Cellular effects of nanosilver in human macrophages: Uptake, oxidative stress, lipid alterations and functional impairment

Haase,A.; Tentschert,J.; Graf,P.; Mantion,A.; Draude,F.; Jungnickel,H.; Galla,S.; Plendl,J.; Arlinghaus,H.F.; Thünemann,A.F.; Taubert,A.; Luch,A.

Silver nanoparticles (SNP) are among the most commercialized nanoparticles. Sincemacrophages represent a physiological relevant cell system, we performed in vitro studies inhuman macrophages derived from THP-1 cell line. We studied SNP of different sizes (20 nmto 40 nm) and different coatings (citrate, peptide). Apart from clear dose- and time-dependenteffects, the toxicity strongly relied on size and the type of coating, with citrate coated particlesbeing less toxic. Gold nanoparticles, which served as control, showed nearly no adverseeffects.The uptake was studied with Confocal Raman Spectroscopy as well as by TEM. SNP couldbe detected as aggregates in the cytoplasm but also as individual particles throughout wholecells, also in nucleus and lysosomes. Here we used a novel time-of-flight secondary ionmass spectrometry (TOF-SIMS) and Laser postionization secondary neutral massspectrometry (Laser-SNMS) approach to visualize intracellular SNP and to study cellulareffects in parallel. We could detect significant changes in lipid composition of the outercellular membrane leaflet, which were indicative for oxidative stress and alterations inmembrane fluidity. We further supported this finding by different biochemical methods. Wecould detect a time- and dose-dependent induction of heme oxygenase 1 and proteincarbonylation, both of which are established markers for oxidative stress. Each processfollows distinct kinetics, supporting a hierarchical model of oxdiative stress. We analyzed thecarbonylated proteins on 2D gels, thereby enabling visualization and separation of a complexpattern of modified proteins. Importantly, with the 2D gel approach we were able to clearlydistinguish between the effects of different nanoparticles as they induce differentcarbonylation patterns. In addition we detected functional alterations of the macrophages.Even very low concentrations reduced the phagocytic activity considerably. Both endpoints,impaired phagocytosis and lipid alterations could be linked to the particle-mediatedgeneration of oxidative stress. Some of these effects could be reversed depending on thetime and dose of nanoparticles used during treatment of cells. Thus we were able todetermine a "point of no return" that was found nicely fitting to that what was asserted asoverload dose for macrophages based on in vivo studies.We conclude that SNP exert adverse effects in human macrophages also at subcytotoxicconcentrations via oxidative stress, leading to changes in membrane lipid composition and tocell's functional impairment. While all SNP were effectively taken up by macrophages,different SNP induce distinguishable effects.

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Haase,A. / Tentschert,J. / Graf,P. / et al: Cellular effects of nanosilver in human macrophages: Uptake, oxidative stress, lipid alterations and functional impairment. 2011.

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