Effect of Titanium dioxide dietary particles on host-gut microbiota interactions: role of aryl hydrocarbon receptor and impact on the development of metabolic disorders and colon cancer. – TitADiet

The TitADiet project combined several approaches to study the effects of E171 at doses relevant to humans. The direct impact of E171 on the human gut microbiota was evaluated in vitro using a mini-bioreactor array (MBRA) system. The effects on microbial diversity and the production of microbial metabolites were measured. To explore the link between intestinal dysbiosis, E171 exposure, and the development of metabolic disorders, fecal titanium concentrations and the production of pro- or anti-inflammatory metabolites by the microbiota were analyzed in stool samples from obese or normal-weight children.

A second series of experiments was conducted in mice. Female mice (F0) were fed either a control diet or a diet containing E171 from gestation through lactation. After weaning, some of the offspring (F1) continued on the same diet as their mothers, while others received a high-fat diet, supplemented or not with E171, until postnatal day 150 (J150). In these offspring, intestinal inflammation, microbiota composition and function, metabolic status, and the development of preneoplastic colonic lesions were assessed. To study the role of the microbiota in the effects of E171, intestinal microbiota transfer approaches were performed. All these parameters were also determined in male mice fed a normal or high-fat diet and exposed only in adulthood to E171 for 115 days.

Finally, the direct effects of E171 on the intestinal epithelium were evaluated using enteroid-derived monolayers (EDMs) obtained from mouse intestinal organoids. EDMs were exposed to E171 for 24 hours, and the expression of genes involved in cell differentiation, genotoxicity, antimicrobial peptide production, epithelial permeability, oxidative stress, Toll-like receptors, and the inflammatory response (NFκB, chemokines) was studied by qPCR. Apoptosis and genotoxicity were assessed by immunofluorescence.

The results obtained using the EDM model show that the integrity of the intestinal epithelial barrier in terms of cell proliferation/differentiation, genotoxicity, innate defenses, and permeability was significantly affected after exposure to E171. These findings are consistent with in vivo data available in the literature and provide proof of concept that EDMs are a relevant tool for assessing the toxicity of inorganic (nano)particles on the intestinal epithelium.

In parallel, the use of an in vitro human gut microbiota culture model demonstrated that direct interaction between E171 and commensal bacteria is sufficient to induce dysbiosis. In obese children, higher fecal titanium levels were correlated with increased fecal flagellin, a biomarker of metabolic dysfunction. While these human data suggest a link between E171 exposure, dysbiosis, and metabolic disorders, causality remained to be established in animal models.

In male F1 mice on a normal diet, exposure to E171 induced mild intestinal inflammation and altered microbiota composition, notably with decreased production of AhR ligands. These changes were accompanied by glucose intolerance and fasting hyperinsulinemia, and were exacerbated by a high-fat diet. Microbiota transfer experiments showed that the metabolic disorders induced by E171 in male F1 mice on a normal diet were microbiota-dependent. In contrast, in female F1 mice on a normal diet, E171 reduced the production of both pro- and anti-inflammatory cytokines in the colon, while under a high-fat diet, intestinal and metabolic alterations were not worsened by the additive. Notably, due to a tissue fixation issue, it was not possible to assess the effect of E171 on the development of preneoplastic lesions. Furthermore, E171 exposure limited to adulthood in males led only to minor changes in the microbiota, without affecting immune or metabolic functions. These data highlight that exposure to E171 from in utero is crucial for the initiation and promotion of metabolic disorders observed exclusively in males.

In conclusion, our results in humans revealed a link between E171 exposure, dysbiosis, and metabolic dysfunction, while the mouse data demonstrated a causal role for the gut microbiota in the development of metabolic disorders. Altogether, these findings suggest that chronic exposure to E171 could be an environmental factor promoting the emergence of metabolic diseases in industrialized countries.

This project opens up numerous perspectives on scientific, regulatory, and societal levels. From a scientific standpoint, the validation of EDM models as relevant tools for assessing the toxicity of inorganic (nano)particles on the intestinal barrier represents a major methodological advance, enabling a reduction in the use of animal experimentation. The identification of a link between dysbiosis, metabolic disorders, and chronic exposure to E171, particularly during early exposure or when combined with a high-fat diet, highlights the importance of considering the nutritional environment and microbiota status in risk assessments by agencies such as Anses and EFSA. Our data support the need for epidemiological studies to further explore this link in humans. Given the increasing use of additives in processed foods, and in light of our results as well as other studies showing microbiota-dependent metabolic disorders after exposure to emulsifiers or sweeteners, it is essential to evaluate the impact of additive mixtures on the prevalence of metabolic diseases. The results of this project pave the way for new research into the mechanisms of action of E171, including the exploration of interactions between TiO₂, gut flora, and the immune system, as well as differences in susceptibility according to sex or the timing of exposure (prenatal versus adult). This work also suggests that the effects of the microbiota should become a criterion in health risk assessment protocols for food additives, which could lead to new recommendations from national and international food safety agencies.

On the regulatory front, the data obtained reinforce the legitimacy of the ban on E171 in the EU and provide a strong argument for a re-evaluation of its use in countries where it remains authorized, including in other sectors such as pharmaceuticals or cosmetics. Furthermore, the observed correlation between fecal titanium load and microbiota alteration in obese children underlines the need for specific nutritional recommendations for the most vulnerable populations, especially children and adolescents, to limit their exposure to this additive.

At the industrial level, these results encourage the development of safer and more innovative alternatives, stimulating research and innovation in the agri-food sector.

Finally, raising public awareness of the potential risks associated with food additives containing nanoparticles could foster changes in purchasing behavior and increase demand for transparency and food safety.

A conference presenting our preliminary results has been selected for the annual congress of the « Société Française de Nutrition (SFN) » during the « Journées Francophones de Nutrition » (JFN, Lille) on November 12, 2021.

Manufactured mineral particles are abundant in daily life products (e.g., cosmetics, textiles, building materials), including foodstuffs. Among them, Titanium dioxide (TiO2) is a common food additive used as whitening and brightening agent in confectionary, processed food, white sauces and icing. TiO2 is also used as antimicrobial agent in packaging in contact with food. In Europe, the use of food-grade TiO2 (referred as E171) is authorized without establishment of an acceptable daily intake. The European Food Safety Authority (EFSA) has estimated the daily exposure levels to food-grade TiO2 ranging from 0.9 to 10.4 mg/kg body weight (bw)/day in children, and 0.3 to 6.8 mg/kg bw/day in adults. Given a high-consumption level, evaluation of the consequences of chronic oral exposure to TiO2 particles has become a major public health issue. Growing concerns were related to their mixed composition of micro- and nanosized particulate matter, due to TiO2 nanoparticles displaying toxicity on intestinal cells, including oxidative stress, genotoxicity, and inflammation. However, the oral route is still poorly studied for TiO2 toxicity, and health authorities conclude the need to develop exposure studies to assess the health risks of food additives containing nanomaterials, mainly with the E171. From animal and in vitro studies, there is evidence that the food-grade TiO2 is able to interact with bacteria from the intestinal microbiota before crossing through the gut epithelial barrier and interacting with local immune cells, setting various deleterious effects for the host. Of importance to the current project, the gut microbiota plays a crucial role in several vital functions such as digestive, metabolic and immune functions, but is often neglected in food toxicology. As microbiota alteration (i.e., dysbiosis) is a critical factor in gastrointestinal and metabolic diseases such as colorectal cancer (CRC), obesity and inflammatory bowel diseases (IBD), sustained gut dysbiosis in response to daily ingestion of TiO2 particles through the diet could contribute to the susceptibility to these diseases.
In Western countries, the prevalence of obesity and CRC has risen in parallel, suggesting shared lifestyle-related risk factors. Among these factors, chronic ingestion of inorganic particulate matter from food additives has been proposed. We recently observed that chronic exposure to food-grade TiO2 induced micro-inflammation in the rat colon, and initiated as well as promoted the expansion of colonic pre-neoplastic lesions. Interestingly, our preliminary results in mice orally exposed to the same TiO2 sample for 60 days, or in rats for 100 days, pointed out an impaired capacity of the gut microbiota to metabolize tryptophan into aryl hydrocarbon receptor (AhR) ligands as well as a decreased intestinal AhR signaling pathways. AhR is a ligand-activated nuclear factor that plays key roles in modulating the immune response as well as intestinal and metabolic functions, and displays an intestinal tumor suppressor role. Importantly, our previous data clearly showed that impaired production of AhR ligands by the gut microbiota is involved in metabolic disorders and IBD pathogenesis, both are risk factors for CRC. Altogether, this suggests that imbalanced host-microbiota interactions in these diseases could be partially mediated by a defect of AhR-ligand production by the gut microbiota, which could be induce by chronic exposure to TiO2.
In this context, the aim of this project is to investigate the potential link between chronic oral exposure to TiO2 (E171) and the occurrence and/or the aggravation of metabolic disorders and CRC, by assessing the impact of TiO2 on AhR ligand production by the microbiota and its consequences on gut barrier function, immune response, metabolic homeostasis and initiation of colonic pre-neoplastic lesions.