The tremendous application potential of nanosized materials stays in sharp contrast to a growing number of critical reports of their potential toxicity. Applications of in vitro methods to assess nanoparticles are severely limited through difficulties in exposing cells of the respiratory tract directly to airborne engineered nanoparticles. We present a completely new approach to expose lung cells to particles generated in situ by flame spray synthesis. Cerium oxide nanoparticles from a single run were produced and simultaneously exposed to the surface of cultured lung cells inside a glovebox. Separately collected samples were used to measure hydrodynamic particle size distribution, shape, and agglomerate morphology. Cell viability was not impaired by the conditions of the glovebox exposure. The tightness of the lung cell monolayer, the mean total lamellar body volume, and the generation of oxidative DNA damage revealed a dose-dependent cellular response to the airborne engineered nanoparticles. The direct combination of production and exposure allows studying particle toxicity in a simple and reproducible way under environmental conditions.
Due to their physicochemical characteristics, metal oxide nanoparticles (NPs) interact differently with cells compared to larger particles or soluble metals. Oxidative stress and cellular metal uptake were quantified in rat type II alveolar epithelial cells in culture exposed to three different NPs: manganese(II,III) oxide nanoparticles (Mn(3)O(4)-NPs), the soluble manganese sulfate (Mn-salt) at corresponding equivalent doses, titanium dioxide (TiO(2)-NPs) and cerium dioxide nanoparticles (CeO(2)-NPs). In the presence of reactive oxygen species an increased apoptosis rate was hypothesized. Oxidative stress was assessed by detection of fluorescently labeled reactive oxygen species and by measuring intracellular oxidized glutathione. Catalytic activity was determined by measuring catalyst-dependent oxidation of thiols (DTT-assay) in a cell free environment. Inductively coupled plasma mass spectrometry was used to quantify cellular metal uptake. Apoptosis rate was determined assessing the activity of caspase-3 and by fluorescence microscopic quantification of apoptotic nuclei. Reactive oxygen species were mainly generated in cells treated with Mn(3)O(4)-NPs. Only Mn(3)O(4)-NPs oxidized intracellular glutathione. Catalytic activity could be exclusively shown for Mn(3)O(4)-NPs. Cellular metal uptake was similar for all particles, whereas Mn-salt could hardly be detected within the cell. Apoptosis was induced by both, Mn(3)O(4)-NPs and Mn-salt. The combination of catalytic activity and capability of passing the cell membrane contributes to the toxicity of Mn (3)O (4)
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