Activation of stress-related signalling pathway in human cells upon SiO2 nanoparticles exposure as an early indicator of cytotoxicity

Abstract

BackgroundNanomaterials such as SiO2 nanoparticles (SiO2NP) are finding increasing applications in the biomedical and biotechnological fields such as disease diagnostics, imaging, drug delivery, food, cosmetics and biosensors development. Thus, a mechanistic and systematic evaluation of the potential biological and toxic effects of SiO2NP becomes crucial in order to assess their complete safe applicability limits.ResultsIn this study, human monocytic leukemia cell line THP-1 and human alveolar epithelial cell line A549 were exposed to a range of amorphous SiO2NP of various sizes and concentrations (0.01, 0.1 and 0.5 mg/ml). Key biological indicators of cellular functions including cell population density, cellular morphology, membrane permeability, lysosomal mass/pH and activation of transcription factor-2 (ATF-2) were evaluated utilizing quantitative high content screening (HCS) approach and biochemical techniques. Despite the use of extremely high nanoparticle concentrations, our findings showed a low degree of cytotoxicity within the panel of SiO2NP investigated. However, at these concentrations, we observed the onset of stress-related cellular response induced by SiO2NP. Interestingly, cells exposed to alumina-coated SiO2NP showed low level, and in some cases complete absence, of stress response and this was consistent up to the highest dose of 0.5 mg/ml.ConclusionsThe present study demonstrates and highlights the importance of subtle biological changes downstream of primary membrane and endocytosis-associated phenomena resulting from high dose SiO2NP exposure. Increased activation of transcription factors, such as ATF-2, was quantitatively assessed as a function of i) human cell line specific stress-response, ii) SiO2NP size and iii) concentration. Despite the low level of cytotoxicity detected for the amorphous SiO2NP investigated, these findings prompt an in-depth focus for future SiO2NP-cell/tissue investigations based on the combined analysis of more subtle signalling pathways associated with accumulation mechanisms, which is essential for establishing the bio-safety of existing and new nanomaterials.

The microtubule targeting agent PBOX-15 inhibits integrin-mediated cell adhesion and induces apoptosis in acute lymphoblastic leukaemia cells

“…Adhesion of cells on anti-integrin antibody or fibronectin coated surfaces was detected and subsequently quantified using a cell based high content analysis (HCA) system. The assay operates on the principle of fully automated fluorescent microscopy described previously (30)(31)(32) and allows multiplexing of key reporter parameters including cell-adhesion. Untreated or PBOX-15 pre-treated cells were added to fibronectin, anti-α4-, β1-, or β2-integrin antibody coated 24-well or 96-well plates (Nunc, Roskilide, Denmark) at a density of 2x10 5 or 1x10 4 cells/well respectively.…”

Evaluation of Viability and Proliferation Profiles on Macrophages Treated with Silica Nanoparticles In Vitro via Plate-Based, Flow Cytometry, and Coulter Counter Assays

“…SiO 2 NPs may exist in many different forms, including different crystallinity, morphology, and pore structure, with each form exhibiting unique characteristics [31][32][33]. Although several in vitro studies using SiO 2 NPs have been performed to show either cytotoxicity [34][35][36] or no effect on viability in vitro [34,37,38], there is limited information regarding SiO 2 NPs interference with plate-based assays. For example, it is known that ViaLight substrate (fluorescent ATP) caused precipitation of 15 nm SiO 2 NP resulting in a cloudy appearance of NP, but without ultimately affecting luminescence signal [22], and SiO 2 NPs (15 nm) caused precipitation of LDH resulting in high absorbance values and false-positive results [22]; astrocytes treated with mesoporous silica NPs (SiO 2 NP) show decreased MTT signal due to SiO 2 NP-mediated formazan exocytosis as opposed to decreased viability [39].…”

Multifactorial determinants that govern nanoparticle uptake by human endothelial cells under flow

“…Before the start of the experiments, the nuclei were stained with Hoechst (1:1000 from stock of 1 mg/mL) for 5 minutes. After washing the cells with medium, endothelial monolayers (normal or supplemented with tumor necrosis factor-α [TNF-α] at 10 ng/mL for 12 hours) were exposed to QD2.7, QD4.7, or NP50 suspended in PBS under low, medium, and high SS rates (0.05, 0.10, and 0.50 Pa, respectively) for 20 minutes using 41 …”