Protein carbonylation as a marker of oxidative stress induced by nanoparticles: Analysis of 16 inorganic nanoparticles

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. Here we focused on protein carbonylation as a consequence of oxidative stress for a set of 16 different nanoparticles, used as either plane materials or with different surface coatings. In parallel several in vitro and in vivo toxicity endpoints have been analyzed. We used NP of 10 nm (ZrO2), 15 nm (SiO2) and 50 nm or 200 nm (Ag), furnished either with acidic, basic or polymeric functionalities and TiO2, ZnO, BaSO4 and AlOOH as references. In a screening approach we studied time- and dose-dependent carbonylation of all 16 NP in NRK-52E cells via 1D immunoblots. Data were correlated with cytotoxicity (WST-8, LDH assay) and ROS formation (DCFDA assay). Furthermore we applied a 2D proteomics approach combined with MALDI-MS/MS to identify the proteins modified. Finally, for several NP we analyzed lung tissues after in vivo instillation in rats. Eight out of 16 NP induced protein carbonylation in NRK-52E cells. Observed protein carbonylation correlated well with overall toxicity. The 2D approach revealed a complex and distinct pattern of carbonyls. Modified proteins were identified as cytoskeleton proteins, heat shock proteins or proteins of major cellular pathways (i.e. glycolysis). We also observed carbonyl modifications in lung tissue homogenates of rats intratracheally instilled with the same NP. Taken together, analysis of protein carbonylation is a useful tool for the analysis and mechanistic understanding of ROS dependent NM toxicity.