Oral exposure to food grade titanium dioxide (E171) induces intestinal and behavioural alterations in adult mice but limited effects in young mice.

BACKGROUND: Food-grade titanium dioxide (E171), a white colourant widely used in ultra-processed food products, has been banned in the European Union. However, its usage is still permitted in medicines, and in several other countries. The estimated intake of E171 in children is higher than in adults, which led us to hypothesise that E171 induces differential effects depending on age, with adult mice being the most susceptible due to age, despite the lower dose. AIM: To evaluate the effects of oral administration of E171 on intestinal permeability, ileum, and colon histology, and how these effects impact anxious and depressive behaviour in young and adult mice of both sexes. METHODS: Young and adult mice of both sexes C57BL/6 mice received 10 mg/kgbw E171/3 times per week for 3 months. E171 was administered orally in water by pipetting, while control groups only received drinking water, then intestinal permeability, histology and animal behaviour were analysed. RESULTS: E171 showed an amorphous shape, primary particles sized below 1 µm and anatase crystalline structure. Oral administration of E171 disrupted the intestinal permeability in adult male and female mice, but no effects were observed in young mice of both sexes. E171 promoted ileal adenoma formation in half of the adult female population, moreover hyperplastic crypts, and hyperplastic goblet cells at histological level in adult mice of both sexes. The colon presented hyperplastic goblet cells, hyperchromatic nuclei, increased proliferation and DNA damage in adult mice of both sexes. The anxiety and depressive behaviour were only altered in adult mice treated with E171, but no changes were detected in young animals of both sexes. CONCLUSIONS: Adult mice displayed higher susceptibility in all parameters analysed in this study compared to young mice of both sexes.

Effects of solvent extracted organic matter from outdoor air pollution on human type II pneumocytes: Molecular and proteomic analysis.

Outdoor air pollution is responsible for the exacerbation of respiratory diseases in humans. Particulate matter with an aerodynamic diameter ≤2.5 µm (PM2.5) is one of the main components of outdoor air pollution, and solvent extracted organic matter (SEOM) is adsorbed to the main PM2.5 core. Some of the biological effects of black carbon and polycyclic aromatic hydrocarbons, which are components of PM2.5, are known, but the response of respiratory cell lineages to SEOM exposure has not been described until now. The aim of this study was to obtain SEOM from PM2.5 and analyze the molecular and proteomic effects on human type II pneumocytes. PM2.5 was collected from Mexico City in the wildfire season and the SEOM was characterized to be exposed on human type II pneumocytes. The effects were compared with benzo [a] pyrene (B[a]P) and hydrogen peroxide (H2O2). The results showed that SEOM induced a decrease in surfactant and deregulation in the molecular protein and lipid pattern analyzed by reflection-Fourier transform infrared (ATR-FTIR) spectroscopy on human type II pneumocytes after 24 h. The molecular alterations induced by SEOM were not shared by those induced by B[a]P nor H2O2, which highlights specific SEOM effects. In addition, proteomic patterns by quantitative MS analysis revealed a downregulation of 171 proteins and upregulation of 134 proteins analyzed in the STRING database. The deregulation was associated with positive regulation of apoptotic clearance, removal of superoxide radicals, and positive regulation of heterotypic cell-cell adhesion processes, while ATP metabolism, nucleotide process, and cellular metabolism were also affected. Through this study, we conclude that SEOM extracted from PM2.5 exerts alterations in molecular patterns of protein and lipids, surfactant expression, and deregulation of metabolic pathways of type II pneumocytes after 24 h of exposure in absence of cytotoxicity, which warns about apparent SEOM silent effects.

Food grade titanium dioxide accumulation leads to cellular alterations in colon cells after removal of a 24-hour exposure.

Titanium dioxide food grade (E171) is one of the most used food additives containing nanoparticles. Recently, the European Food Safety Authority indicated that E171 could no longer be considered safe as a food additive due to the possibility of it being genotoxic and there is evidence that E171 administration exacerbates colon tumor formation in murine models. However, less is known about the effects of E171 accumulation once the exposure stopped, then we hypothesized that toxic effects could be detected even after E171 removal. Therefore, we investigated the effects of E171 exposure after being removed from colon cell cultures. Human colon cancer cell line (HCT116) was exposed to 0, 1, 10 and 50 µg/cm2 of E171. Our results showed that in the absence of cytotoxicity, E171 was accumulated in the cells after 24 of exposure, increasing granularity and reactive oxygen species, inducing alterations in the molecular pattern of nucleic acids and lipids, and causing nuclei enlargement, DNA damage and tubulin depolymerization. After the removal of E171, colon cells were cultured for 48 h more hours to analyze the ability to restore the previously detected alterations. As we hypothesized, the removal of E171 was unable to revert the alterations found after 24 h of exposure in colon cells. In conclusion, exposure to E171 causes alterations that cannot be reverted after 48 h if E171 is removed from colon cells.

Food-grade titanium dioxide decreases hematocrit and hemoglobin and increases compulsive-like behavior in male mice.

Food-grade titanium dioxide (E171) is widely used as a food additive, and it is known that after oral consumption, E171 is translocated into the bloodstream reaching the highest titanium level at 6 h. E171 is accumulated in some organs triggering toxicity, but the effects on the blood parameters after oral consumption have been less studied. Recently, evidence shows that oral exposure to E171 induces behavioral signs of anxiety and depression. The relation between blood alterations and psychiatric disorders has been previously demonstrated. However, the oral exposure to E171 effects on alterations in blood parameters and effects linked to alterations in animal behavior has not been explored. In this short communication, we aimed to investigate the effects of E171 on specific blood parameters (hematocrit, hemoglobin, number of erythrocytes, and leukocytes) and anxiety and compulsive-like behavior in males and females orally exposed to ~5 mg/kg for 4 weeks. The results showed that E171 decreased hematocrit and hemoglobin in male but not in female mice while leukocyte and erythrocyte count remained unaltered. Oral consumption of E171 decreased the levels of anxiety-like behavior in females but not in male mice, while compulsive-like behavior was increased in both male and female mice.

Food-grade titanium dioxide (E171) induces anxiety, adenomas in colon and goblet cells hyperplasia in a regular diet model and microvesicular steatosis in a high fat diet model.

Food-grade titanium dioxide (E171) is a white additive widely used in solid and liquid food products. There is still debate about E171 toxic effects after oral consumption since this additive is deposited in colon, liver, spleen, testis and brain. The consumption of E171 commonly occurs with Western diets that are characterized by a high fat content. Thus, E171 could worsen adverse effects associated with a high fat diet (HFD) such as anxiety, colon diseases and testicular damage. We aimed to evaluate the effects of E171 on anxiety-like behavior, colon, liver and testis and to analyze if the administration of a HFD could exacerbate adverse effects. E171 was administered at ~5 mg/kgbw by drinking water for 16 weeks and mice were fed with a Regular Diet or a HFD. E171 promoted anxiety, induced adenomas in colon, goblet cells hypertrophy and hyperplasia and mucins overexpression, but had no toxic effects on testicular tissue or spermatozoa in regular diet fed-mice. Additionally, E171 promoted microvesicular steatosis in liver in HFD fed-mice and the only HFD administration decreased the spermatozoa concentration and motility. In conclusion, E171 administration increases the number of adenomas in colon, induces hypertrophy and hyperplasia in goblet cells and microvesicular steatosis.

Food additives containing nanoparticles induce gastrotoxicity, hepatotoxicity and alterations in animal behavior: The unknown role of oxidative stress.

Food additives such as titanium dioxide (E171), iron oxides and hydroxides (E172), silver (E174), and gold (E175) are highly used as colorants while silicon dioxide (E551) is generally used as anticaking in ultra-processed foodstuff highly used in the Western diets. These additives contain nanosized particles (1-100 nm) and there is a rising concern since these nanoparticles could exert major adverse effects due to they are not metabolized but are accumulated in several organs. Here, we analyze the evidence of gastrotoxicity, hepatotoxicity and the impact of microbiota on gut-brain and gut-liver axis induced by E171, E172, E174, E175 and E551 and their non-food grade nanosized counterparts after oral consumption. Although, no studies using these food additives have been performed to evaluate neurotoxicity or alterations in animal behavior, their non-food grade nanosized counterparts have been associated with stress, depression, cognitive and eating disorders as signs of animal behavior alterations. We identified that these food additives induce gastrotoxicity, hepatotoxicity and alterations in gut microbiota and most evidence points out oxidative stress as the main mechanism of toxicity, however, the role of oxidative stress as the main mechanism needs to be explored further.

Toxicity of engineered nanomaterials with different physicochemical properties and the role of protein corona on cellular uptake and intrinsic ROS production.

The Organisation for Economic Co-operation and Development has listed thirteen engineered nanomaterials (ENM) in order to investigate their toxicity on human health. Silicon dioxide (SiO2) and titanium dioxide (TiO2) are included on that list and we added indium tin oxide (ITO) nanoparticles (NPs) to our study, which is not listed on OECD suggested ENM to be investigated, however ITO NPs has a high potential of industrial production. We evaluate the physicochemical properties of SiO2 NPs (10-20 nm), TiO2 nanofibers (NFs; 3 µm length) and ITO NPs (<50 nm) and the impact of protein-corona formation on cell internalization. Then, we evaluated the toxicity of uncoated ENM on human lung epithelial cells exposed to 10 and 50 µg/cm2 for 24 h. TiO2 NFs showed the highest capability to adsorb proteins onto the particle surface followed by SiO2 NPs and ITO NPs after acellular incubation with fetal bovine serum. The protein adsorption had no impact on Alizarin Red S conjugation, intrinsic properties for reactive oxygen (ROS) formation or cell uptake for all types of ENM. Moreover, TiO2 NFs induced highest cell alterations in human lung epithelial cells exposed to 10 and 50 µg/cm2 while ITO NPs induced moderated cytotoxicity and SiO2 NPs caused even lower cytotoxicity under the same conditions. DNA, proteins and lipids were mainly affected by TiO2 NFs followed by SiO2 NPs with toxic effects in protein and lipids while limited variations were detected after exposure to ITO NPs on spectra analyzed by Fourier Transform Infrared Spectroscopy.

International landscape of limits and recommendations for occupational exposure to engineered nanomaterials.

The increasing concern of possible adverse effects on human health derived from occupational engineered nanomaterials (ENMs) exposure is an issue addressed by entities related to provide guidelines and/or protocols for ENMs regulation. Here we analysed 17 entities from America, Europe and Asia, and some of these entities provide limits of exposure extrapolated from the non-nanosized counterparts of ENMs. The international landscape shows that recommendations are mostly made for metal oxide based ENMs and tonnage is one of the main criteria for ENMs registration, however, sub-nanometric ENMs are emerging and perhaps a novel category of ENMs will appear soon. We identify that besides the lack of epidemiological evidence of ENMs toxicity in humans and difficulties in analysing the toxicological data derived from experimental models, the lack of information on airborne concentrations of ENMs in occupational settings is an important limitation to improve the experimental designs. The development of regulations related to ENMs exposure would lead to provide safer work places for ENMs production without delaying the nanotechnology progress but will also help to protect the environment by taking opportune and correct measures for nanowaste, considering that this could be a great environmental problem in the coming future.

Food-grade titanium dioxide (E171) by solid or liquid matrix administration induces inflammation, germ cells sloughing in seminiferous tubules and blood-testis barrier disruption in mice.

Food-grade titanium dioxide labeled as E171 has been approved for human consumption by the Food and Drug Administration (USA) and by the European Union for five decades. However, titanium dioxide has been classified as a possible carcinogen for humans by the International Agency of Research in Cancer raising concerns of its oral intake and the translocation to bloodstream, which could disturb barriers such as the blood-testis barrier. There is evidence that titanium dioxide by intragastric/intraperitoneal/intravenous administration induced alterations on testosterone levels, testicular function and architecture, but studies of the E171 effects on the testicle structure and blood-testis barrier are limited. E171 is contained not only in foods in liquid matrix but also in solid ones, which can exert different biological effects. We aimed to compare the effects of E171 consumption in a solid matrix (0.1%, 0.5% and 1% in pellets) and liquid suspension (5 mg/kg body weight) on testis structure, inflammation infiltrate and blood-testis barrier disruption of male BALB/c mice. Results showed that none of the administration routes had influence on body weight but an increase in germ cell sloughing and the infiltrate of inflammatory cells in seminiferous tubules, together with disruption of the blood-testis barrier were similar in testis of both groups even if the dose received in mice in liquid matrix was 136 or 260 times lower than the dose reached by oral intake in solid E171 pellets in 0.5% E171 and 1% E171, respectively. This study highlights the attention on matrix food containing E171 and possible adverse effects on testis when E171 is consumed in a liquid matrix.

Titanium dioxide food additive (E171) induces ROS formation and genotoxicity: contribution of micro and nano-sized fractions.

Since 1969, the European Union approves food-grade titanium dioxide (TiO2), also known as E171 colouring food additive. E171 is a mixture of micro-sized particles (MPs) and nano-sized particles (NPs). Previous studies have indicated adverse effects of oral exposure to E171, i.e. facilitation of colon tumour growth. This could potentially be partially mediated by the capacity to induce reactive oxygen species (ROS). The aim of the present study is to determine whether E171 exposure induces ROS formation and DNA damage in an in vitro model using human Caco-2 and HCT116 cells and to investigate the contribution of the separate MPs and NPs TiO2 fractions to these effects. After suspension of the particles in Hanks' balanced salt solution buffer and cell culture medium with either bovine serum albumin (BSA) or foetal bovine serum, characterization of the particles was performed by dynamic light scattering, ROS formation was determined by electron spin/paramagnetic resonance spectroscopy and DNA damage was determined by the comet and micronucleus assays. The results showed that E171, MPs and NPs are stable in cell culture medium with 0.05% BSA. The capacity for ROS generation in a cell-free environment was highest for E171, followed by NPs and MPs. Only MPs were capable to induce ROS formation in exposed Caco-2 cells. E171, MPs and NPs all induced single-strand DNA breaks. Chromosome damage was shown to be induced by E171, as tested with the micronucleus assay in HCT116 cells. In conclusion, E171 has the capability to induce ROS formation in a cell-free environment and E171, MPs and NPs have genotoxic potential. The capacity of E171 to induce ROS formation and DNA damage raises concerns about potential adverse effects associated with E171 (TiO2) in food.