Since the manufacture and use of nanoparticles are increasing, humans are more likely to be exposed occupationally or via consumer products and the environment. However, so far toxicity data for most manufactured nanoparticles are limited. The aim of this study was to investigate and compare different nanoparticles and nanotubes regarding cytotoxicity and ability to cause DNA damage and oxidative stress. The study was focused on different metal oxide particles (CuO, TiO2, ZnO, CuZnFe2O4, Fe3O4, Fe2O3), and the toxicity was compared to that of carbon nanoparticles and multiwalled carbon nanotubes (MWCNT). The human lung epithelial cell line A549 was exposed to the particles, and cytotoxicity was analyzed using trypan blue staining. DNA damage and oxidative lesions were determined using the comet assay, and intracellular production of reactive oxygen species (ROS) was measured using the oxidation-sensitive fluoroprobe 2',7'-dichlorofluorescin diacetate (DCFH-DA). The results showed that there was a high variation among different nanoparticles concerning their ability to cause toxic effects. CuO nanoparticles were most potent regarding cytotoxicity and DNA damage. The toxicity was likely not explained by Cu ions released to the cell medium. These particles also caused oxidative lesions and were the only particles that induced an almost significant increase (p = 0.058) in intracellular ROS. ZnO showed effects on cell viability as well as DNA damage, whereas the TiO2 particles (a mix of rutile and anatase) only caused DNA damage. For iron oxide particles (Fe3O4, Fe2O3), no or low toxicity was observed, but CuZnFe2O4 particles were rather potent in inducing DNA lesions. Finally, the carbon nanotubes showed cytotoxic effects and caused DNA damage in the lowest dose tested. The effects were not explained by soluble metal impurities. In conclusion, this study highlights the in vitro toxicity of CuO nanoparticles.
An interdisciplinary and multianalytical research effort is undertaken to assess the toxic aspects of thoroughly characterized nano- and micrometer-sized particles of oxidized metallic copper and copper(II) oxide in contact with cultivated lung cells, as well as copper release in relevant media. All particles, except micrometer-sized Cu, release more copper in serum-containing cell medium (supplemented Dulbecco's minimal essential medium) compared to identical exposures in phosphate-buffered saline. Sonication of particles for dispersion prior to exposure has a large effect on the initial copper release from Cu nanoparticles. A clear size-dependent effect is observed from both a copper release and a toxicity perspective. In agreement with greater released amounts of copper per quantity of particles from the nanometer-sized particles compared to the micrometer-sized particles, the nanometer particles cause a higher degree of DNA damage (single-strand breaks) and cause a significantly higher percentage of cell death compared to cytotoxicity induced by micrometer-sized particles. Cytotoxic effects related to the released copper fraction are found to be significantly lower than the effects related to particles. No DNA damage is induced by the released copper fraction.
CuO-nano toxicity is predominantly mediated by intracellular uptake and subsequent release of copper ions, whereas no toxicity is observed for Ag-nano due to low release of silver ions within short time periods.
Cu-containing nanoparticles are used in various applications in order to e.g. achieve antimicrobial activities and to increase the conductivity of fluids and polymers. Several studies have reported on toxic effects of such particles but the mechanisms are not completely clear. The aim of this study was to investigate the interactions between cell membranes and well-characterized nanoparticles of CuO, Cu metal, a binary Cu-Zn alloy and micron-sized Cu metal particles. This was conducted via in vitro investigations of the effects of the nanoparticles on (i) cell membrane damage on lung epithelial cells (A549), (ii) membrane rupture of red blood cells (hemolysis), complemented by (iii) nanoparticle interaction studies with a model lipid membrane using quartz crystal microbalance with dissipation monitoring (QCM-D). The results revealed that nanoparticles of the Cu metal and the Cu-Zn alloy were both highly membrane damaging and caused a rapid (within 1h) increase in membrane damage at a particle mass dose of 20 μg/mL, whereas the CuO nanoparticles and the micron-sized Cu metal particles showed no such effect. At similar nanoparticle surface area doses, the nano and micron-sized Cu particles showed more similar effects. The commonly used LDH (lactate dehydrogenase) assay for analysis of membrane damage was found impossible to use due to nanoparticle-assay interactions. None of the particles induced any hemolytic effects on red blood cells when investigated up to high particle concentrations (1mg/mL). However, both Cu and Cu-Zn nanoparticles caused hemoglobin aggregation/precipitation, a process that would conceal a possible hemolytic effect. Studies on interactions between the nanoparticles and a model membrane using QCM-D indicated a small difference between the investigated particles. Results of this study suggest that the observed membrane damage is caused by the metal release process at the cell membrane surface and highlight differences in reactivity between metallic nanoparticles of Cu and Cu-Zn and nanoparticles of CuO.
Different methodological settings can influence particle characteristics and toxicity in nanotoxicology. The aim of this study was to investigate how serum proteins and sonication of Cu nanoparticle suspensions influence the properties of the nanoparticles and toxicological responses on human lung epithelial cells. This was investigated by using methods for particle characterization (photon correlation spectroscopy and TEM) and Cu release (atomic absorption spectroscopy) in combination with assays for analyzing cell toxicity (MTT-, trypan blue- and Comet assay). The results showed that sonication of Cu nanoparticles caused decreased cell viability and increased Cu release compared to non-sonicated particles. Furthermore, serum in the cell medium resulted in less particle agglomeration and increased Cu release compared with medium without serum, but no clear difference in toxicity was detected. Few cells showed intracellular Cu nanoparticles due to fast release/dissolution processes of Cu. In conclusion; sonication can affect the toxicity of nanoparticles.
Background: There is currently a need to develop and test in vitro systems for predicting the toxicity of nanoparticles. One challenge is to determine the actual cellular dose of nanoparticles after exposure. Methods: In this study, human epithelial lung cells (A549) were exposed to airborne Cu particles at the air-liquid interface (ALI). The cellular dose was determined for two different particle sizes at different deposition conditions, including constant and pulsed Cu aerosol flow. Results: Airborne polydisperse particles with a geometric mean diameter (GMD) of 180 nm [geometric standard deviation (GSD) 1.5, concentration 10 5 particles/mL] deposited at the ALI yielded a cellular dose of 0.4-2.6 lg/ cm 2 at pulsed flow and 1.6-7.6 lg/cm 2 at constant flow. Smaller polydisperse particles in the nanoregime (GMD 80 nm, GSD 1.5, concentration 10 7 particles/mL) resulted in a lower cellular dose of 0.01-0.05 lg/cm 2 at pulsed flow, whereas no deposition was observed at constant flow. Exposure experiments with and without cells showed that the Cu particles were partly dissolved upon deposition on cells and in contact with medium. Conclusions: Different cellular doses were obtained for the different Cu particle sizes (generated with different methods). Furthermore, the cellular doses were affected by the flow conditions in the cell exposure system and the solubility of Cu. The cellular doses of Cu presented here are the amount of Cu that remained on the cells after completion of an experiment. As Cu particles were partly dissolved, Cu (a nonnegligible contribution) was, in addition, present and analyzed in the nourishing medium present beneath the cells. This study presents cellular doses induced by Cu particles and demonstrates difficulties with deposition of nanoparticles at the ALI and of partially soluble particles.
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