Molecular responses of alveolar epithelial A549 cells to chronic exposure to titanium dioxide nanoparticles: A proteomic view

Abstract

Although the biological effects of titanium dioxide nanoparticles (TiO2-NPs) have been studied for more than two decades, the mechanisms governing their toxicity are still unclear. We applied 2D-gel proteomics analysis on A549 epithelial alveolar cells chronically exposed for 2months to 2.5 or 50μg/mL of deeply characterized TiO2-NPs, in order to obtain comprehensive molecular responses that may reflect functional outcomes. We show that exposure to TiO2-NPs impacts the abundance of 30 protein species, corresponding to 22 gene products. These proteins are involved in glucose metabolism, trafficking, gene expression, mitochondrial function, proteasome activity and DNA damage response. Besides, our results suggest that p53 pathway is activated, slowing down cell cycle progression and reducing cell proliferation rate. Moreover, we report increased content of chaperones-related proteins, which suggests homeostasis re-establishment. Finally, our results highlight that chronic exposure to TiO2-NPs affects the same cellular functions as acute exposure to TiO2-NPs, although lower exposure concentrations and longer exposure times induce more intense cellular response.

Biological significance: Our results make possible the identification of new mechanisms that explain TiO2-NP toxicity upon long-term, in vitro exposure of A549 cells. It is the first article describing -omics results obtained with this experimental strategy. We show that this long-term exposure modifies the cellular content of proteins involved in functions including mitochondrial activity, intra- and extracellular trafficking, proteasome activity, glucose metabolism, and gene expression. Moreover we observe modification of content of proteins that activate the p53 pathway, which suggest the induction of a DNA damage response. Technically, our results show that exposure of A549 cells to a high concentration of TiO2-NPs leads to the identification of modulations of the same functional categories than exposure to low, more realistic concentrations. Still the intensity differs between these two exposure scenarios. We also show that chronic exposure to TiO2-NPs induces the modulation of cellular functions that have already been reported in the literature as being impacted in acute exposure scenarios. This proves that the exposure protocol in in vitro experiments related to nanoparticle toxicology might be cautiously chosen since inappropriate scenario may lead to inappropriate and/or incomplete conclusions.

Keywords: A549; Chronic exposure; Long-term exposure; Nanoparticle; Toxicity; Whole-proteome expression.