Evaluation of the cytotoxic and cellular proteome impacts of food-grade TiO2 (E171) using simulated gastrointestinal digestions and a tri-culture small intestinal epithelial model

Engineered nanomaterials (ENMs) are widely used in the food industry; however, regulations for ENMs in food are still in the early stages of development due to insufficient health data. This study investigated the cytotoxicity and changes to the proteomic profile in an in vitro small intestinal epithelium model after exposure to digested food models containing the ubiquitous engineered particulate food additive, TiO2 (E171) with an average size around 110 nm. TiO2 at 0.75% or 1.5% (w/w) concentrations in either a fasting food model (FFM) or a standardized food model (SFM) based on American diet were digested using an in vitro oral-gastric-small intestinal simulator, and the resulting digestas were applied to a small intestinal epithelium tri-culture cellular model. Effects on cell layer integrity, cytotoxicity, and oxidative stress were assessed. In order to explore the impact on cellular processes beyond basic cytotoxicity, mass spectrometry-based quantitative proteomic analyses of control and exposed tri-culture cells was performed. TiO2 in FFM, but not in SFM, produced significant, dose-dependent cytotoxicity (24%, p < 0.001), and at the higher dose caused significant oxidative stress (1.24-fold, p < 0.01), indicative of a food matrix effect. No significant perturbations of the cellular proteome were observed with TiO2 in either FFM or SFM food models. However, proteins involved in energy metabolism and protein synthesis were up-regulated by digestas from SFM compared to those from FFM, indicative of a food matrix effect on the cellular proteome. Interestingly, the differences in profiles between the two food models were more pronounced in the presence of TiO2. Together, these results indicate that TiO2 in a fasting diet may be slightly cytotoxic, and that ingested TiO2 does not significantly alter the epithelial proteome, whereas the food matrix alone can have a dramatic effect on the proteome.