Validating Enteroid-Derived Monolayers from Murine Gut Organoids for Toxicological Testing of Inorganic Particles: Proof-of-Concept with Food-Grade Titanium Dioxide.

Human exposure to foodborne inorganic nanoparticles (NPs) is a growing concern. However, identifying potential hazards linked to NP ingestion often requires long-term exposure in animals. Owing these constraints, intestinal organoids are a promising alternative to in vivo experiments; as such, an in vitro approach should enable a rapid and reliable assessment of the effects of ingested chemicals on the gut. However, this remains to be validated for inorganic substances. In our study, a transcriptomic analysis and immunofluorescence staining were performed to compare the effects of food-grade TiO2 (fg-TiO2) on enteroid-derived monolayers (EDMs) from murine intestinal organoids to the known impacts of TiO2 on intestinal epithelium. After their ability to respond to a pro-inflammatory cytokine cocktail was validated, EDMs were exposed to 0, 0.1, 1, or 10 µg fg-TiO2/mL for 24 h. A dose-related increase of the muc2, vilin 1, and chromogranin A gene markers of cell differentiation was observed. In addition, fg-TiO2 induced apoptosis and dose-dependent genotoxicity, while a decreased expression of genes encoding for antimicrobial peptides, and of genes related to tight junction function, was observed. These results validated the use of EDMs as a reliable model for the toxicity testing of foodborne NPs likely to affect the intestinal barrier.

Chronic exposure to Cytolethal Distending Toxin (CDT) promotes a cGAS-dependent type I interferon response.

The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin produced by pathogenic bacteria causing major foodborne diseases worldwide. CDT activates the DNA Damage Response and modulates the host immune response, but the precise relationship between these outcomes has not been addressed so far. Here, we show that chronic exposure to CDT in HeLa cells or mouse embryonic fibroblasts promotes a strong type I interferon (IFN) response that depends on the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) through the recognition of micronuclei. Indeed, despite active cell cycle checkpoints and in contrast to other DNA damaging agents, cells exposed to CDT reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation in daughter cells. These mitotic phenotypes are observed with CDT from various origins and in cancer or normal cell lines. Finally, we show that CDT exposure in immortalized normal colonic epithelial cells is associated to cGAS protein loss and low type I IFN response, implying that CDT immunomodulatory function may vary depending on tissue and cell type. Thus, our results establish a direct link between CDT-induced DNA damage, genetic instability and the cellular immune response that may be relevant in the context of natural infection associated to chronic inflammation or carcinogenesis.

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

The whitening and opacifying agent titanium dioxide (TiO2) is used worldwide in various foodstuffs, toothpastes and pharmaceutical tablets. Its use as a food additive (E171 in EU) has raised concerns for human health. Although the buccal mucosa is the first area exposed, oral transmucosal passage of TiO2 particles has not been documented. Here we analyzed E171 particle translocation in vivo through the pig buccal mucosa and in vitro on human buccal TR146 cells, and the effects on proliferating and differentiated TR146 cells. In the buccal floor of pigs, isolated TiO2 particles and small aggregates were observed 30 min after sublingual deposition, and were recovered in the submandibular lymph nodes at 4 h. In TR146 cells, kinetic analyses showed high absorption capacities of TiO2 particles. The cytotoxicity, genotoxicity and oxidative stress were investigated in 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 but not following differentiation. Genotoxicity and slight oxidative stress were reported for the E171 and 115 nm TiO2 particles. These data highlight the buccal mucosa as an absorption route for the systemic passage of food-grade TiO2 particles. The greater toxicity on proliferating cells suggest potential impairement of oral epithelium renewal. In conclusion, this study emphasizes 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.