Nanoceramic pulmonary toxicity & reproducible cell line technology
Award Winners Series: Nanoceramic pulmonary toxicity & reproducible cell line technology
Jan 23, 2019 10:00 AM EST
Pairwise toxicity evaluation of ceramic nanoparticles exposure in human alveolar epithelial A549 cells at submerged and air-liquid cultures
Maria Joao Bessa
Portuguese National Institute of Health
Several ceramic industries have already incorporated within their production processes the manufacture of different ceramic nanoparticles (NPs), as well as the application of those nanomaterials on conventional products, which increases the risk of human exposure to these NPs, particularly in occupational settings. To investigate the in vitro toxicity of ceramic NPs (CeO2, ZrO2 and Sb2O3•S_n_O_2 NPs) in human alveolar epithelial cells, a primary target during inhalation exposure, in both submerged and air-liquid cell cultures. For submerged cultures, A549 cells were exposed for 24 h to the NPs, while in case of the air-liquid interface cultures, cells were exposed to aerosolized NPs for 2 and 4 h using a Vitrocell® exposure system (0-150 μg/cm2). Cytotoxicity was assessed by the LDH and WST-1 assays. DNA damage was assessed by the comet assay and the % DNA in the tail was used as a measure of the amount of DNA damage. In submerged conditions, no significant deleterious effects were observed in A549 cells exposed to the nanoceramics, being observed a concentration-dependent decrease in LDH release (clear CeO2 NPs assay interference) and increase in WST-1 metabolization compared to the controls. On the other hand, in air-liquid interface conditions, exposure to the aerosolized ceramic NPs affected plasma membrane integrity and the metabolic activity of A549 cells, being the effects more marked for CeO2 and ZrO2 NPs aerosols. For both in vitro culture scenarios, an increase in primary DNA damage of A549 cells was observed after exposure to the highest tested concentration of any kind of nanoceramics, but in particular to ZrO2 NPs aerosols under ALI conditions. Different toxicity effects were observed for both A549 culture conditions after exposure to the nanoceramics, being the effects more pronounced after exposure to the aerosolized NPs, also less prone to interfere in the cytotoxicity assays conducted. However, these differences in the observed toxicity may also arise from different deposited doses attained under submerged vs ALI conditions. Nevertheless, our findings highlight the potential health risks associated with exposure to this kind of aerosols in the context of the ceramic industry.
Reproducible in vitro toxicology testing using functional immortalized cells
InSCREENeX GmbH, Braunschweig, Germany
In vitro toxicology testing requires reproducible and relevant cellular test system that are available in quantities sufficient for high-throughput approaches. Although primary cells are often considered the gold standard in in vitro toxicology testing, their limited availability and phenotypic instability resulted in the emergence of in vitro expandable cell lines as a viable alternative. However, the establishment of new cell lines is unpredictable and conventional immortalization regimens often result in drastic alterations of cellular physiology. We therefore developed a defined immortalization technology allowing efficient and reproducible
establishment of novel cell lines suitable for in vitro toxicology assays. The technology allows the generation of cell lines from different cell types, from different species and from different donors. The novel cell lines were then analyzed regarding their expansion potentials as well as their functional properties. Novel cell lines including intestinal epithelial cell, alveolar epithelial cells, hepatocytes, fibroblasts, HUVECs and osteoblasts were generated from primary cells within 2 to 3 months. These cell lines showed robust proliferation and could be cultivated for more than 100 cumulative population doublings. Alveolar and intestinal epithelial cells both were polarized, developed cell-cell interactions and showed epithelial-specific structures such as microvilli and desmosomes. They also developed a tight barrier (>2000 Ohm*cm^2) with low passive diffusion. Hepatocytes expressed tissue specific markers and importantly retained their ability to upregulate Phase-1 drug metabolizing enzymes when treated with prototypical xenobiotic inducers. Functional characterization of other cell lines demonstrated that the established cell lines retained the expression of cell type specific marker proteins as well as their specific functions. Importantly, this phenotype was stable throughout the whole cultivation period. Our results demonstrate that the functional immortalization technology provides physiologically relevant cellular test systems in sufficient numbers needed for high throughput ADME and in vitro toxicology studies. These can be used as standalone test systems or easily integrated into advanced systems, e.g. organ-on-a-chip models.