Multiparametric <i>in vitro</i> 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 (aSiO₂ NM) remains uncertain. Herein, we assessed the cyto- and genotoxicity of aSiO₂ 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 aSiO₂ 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 aSiO₂ 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 (SiO₂_7, SiO₂_15_Unmod, SiO₂_15_Amino, and SiO₂_15_Phospho). SiO₂_40 was the only variant that induced significant DNA damage on RLE-6TN cells. On the other hand, all tested variants (SiO₂_7, SiO₂_15_Unmod, SiO₂_15_Amino, and SiO₂_40) significantly increased total γ-H2AX levels. At high concentrations (28 µg/cm²), a decrease in G₀/G₁ subpopulation was accompanied by a significant increase in S and G₂/M sub-populations after exposure to all tested materials except for SiO₂_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: SiO₂_7 = SiO₂_40 = SiO₂_15_Unmod &gt; SiO₂_15_Amino. Our study supports the usefulness of multiparametric approaches to improve the understanding on NM mechanisms of action and hazard prediction.