Agglomeration of titanium dioxide nanoparticles increases toxicological responses in vitro and in vivo

Abstract

Background: The terms agglomerates and aggregates are frequently used in the regulatory definition(s) of nanomaterials (NMs) and hence attract attention in view of their potential influence on health effects. However, the influence of nanoparticle (NP) agglomeration and aggregation on toxicity is poorly understood although it is strongly believed that smaller the size of the NPs greater the toxicity. A toxicologically relevant definition of NMs is therefore not yet available, which affects not only the risk assessment process but also hinders the regulation of nano-products. In this study, we assessed the influence of NP agglomeration on their toxicity/biological responses in vitro and in vivo.

Results: We tested two TiO₂ NPs with different primary sizes (17 and 117 nm) and prepared ad-hoc suspensions composed of small or large agglomerates with similar dispersion medium composition. For in vitro testing, human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic (THP-1) cell lines were exposed to these suspensions for 24 h and endpoints such as cytotoxicity, total glutathione, epithelial barrier integrity, inflammatory mediators and DNA damage were measured. Large agglomerates of 17 nm TiO₂ induced stronger responses than small agglomerates for glutathione depletion, IL-8 and IL-1β increase, and DNA damage in THP-1, while no effect of agglomeration was observed with 117 nm TiO₂. In vivo, C57BL/6JRj mice were exposed via oropharyngeal aspiration or oral gavage to TiO₂ suspensions and, after 3 days, biological parameters including cytotoxicity, inflammatory cell recruitment, DNA damage and biopersistence were measured. Mainly, we observed that large agglomerates of 117 nm TiO₂ induced higher pulmonary responses in aspirated mice and blood DNA damage in gavaged mice compared to small agglomerates.

Conclusion: Agglomeration of TiO₂ NPs influences their toxicity/biological responses and, large agglomerates do not appear less active than small agglomerates. This study provides a deeper insight on the toxicological relevance of NP agglomerates and contributes to the establishment of a toxicologically relevant definition for NMs.

Keywords: Agglomerates; Biological responses; Nanomaterials; Titanium dioxide; Toxicity.

Fig. 1

Representative TEM micrographs of freshly prepared TiO₂ stock suspensions of small (SA) and large agglomerates (LA). 17 nm-SA (a), 17 nm-LA (b), 117 nm-SA (c) and 117 nm-LA (d)

Fig. 2

Estimated TiO₂ dose reaching the bottom of the wells after 24 h as a function of increasing nominal doses applied in exposure media. Dosimetry simulation was performed with a distorted grid (DG) model for 17 (a and c) and 117 nm (b and d) using parameters obtained from exposure media DMEM/F12 (a and b) and RPMI 1640 (c and d). The slope values are indicated near the respective lines. R² > 0.99 for all the suspensions. The percentage of dose delivered to the cells did not differ for 96 and 24 well plates, as the height of the liquid column was similar (6 mm)

Fig. 3

Influence of TiO₂ agglomeration on THP-1 biological responses. Total glutathione (GSH) (a), IL-8 (b) and IL-1β secretion (c), and DNA damage (d) measured in cell cultures after 24 h exposure to different concentrations of small (SA) and large agglomerates (LA) of 17 nm TiO₂. Data are expressed as means ± SD from three independent experiments performed in duplicates. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test). Two-way ANOVA was used to determine the significant differences between suspensions (significant p value indicated at the top left corner)

Fig. 4

Influence of TiO₂ agglomeration on in vivo responses in mice exposed via oropharyngeal aspiration or oral gavage. BAL lymphocytes (a) and Ti persistence in lung tissues (b) in aspirated mice and blood DNA damage in gavaged mice (c and d) measured 3 d after exposure to increasing doses of small (SA) and large agglomerates (LA) of 17 and 117 nm TiO₂. Data are expressed as means ± SD from 4 to 5 mice in each group. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represent significant difference compared to control (One way ANOVA followed by Dunnett’s multiple comparison test). Two-way ANOVA was used to determine the significant differences between suspensions (significant p value indicated at the top left corner)