Proteomic Characterization of Biological Effects Induced by Engineered Nanomaterials

Nanotechnology has been one of the major success stories of the early 21st century. The foundation for this success rests on the discovery that a small size confers completely new properties on materials. Nowadays, engineered nanomaterials (ENMs) are used in a plethora of applications such as paints, cosmetics, food products and electronics. These new properties, however, potentially make ENMs more reactive in biological systems than their large-scale counterparts. Already, asbestos-like effects have been described in mice after exposure to certain forms of carbon nanotubes (CNTs), while nano-sized titanium dioxide (nTiO2) has been shown to evoke inflammation in mouse lung. Therefore, extensive nanosafety studies have to be performed to ensure that no adverse effects are suffered by either workers or end-users of ENM products. This thesis has investigated health effects of ENMs by proteomic methods, first by evaluating the uptake and interactions of ENMs with plasma and cellular proteins followed by an analysis of the effects of ENM exposure on the intracellular proteome and secretome of human primary macrophages. The results revealed that ENM interactions with cellular proteins were governed by the surface reactivity of ENMs, whereas interactions with plasma proteins seemed to depend on the combination of both surface reactivity and active recognition, namely tagging of ENMs by opsonin proteins. The binding of cellular proteins to ENMs and subsequent interference with cellular processes might represent a novel cause of ENM toxicity, especially since transmission electron microscopy (TEM) micrographs indicated that several ENM species could be visualized free in the cytoplasm. The cytoplasmic protein expression changes after exposure to silica coated and uncoated nTiO2 revealed that silica coated TiO2 induced stronger protein expression changes in the macrophages. Most of the proteins with altered expression were related to phagocytosis, oxidative stress and inflammation. These proteome changes indicate that macrophages are actively engulfing ENMs and processing them. Moreover, the up-regulation of oxidative stress related proteins might be an indication of oxidative burst. Finally, nTiO2 treatment evoked acetylation of cytoplasmic proteins, a previously uncharacterized phenomenon in cells exposed to ENMs. The results from the macrophage secretome analysis showed that asbestos and long rigid carbon nanotubes (R CNTs) produced a similar response, while protein secretion profile of macrophages exposed to long tangled carbon nanotubes (T CNTs) exhibited a distinct profile. Bioinformatic analysis revealed that R CNTs evoked secretion of inflammation and apoptosis related proteins, possibly because of lysosomal damage. Functional assay confirmed that R CNT exposure triggered apoptosis in macrophages, while T CNTs and asbestos did not. ABSTRACT This thesis offers new knowledge concerning the biological effects of engineered nanomaterials. Proteomic methods proved to be useful in the ENMprotein interaction studies revealing that it would be beneficial to include the ENM-protein interaction experiments as part of the routine ENM characterization when assessing the health effects of ENMs. By employing quantitative proteomics, we obtained a global view of both cytoplasmic and secreted proteome changes of macrophages exposed to different ENMs.