Effect of\nButyrate on Food-Grade Titanium Dioxide\nToxicity in Different Intestinal In Vitro Models

Short-chain fatty acids (SCFA) are an important energy\nsource for\ncolonocytes and crucial messenger molecules both locally in the intestine\nand systemically. Butyrate, one of the most prominent and best-studied\nSCFA, was demonstrated to exert anti-inflammatory effects, improve\nbarrier integrity, enhance mucus synthesis in the intestine, and promote\ncell differentiation of intestinal epithelial cells in vitro. While\nthe physiological relevance is undisputed, it remains unclear if and\nto what extent butyrate can influence the effects of xenobiotics,\nsuch as food-grade titanium dioxide (E171, <sub>fg</sub>TiO<sub>2</sub>), in the intestine. TiO<sub>2</sub> has been controversially discussed\nfor its DNA-damaging potential and banned as a food additive within\nthe European Union (EU) since 2022. First, we used enterocyte Caco-2\nmonocultures to test if butyrate affects the cytotoxicity and inflammatory\npotential of <sub>fg</sub>TiO<sub>2</sub> in a pristine state or following\npretreatment under simulated gastric and intestinal pH conditions.\nWe then investigated pretreated <sub>fg</sub>TiO<sub>2</sub> in intestinal\ntriple cultures of Caco-2, HT29-MTX-E12, and THP-1 cells in homeostatic\nand inflamed-like state for cytotoxicity, barrier integrity, cytokine\nrelease as well as gene expression of mucins, oxidative stress markers,\nand DNA repair. In Caco-2 monocultures, butyrate had an ambivalent\nrole: pretreated but not pristine <sub>fg</sub>TiO<sub>2</sub> induced\ncytotoxicity in Caco-2 cells, which was not observed in the presence\nof butyrate. Conversely, <sub>fg</sub>TiO<sub>2</sub> induced the\nrelease of interleukin 8 in the presence but not in the absence of\nbutyrate. In the advanced in vitro models, butyrate did not affect\nthe characteristics of the healthy or inflamed states and caused negligible\neffects in the investigated end points following <sub>fg</sub>TiO<sub>2</sub> exposure. Taken together, the effects of <sub>fg</sub>TiO<sub>2</sub> strongly depend on the applied testing approach. Our findings\nunderline the importance of the experimental setup, including the\nchoice of in vitro model and the physiological relevance of the exposure\nscenario, for the hazard testing of food-grade pigments like TiO<sub>2</sub>.