Multiparametric in Vitro Genotoxicity Assessment of Different Variants Of Amorphous Silica Nanomaterials in Rat Alveolar Epithelial Cells

Abstract

[Abstract] 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 c-H2AX levels (DSB) by flow cytom-etry 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 c-H2AX levels. At high concentrations (28 mg/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 ¼ SiO2_40 ¼ SiO2_15_Unmod > SiO2_15_Amino. Our study supports the usefulness of multiparametric approaches to improve the understanding on NM mechanisms of action and haz ard prediction.