#Quantitation of protein carbonylation by dot blot free
Free radicals can be detected in vitro via electron spin resonance (ESR) spectroscopy using reagents called spin traps. The types of ROS generated depend in particular on the NP and its chemical environment. Among these, surface reactions driven by excitation of electrons via UV-light, Fenton-type reactions, catalytic chemistry at the NP surface, or via dissolved (metal) ions are implicated most prominently. Furthermore, ROS are induced in to cells by NPs in different ways. Intracellular ROS are generated as regular byproduct of the respiratory chain and other oxygen consuming reactions. It results from an imbalance between reactive oxygen species (ROS) generation and cellular antioxidants. Oxidative stress has been linked to various adverse outcomes such as inflammation, DNA damage, and general cytotoxicity. One prevailing paradigm explaining NP-mediated toxicity is the induction of oxidative stress. Furthermore, a more detailed knowledge of toxicity mechanisms is helpful for a regulatory prioritization and also for any successful grouping approach. Especially the latter aspect will foster the development of reliable alternative testing methods and may help to guide production of safe-by-design NPs. In particular, fast and reliable toxicity screening methods are urgently needed as well as knowledge on the underlying toxic modes of action. Currently, adaptation of test guidelines for NPs and debates on their validity for NPs are still ongoing. With this ever increasing diverse and common use of NPs it has become important to address concerns regarding possible adverse health effects. the use of ZnO and TiO 2 as UV protection agents in cosmetics and, due to their biocidal activity, silver NPs in packaging or medical devices, and ZrO 2 and SiO 2 as binders in ceramics and fillers in modern polymers. Current uses of nanoparticles (NPs) comprise e.g. The range of industrial processes and products taking advantage of nanotechnology has been growing rapidly in recent years. Furthermore, detailed information about compromised proteins may aid in classifying NPs according to their mode of action. ConclusionsĪnalysis of protein carbonylation is a versatile and sensitive method to describe NP-induced oxidative stress and, therefore, can be used to identify NPs of concern. Furthermore, induction of carbonyls upon silver NP treatment was also verified in rat lung tissue homogenates. Affected proteins comprised cytoskeletal components, proteins being involved in stress response, or cytoplasmic enzymes of central metabolic pathways such as glycolysis and gluconeogenesis. Each NP induced an individual pattern of protein carbonyls on 2D immunoblots. By contrast, results obtained by DCFDA assay were deviating. This was in good agreement with the surface reactivity of the NPs as obtained by ESR and the reduction in cell viability as assessed by WST-1 assay. ResultsĮleven NPs induced elevated levels of carbonylated proteins. In parallel, tissue homogenates from rat lungs intratracheally instilled with silver NPs were studied. Carbonylated proteins were assessed by 1D and/or 2D immunoblotting and identified by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF). NRK-52E cells were treated with all NPs, analyzed for viability (WST-1 assay) and intracellular ROS production (DCFDA assay). Surface reactivities of all NPs were studied in a cell-free system by electron spin resonance (ESR). We investigated a representative panel of 24 NPs including functionalized amorphous silica (6), zirconium dioxide (4), silver (4), titanium dioxide (3), zinc oxide (2), multiwalled carbon nanotubes (3), barium sulfate and boehmite. Thus, the qualitative and quantitative description of protein carbonylation may be used to characterize how biological systems respond to oxidative stress induced by NPs.
As one consequence, protein carbonyl levels may become enhanced. Oxidative stress, a commonly used paradigm to explain nanoparticle (NP)-induced toxicity, results from an imbalance between reactive oxygen species (ROS) generation and detoxification.
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