Most of the atmospheric ultrafine particles (UFPs) in urban areas derive from combustion sources, especially diesel exhaust particles (DEP), but also from non-exhaust sources or from the unintentional release of engineered nanoparticles (NPs) during production and use. Since the environmental exposure to DEP and NPs occur simultaneously, it is necessary to consider their possible interactive effects in biological system. Commercially available (cZnO, cCuO<50 nm) from Sigma-Aldrich) and sonochemically synthesized ZnO and CuO NPs (sZnO, sCuO) from Bar-Ilan University, were used alone or in combination with standard DEP (NIST 2975) to expose A549 cells. After 24–72h exposure to increasing metal oxide NPs concentrations (10–20 µg/ml), with and without DEP at 100 µg/ml, MTT test and Colony Forming Efficiency Assay (CFE) were performed to assess the cytotoxicity. The NP mixtures were characterized by DLS and TEM, while the NP dissolution in cell medium was measured by ICP-OES. In parallel to the cytotoxicity studies, morphological analyses on NPcell interactions were performed by light and transmission electron microscopy. The results suggest that the presence of DEP introduced new physico-chemical interactions able to increase the cytotoxicity of cZnO, but to decrease that of sZnO. For CuO NPs, the presence of DEP significantly reduced the cytotoxicity of cCuO and only slightly that of sCuO. This is probably due to different interferences with the metal oxide NP surface and/or to the modulation of ions release. The results from CFE were coherent with those from MTT. On the basis of the morphology and cell density, four well distinguishable colony types were identified. Cytostatic effects and changes in colony morphology were observed especially after exposure to CuO and DEP+CuO NPs. TEM analyses revealed that both ZnO and CuO NPs, as well as their mixture with DEP, were abundantly internalized in A549 cells, especially in the endo-lysosomal compartments and multilamellar bodies. We are performing additional investigations to discriminate the modality of nano-bio-interactions of CuO and ZnO in presence of DEP and to analyse cell-cell adhesion molecules and epithelial-to-mesenchymal transition mechanisms. Acknowledgements: EU Horizon 2020 project PROTECT (grant agreement No 720851)
Zerboni, A., Catelani, T., Mantecca, P. (2019). In vitro effects of ZnO and CuO NPs in mixture with DEP: different nano-bio-interactions affect viability and colony forming efficiency of A549 cells.. In 55th Congress of the European-Societies-of-Toxicology (EUROTOX) - Toxicology - Science Providing Solutions (pp.S211-S212).
In vitro effects of ZnO and CuO NPs in mixture with DEP: different nano-bio-interactions affect viability and colony forming efficiency of A549 cells.
A. Zerboni
Primo
;T. Catelani;P. ManteccaUltimo
2019
Abstract
Most of the atmospheric ultrafine particles (UFPs) in urban areas derive from combustion sources, especially diesel exhaust particles (DEP), but also from non-exhaust sources or from the unintentional release of engineered nanoparticles (NPs) during production and use. Since the environmental exposure to DEP and NPs occur simultaneously, it is necessary to consider their possible interactive effects in biological system. Commercially available (cZnO, cCuO<50 nm) from Sigma-Aldrich) and sonochemically synthesized ZnO and CuO NPs (sZnO, sCuO) from Bar-Ilan University, were used alone or in combination with standard DEP (NIST 2975) to expose A549 cells. After 24–72h exposure to increasing metal oxide NPs concentrations (10–20 µg/ml), with and without DEP at 100 µg/ml, MTT test and Colony Forming Efficiency Assay (CFE) were performed to assess the cytotoxicity. The NP mixtures were characterized by DLS and TEM, while the NP dissolution in cell medium was measured by ICP-OES. In parallel to the cytotoxicity studies, morphological analyses on NPcell interactions were performed by light and transmission electron microscopy. The results suggest that the presence of DEP introduced new physico-chemical interactions able to increase the cytotoxicity of cZnO, but to decrease that of sZnO. For CuO NPs, the presence of DEP significantly reduced the cytotoxicity of cCuO and only slightly that of sCuO. This is probably due to different interferences with the metal oxide NP surface and/or to the modulation of ions release. The results from CFE were coherent with those from MTT. On the basis of the morphology and cell density, four well distinguishable colony types were identified. Cytostatic effects and changes in colony morphology were observed especially after exposure to CuO and DEP+CuO NPs. TEM analyses revealed that both ZnO and CuO NPs, as well as their mixture with DEP, were abundantly internalized in A549 cells, especially in the endo-lysosomal compartments and multilamellar bodies. We are performing additional investigations to discriminate the modality of nano-bio-interactions of CuO and ZnO in presence of DEP and to analyse cell-cell adhesion molecules and epithelial-to-mesenchymal transition mechanisms. Acknowledgements: EU Horizon 2020 project PROTECT (grant agreement No 720851)
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