Nanoparticles: biological effects on in vitro and in vivo systems

The advent of nanotechnology and the commercialization of several nanoparticle-containing products call to a serious assessment of the environmental and human risks derived from the exposure to these new materials. The emerging properties, the increasing world market and the several exposure scenarios are some of the main criticisms related to distribution of these products. Among nanomaterials metal oxides nanoparticles are considered at the same time an interesting and critical group; in particular titanium dioxide, zinc oxide and cupric oxide (nTiO2, nZnO, nCuO) have received particular attention due to their unique physical and chemical properties and their environmental and economical relevance. Previous studies focused on toxicity of these materials highlighted different adverse effects on the biological systems exposed but there is still a lack of information about the mechanisms involved. These research aims to evaluate the potential biological effects of these commercial transition metal nanoxides on two different experimental models: an in vivo model and an in vitro one. The in vivo model has been used to assess the aquatic toxicity, which has been evaluated by the use of the FETAX assay (Frog embryo teratogenesis assay - Xenopus), a sensitive test commonly used to investigate the embryotoxic effects induced by chemicals. After a preliminary physico-chemical characterization, nTiO2, nZnO, nCuO were administered to Xenopus embryos as suspensions in FETAX medium; at the end of the test the main end points of growth retardation, mortality, absolute and relative frequency of malformations have been analysed. In parallel, different microscopic techniques were used to better characterise the effects observed and to localise the particles inside the organisms. Our results revealed that even high NP concentrations (up to 500 mg/ml) were not lethal for such NPs with the exception of nCuO but all NPs induced relevant malformations. Exposure to titanium dioxide did produce only slight embryotoxic effects, but these NPs were able to penetrate inside the embryo tissues and translocate throughout the body. The intestine appeared to be the main target of nZnO: it induced severe histological alterations of the gut epithelium and over passed the intestinal barrier. These effects were not observed in embryo exposed to soluble Zn, confirming the key role played by nanoparticles themselves. Preliminary results regarding the potential mechanisms involved in toxicity of nZnO indicate lipid peroxidation and alteration of cell junctions as mainly causes of epithelial lesions and NP paracellular translocation. The human alveolar epithelial cell line A459 has been used as in vitro model to assess the potential toxicity related to human inhalation of MONs. The nanotoxicological study has been approached with the aim to define a correlation between the biochemical analyses and the morphological evidences, to better characterize the phenomena at the nano-level. Thus a comparative study on the cytotoxicity and cell-particle interactions of nTiO2 and nCuO was performed. Then we focused the attention on nCuO in order to investigate sensitive cellular targets and the potential mechanisms involved in nCuO toxicity. Ion dissolution, oxidative stress, mitochondrial and lysosomal perturbation and cell cycle alterations were analysed as potential causes of the cytotoxic effects observed. Our study demonstrates that the different effects promoted by MONs on A549 cells depend on the chemical composition, aggregation state, particle dissolution and surface reactivity. Large nTiO2 particles or aggregates were easily internalised by cells trough the endocytic process. They distributed at different level but they didn’t induce structural alterations even at 24 hours of exposure, despite their massive uptake. At this time, the plasma membrane appeared as the major target structure, even cytotoxicity was not significant. nCuO resulted to be a very toxic compound and a dose- and time- dependent effects were observed. Surface reactivity and copper release from particles differently contributed to the registered cytotoxicity. After few hours of exposure, particles themselves were responsible for significant alterations such as increase in reactive oxygen species, formation of lipid peroxidation products and ultrastructural damages at the mitochondria and plasma membrane. A prolonged exposure, instead, allows particles to dissolve in the extra and intracellular milieu. Extracellular release of Cu from particles contributed for the significant decrease in cell viability observed after 24h. At the same time point the intracellular dissolution of copper ions induced high levels of lipid peroxidation and was responsible for significant ultrastructural alterations. In conclusion this study contributed to improve the current knowledge of the (eco)toxicological profile of three widely used MONs: nTiO2, nCuO, nZnO; in particular, in both the models investigated, the key role played by particle themselves was highlighted. Nevertheless, due to the complexity of intrinsic variables and the plurality of potential interactions between MONs and the biological systems, harmonised protocols for testing toxicity of nanoparticles should be adopted and interlaboratory comparisons of results are suggested in order to define adequate guidelines for toxicity studies and to harmonise the production of new and safe materials.