Different genotoxic effects of multi-walled carbon nanotubes in A549 cells: implications for nanomaterials safety investigation

Human exposure to nanomaterials (NM) has been increasing worldwide, either due to the growing of environmental sources or from increased deliberated production for application in consumer products and nanomedicine. In particular, single- and multi-walled carbon nanotubes (MWCNT) have been developed for industrial purposes, and their safety must be assured. The same properties that render MWCNT-based materials so attractive may also cause higher toxicity. In particular, the similarity, in size and shape, between MWCNTs and asbestos fibres has raised concerns about their potential effects in human health. Moreover, contradictory results concerning their genotoxicity and carcinogenicity have been reported and further safety assessment is urgent. The objective of the present work was to characterize the potential cyto- and genotoxic effects of two MWCNTs (NM402 and NM403) in a human type-II alveolar epithelial cell line (A549). Dispersions of each NM were freshly prepared and cultures were exposed to NMs concentrations ranging from 0.52-52.08 μg/cm2. The clonogenic assay was used to determine in situ cell survival (8-days exposure) and the cytokinesis-block micronucleus assay was carried out (48h-exposure) to evaluate genotoxicity. Concurrent control cultures were also analysed: vehicle control, positive control (mitomycin C, MMC) and reference NM (ZnO-NM110). The results of the clonogenic assay showed that both NMs induced a concentration-dependent reduction of the cell survival with IC50 of 25.15 and 27.63 μg/cm2 for NM402 and NM403, respectively. The highest concentrations of NM402, 26.04 and 52.08 μg/cm2, induced a 2-fold significant increase in micronucleated binucleate cells (MNBCs) compared with the vehicle controls (P=0.006 and 0.019, respectively). Regression analysis indicated a concentration-response relationship that was best fitted to a linear-quadratic model (R2= 0.861). However, no concentration-response relationship in MNBCs was observed for NM403. The cytokinesis-block proliferation index (CBPI) remained unaltered following A549 cells exposure to NM402 or NM403. The positive controls, MMC and ZnO significantly increased MNBCs frequency and concomitantly decreased CBPI. In summary, while both NMs were cytotoxic for A549 cells, their ability to cause DNA damage was distinct. NM403 was not genotoxic while NM402 caused a dose-dependent genotoxic effect, which may be related to a potential carcinogenic activity. The differences observed may be explained by structural differences between the two MWCNTs. Although both present low diameter, they differ in length, being NM402 the longest. Thus, the result of lower genotoxicity of NM403 is in line with the fibre paradigm of CNT toxicity, whereby the length would be critical to their toxic potential. However, the NMs also differ in the types and contents of impurities, being NM402 the less pure (>90%), which may contribute to the observed genotoxicity. Regarding safety assessment, the different genotoxicity observed for these two closely related NMs highlights the importance of investigating the toxic potential of each NM individually, instead of considering a common mechanism responsible for CNT toxicity, since physical-chemical characteristics are recognized as important toxicity determinants. Co-funded by EU Grant Agreement 2009 21 01 (NANOGENOTOX), in the framework of the Health Programme and INSA