Genotoxicity assessment of titanium dioxide nanoparticle accumulation of 90 days in the liver of gpt delta transgenic mice

Backgound A variety of in vivo and in vitro studies to assess the genotoxicity of titanium dioxide nanoparticles (TiO2 NPs) have been reported, but the results are inconsistent. Recently, we reported that TiO2 NPs exhibit no genotoxic effects in the liver and erythrocytes during a relatively brief period following intravenous injection into mice. However, there is no information about long-term genotoxicity due to TiO2 NP accumulation in tissues. In this study, we investigated the long-term mutagenic effects of TiO2 NPs and the localization of residual TiO2 NPs in mouse liver after multiple intravenous injections. Results Male gpt delta C57BL/6 J mice were administered with various doses of TiO2 NPs weekly for 4 consecutive weeks. The long-term mutagenic effects on the liver were analyzed using gpt and Spi− mutation assays 90 days after the final injection. We also quantified the amount of titanium in the liver using inductively coupled plasma mass spectrometry and observed the localization of TiO2 NPs in the liver using transmission electron microscopy. Although TiO2 NPs were found in the liver cells, the gpt and Spi− mutation frequencies in the liver were not significantly increased by the TiO2 NP administration. Conclusions These results clearly show that TiO2 NPs have no mutagenic effects on the liver, even though the particles remain in the liver long-term.


Introduction
Titanium dioxide nanoparticles (TiO 2 NPs) have become widely used in several industrial applications. Ultrafine TiO 2 NPs (10-50 nm) cause lung cancer in rats through chronic inhalation [1]. Therefore, TiO 2 NPs are classified as an IARC Group 2B carcinogen (possibly carcinogenic to humans) [1,2]. Genotoxicity is one of the key factors to assess the carcinogenic risk to humans. Several studies in mice and rats have reported conflicting results of various genotoxic endpoint analyses [3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Recently, we reported that TiO 2 NPs have no genotoxic effects in the liver and erythrocytes when intravenously injected into gpt delta mice [17], but there are still reports about their positive effect [18,19]. Thus, the genotoxicity of TiO 2 NPs remains unclear [20,21]. TiO 2 NPs in the rodent bloodstream are translocated to the liver and remain in the tissue long-term [22][23][24][25]. However, the long-term genotoxic effects of TiO 2 NPs in the liver are not well understood. To investigate the long-term genotoxic effects, the mutagenicity is the optimum endpoint among various genotoxic endpoints because of the accumulation potential of TiO 2 NPs. In this study, we examined the long-term mutagenicity of TiO 2 NPs and the amount and localization of the remaining particles in the liver after intravenous injection in gpt delta mice [26]. Our results indicated that TiO 2 NPs show no mutagenicity in the tissue, although they remain within the liver cells for an extended period of time.

Animals and reagents
The guidelines for the care and use of laboratory animals set forth by the Institutional Animal Care and Use Committee of the Japan National Institute of Occupational Safety and Health were followed. Male C57BL/6 J gpt delta mice were obtained from Japan SLC (Shizuoka, Japan). They were housed under specific pathogen-free conditions with a 12 h light-dark cycle and provided tap water and sterile CE-2 pellets (CLEA Japan Inc., Tokyo, Japan) ad libitum. Aeroxide® P25 titanium dioxide (TiO 2 -P25) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of the TiO 2 NP suspension and its administration to mice
The TiO 2 -P25 suspension was prepared as previously described [17]. Eight-week-old male gpt delta mice were randomly divided into four groups, with 6 mice per group. Mice were administrated by tail vein injection with the TiO 2 -P25 NP suspension at doses of 2, 10, and 50 mg/kg body weight once a week for 4 consecutive weeks. Mice were euthanized on day 90 after the final injection of TiO 2 -P25. Portions of the middle liver lobe were removed for gpt and Spi − mutation assays, quantification of titanium by inductively coupled plasma mass spectrometry (ICP-MS), and observation of TiO 2 -P25 particles by transmission electron microscopy (TEM).
gpt and Spi − mutation assay The gpt and Spi − mutation assays were conducted as previously described [17].

Quantification of titanium in the liver by ICP-MS
Liver samples were weighed and digested with nitric acid and hydrogen peroxide. The concentration of titanium in each sample was measured using ICP-MS (Agi-lent7900 ICP-MS, Agilent Technologies, Tokyo, Japan). The titanium concentration was determined at the mass number of 47 m/z as previously reported [27].

Observation of hepatocyte ultrastructure by TEM
Liver samples taken at day 90 after the final injection of TiO 2 -P25 were analyzed using a JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan) as previously described [17].

Statistical analysis
Statistical significance was examined using Dunnett's test after one-way ANOVA. Values of P < 0.05 were considered significant.

Characterization of TiO 2 suspensions
TiO 2 -P25 was dispersed in disodium phosphate by sonication, as previously reported [17], then diluted to the concentration corresponding to each dose. The hydrodynamic sizes of TiO 2 -P25 in these diluents were measured each time before the injection by dynamic light scattering, and the average of four time determinations was showed in Table 1. The Z-average of the TiO 2 -P25 particles in suspension was about 150 d.nm, regardless of the different concentrations in these diluents.

General observations of the animals
The body weight of mice in each group did not differ at weekly measurement for the first 4 weeks and thereafter to the end of the experiment (data not shown). However, one mouse was dead immediately after the injection at the third week, but the cause for the death could not be identified. Some of the mice got astounded for a short time immediately after the injection, but the mice in all groups were basically not found with abnormal behaviors and appearance.

Mutation frequencies of gpt and Spi − in the liver
The gpt and Spi − mutation frequencies in the liver were determined on day 90 after the last administration of TiO 2 -P25 (Tables 2 and 3). Either the gpt or Spi − mutation frequencies were not significantly different between the vehicle control group and TiO 2 administration groups at any dose. These results suggest that TiO 2 -P25 has no mutagenic effect on hepatocytes in mice at 90 d after the last administration.

Quantification and localization of TiO 2 NPs in the liver
The amount of titanium in the liver of mice administered TiO 2 -P25 was quantified via ICP-MS. The average amount of titanium in the liver was dose dependent (Table 4). To clarify the localization of TiO 2 particles, liver sections were obtained from mice treated with 50 mg/kg TiO 2 -P25, and the sections were observed by TEM. Large clusters containing the TiO 2 NPs were found in the parenchymal hepatocytes (Fig. 1c) and Kupffer cells (Fig. 1b), although the clusters were much more prevalent in the latter. The particles were extremely agglomerated within the cytoplasm of both cell types. These results indicate that TiO 2 NPs remained in the liver 90 d after the last injection and were mainly localized in the cytoplasm of Kupffer cells.

Discussion
TiO 2 NPs have been classified as an IARC Group 2B potential carcinogen. Therefore, their genotoxicity is an important property for risk assessment. Several in vivo studies related to genotoxic effects of TiO 2 NPs have been reported, but their results are inconsistent [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. Almost all of these reports analyzed the micronuclei and DNA damage by the comet assay, which reveals transient genotoxic consequences that occur shortly after exposure. In addition, the mutagenicity in TiO 2 -accumulating tissues such as the liver and spleen has been examined in transgenic mice for a relatively brief period [8,17]. Thus, the long-term mutagenic effects of TiO 2 NPs remain unclear. In this study, long-term genotoxic effects were examined in the liver of mice intravenously administered TiO 2 NPs.
Recently, we reported that TiO 2 -P25 exhibits no mutagenicity in the liver of mice 9 d after the final injection based on gpt and Spi − mutation assays [17]. However, TiO 2 NPs translocated to the liver are known to accumulate for a long period [22][23][24][25]. Thus, we have examined the mutagenicity of TiO 2 -P25 in the liver of longterm housed mice after the last administration. TiO 2 -P25 caused no mutagenic effects in the liver 90 d after the final injection, even though the particles were observed in the liver cells. The state of the cells that   incorporated TiO 2 NPs after 90 d seemed mostly unchanged compared to that after 9 d [17]. In addition, the amount of titanium in the liver at 90 d after the last administration was similar to that at 9 d [17]. This result indicates that TiO 2 NPs are not easily removed from the liver, even after a long period, but their presence does not cause any adverse effects.
There were a few in vivo studies reported the positive genotoxic effect of TiO 2 -NPs. For example, TiO 2 (Aeroxide P25®) (same material as used in our present study) induced micronuclei and DNA strand breaks in peripheral blood in adult male mice exposed to 500 mg/kg TiO 2 NPs of 21 nm size through drinking water for 5 d [14], but the effect was not analyzed in liver. With the same material and intravenous injection route, Dobrzynska et al [4] detected increase of micronuclei in bone marrow polychromatic erythrocytes of mice only at 24 h but not later at 7 and 28 d. Modrzynska et al [28] investigated the DNA strand breaks in the liver of mice treated with TiO 2 NPs (NanoAmor, 10.5 nm) by intratracheal instillation, intravenous injection or oral gavage at a single dose of 162 μg/mouse, and did not find DNA damages in liver tissue on day 1, 28 or 180 after the exposure by any administration routes, though there was a significant increase in the level of DNA damages in lung tissue on day 180 following intratracheal instillation. These reports suggested that TiO 2 NPs may induce transient DNA damages in tissue such as blood cells, but not in liver. The endpoint of genotoxicity in this study was gene mutations, which are basically not repairable and could indicate the long term effect. The negative findings in this study are supported by many other reports [6-9, 12, 28]. It is known that physicochemical characteristics (primary size, shape, etc.) and study designs (dose, dispersion method, recovery time, models) can influence the toxicity of nanoparticles in the assay system. More studies with different TiO 2 NPs are needed to better understand the health effects of this new material.

Conclusion
TiO 2 NPs accumulate in the liver cells for long term. However, they do not induce genotoxic effect in the tissue. Therefore, the long-term genotoxic effects of TiO 2 NPs administered by inhalation and ingestion which may introduce a small portion of the particles into liver, may be negligible in the liver.