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.

Differential response of immobile (pneumocytes) and mobile (monocytes) barriers against 2 types of metal oxide nanoparticles.

BACKGROUND: Inhaled nanoparticles (NPs) challenges mobile and immobile barriers in the respiratory tract, which can be represented by type II pneumocytes (immobile) and monocytes (mobile) but what is more important for biological effects, the cell linage, or the type of nanoparticle? Here, we addressed these questions and we demonstrated that the type of NPs exerts a higher influence on biological effects, but cell linages also respond differently against similar type of NPs. DESIGN: Type II pneumocytes and monocytes were exposed to tin dioxide (SnO2) NPs and titanium dioxide (TiO2) NPs (1, 10 and 50 µg/cm2) for 24 h and cell viability, ultrastructure, cell granularity, molecular spectra of lipids, proteins and nucleic acids and cytoskeleton architecture were evaluated. RESULTS: SnO2 NPs and TiO2 NPs are metal oxides with similar physicochemical properties. However, in the absence of cytotoxicity, SnO2 NPs uptake was low in monocytes and higher in type II pneumocytes, while TiO2 NPs were highly internalized by both types of cells. Monocytes exposed to both types of NPs displayed higher number of alterations in the molecular patterns of proteins and nuclei acids analyzed by Fourier-transform infrared spectroscopy (FTIR) than type II pneumocytes. In addition, cells exposed to TiO2 NPs showed more displacements in FTIR spectra of biomolecules than cells exposed to SnO2 NPs. Regarding cell architecture, microtubules were stable in type II pneumocytes exposed to both types of NPs but actin filaments displayed a higher number of alterations in type II pneumocytes and monocytes exposed to SnO2 NPs and TiO2 NPs. NPs exposure induced the formation of large vacuoles only in monocytes, which were not seen in type II pneumocytes. CONCLUSIONS: Most of the cellular effects are influenced by the NPs exposure rather than by the cell type. However, mobile, and immobile barriers in the respiratory tract displayed differential response against SnO2 NPs and TiO2 NPs in absence of cytotoxicity, in which monocytes were more susceptible than type II pneumocytes to NPs exposure.

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.

Astrocytes Are More Vulnerable than Neurons to Silicon Dioxide Nanoparticle Toxicity in Vitro.

Some studies have shown that silicon dioxide nanoparticles (SiO2-NPs) can reach different regions of the brain and cause toxicity; however, the consequences of SiO2-NPs exposure on the diverse brain cell lineages is limited. We aimed to investigate the neurotoxic effects of SiO2-NP (0-100 µg/mL) on rat astrocyte-rich cultures or neuron-rich cultures using scanning electron microscopy, Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR), FTIR microspectroscopy mapping (IQ mapping), and cell viability tests. SiO2-NPs were amorphous particles and aggregated in saline and culture media. Both astrocytes and neurons treated with SiO2-NPs showed alterations in cell morphology and changes in the IR spectral regions corresponding to nucleic acids, proteins, and lipids. The analysis by the second derivative revealed a significant decrease in the signal of the amide I (α-helix, parallel ß-strand, and random coil) at the concentration of 10 µg/mL in astrocytes but not in neurons. IQ mapping confirmed changes in nucleic acids, proteins, and lipids in astrocytes; cell death was higher in astrocytes than in neurons (10-100 µg/mL). We conclude that astrocytes were more vulnerable than neurons to SiO2-NPs toxicity. Therefore, the evaluation of human exposure to SiO2-NPs and possible neurotoxic effects must be followed up.

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.

Irreversible disruption of the cytoskeleton as induced by non-cytotoxic exposure to titanium dioxide nanoparticles in lung epithelial cells.

Exposure to TiO2 NPs induces several cellular alterations after NPs uptake including disruption of cytoskeleton that is crucial for lung physiology but is not considered as a footprint of cell damage. We aimed to investigate cytoskeleton disturbances and the impact on cell migration induced by an acute TiO2 NPs exposure (24 h) and the recovery capability after 6 days of NPs-free treatment, which allowed investigating if cytoskeleton damage was reversible. Exposure to TiO2 NPs (10 µg/cm2) for 24 h induced a decrease 20.2% and 25.1% in tubulin and actin polymerization. Exposure to TiO2 NPs (10 µg/cm2) for 24 h followed by 6 days of NPs-free had a decrease of 26.6% and 21.3% in tubulin and actin polymerization, respectively. The sustained exposure for 7 days to 1 µg/cm2 and 10 µg/cm2 induced a decrease of 22.4% and 30.7% of tubulin polymerization respectively, and 28.7% and 46.2% in actin polymerization. In addition, 24 h followed 6 days of NPs-free exposure of TiO2 NPs (1 µg/cm2 and 10 µg/cm2) decreased cell migration 40.7% and 59.2%, respectively. Cells exposed (10 µg/cm2) for 7 days had a decrease of 65.5% in cell migration. Ki67, protein surfactant B (SFTPB) and matrix metalloprotease 2 (MMP2) were analyzed as genes related to lung epithelial function. The results showed a 20% of Ki67 upregulation in cells exposed for 24 h to 10 µg/cm2 TiO2 NPs while a downregulation of 20% and 25.8% in cells exposed to 1 µg/cm2 and 10 µg/cm2 for 24 h followed by 6 days of NPs-free exposure. Exposure to 1 µg/cm2 and 10 µg/cm2 for 24 h and 7 days upregulates SFTPB expression in 53% and 59% respectively, MMP2 expression remain unchanged. In conclusion, exposure of TiO2 NPs affected cytoskeleton of lung epithelial cells irreversibly but this damage was not cumulative.

Differences in cytotoxicity of lung epithelial cells exposed to titanium dioxide nanofibers and nanoparticles: Comparison of air-liquid interface and submerged cell cultures.

Air Liquid Interface (ALI) system has emerged as a useful tool for toxicity evaluation of nanomaterials related to inhalation since the system mimics the aerosol exposure. We compared the biological responses of lung epithelial cells exposed to titanium dioxide (TiO2) nanofibers and nanoparticles in ALI and submerged cell cultures systems. Cells were exposed to 2 and 10 µg/cm2 for 24 h, 48 h and 72 h and LDH release, TiO2 internalization, DNA-double strand breaks (DSBs) and ROS production were assessed. LDH release was similar in both systems and particles had higher cytoplasmic uptake in submerged systems. Both TiO2 types were located in the cytoplasm but nanofibers had nuclear uptake regardless to the system tested. Cells exposed to TiO2 nanofibers had higher DSBs in the ALI system than in submerged cell cultures but cells exposed to TiO2 nanoparticles had similar DSBs in both systems. ROS production was higher in cells exposed to TiO2 nanofibers compared to cells exposed to TiO2 nanoparticles. In conclusion, cytotoxicity of lung epithelial cells was similar in ALI or submerged cell cultures, however cells exposed to TiO2 nanofibers displayed higher toxicity than cells exposed to TiO2 nanoparticles.

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.

Role of Wasp and the small GTPases RhoA, RhoB, and Cdc42 during capacitation and acrosome reaction in spermatozoa of English guinea pigs.

Cytoskeleton remodeling is necessary for capacitation and the acrosome reaction in spermatozoa. F-actin is located in the acrosome and equatorial region during capacitation, but is relocated in the post-acrosomal region during the acrosome reaction in spermatozoa from bull, rat, mice, and guinea pig. Actin polymerization and relocalization are generally regulated by small GTPases that activate Wasp protein, which coordinates with Arp2/3, profilin I, and profilin II to complete cytoskeletal remodeling. This sequence of events is not completely described in spermatozoa, though. Therefore, the aim of this study was to determine if Wasp interacts with small GTPases (RhoA, RhoB, and Cdc42) and proteins (Arp2/3, profilin I, and profilin II) that co-localize with F-actin during capacitation and the acrosome reaction in English guinea pig spermatozoa obtained from the vas deferens. The spermatozoa were capacitated in calcium-free medium, incubated with an activator or an inhibitor of GTPases, and then induced to acrosome react using calcium. The distribution patterns of F-actin were compared to the patterns of Wasp and its putative interaction partners: Wasp and RhoB, but not RhoA or Cdc42, localization overlap with F-actin during capacitation and the acrosome reaction. Activation of small GTPases localized RhoB to the post-acrosomal region whereas their inhibition prevented acrosome exocytosis. Arp2/3 and profilin II appear to interact with Wasp in the post-acrosomal region and flagellum, while profilin I and Wasp could be found in the equatorial region. Thus, Wasp and F-actin distribution overlap during capacitation and acrosome reaction, and small GTPases play an important role in cytoskeleton remodeling during these processes in spermatozoa. Mol. Reprod. Dev. 83: 927-937, 2016 © 2016 Wiley Periodicals, Inc.

Titanium dioxide nanoparticles induce an adaptive inflammatory response and invasion and proliferation of lung epithelial cells in chorioallantoic membrane.

Titanium dioxide nanoparticles (TiO2 NPs) studies have been performed using relatively high NPs concentration under acute exposure and limited studies have compared shape effects. We hypothesized that midterm exposure to low TiO2 NPs concentration in lung epithelial cells induces carcinogenic characteristics modulated partially by NPs shape. To test our hypothesis we synthesized NPs shaped as belts (TiO2-B) using TiO2 spheres (TiO2-SP) purchased from Sigma Aldrich Co. Then, lung epithelial A549 cells were low-exposed (10 µg/cm(2)) to both shapes during 7 days and internalization, cytokine release and invasive potential were determined. Results showed greater TiO2-B effect on agglomerates size, cell size and granularity than TiO2-SP. Agglomerates size in cell culture medium was 310 nm and 454 nm for TiO2-SP and TiO2-B, respectively; TiO2-SP and TiO2-B induced 23% and 70% cell size decrease, respectively, whilst TiO2-SP and TiO2-B induced 7 and 14-fold of granularity increase. NOx production was down-regulated (31%) by TiO2-SP and up-regulated (70%) by TiO2-B. Both NPs induced a transient cytokine release (IL-2, IL-6, IL-8, IL-4, IFN-γ, and TNF-α) after 4 days, but cytokines returned to basal levels in TiO2-SP exposed cells while TiO2-B induced a down-regulation after 7 days. Midterm exposure to both shapes of NPs induced capability to degrade cellular extracellular matrix components from chorioallantoic membrane and Ki-67 marker showed that TiO2-B had higher proliferative potential than TiO2-SP. We conclude that midterm exposure to low NPs concentration of NPs has an impact in the acquisition of new characteristics of exposed cells and NPs shape influences cellular outcome.