(209d) Genomic-Level Effects of Carbon Nanotubes

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. 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. 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.

            This study indicates that CNT functionalization modulates the advent of early biological events affecting their health effects. One of the goals of functionalizing CNTs is to increase their solubility in aqueous media, a feature that may make CNTs more compatible with physiological systems. Differential modulation in gene expression was observed as a function of single or multiple wall geometry and presence of specific functional groups.

            Comparison of gene expression between in vitro and in vivo exposure to CNTs revealed significant differences in the level of biological response induced towards endpoints such as oxidative stress or inflammation. In particular, MW-COOH induced the excessive coding for specific proteins associated clinically with lung fibrosis and other inflammatory processes. Toxicogenomics by Total Gene Expression Analysis is a powerful approach to identify early biological responses in in vitro and in vivo systems following exposure to carbon nanotubes.

            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 way 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.

            Thus, this study opens the way towards "intelligent" CNT functionalization that aims at reducing or eliminating potential health risks while delivering the added value with regard to applications such as enhanced drug delivery. While systematic approaches for evaluating potential adverse effects of nanomaterials are still to be implemented, the combination of cell culture systems with in vivo experiments designed to minimize the use of laboratory animals in toxicity testing through the use of whole genome microarrays are expected to become a mainstay.