Effetto del siero bovino fetale sulla tossicità di nanoparticelle di TiO2 verso cellule intestinali umane (Caco-2)

The use of engineered nanoparticles (ENPs) in the food sector is anticipated to increase dramatically, whereas their potential hazards, especially for the gastrointestinal tract, are still largely unknown, as well as poorly investigated. Titanium dioxide (TiO2) micrometric powders, have a long-standing use as food additives, e.g. applied as whiteners in confectionary or dairy products. Conversely, in the last years, nanometric TiO2 powders have generated some concern since the International Agency of Research on Cancer (IARC) has recently reclassified it in group 2B (possibly carcinogenic to humans). The cellular responses to insoluble inorganic particulates are considered to be mainly mediated by the surface of particles as well as by their physico-chemical properties. However, recently the interaction of nanoparticles with the components of cellular media, in particular with proteins, has been shown to alter ENPs properties such as agglomeration state and surface charge and, consequently, their effect on cells. In the present study the effect of Fetal Calf Serum (FCS), which is commonly added to the cellular media, on the cytotoxicity of TiO2 ENPs on a cell line derived from the human colon epithelium (Caco-2) has been evaluated. The presence of FCS may affect the effects of TiO2 ENPs in two different ways: (1) FCS will improve cell viability, growth and division and serves as a pool of extracellular antioxidants that may offer enhanced protection of the cells to toxic effects of test compounds; (2) FCS will provide large extracellular pool of proteins and other factors which may modify various physico-chemical properties of particles, leading to altered surface activity, altered agglomeration and, possibly, altered internalization into the cells after treatment. To address this issue four types of TiO2 ENPs and one micrometric powders have been prepared and characterized and have tested with or without FCS for their toxicity toward Caco-2 cells. The cytotoxicity has been evaluated by two different assays (LDH and WST-1), whereas as markers of oxidative stress and inflammation, intracellular GSH level and pro-inflammatory IL-8 cytokine release and the mRNA expression of the genes that encode IL-8 and γ-GCS, the rate limiting enzyme of GSH synthesis, have been used. The data obtained from this study confirm that TiO2 NPs, whatever the crystalline phase, have an overall poor cytotoxic effect on Caco-2 cells and the observed toxicity is not mediated by oxidative stress or pro-inflammatory effects, but should be ascribed therefore to alternative mechanisms. Furthermore the presence of FCS appears to modulate the toxicity of NPs. This needs to be considered during the set-up of protocols for in vitro testing of TiO2 NPs. Finally a distinctive behavior of P25 samples has been confirmed. Surface features related to the synthetic method of this sample are likely responsible of the higher toxicity more than a mere coexistence of the anatase and rutile phases in the same sample.