78 Fibre-like behaviour of thin nanotubes: <i>in vitro</i> toxicological and proteomic evidence

Abstract Inhalation of rigid, biopersistent mineral fibres may induce fibrosis, lung cancer, and mesothelioma. However, the toxicological effects of nanofibres, including carbon nanotubes (CNTs), remain less understood. Rigid nanofibres are known to damage alveolar macrophages and cause characteristic proteomic alterations, whereas CNTs with diameters (Ø) &amp;lt;30 nm cause differential effects. Here we investigated toxicological and proteomic responses of alveolar macrophages exposed to CNTs with varying diameter and rigidity. NR8383 cells were exposed for 18 h to CNTs with different diameters: two rigid CNTs (Ø 66 to 95 nm), one CNT (Ø 41 to 50 nm), and two CNTs (Ø &amp;lt;30 nm). Dispersions were produced by ultrasonic energy following the NanoGenoTox protocol and characterised by electron microscopy. Uptake was confirmed by light microscopy. Release of lactate dehydrogenase, glucuronidase and TNFα were analysed after 18 h exposure to identify the non-cytotoxic condition for proteomic profiling (22.5 µg/mL) which was conducted on cell lysates and cell culture supernatants using Tandem Mass Tag technology. CNTs Ø &amp;gt;30 nm induced greater cytotoxicity, consistent with existing literature. Electron microscopy showed that flexible CNTs Ø &amp;lt;30 nm formed secondary high–aspect ratio structures, which may underlie the observed fibre-like proteomic responses, including significant alterations in the lysosomal KEGG pathway. These findings highlight the need to further elucidate toxicity mechanisms of CNTs &amp;lt;30 nm, placing emphasis on fibre morphology and agglomeration under specific exposure conditions. To better control fibre morphology and circumvent agglomeration in media, the exposure of macrophages to aerosolised CNTs on cell culture dishes is currently explored.