Responses of A549 human lung epithelial cells to cristobalite and α-quartz exposures assessed by toxicoproteomics and gene expression analysis

Abstract

In this study, we used cytotoxicity assays, proteomic and gene expression analyses to examine the difference in response of A549 cells to two silica particles that differ in physical properties, namely cristobalite (CR) and α‐quartz (Min‐U‐Sil 5, MI). Cytotoxicity assays such as lactate dehydrogenase release, 5‐bromo‐2′‐deoxyuridine incorporation and cellular ATP showed that both silica particles could cause cell death, decreased cell proliferation and metabolism in the A549 human lung epithelial cells. While cytotoxicity assays revealed little difference between CR and MI exposures, proteomic and gene expression analyses unveiled both similar and unique molecular changes in A549 cells. For instance, two‐dimensional gel electrophoresis data indicated that the expression of proteins in the cell death (e.g., ALDH1A1, HTRA2 and PRDX6) and cell proliferation (e.g., FSCN1, HNRNPAB and PGK1) pathways were significantly different between the two silica particles. Reverse transcription–polymerase chain reaction data provided additional evidence supporting the proteomic findings. Preliminary assessment of the physical differences between CR and MI suggested that the extent of surface interaction between particles and cells could explain some of the observed biological effects. However, the differential dose–response curves for some other genes and proteins suggest that other physical attributes of particulate matter can also contribute to particulate matter‐related cellular toxicity. Our results demonstrated that toxicoproteomic and gene expression analyses are sensitive in distinguishing subtle toxicity differences associated with silica particles of varying physical properties compared to traditional cytotoxicity endpoints. Copyright © 2016 Her Majesty the Queen in Right of Canada. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.

A matrix‐assisted laser desorption ionization–time‐of‐flight–time‐of‐flight–mass spectrometry‐based toxicoproteomic screening method to assess in vitro particle potencies

“…Although, the primary aim of this work was to develop high‐content MS‐based proteomic profiling for PM‐related changes and for reliable assessment of relative PM potencies, nevertheless, we tentatively identified some (false discovery rate < 5% with at least one unique tryptic peptide) candidate biomarkers of PM exposures for both cell types. The candidate marker proteins identified in these cell types were associated with cell proliferation, apoptosis, energy metabolism, membrane and mitochondrial protein changes in line with what we have observed before (Vuong et al, ). For instance, one of the identified candidate markers of PM exposure in J774 cells was caspase‐7, which is associated with a mitochondrial apoptosis pathway (Bastiani, Vidotto, & Horn, ).…”

“…For proteomic processing, only the 0, 30 and 100 μg cm −2 samples were selected due to the frank cytotoxicity observed at the 300 μg cm −2 dose based on cytotoxicity results, and the high likelihood for this dose to be associated with mostly cell death mechanisms (Vuong et al, ). The goal of this study was not to identify, exhaustively and comprehensively, the entire cellular proteome following exposure to airborne particles, but rather to develop a proteomic method that will generate information with an acceptable sensitivity and specificity to conduct toxicity screening for environmental PM in small volumes of in vitro cell lysate samples.…”

An updated review of the genotoxicity of respirable crystalline silica

“…Gene expression was also investigated by Vuong et al [ 39 ], alongside proteomic analysis, in cultured A549 cells in vitro after exposure to cristobalite and quartz (Min-U-Sil 5). Particles were prepared in buffer (0.19% NaCl, 25 μg/mL Tween-80) at a concentration of 10 mg/mL and diluted in culture media (DMEM) for treatment.…”

Comparative cytotoxicity of respirable surface-treated/untreated calcium carbonate rock dust particles in vitro

“…In this paper we investigate the comparative cytotoxicity of respirable calcium carbonate rock dust particles with or without specific anti-caking surface treatments, using a surrogate for lung epithelial cells A549, a human lung epithelial adenocarcinoma and widely used cell model for alveolar epithelial function (Hetland et al, 2001; Ovrevik et al, 2006; Vuong et al, 2017). We also used the human promyelocytic cells (THP-1) differentiated with phorbol 12-myristate 13-acetate (PMA), to study the macrophage responses to external stimuli (Chanput, Mes, and Wichers, 2014; Riendeau and Kornfeld, 2003; Tedesco et al, 2018; Van der Meeren, Moureau, Laurent, Laroche, and Angulo, 2016; Voth, Howe, and Heinzen, 2007).…”