Multiparametric in vitro genotoxicity assessment of different variants of amorphous silica nanomaterials in rat alveolar epithelial cells.

The hazard posed to human health by inhaled amorphous silica nanomaterials (aSiO 2 NM) remains uncertain. Herein, we assessed the cyto- and genotoxicity of aSiO 2 NM variants covering different sizes (7, 15, and 40 nm) and surface modifications (unmodified, phosphonate-, amino- and trimethylsilyl-modified) on rat alveolar epithelial (RLE-6TN) cells. Cytotoxicity was evaluated at 24 h after exposure to the aSiO 2 NM variants by the lactate dehydrogenase (LDH) release and WST-1 reduction assays, while genotoxicity was assessed using different endpoints: DNA damage (single- and double-strand breaks [SSB and DSB]) by the comet assay for all aSiO 2 NM variants; cell cycle progression and γ-H2AX levels (DSB) by flow cytometry for those variants that presented higher cytotoxic and DNA damaging potential. The variants with higher surface area demonstrated a higher cytotoxic potential (SiO 2 _7, SiO 2 _15_Unmod, SiO 2 _15_Amino, and SiO 2 _15_Phospho). SiO 2 _40 was the only variant that induced significant DNA damage on RLE-6TN cells. On the other hand, all tested variants (SiO 2 _7, SiO 2 _15_Unmod, SiO 2 _15_Amino, and SiO 2 _40) significantly increased total γ-H2AX levels. At high concentrations (28 µg/cm 2 ), a decrease in G 0 /G 1 subpopulation was accompanied by a significant increase in S and G 2 /M sub-populations after exposure to all tested materials except for SiO 2 _40 which did not affect cell cycle progression. Based on the obtained data, the tested variants can be ranked for its genotoxic DNA damage potential as follows: SiO 2 _7 = SiO 2 _40 = SiO 2 _15_Unmod > SiO 2 _15_Amino. Our study supports the usefulness of multiparametric approaches to improve the understanding on NM mechanisms of action and hazard prediction.