Exploring potential genotoxic effects of nanocelluloses versus multi-walled carbon nanotubes in co-cultures of human lung epithelial cells and monocyte-derived macrophages

Cellulose nanomaterials (CNMs) are advanced materials exhibiting unique properties for innumerous industrial and biomedical applications. Human exposure to CNMs has been equally growing, which raises some concern, given the similarity of some CNMs size and morphology with that of multiwalled carbon nanotubes (MWCNTs) that induce lung toxicity. The genotoxic effects of three CNMs produced from Eucalyptus globulus bleached kraft pulp through different methods and, hence, harbouring different physicochemical properties (two micro/nano- fibrillated and one nanocrystalline), were investigated in human alveolar (A549) cells co-cultured with monocyte- derived (THP-1) macrophages and in conventional A549 cultures. Two MWCNT differing in diameter, length and flexibility, NM 401 and NM 402 (JRC Repository), were used as references. None of the CNMs was toxic to A549 cells. The results of the in vitro micronucleus assay showed that exposure of A549 cells (1.5 to 50 μg/cm2 48h) to each CNM or to NM 402), either in monoculture or in co-culture, did not produce significant alterations in the frequency of micronucleated binucleated cells (MNBNC), as compared to the control. In contrast, NM-401, the thickest, longest and more rigid nanofiber, was able to significantly increase the frequencies of MNBNC. The cytokinesis-block proliferation index of A549 cells was not affected by CNMs or MWNTs exposure. To conclude, although data from other endpoints is needed, the present in vitro data suggests that the studied CNMs are neither toxic nor genotoxic to lung cells, increasing the weight of evidence in favor of their biocompatibility.The work was funded by FCT/MCTES through national funds (PIDDAC), project ToxApp4NanoCELFI (PTDC/SAU-PUB/32587/2017), UIDB/00102/2020 and UIDB/00009/2020; UIDP/00009/2020; FEDER, EU funds, through the COMPETE 2020 POCI and PORL, FCT/MCTES and POR Lisboa2020, under PIDDAC (POCI-01-0145-FEDER-007688) to projects UIDB/50025/2020-2023, PTDC/CTM-REF/30529/2017 and EUTOPIA CA17139.The authors acknowledge CYTED network NANOCELIA (Transferencia Tecnológica sobre aplicaciones de nanocelulosa en iberoamerica).N/