Identifying contact-mediated, localized toxic effects of MWCNT aggregates on epithelial monolayers: a single-cell monitoring toxicity assay

Abstract

Aggregates of multiwalled carbon nanotubes (MWCNT) impair the barrier properties of human airway cell monolayers. To resolve the mechanism of the barrier alteration, monolayers of Calu-3 human airway epithelial cells were exposed to aggregated MWCNT. At the cell-population level, trans-epithelial electrical resistance (TEER) was used as an indicator of barrier competence, caspase activity was assessed with standard biochemical assays, and cell viability was investigated by biochemical techniques and high-throughput screening (HTS) technique based on automated epifluorescence microscopy. At cell level, the response to MWCNT was investigated with confocal microscopy, by evaluating cell death (calcein/propidium iodide (PI)), proliferation (Ki-67), and apoptosis (caspase activity). At the cell-population level, exposure to aggregated MWCNT caused a decrease in TEER, which was not associated with a decrease in cell viability or onset of apoptosis even after an 8-d exposure. In contrast, confocal imaging demonstrated contact with MWCNT aggregates triggered cell death after 24 h of exposure. In the presence of a natural surfactant, both TEER decrease and contact-mediated toxicity were mitigated. With confocal imaging, increased proliferation and apoptosis were detected in Calu-3 cells next to the aggregates. Contact-mediated cytotoxicity was recorded in two additional cell lines (BEAS-2B and A549) derived from human airways. Similar results were confirmed by adopting two additional MWCNT preparations with different physico-chemical features. This indicates MWCNT caused localized damage to airway epithelial monolayers in vitro and altered the apoptotic and proliferative rate of epithelial cells in close proximity to the aggregates. These findings provide evidence on the pathway by which MWCNT aggregates impair airway barrier function, and support the use of imaging techniques as a possible regulatory-decision supporting tool to identify effects of aggregated nanomaterials not readily detected at cell population level.

• Carbon nanotubes
• cytotoxicity
• lung epithelial barrier
• proliferation
• trans-epithelial electrical resistance

Supplementary material available online Supplementary Figure S1–S4

Acknowledgements

The authors would like to thank Prof. Petronini (University of Parma) for the supply of BEAS-2B and A549 cell lines. We would also like to thank Chiesi Farmaceutici SpA, Parma, Italy, for the kind donation of the natural surfactant Curosurf®.

Declaration of interest

The authors declare that they have no competing interests. This work was supported by EU FP7 Sanowork (Ref. 280716) and Marina (Ref. 263215) to E. B.; D. M. was partially supported by EU FP7 NANoREG project (Ref. 310584), whereas A. P. M. was partially supported by the EU FP7 project NAMDIATREAM (Ref. 246479). M. G. B. is supported by a research scholarship of the University of Parma Medical School.

Authors' contributions

O. B., E. B., and A. P. M. conceived and designed the study, participated in its coordination, drafted the manuscript, and critically reviewed the final version of the manuscript. B. M. R. participated in the design of the study, carried out cell viability, and TEER measurements and critically reviewed the results. R. G. carried out confocal studies and critically reviewed the results. M. G. B. and L. D. C. cooperated in the confocal observations. I. F. and F. S. carried out part of the characterization experiments and critically evaluated the results. D. M. and A. P. M. carried out HR-TEM, TGA, and HTS experiments and actively reviewed the manuscript. All authors read and approved the final manuscript. B. M. R., R. G., and D. M. equally contributed to this work.