Chapter 6
Chapter 6
114
Summary of results
Engineered nanoparticles enter several types of mammalian cells
Toxic effects of nanoparticles may arise either from interactions with the cell surface or by internalisation into the cytoplasm and sub‐cellular structures. Thus, at first it was investigated whether engineered nanoparticles are principally able to penetrate mammalian cells. Several cell types and six types of nanoparticles with different properties were considered for this study (Chapter 2). Results of light‐ and electron microscopy experiments showed that all investigated types of particles were able to enter all of the different cell types. The particles were always found within the cytoplasm but never in cell nuclei. Furthermore, it could be shown that nanoparticles within cells co‐
localise with lysosomal structures. A relative quantification of particle uptake was established by flow cytometry measurements. Changes of cellular granularity were generally more dependent on the particle than on the cell type; indicating an influence of particle properties either on their internalisation ability or the cellular granularity measurements itself. After short exposure times, a slight correlation was found for the granularity data with the primary particles sizes. However, particle properties which were not considered in the study might influence the cellular granularity measurements as well. Further research is necessary to elucidate these interactions.
Flow cytometry was also used to investigate the impact of the actin‐ (i.e. a cytoskeleton‐) inhibitor Cytochalasin D on the particle incorporation into cells. The ability to incorporate nanoparticles was not affected by the inhibitor in most of the cell types but a reduction of the particle internalisation was found in a few cases. This study illustrated that technical nanoparticles are able to enter all kinds of mammalian cells independent of the cellular phagocytosis ability. However, the underlying mechanisms of incorporation remain to be elucidated. An export of particles out of the cells was not observed in vitro in this study, but translocation aspects within and out of whole organisms should be considered for future research projects.
Toxicity of tungsten based nanoparticles
Acute toxicity of technical nanoparticles was assessed with focus on WC (tungsten carbide) and WC‐Co (tungsten carbide cobalt) nanoparticles and their impact on human
(Chapter 3) and fish cells (Chapter 4). By physico‐chemical characterisation of the particles it was found that serum proteins (especially albumin) bound to the particles. The inclusion of serum proteins in cell culture media had a stabilising effect and prevented agglomeration of particles. Furthermore it was shown that cobalt leached out of WC‐Co nanoparticles to some extent. This was considered by implementing comparative experiments with CoCl2. Cell viability and membrane integrity of human lung, skin, and colon cells were not affected by WC nanoparticles up to a concentration of 30 μg/ml. In contrast, WC‐Co nanoparticles were slightly toxic to all cells; the colon cells were the most sensitive type of the human cells. The effects of WC‐Co were always stronger than effects of CoCl2 or WC+CoCl2 applied in equivalent concentrations (Chapter 3). An increased toxicity for WC‐Co was also found with trout gill cells (Chapter 4). WC and WC‐Co nanoparticle were detected within the cytoplasm of the gill cell line; media of different complexity, whose constituents (e.g. proteins) influence the agglomeration of the particles, had no effects on the particle internalisation. However, the toxicological effects of CoCl2 as well as the nanoparticle effects were found to vary depending on the composition of exposure media (Chapter 4).
Altered gene expression and the role of leached ions
In order to analyse effects of WC and WC‐Co nanoparticles in more detail and with the aim to elucidate the modes of action of the enhanced toxicity of WC‐Co nanoparticles, global gene expression in human skin cells (HaCaT) was investigated also in comparison to dissolved cobalt ions (Chapter 5). Therefore, whole genome microarrays were used and patterns of the transcriptome were compared between WC, WC‐Co or CoCl2 treated cells.
Extensive data and pathway analyses showed that most genes were differentially expressed after the CoCl2 treatment. Exposure to WC‐Co nanoparticles resulted also in a large number of genes with altered expression. By contrast, gene expression patterns of WC treated cells were hardly influenced by the nanoparticles. Most of the genes affected by WC‐Co nanoparticles were also found to be altered in the CoCl2 treatment. Within these genes, a significant enrichment of genes known as targets of the HIF1α (hypoxia inducible factor) transcription factor or genes coding proteins that are involved in response to hypoxia could be identified by pathway and gene set enrichment analyses.
Cobalt ions are known to stabilise the transcription factor HIF1α, which affects
Chapter 6
116
appropriate downstream target genes. Hence, this study clearly indicates that leached cobalt ions play an important role in toxicity of WC‐Co nanoparticles. The underlying mechanism for the enhanced toxicity of WC‐Co in comparison to WC particles and dissolved cobalt, however, could not be explained by the genome wide transcription analysis (Chapter 5).
Concluding remarks and future directions
Nanotechnology industry, focussing on the development and production of new products for many fields of application, is rapidly growing. The emerging use of nanoparticles could lead to their release and emissions with potential negative consequences for occupational and environmental health. So far, nanoparticles are not specifically regulated and labelling of nanoparticle‐containing products is not required.
Potential health or environmental effects are assessed based on the properties of the bulk materials. The need to establish nanoparticle‐specific regulations is controversially discussed. Finally, for risk assessment, both the hazardous potential and the exposure probability have to be considered. Therefore, successive analyses are necessary at which in vitro experiments contribute to identify acute toxic nanoparticles and potential modes of action as initial steps for the risk assessment. In vitro studies are economically and ethically desirable and provide evidence about possible hazardous potential. Especially for the toxicological screening of the huge palette of different nanoparticles, in vivo studies are inappropriate or not even feasible. Nevertheless, in vitro methods based on cell cultures still have a limited capacity to predict in vivo effects (Sayes et al. 2007). This is primarily caused by the specific uptake and translocation routes in vivo, which are difficult to be mimicked in vitro. Particles that enter an organism via the lung may lead to reactions that are a consequence of communication between several cell types and are not detectable in a single cell culture (e.g. inflammation due to immunological and defence responses independent of particle translocation). Co‐cultures of several cell types, as recently demonstrated by a few studies (Brandenberger et al. 2010, Burguera et al. 2010), might provide an in vitro approach for future activities addressing these problems of systemic interactions. However, considering our knowledge so far, particles that are toxic in vitro should be handled accordingly and exposure should be avoided generally. Non
toxic particles (such as WC), that have been found to enter cells easily, have to be assessed in vivo due to their possible long term and systemic impacts on organisms.
Besides toxicological issues, estimating and analysing the particle uptake into cells is rather difficult, especially in a quantitative manner. Initial studies show that “Inductively Coupled Plasma ‐ Mass Spectrometry” (ICP‐MS) might be a useful tool to measure intracellular particle contents (Limbach et al. 2005, Wang et al. 2008a, Xia et al. 2008).
However, for this method the chemical destruction of the particles is necessary, which is not trivial for hard materials such as tungsten. Furthermore, the detection and distinction of free or particle‐bound ions in comparison to solid particles is not possible. Therefore, further investigations are necessary to establish a system to measure absolute amounts of particles within cells. Studies should focus on the development or adaptation of methods for the quantitative determination of intracellular nanoparticles and leached components to better link toxicological effects to internal particle or ion concentrations. Furthermore, exposure scenarios for humans and the environment have to be generated to estimate relevant exposure concentrations and the abilities to measure nanoparticles qualitatively and quantitatively in air and the environment have to be improved.
The research of this thesis shows that toxicological investigations of engineered nanoparticles in vitro are possible and appropriate for the understanding of interactions between technical nanoparticles and cells. Based on these data, further activities should focus on the role of the potential carrier function of nanoparticles and consider questions of bioaccumulation and intracellular effect concentrations.
118