ␮m nylon meshes and maintained on ice. The cell suspension was centrifuged in a Percoll gradient (heavy density 1.089, light density 1.040; Amersham Biosciences, Uppsala, Sweden), and the isolated cells were plated on Petri dishes and incubated for 1 h at 37 ◦ C, to allow fibroblasts and macrophages to attach. Then, the cell suspension was collected and centrifuged at 400 × g for 5 min. The comet assay was performed in alkaline conditions (pH > 13) as described previously [44]. Briefly, 1–3 × 104 cells were resuspended in 75 ␮L molten (37 ◦ C) 0.5% low-melting-point agarose (LMPA; Bio-Rad Laboratories, Hercules, CA). The resuspended cells in agarose were put onto dry microscope slides (Assistant, Sondhiem/Röhn, Germany) pre-coated with 1% normal-melting agarose (International Biotechnologies, New Haven, CT, USA), and the agar was allowed to solidify for 10 min. The slides were thereafter immersed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100) for at least 1 h at 4 ◦ C, after which they were transferred to an electrophoresis tank containing freshly made electrophoresis buffer (1 mM EDTA, 300 mM NaOH; pH > 13), where they were kept for 20 min at room temperature to allow DNA unwinding. Electrophoresis was performed in the same buffer at room temperature for 15 min at 24 V and 300 mA (0.8 V/cm). The slides were then neutralized 3 times with 0.4 M Tris buffer (pH 7.5), air-dried, and fixed in methanol. DNA was stained with ethidium bromide (2 ␮g/mL) in water for 5 min. The slides were coded, and one scorer performed the comet analysis using a fluorescence microscope (Axioplan 2, Zeiss, Jena, Germany) and an interactive automated comet counter (Komet 4.0, Kinetic Imaging Ltd., Liverpool, UK). The percentage of DNA in the comet tail from 100 cells per animal (two replicates, 50 cells each) was used as a measure of the amount of DNA damage. 2.5. Micronuclei in peripheral blood erythrocytes The micronucleus assay was performed primarily as described before [45]. Because exposed PCEs are expected to appear in peripheral blood about 48 h after exposure, the animals were different than those used for the comet assay. Blood samples were collected from the tails of the mice 48 h after the last exposure, and whole blood smears were prepared. The next day, the same mice were used for the lung deposition analyses [see 39] and for the BAL cell analysis. The smear slides were stained with acridine orange fluorescent dye (32 ␮g/mL, 1 min). Microscopic analysis was performed with a Zeiss Axioplan 2E Universal Microscope using 63×/1.25 and 100×/1.30 Zeiss Plan-Neofluar objectives and a FITC/TRITC double filter (Chroma, Rockingham, VT, USA); in these conditions DNA showed green fluorescence and RNA red fluorescence. The slides were coded, and one microscopist scored the frequency of micronucleated erythrocytes in 2000 PCEs (stained red by acridine orange) and in 2000 normochromatic erythrocytes (NCEs; weakly stained by acridine orange but outline visible in the microscope) per mouse. Also the percentage of PCEs was assessed (scored until the number of PCEs or NCEs reached 2000) to indicate possible bone marrow toxicity. 2.6. Statistical tests The unpaired two-sample t-test was applied to determine whether the exposure to the three doses of TiO2 (two-tailed test) or to the positive control, ethylene oxide (one-tailed test), induced a statistically significant difference as compared with the corresponding untreated control groups for the percentage of DNA in tail, the frequency of micronucleated erythrocytes, and the percentage of PCEs. The Mann–Whitney test (two-tailed) was used to examine if the percentage of neutrophils in BAL cells differed between a treated group and the control group. The differences were interpreted to be significant if p was below 0.05. All the statistical analyses were performed by the Statistix for Windows 2.0 programme (Tallahassee, FL, USA).

2.4. Isolation of alveolar type II/Clara cells and comet assay

3. Results

The comet assay was performed on alveolar type II and Clara cells collected immediately after the last exposure. Alveolar type II cells and Clara cells were isolated according to method 2 reported by Lindberg et al. [42], based on a technique originally described for rat alveolar type II cells [43]. After the BAL sample had been

The results of the comet assay and BAL cell analysis performed immediately after the last exposure are shown in Table 1. No significant induction of DNA damage was seen in the comet assay at

H.K. Lindberg et al. / Mutation Research 745 (2012) 58–64

61

Table 1 Percentage of DNA in tail (DNA damage) in alveolar type II/Clara cells and proportion of neutrophils in bronchoalveolar lavage (BAL) cells of C57BL/6J mice exposed by inhalation (5 days, 4 h/day) to nanosized TiO2 and sampled immediately after the last exposure. Exposure concentration by

Total TiO2 dose per animal (␮g)a

Mass (mg/m3 )

Inhaled

Surface area (m2 /m3 )

Nanosized TiO2 0.8 0.049 Controls 7.2 0.44 Controls 28.5 1.74 Controls Ethylene oxide (positive control)d 600 Controls a b c d e f

30

No. animals

Mean (SE) % DNA in tail in alveolar type II/Clara cellsb

Mean (SE) % neutrophils among BAL cells

6 6 6 4 6 6

10.0 (1.8) 6.4 (1.0) 8.2 (1.0) 7.4 (1.7) 15.8 (3.0) 19.3 (8.7)

0.3 (0.1) 0.4 (0.2) 0.5 (0.2) 0.0 (0.0) 15.0 (5.5)c 0.3 (0.2)

6 6

43.0 (9.1)e 25.4 (12.2)

NDf ND

Retained in lungs

2.7

330

18

1020

84

Based on estimated TiO2 mass in volume inhaled during the experiment and Ti in the lungs determined by inductively coupled plasma mass spectrometry [39]. 100 cells were scored per animal. p = 0.006, in comparison with concurrent controls, Mann–Whitney test (two-tailed). Inhalation exposure for 4 h. p = 0.004, in comparison with concurrent controls, t-test (one-tailed). ND, not determined.

any of the three doses of nanosized TiO2, when the exposed mice were compared with the corresponding negative controls. However, the ethylene oxide-treated mice (the positive control group) showed a statistically significant 1.7-fold increase in the mean percentage of DNA in tail in comparison with the concurrent negative control group, despite the high inter-individual variation in DNA damage levels seen in the concurrent control animals (Table 1). A significant increase in inflammatory response, as measured by the percentage of neutrophils among BAL cells, was observed at the highest tested dose of nanosized TiO2 (mean 15.0%), as compared with the controls (0.3%; p = 0.006). Table 2 shows the results of the micronucleus assay in peripheral blood erythrocytes collected 48 h after the last exposure. None of the tested doses of nanosized TiO2 caused a significant induction of micronucleated PCEs or NCEs as compared with the corresponding negative controls. A statistically significantly higher frequency of micronucleated PCEs (8.3/1000 cells) was observed in mice exposed to ethylene oxide as compared with the control mice (4.0/1000 cells; p < 0.05). No decrease in the percentage of PCEs was seen

with any exposure, indicating an absence of a toxic effect on bone marrow. Mice exposed to 0.8 mg/m3 of nanosized TiO2 actually showed a significantly higher percentage of PCEs (2.7%) than their corresponding controls (1.9%; p = 0.004); however, as the value was within the control variation observed in the study, this result was probably due to chance. In BAL fluid collected 24 h after the tail blood samples (72 h after the last exposure), a significant increase in the mean percentage of neutrophils was seen at the highest dose (28.5 mg/m3 ) of nanosized TiO2 (9.6%) in comparison with the control group (0.5%; p < 0.05) (Table 2). Thus, the increase in neutrophils observed immediately after the last exposure still remained 3 days later; although the proportion of neutrophils in BAL fluid was lower in the later than the earlier sampling, the difference was not statistically significant. 4. Discussion Most data available on the genotoxicity of TiO2 nanoparticles are from in vitro studies. Various types of nanosized and fine TiO2

Table 2 Frequency of micronucleated polychromatic (PCEs) and normochromatic (NCEs) erythrocytes and percentage of PCEs in peripheral blood 48 h after the last exposure, and proportion of neutrophils among bronchoalveolar lavage (BAL) cells (means and standard errors) 72 h after exposure of C57BL/6 J mice by inhalation (5 days, 4 h/day) to nanosized TiO2 . Exposure concentration (mg/m3 )

Nanosized TiO2 0.8 Controls 7.2 Controls 28.5 Controls Ethylene oxide (positive control)e 600 Controls a b c d e f g

No. animals

Mean (SE) no. micronucleated cells/1000 cellsa

Mean (SE) % PCEs among all erythrocytesb

Mean (SE) % neutrophils among BAL cells

PCEs

NCEs

6 6 6 4 6 6

3.9 (0.5) 3.8 (0.8) 3.9 (0.4) 4.9 (0.9) 4.3 (0.7) 3.7 (0.6)

1.6 (0.2) 1.0 (0.3) 1.2 (0.2) 1.0 (0.4) 1.3 (0.2) 0.9 (0.3)

2.7 (0.1)c 1.9 (0.2) 2.3 (0.2) 3.1 (0.3) 2.1 (0.4) 2.4 (0.3)

0.0 (0.0) 0.0 (0.0) 0.2 (0.2) 0.1 (0.1) 9.6 (5.2)d 0.5 (0.4)

6 6

8.3 (0.7)f 4.0 (0.7)

1.4 (0.2) 1.5 (0.4)

2.5 (0.1) 2.6 (0.3)

NDg ND

2000 PCEs and 2000 NCEs scored per animal for micronuclei. Corresponding to 2000 NCEs scored. p = 0.004, in comparison with concurrent controls, t-test (two-tailed). p = 0.006, in comparison with concurrent controls, Mann–Whitney test (two-tailed). Inhalation exposure for 4 h. p < 0.001, in comparison with concurrent controls, t-test (one-tailed). ND, not determined.

62

H.K. Lindberg et al. / Mutation Research 745 (2012) 58–64

have shown genotoxic activity in different cell culture systems [2,10–16]. Although the mechanisms are not clear, the positive results have often been assumed to reflect particle-mediated generation of reactive oxygen species (ROS). ROS production has also been described for TiO2 in vivo, particularly in connection with inflammation [22,30], but it is unclear whether in vitro assays can correctly identify secondary genotoxicity due to inflammation. Many of the components of the complex process of inflammation are not available in vitro. To evaluate the performance of in vitro assays in assessing the genotoxicity of nanoparticles, comparative data from suitable in vivo assays are required. The mammalian erythrocyte micronucleus test [46,47] used as the primary genotoxicity assay in vivo, is able to reveal agents that have a systemic genotoxic effect or that target the bone marrow. Several possibilities to study genotoxic effects in other organs also exist [48–51], but there are only a few studies assessing genotoxicity in the target cells of lung carcinogenesis [42,43,51–53]. In the present study, we exposed mice by inhalation to freshly generated nanosized TiO2 anatase–brookite (74%/26%) for 5 days and examined systemic genotoxic effects in blood PCEs by the micronucleus assay and local genotoxic effects in alveolar type II and Clara cells (presumed target cells of lung carcinogenesis) by the comet assay. The latter assay, based on a method developed for rat alveolar type II cells [43], was applied here for the first time to the comet assay in mice. Our previous study [42] showed that both alveolar type II cells and Clara cells of mice are isolated by this procedure. The technique seems to work well, although further development is required to get rid of occasional high %DNA in tail values in control animals. The highest exposure level used (28.5 mg/m3 ), chosen on the principle that nanoparticulate TiO2 could still be produced at stable conditions without excessive agglomeration [39,41], was able to induce a clear inflammatory response, seen as an influx of neutrophils to the lungs. Our earlier experiments with BALB/c mice exposed for 2 h, 4 days (2 h/day), or 4 weeks (4 days/week, 2 h/day) to 10 mg/m3 of the same nanosized TiO2 anatase–brookite as studied here did not produce neutrophilia [41]. The differential response may reflect a sensitivity difference between the two strains of mice or the lower TiO2 concentration used in the first study. The two lower exposure levels (0.8 and 7.2 mg/m3 ) did not induce neutrophilia in the present study either. The only form of TiO2 that showed an inflammatory effect in the previous study, after the 4-day and 4-week exposures to 10 mg/m3 , was a nanosized rutile coated with amorphous silica [41]. Another acute study on C57BL/6 mice exposed by inhalation to nanosized TiO2 anatase for 4 h (0.77 and 7.22 mg/m3 ) or 10 days (4 h/day; 8.88 mg/m3 ) reported a modest inflammatory response, seen as an increase in total cells and macrophages in BAL fluid after the 10-day exposure, but no neutrophilia [54]. Neutrophilia was, however, observed after a subchronic 13-week inhalation exposure of B3C3F1/CrlBR mice to 10 mg/m3 of nanosized TiO2 P25 [55] and to 50 or 250 mg/m3 of pigmentary TiO2 rutile [56]; the effect remained through week 52 postexposure. Despite the inflammatory response at 28.5 mg/m3 of anatase–brookite, we observed no increase in DNA damage in lung cells or micronuclei in PCEs of the exposed animals. Our findings disagree with the recent study where TiO2 P25 nanoparticles administered in drinking water for 5 days were observed to have genotoxic effects in various organs of C57BL/6Jpun /pun (essentially the same as C57BL/6 J) mice, including similar endpoints as we studied – DNA damage by the comet assay in blood leukocytes and micronuclei in blood PCEs [34]. The findings were explained by a secondary effect of inflammation, suggested by the induction of mRNA transcripts of proinflammatory cytokines in blood [34]. There are obvious differences between the two studies which could explain the differential results, such as the cells examined for the comet assay (type II alveolar cells and Clara cells vs blood

leukocytes), the type of TiO2 anatase nanoparticles (containing brookite vs rutile), and the route of exposure (inhalation vs drinking water). TiO2 is probably better distributed systemically when administered orally than by inhalation [26]. Although lung clearance may not be as efficient for nanoparticles as it is for larger particles [27], macrophages are expected to remove much of the inhaled TiO2 agglomerates, reducing the exposure of epithelial cells and the rest of the body. Furthermore, P25 anatase–rutile may be a more effective genotoxicant than anatase–brookite. Nevertheless, the greatest difference between our study and the previous one [34] concerns TiO2 dose. Micronuclei and DNA damage were significantly increased in the drinking water study at 500 mg/kg b.wt [3], which corresponded to a total dose of 12.5 mg for a 25 g mouse. If our mice had retained all the TiO2 they inhaled, their total dose after the 5-day exposure at 28.5 mg/m3 would have been about 1 mg [39], i.e., 8% of the oral dose. Furthermore, the TiO2 content in the lungs of our mice after BAL was only 8% of the maximum inhaled TiO2 dose, suggesting that most of the initial TiO2 was exhaled, cleared from the lungs, or retained in the upper airways. Therefore, our mice received a much lower dose of TiO2 than the mice treated via drinking water. Also the two positive genotoxicity studies performed with non-nanosized TiO2 using the bone marrow and blood erythrocyte micronucleus assay in mice [35] and the hprt mutation assay in rats [22] used high doses of 250–1000 mg/kg b.wt i.p. and 100 mg/kg b.wt i.t., respectively; in the latter study, TiO2 dose to the lungs was about 2.5 times higher than our maximum inhaled dose, but TiO2 lung deposition was probably much more effective in the i.t. exposure than in our inhalation experiment. With the aerosol generator we used, it was not possible to increase TiO2 concentration above 28.5 mg/m3 without seriously compromising aerosol stability and increasing agglomerate size above nano-range. This concentration, which induced an inflammatory response, is 2–3 times higher than the present OELs for total inorganic dusts applied in most countries to regulate TiO2 . For instance, the OEL (8 h time-weighted average concentration) for inorganic total dust is presently 10 mg/m3 in Finland [40] and 15 mg/m3 (permissible exposure level, Occupational Safety and Health Administration) in the USA [57]. These OELs are high, considering that inhalation exposure to about 10 mg/m3 of nanoscale TiO2 P25 for 24 months induced lung cancer in rats [29]. The new recommendations of NIOSH [5] for OELs are 0.3 mg/m3 for ultrafine and 2.4 mg/m3 for fine TiO2 , regarding ultrafine TiO2 as a potential occupational carcinogen. One way of increasing total TiO2 dose in the inhalation exposure would have been to prolong the exposure duration beyond the 5 days we used. However, we would have had to continue the exposure for 3 months (assuming 100% retention) to reach a maximum total dose of 500 mg/kg. Considering that only a minority of this amount is retained in the body, we should have extended the inhalation still much more. This would not anymore have been a short-term assay. The results of Saber et al. [36] differed from the other positive in vivo genotoxicity studies with TiO2 in that a 1.4-fold induction of DNA damage in BAL cells was observed 24 h after an i.t. administration of a low dose of 54 ␮g/mouse (2.8 mg/kg) of coated fine and nanosized TiO2 rutile. This i.t. dose was 64% of the estimated TiO2 dose retained in the lungs at the 28.5 mg/m3 concentration in the present study (Table 1). However, intracellular TiO2 dose was probably much higher in the BAL cells [36] than in epithelial cells, because most cells in BAL fluid are macrophages which effectively take up particles. The nanosized rutile also induced pulmonary neutrophilia while the fine rutile did not, suggesting that the DNA damage was not associated with neutrophils. It is noteworthy that uncoated nanosized anatase did not increase DNA damage in BAL cells at the 54 ␮g i.t. dose, despite effective induction of

H.K. Lindberg et al. / Mutation Research 745 (2012) 58–64

pulmonary neutrophilia [36], in agreement with our negative comet assay results with nanosized anatase–brookite in lung epithelial cells. Saber et al. [36] concluded that there is no simple correlation between inflammation and DNA damage. On the other hand, the proportion of neutrophils among BAL cells was 68% and 39% after i.t. treatment with nanosized anatase and rutile, respectively [36]. The higher proportion of phagocytosing macrophages (apparently with a high intracellular TiO2 dose) among BAL cells in animals treated with rutile than in those treated with anatase may have contributed to the fact that an induction of DNA damage was seen with rutile but not with anatase [36]. In summary, previous in vivo studies have suggested that nanosized or fine TiO2 can induce a genotoxic effect after high oral or i.p. dosage in mice [34,35] and i.t. instillation in rats [22], which has been suggested to reflect secondary effects due to inflammation and oxidative stress. In BAL cells, which mostly consist of phagocytosing macrophages, DNA damage has also been observed at a low i.t. dose of nanosized and fine TiO2 , apparently without a connection to inflammation [36]. As the genotoxicity of TiO2 has not previously been assessed by inhalation, we studied here the genotoxicity of inhaled nanosized TiO2 anatase–brookite aerosols in mice in a 5-day experiment. Despite the pulmonary neutrophilia observed, we did not see any genotoxic effects locally in the lungs or systemically in the blood, probably because the maximum particle concentration still allowing stable production of nanoscale aerosol delivered a clearly lower (systemic or intracellular) TiO2 dose than occurred in the previous studies using other exposure routes or target cells (BAL cells). Possibly, the inflammatory response induced in our experiments was not adequately high or did not prevail long enough to provoke a genotoxic effect. It may also be that there is no simple association between the inflammatory and genotoxic effects of TiO2 [36]. Besides producing pulmonary inflammation, inhalation of nanosized TiO2 seems to reduce lung clearance, increase particle retention, and induce epithelial cell proliferation in both rats and mice [4,5,55,56], but fibrosis and lung cancer has thus far only been described in rats [4,5,29,55,56]. However, as there is presently only one (negative) chronic cancer bioassay on inhaled nanoscale TiO2 (P25; 10 mg/m3 for 13.5 months) in mice [29], data on the carcinogenicity of nanosized TiO2 in mice are not conclusive. The role of genotoxic events in lung carcinogenesis by nano-TiO2 remains an open question. Further studies are required to examine whether longer-term inhalation to nanoscale TiO2 could be genotoxic and to better understand the genotoxic mechanisms of nanoparticles in vitro and in vivo and the tentative relationship between inflammation and genotoxic effects. Conflict of interest The authors declare that there are no conflict of interest. Acknowledgements This work was supported by the FinNano Program of the Academy of Finland (NANOHEALTH, Engineered Nanoparticles: Synthesis, Characterization, Exposure and Health Hazards) and the European Commission FP6 (NANOSH, NMP4-CT-2006-032777, Inflammatory and Genotoxic Effects of Engineered Nanomaterials). The views and opinions expressed in this paper do not necessarily reflect those of the European Commission. References [1] R. Baan, K. Straif, Y. Grosse, B. Secretan, F. El Ghissassi, V. Cogliano, Carcinogenicity of carbon black, titanium dioxide, and talc, Lancet Oncol. 7 (2006) 295–296.

63

[2] G.C.-M. Falck, H.K. Lindberg, S. Suhonen, M. Vippola, E. Vanhala, J. Catalán, K. Savolainen, H. Norppa, Genotoxic effects of nanosized and fine TiO2 , Hum. Exp. Toxicol. 28 (2009) 339–352. [3] A.M. Schrand, M.F. Rahman, S.M. Hussain, J.J. Schlager, D.A. Smith, A.F. Syed, Metal-based nanoparticles and their toxicity assessment, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (2010) 544–568. [4] IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 93, Carbon Black, Titanium Dioxide, and Talc, Lyon, France, 2010. [5] National Institute for Occupational Safety and Health, Occupational exposure to titanium dioxide, Department of Health and Human Services, Centers for Disease Control and Prevention, Curr. Intell. Bull. 63 (2011). [6] R. Dunford, A. Salinaro, L. Cai, N. Serpone, S. Horikoshi, H. Hidaka, J. Knowland, Chemical oxidation and DNA damage catalysed by inorganic sunscreen ingredients, FEBS Lett. 418 (1997) 87–90. [7] C.M. Sayes, R. Wahi, P.A. Kurian, Y. Liu, J.L. West, K.D. Ausman, D.B. Warheit, V.L. Colvin, Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells, Toxicol. Sci. 92 (2006) 174–185. [8] L.K. Braydich-Stolle, N.M. Schaeublin, R.C. Murdock, J. Jiang, P. Biswas, J.J. Schlager, S.M. Hussain, Crystal structure mediates mode of cell death in TiO2 nanotoxicity, J. Nanopart. Res. 11 (2009) 1361–1374. [9] A.A. Shvedova, V.E. Kagan, B. Fadeel, Close encounters of the small kind: adverse effects of man-made materials interfacing with the nano-cosmos of biological systems, Ann. Rev. Pharmacol. Toxicol. 50 (2010) 63–88. [10] S. Barillet, A. Simon-Deckers, N. Herlin-Boime, M. Mayne-L’Hermite, C. Reynaud, D. Cassio, B. Gouget, M. Carrière, Toxicological consequences of TiO2 , SiC nanoparticles and multi-walled carbon nanotubes exposure in several mammalian cell types: an in vitro study, J. Nanopart. Res. 12 (2010) 61–73. [11] J.R. Gurr, A.S. Wang, C.H. Chen, K.Y. Jan, Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells, Toxicology 213 (2005) 66–73. [12] J.J. Wang, B.J.S. Sanderson, H. Wang, Cyto- and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells, Mutat. Res. 628 (2007) 99–106. [13] I.F. Osman, A. Baumgartner, E. Cemeli, J.N. Fletcher, D. Anderson, Genotoxicity and cytotoxicity of zinc oxide and titanium dioxide in HEp-2 cells, Nanomedicine 5 (2010) 1193–1203. [14] R.K. Shukla, V. Sharma, A.K. Pandey, S. Singh, S. Sultana, A. Dhawan, ROSmediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells, Toxicol. In Vitro 25 (2011) 231–241. [15] M. Roller, In vitro genotoxicity data of nanomaterials compared to carcinogenic potency of inorganic substances after inhalational exposure, Mutat. Res. 727 (2011) 72–95. [16] S.J. Kang, B.M. Kim, Y.J. Lee, H.W. Chung, Titanium dioxide nanoparticles trigger p53-mediated damage response in peripheral blood lymphocytes, Environ. Mol. Mutagen. 49 (2008) 399–405. [17] J. Catalán, H. Järventaus, M. Vippola, K. Savolainen, H. Norppa, Induction of chromosomal aberrations by carbon nanotubes and titanium dioxide nanoparticles in human lymphocytes in vitro, Nanotoxicology, doi:10.3109/17435390.2011.625130. [18] Y. Nakagawa, S. Wakuri, K. Sakamoto, N. Tanaka, The photogenotoxicity of titanium dioxide particles, Mutat. Res. 394 (1997) 125–132. [19] E. Theogaraj, S. Riley, L. Hughes, M. Maier, D.D. Kirkland, An investigation of the photo-clastogenic potential of ultrafine titanium dioxide particles, Mutat. Res. 634 (2007) 205–219. [20] D.B. Warheit, R.A. Hoke, C. Finlay, E.M. Donner, K.L. Reed, C.M. Sayes, Development of a base set of toxicity tests using ultrafine TiO2 particles as a component of nanoparticle risk management, Toxicol. Lett. 171 (2007) 99–110. [21] ILSI Risk Science Institute Workshop Participants, The relevance of the rat lung response to particle overload for human risk assessment: a workshop consensus report, Inhal. Toxicol. 12 (2000) 1–17. [22] K.E. Driscoll, L.C. Deyo, J.M. Carter, B.W. Howard, D.G. Hassenbein, T.A. Bertram, Effects of particle exposure and particle-elicited inflammatory cells on mutation in rat alveolar epithelial cells, Carcinogenesis 18 (1997) 423–430. [23] R. Landsiedel, L. Ma-Hock, B. Van Ravenzwaay, M. Schulz, K. Wiench, S. Champ, S. Schulte, W. Wohlleben, F. Oesch, Gene toxicity studies on titanium dioxide and zinc oxide nanomaterials used for UV-protection in cosmetic formulations, Nanotoxicology 4 (2010) 364–381. [24] B. Rehn, F. Seiler, S. Rehn, J. Bruch, M. Maier, Investigations on the inflammatory and genotoxic lung effects of two types of titanium dioxide: untreated and surface treated, Toxicol. Appl. Pharmacol. 189 (2003) 84–95. [25] L.C. Renwick, D. Brown, A. Clouter, K. Donaldson, Increased inflammation and altered macrophage chemotactic responses caused by two ultrafine particle types, Occup. Environ. Med. 61 (2004) 442–447. [26] J. Wang, G. Zhou, C. Chen, H. Yu, T. Wang, Y. Ma, G. Jia, Y. Gao, B. Li, J.J. Sun, Y. Li, F. Jiao, Y. Zhaoa, Z. Chaiet, Acute toxicity and biodistribution of different sized titanium dioxide particles in mice after oral administration, Toxicol. Lett. 168 (2007) 176–185. [27] M. Geiser, M. Casaulta, B. Kupferschmid, H. Schulz, M. Semmler-Behnke, W. Kreyling, The role of macrophages in the clearance of inhaled ultrafine titanium dioxide particles, Am. J. Respir. Cell Mol. Biol. 38 (2008) 371–376. [28] F. Pott, M. Roller, Carcinogenicity study with nineteen granular dusts in rats, Eur. J. Oncol. 10 (2005) 249–281. [29] U. Heinrich, R. Fuhst, S. Rittinghausen, O. Creutzenberg, B. Bellmann, W. Koch, K. Levsen, Chronic inhalation exposure of Wistar rats and two different strains

64

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40] [41]

[42]

H.K. Lindberg et al. / Mutation Research 745 (2012) 58–64 of mice to diesel engine exhaust, carbon black, and titanium dioxide, Inhal. Toxicol. 7 (1995) 533–556. F. Bischoff, G. Bryson, Tissue reaction to and fate of parenterally administered titanium dioxide. I. The intraperitoneal site in male Marsh-Buffalo mice, Res. Commun. Chem. Pathol. Pharmacol. 38 (1982) 279–290. F. Furukawa, Y. Doi, M. Suguro, O. Morita, H. Kuwahara, T. Masunaga, Y. Hatakeyama, F. Mori, Lack of skin carcinogenicity of topically applied titanium dioxide nanoparticles in the mouse, Food Chem. Toxicol. 49 (2011) 744–749. K. Onuma, Y. Sato, S. Ogawara, N. Shirasawa, M. Kobayashi, J. Yoshitake, T. Yoshimura, M. Iigo, J. Fujii, F. Okada, Nano-scaled particles of titanium dioxide convert benign mouse fibrosarcoma cells into aggressive tumor cells, Am. J. Pathol. 175 (2009) 2171–2183. E.-Y. Moon, G.-H. Yi, J.-S. Kang, J.-S. Lim, H.-M. Kim, S. Pyo, An increase in mouse tumor growth by an in vivo immunomodulating effect of titanium dioxide nanoparticles, J. Immunotoxicol. 8 (2011) 56–67. B. Trouiller, R. Reliene, A. Westbrook, P. Solaimani, R.H. Schiestl, Titanium dioxide nanoparticles induce DNA damage and genetic instability in vivo in mice, Cancer Res. 69 (2009) 8784–8789. M.D. Shelby, G.L. Erexson, G.J. Hook, R.R. Tice, Evaluation of a three-exposure mouse bone marrow micronucleus protocol: results with 49 chemicals, Environ. Mol. Mutagen. 21 (1993) 160–179. A.T. Saber, K.A. Jensen, N.R. Jacobsen, R. Birkedal, L. Mikkelsen, P. Møller, S. Loft, H. Wallin, U. Vogel, Inflammatory and genotoxic effects of nanoparticles designed for inclusion in paints and lacquers, Nanotoxicology, doi:10.3109/17435390.2011.587900. K. Okuyama, Y. Kousaka, N. Tohge, S. Yamamoto, J.J. Wu, R.C. Flagan, J.H. Seinfeld, Production of ultrafine metal oxide aerosol particles by thermal decomposition of metal alkoxide vapors, AIChE J. 32 (1986) 2010–2019. M. Miettinen, J. Riikonen, U. Tapper, U. Backman, J. Joutsensaari, A. Auvinen, V.-P. Lehto, J. Jokiniemi, Development of a highly controlled gas-phase nanoparticle generator for inhalation exposure studies, Hum. Exp. Toxicol. 28 (2009) 413–419. A.J. Koivisto, M. Mäkinen, E.M. Rossi, H.K. Lindberg, M. Miettinen, G.C.-M. Falck, H. Norppa, H. Alenius, A. Korpi, J. Riikonen, E. Vanhala, M. Vippola, P. Pasanen, V.-P. Lehto, K. Savolainen, J. Jokiniemi, K. Hämeri, Aerosol characterization and lung deposition of synthesized TiO2 nanoparticles for murine inhalation studies, J. Nanopart. Res. 13 (2011) 2949–2961. HTP-arvot, Haitallisiksi tunnetut pitoisuudet, Sosiaali-ja terveysministeriön julkaisuja 2009 (2009) 11–20. E.M. Rossi, L. Pylkkänen, A.J. Koivisto, M. Vippola, K.A. Jensen, M. Miettinen, K. Sirola, H. Nykäsenoja, P. Karisola, T. Stjernvall, E. Vanhala, M. Kiilunen, P. Pasanen, M. Mäkinen, K. Hämeri, J. Joutsensaari, T. Tuomi, J. Jokiniemi, H. Wolff, K. Savolainen, S. Matikainen, H. Alenius, Airway exposure to silica-coated TiO2 nanoparticles induces pulmonary neutrophilia in mice, Toxicol. Sci. 113 (2010) 422–433. H.K. Lindberg, G.C.-M. Falck, J. Catalán, T. Santonen, H. Norppa, Micronucleus assay for mouse alveolar type II and Clara cells, Environ. Mol. Mutagen. 51 (2010) 164–172.

[43] M. De Boeck, P. Hoet, N. Lombaert, B. Nemery, M. Kirsch-Volders, D. Lison, In vivo genotoxicity of hard metal dust: induction of micronuclei in rat type II epithelial lung cells, Carcinogenesis 24 (2003) 1793–1800. [44] J. Nygren, S. Suhonen, H. Norppa, K. Linnainmaa, DNA damage in bronchial epithelial and mesothelial cells with and without associated crocidolite asbestos fibers, Environ. Mol. Mutagen. 44 (2004) 477–482. [45] H.K. Lindberg, A. Korpi, T. Santonen, K. Säkkinen, M. Järvelä, J. Tornaeus, N. Ahonen, H. Järventaus, A.-L. Pasanen, C. Rosenberg, H. Norppa, Micronuclei, hemoglobin adducts and respiratory tract irritation in mice after inhalation of toluene diisocyanate (TDI) and 4-4 -methylenediphenyl diisocyanate (MDI), Mutat. Res. 723 (2011) 1–10. [46] OECD guidelines for the testing of chemicals, no. 474, Mammalian Erythrocyte Micronucleus Test, 1997. [47] G. Krishna, M. Hayashi, In vivo rodent micronucleus assay: protocol, conduct and data interpretation, Mutat. Res. 455 (2000) 155–166. [48] D.R. Boverhof, M.P. Chamberlain, C.R. Elcombe, F.J. Gonzalez, R.H. Heflich, L.G. Hernández, A.C. Jacobs, D. Jacobson-Kram, M. Luijten, A. Maggi, M.G. Manjanatha, J. Benthem, B.B. Gollapudi, Transgenic animal models in toxicology: historical perspectives and future outlook, Toxicol. Sci. 121 (2011) 207–233. [49] S. Brendler-Schwaab, A. Hartmann, S. Pfuhler, G. Speit, The in vivo comet assay: use and status in genotoxicity testing, Mutagenesis 20 (2005) 245–254. [50] T. Morita, J.T. MacGregor, M. Hayashi, Micronucleus assays in rodent tissues other than bone marrow, Mutagenesis 26 (2011) 223–230. [51] H.L. Karlsson, The comet assay in nanotoxicology research, Anal. Bioanal. Chem 398 (2010) 651–666. [52] J. Muller, I. Decordier, P. Hoet, N. Lombaert, L. Thomassen, F. Huaux, D. Lison, M. Kirsch-Volders, Clastogenic and aneugenic effects of multi-wall carbon nanotubes in epithelial cells, Carcinogenesis 29 (2008) 427–433. [53] A. Wessels, D. Van Berlo, A.W. Boots, K. Gerloff, A.M. Scherbart, F.R. Cassee, M.E. Gerlofs-Nijland, E.J. Van Schooten, C. Albrecht, R.P. Schins, Oxidative stress and DNA damage responses in rat and mouse lung to inhaled carbon nanoparticles, Nanotoxicology 5 (2011) 66–78. [54] V.H. Grassian, P.T. O‘Shaughnessy, A. Adamcakova-Dodd, J.M. Pettibone, P.S. Thorne, Inhalation exposure study of titanium dioxide nanoparticles with a primary particle size of 2 to 5 nm, Environ. Health Perspect. 115 (2007) 397–402. [55] E. Bermudez, J.B. Mangum, B. Asgharian, B.A. Wong, E.E. Reverdy, D.B. Janszen, P.M. Hext, D.B. Warheit, J.I. Everitt, Long-term pulmonary responses of three laboratory rodent species to subchronic inhalation of pigmentary titanium dioxide particles, Toxicol. Sci. 70 (2002) 86–97. [56] E. Bermudez, J.B. Mangum, B.A. Wong, B. Asgharian, P.M. Hext, D.B. Warheit, J.I. Everitt, Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles, Toxicol. Sci. 77 (2004) 347–357. [57] Occupational Safety and Health Administration, Occupational safety and health standards, Subpart Z, Toxic and hazardous substances, Limits for air contaminants, http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=standards&p id=9992.