Nano-TiO2 stability in medium and size as important factors of toxicity in macrophage-like cells
Graphical abstract
Introduction
Nano-TiO2 is commonly used in products because of their distinct properties (e.g. ultraviolet radiation absorption, photocatalytic properties or higher specific surface connected with catalytic properties) when compared to those consisting of larger particles. TiO2 is one of the most widely used materials in nanoparticles production (Vance et al., 2015). Nano-TiO2 is used in specialised products, mainly in cosmetics for UV absorption or in catalysts. However pigmentary material, used also for food colouring, always contains a part of particles with size below 100 nm; usually <2% of mass or up to 40% of particle number (EFSA ANS Panel, 2016). Nano-forms of various materials could affect organisms in a different way than micro-forms, which have been considered to be inert and safe for human health for decades (Ophus et al., 1979; Hext et al. 2005; Chen et al., 1988). Nanoparticles are small enough to enter the cells, the blood stream, or to be transferred along the nerve (Elder et al. 2006; Kermanizadeh et al. 2015). TiO2 nanoparticles administered through various routes can translocate via systemic circulation to different organs (Shakeel et al. 2016). There is evidence that nano-TiO2 is more toxic than bulk material (Guichard et al., 2012). OECD identified nano-TiO2 as one of the priority manufactured nanomaterials for toxicology and risk assessment so as to avoid adverse effects from the use of this material (OECD 2010). Some evidence is arising that broad usage of nano-TiO2in consumer products (Guo et al., 2017) or as a photocatalyst (Kebede et al., 2013) might not be safe.
The toxicity of various forms of nano-TiO2 depends on their characteristics, such as shape, size, crystal structure, zeta potential, aggregation and agglomeration tendency, surface characteristics and coatings (Sha et al., 2015; Zhang et al., 2015). However, their contribution to toxicity remains unclear, as evident from inconsistent results from different studies (Shi et al., 2013). For instance, toxicity inversely associated with size was shown for TiO2 nanoparticles (Xiong et al., 2013); but opposite observations, i.e. higher toxicity of 21 nm TiO2 nanoparticles in comparison with 12 nm and 98 nm, were also observed (Zhang et al. 2012). Anatase, the less stable crystalline structure (Zhang et al., 2015) of TiO2, was found to be more toxic to mouse macrophages (Zhang et al., 2012), human lung epithelial cells, human dermal fibroblasts, and human breast cancer cells (Sayes et al., 2006; De Matteiset al., 2016). Although in general, anatase is regarded to be more cytotoxic, De Matteiset al., (2016), Guichard et al. (2012) and Numano et al. (2014) found higher cytotoxicity for rutile rods than anatase particles. In this particular case, however, the result might be affected by the elongated shape of rutile nanomaterial (NM). Particles were also found to be less toxic then nano-belts (Silva et al., 2013; Hamilton et al., 2009), and fibrous nano-TiO2 (Watanabe et al., 2002). The mechanisms of adverse effects of nano-TiO2 are associated with reactive oxygen species (ROS) generation, as pointed out by Sayes et al. (2006). ROS generation can be enhanced by nontoxic UV illumination which triggers hydroxyl radical generation (Yin et al., 2012; Zhang et al., 2015). On the other hand, there are opposed findings of the cytotoxic effect of photoactivated (UV irradiated) nano-TiO2 on human alveolar cell lines (A549) (Numano et al., 2014; Sayes et al., 2006). The characteristics possibly responsible for cytotoxicity of nano-TiO2 have still not been fully understood.
In vivo studies have revealed the distribution of nano-TiO2 in the organism. Intravenously administered nano-TiO2 are mainly accumulated in the liver and spleen of the rat (Xie et al. 2011; Shinohara et al. 2014) due to the high population of macrophages in these organs. High levels of nano-TiO2 retained in the organs 30 days after the administration indicated accumulation of nano-TiO2 in these cells. Macrophages, nonspecific immune cells, are responsible for the uptake and degradation of foreign material (Saba, 1970) mainly through phagocytosis. As cleaners of the body environment, they play an irreplaceable role in NMs removal, immune response, and inflammation development, and could be considerably affected by the presence of nano-TiO2 in the organism.
In the present study, we analyzed the cytotoxic effects of fourteen diverse nano-TiO2 in the human monocytic cell line THP-1, differentiated into macrophage-like cells. The physical properties possibly affecting cytotoxicity were chosen using LASSO (least absolute shrinkage and selectionoperator), which in contrast to stepwise regression techniques gives consistent results (small changes in samples properties would not result in entirely different models) due to a model complexity penalty and cross-validation (Tibshirani,1996). Former LASSO applications in toxicology can be found in Hamon et al. (2015) or Laaksonen et al. (2006).
To enhance the comparability of our results with the data produced by other European laboratories, as well as to ensure their compatibility with nanotoxicological databases prepared in the scope of several ongoing EU projects (e.g. NANoREG, COST Action TD1204 MODENA), standardised protocols for cell cultivation, preparation of NM dispersion and cytotoxicity assays developed within NANOGENOTOX (https://www.anses.fr/fr/node/120284, Jensen et al. 2011) and NANOVALID projects (http://www.nanovalid.eu/) were used.
Section snippets
TiO2 nanomaterials
To comprehensively assess the cytotoxic potential of various types of nano-TiO2, 14 commercially available nano-TiO2 were used in this study. The set consisted of 5 variants of anatase and 5 variants of rutile nanoparticles differing in their diameter (ranging from 3 to 165 nm), 3 variants of anatase with high aspect ratio (tubes and wires) of different widths and lengths, and one silicon coated (hydrophobic) rutile particle. Anatase tubes and the smallest rutile particles were delivered in a
Nanomaterials characteristics
Results of raw particles characterisation are listed in Table 2. As nanomaterials in the paste form (R1-3P-, A10x1T-, A10x10W-, and A100x20W-) were accessible only in a limited amount, they were characterised only dissolved in the cell culture medium. Their thermogravimetric analysis was utilised for determination of the equivalent mass corresponding to powder nano-TiO2. Analyses confirmed anatase structure in all samples declared as being anatase by the suppliers. However, one of the rutile
Discussion
As recommended by Warheit (2008), a combination of several techniques for NMs characterisation was performed in our study. The results showed differences between the manufacturers' data and real measured characteristics. Overall, there were inconsistencies in the nanoparticle size; moreover, the R30PS sample had different crystallite content. These inconsistencies showed how crucial proper NM characterisation is for overall understanding of nanotoxicity.
Hydrodynamic size and zeta potential were
Conclusions
We evaluated the cytotoxic effect of 14 diverse nano-TiO2 on model macrophage cells, i.e. cells that potentially capture nano-TIO2 in the body and internalise them. Three assays were used to generate the cytotoxicity data, which were then statistically analysed by LASSO. As LASSO employs cross validation, this approach generates more relevant variables in comparison with stepwise regression.
Except for concentration, polydispersity index (PDI) in media measured within 1 h after exposure was
Acknowledgements
Supported by the Ministry of Education, Youth and Sports of the Czech Republic (LO1508; CZ.02.1.01/0.0/0.0/16_019/0000765) and the Czech Science Foundation (P503-12-G147). The authors acknowledge the assistance provided by the Research Infrastructure NanoEnviCz, supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project No. LM2015073.
The Microscopy Centre - Electron Microscopy CF, IMG AS CR is supported by the Czech-BioImaging large RI project (LM2015062 funded
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These authors contributed equally to this work.