1A and B). The activation of the IRE1 and ATF-6 pathways occurred at all SiO2-NP concentrations, whereas the activation of the PERK pathway occurred at the

two higher concentrations. The induction of ER stress can have several consequences for the cell. Either the cell can cope with the stress and restore normal cellular functions, or it will undergo apoptosis. Y27632 To restore cellular functions and remove the unfolded proteins from the ER, chaperons become up-regulated, protein translation is inhibited and protein degradation increases. In case the ER stress is too strong and the cell cannot restore normal ER function, apoptotic pathways will be activated [37]. Therefore, ER stress is one mechanism contributing to the cytotoxicity of NPs. One important consequence of ER stress is the release of calcium from the ER lumen into the cytosol [11]. Increased calcium concentration in turn can have important consequences. One effect is the phosphorylation of the transcription factor CREB, which induces the transcription of protein phosphatase 2A (PP2A). Our data demonstrate the up-regulation of PP2A on the mRNA and

on the protein level by SiO2-NPs (Figs. 2 C and D). PP2A is involved in a wide range of cellular processes including cell cycle regulation, cell morphology, development, signal PI3K inhibitor transduction, apoptosis and stress response [23]. Therefore, the induction of ER stress followed by up-regulation of PP2A has marked cellular effects. Previously, increased cytosolic calcium concentrations were reported in neuronal cells after silica NP exposure [3], and interpreted as an influence of the

nanoparticles on influx pumps. However, based on our data, the increased calcium concentration may also originate from the ER stress response. Induction of intracellular calcium transients was also found in human Nabilone lung fibroblasts after exposure to silver nanoparticles [4]. Additionally, an increase in intracellular free calcium was observed after exposure of cells with TiO2-NPs [29]. Consequently, ER stress and associated alteration of calcium homeostasis triggering cellular toxicity may be an important effect underlying cytotoxicity of NPs. Furthermore, ER stress was also shown for other nanoparticles, including ZnO-NPs in human umbilical vein endothelial cells [8], poly(lactic-co-glycolic acid)-nanoparticles [22] and gold nanoparticles in human chronic myelogenous leukemia cells [43]. Activation of both the PERK and IRE1 pathways leads to regulation of the NFκB-IKK signalling pathway during ER stress through activation of IκB kinase (IKK) or degradation of the p65 unit [1]. The ATF6 branch of the ER stress response can also regulate NFκB activity [46]. We could also show the activation of NFκB in Huh7 cells after SiO2-NP exposure (Fig. 3A). Consequences of the activation of NFκB are the induction of INF-α [1] and TNF-α [30]. This was also observed in our experiments (Fig.