Nanoparticles and the intestine: in vitro and in vivo investigations on genotoxic and inflammatory effects

The toxicological mechanisms of nanoparticles (NP) in the respiratory tract are relatively well understood and are thought to arise mainly from their unique ability to generate reactive oxygen species (ROS). NP are characterised by a size range between 1 and 100 nm and can directly induce intracellular oxidative stress, e.g. resulting in cell death or oxidative DNA damage. Induction of pro-inflammatory signalling pathways, which is also triggered by ROS, can initiate or exacerbate inflammatory events by recruitment and activation of phagocytes such as neutrophils or macrophages. NP induced oxidative stress and inflammation in the lung is known to play a role in various lung diseases including chronic obstructive pulmonary disease (COPD) and asthma. Elevated DNA strand breakage and oxidative DNA damage by ROS is implicated in carcinogenesis and may provide an explanation for the associations between inhalation of ambient particulate matter, diesel exhaust particles or nano-sized metal oxide particles and lung cancer. Industrial applications of nano-sized materials in food and food products have increased dramatically. However, possible adverse effects of particles in the human intestine are poorly investigated to date and these may play a role in chronic inflammatory bowel diseases or bowel cancer. In the present thesis, a selection of five NP relevant as likely food additives or contaminants (e.g. for use in food packaging) was analysed for the potential to induce cytotoxicity and DNA damage in the widely used human intestinal epithelial cell line Caco-2. Carbon black, a model for environmental NP, as well as magnesium oxide did only slightly reduce the metabolic activity of the cells, and no DNA damaging potential was found. In contrast, titanium dioxide (TiO2) revealed both cytotoxic and light-dependent DNA damaging potential. Upon detailed particle characterisation, these effects on intestinal cells were found to be mainly driven by the crystalline structure of the different TiO2 samples and less on their particle size or specific surface area, both being major factors in TiO2 induced lung toxicity. Silicon dioxide (SiO2) and zinc oxide (ZnO) were both found to induce not only strong cytotoxic effects in Caco-2 cells, but also oxidative stress accompanied by induction of (oxidative) DNA damage: Moreover, they showed a strong potential to upregulate the pro-inflammatory cytokine interleukin-8, which is known as a potent recruitment and activating factor for neutrophils. Both materials tended to form aggregates and/or agglomerates in suspension and were found to be taken up into Caco-2 cells independent of microtubule- and clathrin/AP2-mediated endocytosis. A simulated gastrointestinal digestion procedure was established; however, this treatment did not alter the cytotoxic or pro-inflammatory potentials of both SiO2 and ZnO. This is relevant information for hazard assessment, when these particles are used in food products and as such enter the human intestinal tract. Potential interactions between NP and neutrophils were investigated using a co-culture-model of freshly isolated primary human neutrophils with Caco-2 cells. In this model enhanced superoxide anion production by the neutrophils was observed and, in association with this, increased DNA damage and oxidative lesions in the Caco-2 cells. However, SiO2 did not directly activate the neutrophils, and no induction of DNA strand breakage was detected when SiO2 was applied in the co-culture model. The effect of ingested SiO2 was therefore also determined in vivo in healthy as well as colitis induced mice. The induction of DNA strand breakage and oxidative lesions were analysed by using the highly sensitive Fpg-modified comet assay and immunohistochemical staining. Interestingly, SiO2 only appeared to induce slight oxidative DNA damage after chronic but not after acute treatment and played no role in the modification of the genome integrity in colitic colons. Taken together, the present thesis contributed to a better understanding of various adverse effects of relevant NP in the intestinal epithelium and thus offers valuable information for improved hazard and risk assessment. Furthermore, specific experimental models used in the framework of this thesis may serve as tools to screen for the potential hazards of novel engineered NP for which oral exposure can be anticipated.