Evaluating the Use of TiO2 Nanoparticles for Toxicity Testing in Pulmonary A549 Cells
Jana Báčová, Petr Knotek, Kateřina Kopecká, Luděk Hromádko, Jan Čapek, Pavlina Nyvltová, Lenka Brůčková, Ladislava Schröterová, Blanka Šestáková, Jiří Palarčík, Martin Motola, Dana Čížková, Aleš Bezrouk, Jiří Handl, Zdeněk Fiala, Emil Rudolf, Zuzana Bı́lková, Jan M. Macák, Tomáš Roušar
Abstract
Purpose: Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO 2 P25), are among the most produced nanomaterials worldwide. The broad use of TiO 2 P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO 2 P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO 2 P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO 2 P25 for biological testing in A549 cells and to evaluate their biological effects. Methods: We determined the size and crystallinity of TiO 2 P25. We used four techniques for TiO 2 P25 dispersion. We estimated the colloid stability of TiO 2 P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO 2 P25 (0– 100 μg.mL − 1 ) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. Results: We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO 2 P25. Under these conditions, TiO 2 P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO 2 P25 in A549 cells were met, similar results on the biological effects of TiO 2 P25 were obtained in two independent cell laboratories. Conclusion: We optimized the experimental conditions of TiO 2 P25 preparation for toxicity testing in A549 cells. The results presented here on TiO 2 P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO 2 P25 as a reference material. Keywords: titanium dioxide, nanoparticles, P25, nanotoxicity, A549 cells, dispersion