Exposome-Based Risk Assessment of Carbon Nanotube Functionalisation

The exposome encompasses all the exposures of a human during his/her lifetime. As such it promises to overhaul the current regulatory risk assessment paradigm for chemicals and pharmaceuticals by considering co-exposures during critical life windows. This paper presents a first-of-its-kind exposome study of engineered carbon nanotubes in order to incorporate risk-based considerations in the design process. The widespread use of functionalized carbon nanotubes (CNTs) makes the understanding of potential harmful effects highly important. Two cell culture systems, human A549 pneumocytes and HaCaT keratinocytes, were used to assess the modulation of gene expression due to exposure to single and multi-walled CNTs. Moreover, CD-1 male mice were exposed to the CNTs tested by intra-tracheal instillation and lung samples were taken and analyzed after 1 day of exposure. Twelve healthy volunteers between 25 and 30 years old were exposured to low doses of CNTs and blood and urine samples were analysed using advanced metabolomics and lipidomics. Differentially functionalized CNTs (MW-COOH and MW-NH2) were tested in comparison with pristine multi-wall carbon nanotubes (MWCNTs) and single-wall carbon nanotubes (SWCNTs). Toxicogenomic analysis included whole genome micro-array analysis and quantitative PCR using micro-fluidic cards for inflammation genes. Comparison of gene expression between in vitro and in vivo exposure to CNTs revealed differences in the level of biological response induced towards oxidative stress, inflammation and apoptosis. Differential modulation in gene expression after in vivo exposure was observed as a function of single or multiple wall geometry and presence of specific functional groups. MW-COOH showed a very high degree of up-modulation of the genes coding for chemokine ligands clinically associated with the onset of lung fibrosis in humans. The This effect was much less pronounced with MW-NH2 or SWCNT, whereas pristine MWVNT did not show any statistically significant modulation in gene expression. The main biological pathways induced by the tested CNTs were chemokine and cytokine induced inflammation and oxidative stress. Activation of the NFkB pathway is the key biological process initiating a cascade of effects. This may cause a perturbation of the IL-6 pathway that aims to regulate the inflammatory processes and compensate apoptotic changes. Overall the immunological responses related toMWCNT exposure are considered as the result of the synergistic effect of systemic (mediated by cells of the routes of exposure) and local inflammation (blood cells).

The complexity of biological responses following exposure to engineered nanotubes was shown and the need for development of dedicated, comprehensive methodologies to approach the potential health effects of nanomaterials underlined. Before considering chemical functionalization as a general approach to improve biocompatibility and safety characteristics of CNTs, a number of toxicological issues must be addressed and more extensive investigations on effects of functionalized CNTs in biological systems are warranted. This opens the way towards "intelligent" CNT functionalization that aims at reducing or eliminating potential health risks while delivering the added value of CNTs that are proposed for diagnostic or therapeutic applications bringing patients into direct contact with them.