Synthesis and characterization of copper oxide nanoparticles and investigation of their effects on the vitality and metabolism of astrocytes

Copper oxide nanoparticles (CuO-NPs) recently raised the industry s interest due to their interesting chemical and physical properties. The continuous increase of products containing CuO-NPs and the unintentional generation of CuO-NPs by technical processes establish an increased risk of human exposure. Since nanoparticles can reach the brain upon exposure, it is of high interest to evaluate the uptake and potential adverse effects on brain cells. In this context astrocytes are of special interest due to their central role in the brain homeostasis and in defence processes. In the presented thesis, a method for the synthesis of CuO-NPs was established. After a detailed analysis of the physico-chemical properties of the synthesized CuO-NPs, primary astrocytes cultures were exposed to these CuO-NPs. The accumulation and uptake mechanism of CuO-NPs by cultured astrocytes as well as the resulting effects on the cell vitality and metabolism were investigated. It was shown that cultured astrocytes strongly accumulated CuO-NPs in a time-, concentration-, temperature- and media-dependent manner. Results from experiments with pharmacological inhibitors of different endocytotic pathways suggest that clathrin-mediated endocytosis, macropinocytosis and the recycling of membranes are involved in the uptake of CuO-NPs by astrocytes. Accumulated CuO-NPs exerted a strong toxicity when the specific cellular copper contents reached values above 10 nmol copper per mg protein. The mechanism of toxicity was assigned to a strong increase in reactive oxygen species in the treated cells. Cultured astrocytes treated with subtoxic concentrations of CuO-NPs over a time period of 24 h strongly increased their gycolytic flux, their glutathione content as well as the levels of the copper storage protein metallothioneine. The observed increase in glycolytic flux and metallothioneine levels was prevented in presence of the cell permeable copper chelator tetrathiomolybdate. This chelator was also capable of preventing the strong toxicity and the increased generation of reactive oxygen species in acute exposure scenarios where high concentrations of CuO-NPs were applied to cultured astrocytes. Similarly, toxicity observed after the exposure of cultured astrocytes with CuCl2 was prevented by copper chelators while copper reducing antioxidants increased copper uptake and copper derived toxicity. The data presented in this thesis reveal that CuO-NPs can have severe deleterious effects on astrocytes, which otherwise are very robust against several toxins. The vulnerability of astrocytes to CuO-NPs suggests that brain cells may be severely damaged if they encounter such nanoparticles. Hence, the exposure of humans to CuO-NPs should be minimized and carefully evaluated to prevent potential health hazards.