Food-grade titanium dioxide translocates across the oral mucosa in pigs and induces genotoxicity in an in vitro model of human oral epithelium

Abstract

Background Food-grade titanium dioxide (TiO2), composed of nano- and submicron-sized particles, is used worldwide in various foodstuffs, toothpastes and pharmaceutical tablets as a whitening and opacifying agent. Its use as a food additive (E171 in EU) has raised concerns for human health regarding its systemic availability, tissue accumulation, genotoxicity and promotion of precancerous lesions. However, although the buccal mucosa is the first area exposed, oral transmucosal passage of TiO2 particles has not been documented. Here we analyzed TiO2 (E171) particle translocation in vivo through the pig buccal mucosa and in vitro on human buccal TR146 cells, and the effects of E171 on proliferating and differentiated human oral epithelial cells. Results Using transmission electronic microscopy (TEM) coupled to energy-dispersive X-ray spectroscopy (EDX), isolated TiO2 particles and small aggregates were observed in the buccal floor of pigs starting 30 min after the sublingual deposition of E171 suspended in water, and recovered in the submandibular lymph nodes at 4 h. In human TR146 cells exposed to E171, kinetic analyses using confocal microscopy, TEM and high-resolution secondary ion mass spectrometry (SIMS) imaging showed high uptake capacities of both the nano- and submicron-sized TiO2 particles. At 2 h, the cytotoxicity, genotoxicity and oxidative stress were investigated in both proliferating and differentiated TR146 cells exposed to E171 in comparison with two TiO2 size standards of 115 and 21 nm in diameter. All TiO2 samples were reported cytotoxic in proliferating cells, an effect almost abolished following differentiation. Genotoxicity (γH2AX or 53BP1 foci formation and comet assays) and oxidative stress (CellRox reagent) were only reported for the E171 and 115 nm TiO2 particles, and mainly in proliferating cells. Conclusions These data showed that the buccal mucosa is an important absorption route for the systemic passage of food-grade TiO2 particles. In human cells, TiO2 particles are cytotoxic and generate size-dependent oxidative and genotoxic stresses in proliferating cells, potentially impairing oral epithelium renewal. Altogether, these data emphasize that buccal exposure should be considered during toxicokinetic studies and for risk assessment of TiO2 in human when used as food additive, including in toothpastes and pharmaceutical formulations.