Cytotoxicity of carbon nanotube variants: A comparative in vitro exposure study with A549 epithelial and J774 macrophage cells

Abstract

While production of engineered carbon nanotubes (CNTs) has escalated in recent years, knowledge of risk associated with exposure to these materials remains unclear. We report on the cytotoxicity of four CNT variants in human lung epithelial cells (A549) and murine macrophages (J774). Morphology, metal content, aggregation/agglomeration state, pore volume, surface area and modifications were determined for the pristine and oxidized single-walled (SW) and multi-walled (MW) CNTs. Cytotoxicity was evaluated by cellular ATP content, BrdU incorporation, lactate dehydrogenase (LDH) release, and CellTiter-Blue (CTB) reduction assays. All CNTs were more cytotoxic than respirable TiO₂ and SiO₂ reference particles. Oxidation of CNTs removed most metallic impurities but introduced surface polar functionalities. Although slopes of fold changes for cytotoxicity endpoints were steeper with J774 compared to A549 cells, CNT cytotoxicity ranking in both cell types was assay-dependent. Based on CTB reduction and BrdU incorporation, the cytotoxicity of the polar oxidized CNTs was higher compared to the pristine CNTs. In contrast, pristine CNTs were more cytotoxic than oxidized CNTs when assessed for cellular ATP and LDH. Correlation analyses between CNTs’ physico–chemical properties and average relative potency revealed the impact of metal content and surface area on the potency values estimated using ATP and LDH assays, while surface polarity affected the potency values estimated from CTB and BrdU assays. We show that in order to reliably estimate the risk posed by these materials, in vitro toxicity assessment of CNTs should be conducted with well characterized materials, in multiple cellular models using several cytotoxicity assays that report on distinct cellular processes.

• Carbon nanotubes
• cytotoxicity
• in vitro exposure
• lung epithelial cells
• macrophages
• physico–chemical characteristics

Acknowledgements

The authors wish to thank Peter Rippstein from the Ottawa Heart Research Institute for the electron microscopy of cells. Dr Mohamed Abdel Salam was a Natural Sciences and Engineering Research Council (NSERC) Postdoctoral Fellow in Canadian Government Laboratories and is currently at the Chemistry Department, Faculty of Science, King Abdulaziz University, Kingdom of Saudi Arabia.

Declaration of interest

This work was supported by Health Canada (4320105 and Chemical Management Plan). The authors declare no competing interests. Although the content of this communication reflects the work and views of the authors, Health Canada managers review, comment and approve manuscripts before publication.