aDepartment of Environmental Health, Portuguese National Institute of Health, Rua Alexandre Herculano, 321, 4000-055 Porto, PortugalbEPIUnit - Institute of Public Health, University of Porto, Rua das Taipas 135, 4050-600, Porto, PortugalcElectroceramic Department, Instituto de Cerámica y Vidrio, CSIC, Campus de Cantoblanco, Calle de Kelson, 5, 28049 Madrid, SpaindCatalytic Spectroscopy Laboratory, Instituto de Catálisis y Petroleoquímica, ICP-CSIC, Madrid, Spain
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Immobilization of nanoparticles on inorganic supports has been recently developed, resulting in the creation of nanocomposites. Concerning titanium dioxide nanoparticles (TiO2 NPs1), these have already been developed in conjugation with clays, but so far there are no available toxicological studies on these nanocomposites. The present work intended to evaluate the hepatic toxicity of nanocomposites (C-TiO22), constituted by rutile TiO2 NPs immobilized in nanokaolin (NK3) clay, and its individual components.These nanomaterials were analysed by means of FE-SEM4 and DLS5 analysis for physicochemical characterization. HepG2 cells were exposed to rutile TiO2 NPs, NK clay and C-TiO2 nanocomposite, in the presence and absence of serum for different exposure periods. Possible interferences with the methodological procedures were determined for MTT,6 neutral red uptake, alamar blue (AB), LDH,7 and comet assays, for all studied nanomaterials. Results showed that MTT, AB and alkaline comet assay were suitable for toxicity analysis of the present materials after slight modifications to the protocol. Significant decreases in cell viability were observed after exposure to all studied nanomaterials. Furthermore, an increase in HepG2 DNA damage was observed after shorter periods of exposure in the absence of serum proteins and longer periods of exposure in their presence. Although the immobilization of nanoparticles in micron-sized supports could, in theory, decrease the toxicity of single nanoparticles, the selection of a suitable support is essential. The present results suggest that NK clay is not the appropriate substrate to decrease TiO2 NPs toxicity. Therefore, for future studies, it is critical to select a more appropriate substrate for the immobilization of TiO2 NPs.HighlightsOnly the MTT and AB assays were found to be suitable for cytotoxicity assessment.Alkaline comet assay was also appropriate for genotoxicity evaluation.All nanomaterials decreased the HepG2 cell viability and caused DNA damage.Nanokaolin is not a suitable clay substrate for the immobilization of TiO2 NPs.Further toxicity studies must be performed in other clays to support nanoparticles.