Cellular and Molecular Mechanisms of Toxicity of Ingested Titanium Dioxide Nanomaterials.

An exponential increase in products containing titanium dioxide nanomaterials (TiO2), in agriculture, food and feed industry, lead to increased oral exposure to these nanomaterials (NMs). Thus, the gastrointestinal tract (GIT) emerges as a possible route of exposure that may drive systemic exposure, if the intestinal barrier is surpassed. NMs have been suggested to produce adverse outcomes, such as genotoxic effects, that are associated with increased risk of cancer, leading to a concern for public health. However, to date, the differences in the physicochemical characteristics of the NMs studied and other variables in the test systems have generated contradictory results in the literature. Processes like human digestion may change the NMs characteristics, inducing unexpected toxic effects in the intestine. Using TiO2 as case-study, this chapter provides a review of the works addressing the interactions of NMs with biological systems in the context of intestinal tract and digestion processes, at cellular and molecular level. The knowledge gaps identified suggest that the incorporation of a simulated digestion process for in vitro studies has the potential to improve the model for elucidating key events elicited by these NMs, advancing the nanosafety studies towards the development of an adverse outcome pathway for intestinal effects.

Challenges of the Application of In Vitro Digestion for Nanomaterials Safety Assessment.

Considering the increase in the production and use of nanomaterials (NM) in food/feed and food contact materials, novel strategies for efficient and sustainable hazard characterization, especially in the early stages of NM development, have been proposed. Some of these strategies encompass the utilization of in vitro simulated digestion prior to cytotoxic and genotoxic assessment. This entails exposing NM to fluids that replicate the three successive phases of digestion: oral, gastric, and intestinal. Subsequently, the resulting digestion products are added to models of intestinal cells to conduct toxicological assays, analyzing multiple endpoints. Nonetheless, exposure of intestinal cells to the digested products may induce cytotoxicity effects, thereby posing a challenge to this strategy. The aim of this work was to describe the challenges encountered with the in vitro digestion INFOGEST 2.0 protocol when using the digestion product in toxicological studies of NM, and the adjustments implemented to enable its use in subsequent in vitro biological assays with intestinal cell models. The adaptation of the digestion fluids, in particular the reduction of the final bile concentration, resulted in a reduced toxic impact of digestion products.

Analysis of the Characteristics and Cytotoxicity of Titanium Dioxide Nanomaterials Following Simulated In Vitro Digestion.

Several metallic nanomaterials (NMs), such as titanium dioxide nanomaterials (TiO2), present beneficial properties with a broad range of innovative applications. The human population is exposed to TiO2, particularly by ingestion, due to its increasing use as a food additive and inclusion in dietary supplements and food packaging materials. Whether this oral exposure may lead to adverse local or systemic outcomes has been the subject of research, but studies have generated contradictory results, reflecting differences in the physicochemical properties of the TiO2 studied, effects of the surrounding matrix, and modifications during digestion. This work aimed to investigate the toxic effects of three different TiO2 NMs (NM-103, NM-103 and NM-105) on the gastrointestinal tract cells, Caco-2 and HT29-MTX-E12, after the use of the standardized static INFOGEST 2.0 in vitro digestion method to mimic human digestion of TiO2, contributing to hazard assessment. The results show that, for one of the digested TiO2 NMs studied (NM-105), a more pronounced toxicity occurs after exposure of HT29-MTX-E12 intestinal cells, as compared to undigested NM, concomitantly with subtle changes in characteristics of the NM. Thus, the inclusion of the digestion simulation in the safety evaluation of ingested NMs through in vitro bioassays can better integrate the modifications that NMs suffer in the organism. It is expected that such an approach will reduce uncertainties in the hazard assessment of ingested NMs for human health.