Nanoparticle-induced oxidative stress alters phospho-tyrosine patterns in mammalian cells: Results of an SH2 profiling approach

The BMBF-funded project “nanoGEM” follows a systematic approach to understand hazards associated with different types of nanoparticles (NP). Oxidative stress is considered to be a major paradigm to explain NP toxicity and can result in altered levels of tyrosine phosphorylation, probably by protein tyrosine phosphatase inhibition which are major components of various cellular signaling pathways. Here we focused on global changes of tyrosine phosphorylation resulting from treatment with 16 NP bearing different surface coatings. In parallel several well established in vitro and in vivo toxicity endpoints were analyzed. We used ZrO2, SiO2 Ag NP, either plane or acidic, basic or polymeric functionalisation and in addition TiO2, ZnO, BaSO4 and AlOOH as references. We analyzed NP treated NRK-52E cells by far western blot analysis with a set of 70 src homolgy 2 (SH2) domains known to differentially bind phospho-tyrosine sites. From these 70 SH2 domains, a set of 9 domains was selected and cellular reactions after SiO2 and Ag NP treatment were studied in detail. Furthermore we used these 9 SH2 domains to study changes in signaling pathways in lung tissue lysates after in vivo instillation in rats. Most NP caused alterations in phospho-tyrosine dependent cell signaling. Strongest responses were observed for cytotoxic NP (SiO2naked, ZnO), fitting very well to overall observed toxicity, additionally enabling us to detect subtle changes in phospho-tyrosine dependent signaling also for non-toxic NP. SH2 profiling appears to be a powerful tool to study cellular responses after NP treatment and opens insights into signaling mechanisms underlying toxicty.